Bioluminescence Fundamentals and Applications in Biotechnology - 2014

Advances in Biochemical Engineering/Biotechnology 144
Series Editor: T. Scheper
Gérald Thouand
Robert Marks Editors
Bioluminescence:
Fundamentals and
Applications in
Biotechnology–
Volume 1
144
Advances in Biochemical
Engineering/Biotechnology
Series editor
T. Scheper, Hannover, Germany
Editorial Board
S. Belkin, Jerusalem, Israel
P. M. Doran, Hawthorn, Australia
I. Endo, Saitama, Japan
M. B. Gu, Seoul, Korea
S. Harald, Potsdam, Germany
W. S. Hu, Minneapolis, MN, USA
B. Mattiasson, Lund, Sweden
J. Nielsen, Gothenburg, Sweden
G. N. Stephanopoulos, Cambridge, MA, USA
R. Ulber, Kaiserslautern, Germany
A.-P. Zeng, Hamburg-Harburg, Germany
J.-J. Zhong, Shanghai, China
W. Zhou, Framingham, MA, USA
For further volumes:

http://www.springer.com/series/10
Aims and Scope
This book series reviews current trends in modern biotechnology and biochemical
engineering. Its aim is to cover all aspects of these interdisciplinary disciplines,
where knowledge, methods and expertise are required from chemistry, biochem-
istry, microbiology, molecular biology, chemical engineering and computer
science.
Volumes are organized topically and provide a comprehensive discussion of

developments in the field over the past 3–5 years. The series also discusses new
discoveries and applications. Special volumes are dedicated to selected topics
which focus on new biotechnological products and new processes for their syn-
thesis and purification.
In general, volumes are edited by well-known guest editors. The series editor and
publisher will, however, always be pleased to receive suggestions and supple-
mentary information. Manuscripts are accepted in English.
In references, Advances in Biochemical Engineering/Biotechnology is abbreviated

as Adv. Biochem. Engin./Biotechnol. and cited as a journal.
Gérald Thouand Robert Marks
•
Editors
Bioluminescence:
Fundamentals and
Applications in
Biotechnology–Volume 1
With contributions by
Dan Close Elena Esimbekova Paul Dunlap Ludmila A. Frank

Jochen Klumpp Vasilisa V. Krasitskaya Valentina Kratasyuk
Martin J. Loessner Yuichi Oba Steven Ripp Gary Sayler
Darrin T. Schultz Osamu Shimomura Abby Smartt
Tingting Xu
123
Editors
Gérald Thouand
UMR CNRS 6144
University of Nantes
La Roche-sur-Yon
France
Robert Marks
National Institute for Biotechnology
in the Negev
Ben Gurion University
Beersheba
Israel
and
Nanyang Technological University

Singapore
Singapore
ISSN 0724-6145 ISSN 1616-8542 (electronic)

ISBN 978-3-662-43384-3 ISBN 978-3-662-43385-0 (eBook)
DOI 10.1007/978-3-662-43385-0
Springer Heidelberg New York Dordrecht London
Library of Congress Control Number: 2014943751
Ó Springer-Verlag Berlin Heidelberg 2014

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of
the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or
information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar
methodology now known or hereafter developed. Exempted from this legal reservation are brief
excerpts in connection with reviews or scholarly analysis or material supplied specifically for the
purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the
work. Duplication of this publication or parts thereof is permitted only under the provisions of
the Copyright Law of the Publisher’s location, in its current version, and permission for use must
always be obtained from Springer. Permissions for use may be obtained through RightsLink at the
Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
publication does not imply, even in the absence of a specific statement, that such names are exempt
from the relevant protective laws and regulations and therefore free for general use.
While the advice and information in this book are believed to be true and accurate at the date of
publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for
any errors or omissions that may be made. The publisher makes no warranty, express or implied, with
respect to the material contained herein.
Printed on acid-free paper
Springer is part of Springer Science+Business Media (www.springer.com)

Contents
Part I Fundamentals of Bioluminescence
Eco-Evo Bioluminescence on Land and in the Sea . . . . . . . . . . . . . . . 3

Yuichi Oba and Darrin T. Schultz
Biochemistry and Genetics of Bacterial Bioluminescence . . . . . . . . . . 37

Paul Dunlap
Part II Applications of Bioluminescence in Environment

and Security
Application of Enzyme Bioluminescence in Ecology . . . . . . . . . . . . . . 67

Elena Esimbekova, Valentina Kratasyuk and Osamu Shimomura
Detection of Organic Compounds with Whole-Cell Bioluminescent

Bioassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Tingting Xu, Dan Close, Abby Smartt, Steven Ripp and Gary Sayler
Part III Applications of Bioluminescence in Agriculture

and Bioprocess
Detection of Bacteria with Bioluminescent Reporter Bacteriophage . . . 155

Jochen Klumpp and Martin J. Loessner
Part IV Applications of Bioluminescence in Health
Application of Enzyme Bioluminescence for Medical Diagnostics . . . . 175

Ludmila A. Frank and Vasilisa V. Krasitskaya
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
v
Part I
Fundamentals of Bioluminescence
Eco-Evo Bioluminescence on Land
and in the Sea
Yuichi Oba and Darrin T. Schultz
Abstract This review discusses the evolution of bioluminescence organisms that

inhabit various environments based on the current understanding of their unique
ecologies and biochemistries. As shown here, however, there are still many
unanswered questions regarding the functions and mechanisms of biolumines-
cence, which should be investigated in further studies. To facilitate future research
in this field, we introduce our recent attempt, the bioluminescent organism DNA
barcode initiative. This genetic reference library will provide resources for other
scientists to efficiently identify unstudied bioluminescent organisms, focus their
biochemical and genetic research goals, and will generally promote biolumines-
cence as a field of scientific study.

Keywords Aposematism Coelenterazine Counter-illumination Cypridinid

luciferin DNA barcoding Ecology Evolution Symbiotic luminescence
Contents
1 Introduction.......................................................................................................................... 4
2 Function of Bioluminescence.............................................................................................. 4
2.1 Self-defense................................................................................................................. 4
2.2 Light Organ Lure........................................................................................................ 10
2.3 Intraspecific Communication ..................................................................................... 12
2.4 Camouflage ................................................................................................................. 12
2.5 Searchlight .................................................................................................................. 13
2.6 Functionless Bioluminescence.................................................................................... 14
3 Terrestrial Taxa ................................................................................................................... 14
3.1 On Land ...................................................................................................................... 14
Y. Oba (&) D. T. Schultz

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan
e-mail: oba@agr.nagoya-u.ac.jp
G. Thouand and R. Marks (eds.), Bioluminescence: Fundamentals and Applications 3

in Biotechnology–Volume 1, Advances in Biochemical Engineering/Biotechnology 144,
DOI: 10.1007/978-3-662-43385-0_1, Ó Springer-Verlag Berlin Heidelberg 2014
4 Y. Oba and D. T. Schultz
3.2 In the Air..................................................................................................................... 16

3.3 Underground: Why in Soil? ....................................................................................... 17
3.4 Fresh Water: Why so Rare? ....................................................................................... 17
4 Marine Taxa......................................................................................................................... 19
4.1 Intertidal Zone ............................................................................................................ 19
4.2 Coastal Water.............................................................................................................. 21
4.3 Midwater: Why so Species-Rich?.............................................................................. 23
4.4 Deep-Sea Benthos....................................................................................................... 26
5 Conclusion ........................................................................................................................... 27
6 Perspectives ......................................................................................................................... 28
References.................................................................................................................................. 29
1 Introduction
Bioluminescent organisms occur in a wide variety of habitats, from tropical jun-

gles to barren fields, from sunny beaches to dark caves, and from oceanic surface
waters to abyssal depths. Those species that are aquatic are largely confined to the
oceans whereas only a few occur in freshwater. Bioluminescent taxa have been
reported from bacteria to vertebrates, in fact from most branches on the tree of life.
However, some branches are species-rich, such as teleosts, crustaceans, cnidarians,
and coleopterans, whereas others contain few or, like plants, tetrapods, arachnids,
and lepidopterans, have no known bioluminescent representatives (Fig. 1). The
biological and ecological functions of bioluminescence vary from species to
species, but it seems likely that counter-illumination is predominant in marine
species, and aposematism is abundant in terrestrial species. Coelenterazine, a
substance involved in the luminescence reaction, is commonly used by disparate
marine taxa, whereas firefly luciferin is used only in the terrestrial elateroid bee-
tles. In this chapter, we review the ecology and evolution of luminous organisms,
and discuss the possible reasons why some functions, habitats, chemical substrates,
and taxa are preferred or avoided in the world of bioluminescence.
2 Function of Bioluminescence
2.1 Self-defense
Many different biological and ecological functions have been proposed for bio-
luminescence [1, 2], but the most parsimonious explanation is that it is used as an
antipredator defensive device. Here we categorize the various defensive reactions
by luminous organisms aimed at predators into three types: startling (or deterring),
misdirection (or distraction/obstruction of sight), and aposematism (warning
display).
Eco-Evo Bioluminescence on Land and in the Sea 5
Fig. 1 The diversity of bioluminescent organisms as proportions of the named 746 genera. The
fraction indicates the number of terrestrial genera relative to the total number of genera. Numbers
are based on Herring [1] and the supplemental material in Haddock et al. [2]. The genera not
certain to be luminous in Herring [1], and ‘‘doubtfully’’ luminous genera of sponge and bryzoan
[2] are excluded. Numbers of genera in some groups are modified according to recent
publications: Bacteria [139], Lampyridae [173], Phengodidae [8], Rhagophthalmidae [174],
Gastropoda [109], Oligochaeta [102], Homalisidae (Omalisidae) [81], and Fungi [97]. Nematode
Heterorhabditis is excluded because symbiotic luminous bacteria Photorhabdus does not emit
visible light in the nematode [100]. For compatibility with the list by Herring [1], the
classification of Division and Phylum is followed in the systematics by Parker [95]
Sudden flashes in dark surroundings have been shown to startle, deter, and stun
or temporarily blind predators regardless of the prey’s palatability. For example,
the mesopelagic squid, Taningia danae, uses its arm-tip photophores to flash in
bursts while attacking bait rigs [3]. Moreover, in the firefly squid Watasenia
scintillans (Fig. 2), the nervous-system controlled arm-tip photophores are thought
to flash in bursts when attacked by predators [4, 5]. This seems to be similar to
cases in which the eye-spots on the surfaces of the hind wings of some Lepidoptera
startle predators by their sudden exposure [6], an example of Batesian mimicry in
which reflective owl eyes are the model. However, startling bioluminescence is
probably not Batesian mimicry, as it does not imitate any predator models. The
exceptions include some phengodid beetle larviform female adults, and the adults
of click beetles. The female phengodids Diplocladon and Rhagophthalmus curl
their bodies around their eggs to protect the brood, and when disturbed emit light
from small circular photophores lining the body (Fig. 3).
Fig. 2 Firefly squid Watasenia scintillans collected in Toyama Bay, Japan. This picture shows
the interspersed photophores on the ventral side of the mantle that face down as the creature
swims. The larger light organ on the tip of arms is assumed to be used for stunning predators.
Photo courtesy of So Yamashita
Fig. 3 Luminescence of the phengodid beetle Rhagophthalmus ohbai female adult at Ishigaki
Island, Japan. Attracting a male (left) and after copulation (right)
Ohba [7] hypothesized that the light emission may function to startle predators
by mimicking the shining eyes of nocturnal birds. The adults of the luminous click
beetles (e.g., Jamaican Pyrophorus species) possess a pair of oval photophores at
the outward-angled posterior margins of the prothorax [8], and the luminescence
looks like the shining eyes of nocturnal animals found in forests at night (VB
Meyer-Rochow, personal communication). Thus, the luminescence of phengodid
and click beetles may be an example of a startling function evolved in the presence
of a sympatric predator model.
Fig. 4 Coastal ostracod

Vargula hilgendorfii
collected at Seto Inland Sea,
Japan. This blue luminous
cloud was discharged upon
electro-stimulation. Photo
courtesy of Ken-ichi Onodera
Luminescent secretion clouds discharged by agitated bioluminescent organisms

in aphotic or very dim seawater are assumed to cause predators to lose sight of the
prey or become distracted while the creature escapes. This is similar to the squid,
octopus, and other cephalopod behavior of releasing a ‘‘smokescreen’’ of dark ink
to obscure its position from predators when disturbed. Some luminous squid,
shrimp, and ostracods (Fig. 4) discharge luminous clouds into the seawater, pri-
marily to act as a smoke screen against predators while making an escape [2].
Some luminous species discharge luminous droplets or mucus as decoys and flee
when attacked, for example, terrestrial centipedes [9], freshwater limpet Latia (see
Sect. 3.4), and copepods [10, 11]. These behaviors will make predators lose track
of the prey’s path, allowing it to escape safely. However, this strategy is some-
times indistinguishable from that of creating a smokescreen of luminous material
in marine environments.
Aposematism is a signal displayed to potential predators to warn of toxicity
and/or noxiousness. For example, firefly larvae and adults are both noxious and
toxic for various predators, including insects, centipedes, spiders, fish, amphibians,
reptiles, and mammals [12–16], thus a function of firefly luminescence has been
considered to be an aposematic display [17–19]. All known larvae of lampyrid
species are luminous, but the adults of some species are nonluminous [20].
Therefore, it seems that aposematism was a contributing factor in the evolution of
bioluminescence in the Lampyridae, and subsequently became a part of the
courtship behavior in adults [18, 21].
All known phengodid beetles are also luminous in at least the larval stages and
as adult females [8]. Grimaldi and Engel [22] suggested that their bioluminescence
functions as an aposematic signal in most species, but as a courtship signal in only
some species. A brownish substance discharged from Phrixothrix larva is
inflammatory to human skin upon contact [23], and Rhagophthalmus secretes a
caustic odor by stimulation [7, 24]. Toxicity and distastefulness have not been
reported in click beetles, including luminous species. Therefore, luminescence in
these species is not likely to be aposematic in function.
Fig. 5 Luminous millipede Paraspirobolus lucifugus collected in Okinawa, Japan. Blue-green

luminescence was elicited by stimulating with chloroform vapor
The function of the luminescence in millipedes and centipedes has been

interpreted as aposematism [25, 26]. The luminous millipedes Motyxia spp.
gradually intensify the glow of their entire bodies and discharge noxious cyanide-
containing secretions when stimulated [26, 27], and a species of Salpidobolus
similarly sprays caustic substances [28]. When the author stimulated the luminous
millipede Paraspirobolus lucifugus with forceps, the millipede emitted lumines-
cence from gaps between the body segments along with an unpleasant smell
(Fig. 5). Luminous secretions of the centipede Otostigmus aculeatus induced
erythema and blisters on human skin [29].
Aposematic coloration, as in the cases of poisonous and colorful butterflies of
the genus Heliconius, usually allows the evolution of palatable mimics (Batesian
mimicry) and/or unpalatable mimics (Müllerian mimicry) [30]. It has been known
that some cantharid and lycid beetles as well as other insects (at least 20 species
belonging to 11 families and four orders of insects) share color patterns with Papua
New Guinea lampyrids, Pteroptyx effulgens [31]. The mimicry involved in these
cases has been interpreted as a combination between Mertensian and Müllerian
mimicry; fireflies may usually be distasteful, but not lethal, whereas numerous
species especially those belonging to the family Lycidae are considerably more
toxic [18, 32]. However, none of firefly-mimics have yet evolved luminescence
properties, and there is the possibility that fireflies mimicked some lycids [31] (VB
Meyer-Rochow, personal communication).
Brightly colored mushrooms are sometimes poisonous: an aposematic display
[33]. Similarly, the persistent glow of bioluminescent mushrooms may be an
aposematic signal of unpalatability toward nocturnal fungivores. However, this
hypothesis has not been proven [34]. The Japanese luminous mushroom Omp-
halotus japonicus is toxic to humans, but the author (YO) has observed staphylinid
beetles frequently consuming this mushroom. On the Japanese island of Hachijo,
the luminous mushroom Mycena chlorophos is an economically important tourist
attraction, but the damage to fruiting bodies by land snails and ants is a problem
(Fig. 6).
Although aposematic luminescence occurs in many terrestrial species, this
strategy seems to be rare in the ocean. One probable example is the luminous
Fig. 6 Luminous fungus Mycena chlorophos fruiting body damaged by ants at nighttime in situ
on Hachijo Island, Japan. Photo courtesy of Masashi Naito
brittle star Ophiopsila (Ophiuroidea, Echinodermata) [35]. Found in shallow reefs,

the brittle star O. riisei is unpalatable for some potential predacious crabs, and
when disturbed by predators it flashes rapidly from its arm-tips. In repeated lab-
oratory trials, the crabs showed increases in avoidance behavior in response to
brittle star luminescence [36]. Wilson and Hastings mentioned in their book [37]
that ostracods are distasteful, thus the luminescence might also function as an
aposematic display. On the other hand, one study showed that ostracods comprised
3–6 % of the gut contents of gobiid fish collected at Odawa Bay of Japan [38],
which suggests that ostracods are palatable for some predators. Haddock et al. [2]
hypothesized that luminescence in cnidarians also plays a role as an aposematic
display, but it has not yet been demonstrated.
In our observations, the Japanese fireworm Odontosyllis released luminous
mucus when attacked by gobiid fish in the aquarium. Lastly, some specimens were
observed breaking off brightly luminescent posterior body segments. A similar
behavior was observed during rough handling (Fig. 7). We also observed that
some gobiid fishes regurgitated the fireworms, whereupon they began to luminesce
brightly. These observations suggest that Odontosyllis employs several of these
luminescent self-defense tactics: startling, decoy, and aposematism.
Fig. 7 Luminous fireworm Odontosyllis sp. after autotomy by stimulation. Detached tail tip
glows continuously. Specimens were collected in Toyama Bay, Japan. Photo courtesy of Masashi
Naito
2.2 Light Organ Lure
Deep-sea anglerfish, such as Himantolophus, are known to attract prey with a light
organ created by cultivating symbiotic luminous bacteria in the tips of their escas,
that is, the lure-like structure created from the modified first spine of the dorsal fin.
However, anglerfish have not been observed in situ luring prey using lumines-
cence. Using bioluminescence to attract prey has also been suggested for other
marine animals, such as stomiid dragonfish, Chiroteuthis squid, Stauroteuthis
octopus, and Siphonophores [2].
There are some hypotheses as to why prey are attracted to luminescence in the
ocean. One plausible explanation is that luminescent particles in the sea visually
indicate food. For example, deep-sea marine snow contains organic detritus and
fecal pellets that host glowing bacteria. Small zooplanktons and fry detect the
luminescence and consume the luminous material [39], and consequently, zoo-
plankton are attracted to the luminescence. Zarubin et al. [39] suggests that the
luminescence of nonsymbiotic bacteria enables them to find a host and to prop-
agate in the nutrient-rich intestines of zooplankton and fish. Previous studies
corroborate the high level of nonsymbiotic luminous bacteria in fecal pellets [40,
41].
The caves and riverbanks of Australia and New Zealand are home to larvae of
fungus gnats, Arachnocampa spp., that attract small phototactic insects with
luminescent posterior light organs into nonluminescent snares of vertical threads
(termed ‘‘fishing-lines’’) coated with mucous [42–44] (Fig. 8). A similar behavior
Fig. 8 Luminescence of the carnivorous fungus gnat Arachnocampa luminosa on a riverbank

near Auckland, New Zealand. Photo courtesy of So Yamashita
Fig. 9 Luminescence of spore-feeding fungus gnat Keroplatus nipponicus on Hachijo Island
was also reported from the North American fungus gnat Orfelia fultoni [45, 46].
Interestingly the larvae of fungus gnats of the genus Keroplatus (Fig. 9), are also
luminescent, despite that these species are likely to be spore-feeders [47]. The
luminescence function of Keroplatus is unclear [9].
Most fireflies do not feed in adult stages. By contrast, it is very unique and
exceptional that female adults of the North American fireflies Photuris and Bi-
cellonycha mimic flash patterns of other fireflies, for example, Photinus and
Pyractomena females, thereby attracting the males of the latter as prey [48].
Photuris cannot produce toxins by themselves, so they obtain the toxins, rather
than nutrition, from prey fireflies and use them for aposematic display [49–51].
Larvae of the luminous click beetle Pyrearinus termitilluminans reside in termite
mounds in Central America, and emit light from their prothorax to attract alate
ants and termites as prey [52].
One of the roles of luminescence in mushrooms may be the attraction of spore-
dispersing organisms. This hypothesis has been widely accepted for many years,
but has never clearly been demonstrated [34].
Luminescence intended for prey sometimes attracts predators instead. The cave
harvestmen Megalopsalis and Hendea possess prominent eyes to detect the
luminescence of larval Arachnocampa luminosa, which are their major prey [53].
It is hypothesized that some large-sized marine predators such as sperm whales
and leatherback turtles may use bioluminescent cues in their search for their food
([2], and references therein).
2.3 Intraspecific Communication
On land, it is well known that some adult fireflies use luminescence signals for
sexual communication between males and females [51, 54, 55]. Larviform females
of R. ohbai display their dorsal photophore toward males with continuous lumi-
nescence (Fig. 3), suggesting the function of luminescence is sexual communi-
cation [55]. The luminescence of the females of other phengodid beetles,
Dioptoma and Diplocladon, also functions to attract their respective males [54,
56]. The function of luminescence in luminous click beetles has also been sug-
gested as a means of sexual communication between males and females [4, 57].
Indeed, the male adults are sometimes attracted to artificial lights, including cig-
arettes [58]. The adults of the New Zealand fungus gnat Arachnocampa luminosa
may use luminescence for sexual communication [42, 59, 60]; this has been
challenged by Broadley [61], who provides evidence in support of pheromonal
attraction.
In the ocean, bioluminescence in some ostracods, including the genus Vargula
and the polychaete genus Odontosyllis has been suspected to possess a role in
sexual communication based on observations of their behavior in the wild [4, 10,
38]. Luminescence of certain shallow water fish, such as leiognathid ponyfish and
the flashlight fish Photoblepharon palpebratus, may function as a means of
intraspecific communication (for mating, dominance display, and/or schooling)
[62–64]. Based on some sexual dimorphism in photophores (light organs) it has
been suggested that various midwater organisms use luminescence for sexual
communication [65, 66].
2.4 Camouflage
Many organisms use bioluminescence to camouflage themselves in the open from

potential predators in a strategy called ‘‘counter-illumination.’’ This strategy is
most prevalent in the marine midwater, a depth down to which dim blue light still
penetrates.
In the open ocean it is rare to find objects to hide in or behind, so mesopelagic
animals become silhouetted against the seawater-penetrating sun and moonlight to
observers below. This silhouette is less advantageous for large animals, such as
Fig. 10 Midwater
hatchetfish Sternoptyx
pseudobscura, with ventral
photophores for counter-
illumination and large eyes
for finding the silhouettes of
prey by looking up. Photo
courtesy of Laboratory of
Marine Biology, Kochi
University
turtles, whales, and sharks. On the other hand, smaller animals are at risk of attack
for many predators look up to find prey (Fig. 10).
Dahlgren [67] first suggested that ventral light organs in luminous squid are
used to negate their silhouette by shining at ambient brightness levels. This
counter-illumination hypothesis has also been used to explain luminescence in
various luminous fish and shrimp; some of their downward-pointing light organs
were shown to dynamically match background light intensity in laboratory studies
[68, 69].
Counter-illumination is common in marine mesopelagic dwellers [70] but not
supported in terrestrial and freshwater species. The myriad shelters and hiding
spots on land as well as in the shallow or cloudy waters of rivers, ponds, and lakes
may in the course of evolution have led to alternatives to bioluminescence as a
survival strategy.
2.5 Searchlight
Species in three genera of deep-sea dragonfish, Malacosteus, Aristostomias, and

Pachystomias, emit red light from a pair of photophores under the eyes. This
unusual red, postorbital luminescence is considered to be akin to ‘‘night-vision’’
illumination. Because most deep-sea organisms are adapted to blue-light sensi-
tivity for dim, blue downwelling light and bioluminescence detection [71], they
are essentially blind to the red light projected by dragon fish. Indeed the vision of
Malacosteus niger is red light sensitive, so it can seek and find unsuspecting prey
[72, 73]. The anomalopid flashlight fish possess a pair of large suborbital light
organs under their eyes, for which Morin et al. [62] have suggested a function to
find and/or attract prey and Meyer-Rochow et al. [74] have reported an increased
light emission activity in Anomalops katoptron during feeding. However, it is
difficult to distinguish whether the photophores located near the eyes and the
mouth function biologically as decoys or searchlights.
2.6 Functionless Bioluminescence
Visible light from some organisms may not have any significance in itself. Bio-
luminescence is considered to be an energetically low-cost mechanism [75, 76],
and thus can potentially evolve as a metabolic byproduct unless the light emission
is disadvantageous. For example, it has been proposed that fungal luminescence is
a byproduct of some unknown metabolic process [34, 77]. Luminescence of
organisms emitting very weak light, such as the Japanese fungus gnat Keroplatus
nipponicus, or glowing underground, such as the annelid Microscolex phospho-
reus, may also be functionless.
3 Terrestrial Taxa
Because of its accessibility to observers, bioluminescence in terrestrial habitats has

received a great deal more attention ecologically and taxonomically than that of
aquatic habitats. Terrestrial environments are varied, and in the following we
discuss various luminous organisms’ life histories and physiologies related to their
habitats: land, air, underground, and freshwater.
3.1 On Land
Terrestrial luminous organisms comprise arthropods and fungi. One exception is

the world’s only known luminous terrestrial mollusk Quantula striata (syn. Dyakia
striata) of Southeast Asia [78]. These land snails, especially the young snails,
produce intracellular light on or near the mouthparts, but do not respond to dis-
turbance. The biological and ecological significance of the luminescence in these
land snails is uncertain, but it has been proposed to facilitate aggregation behavior
[79]. In the Plantae, no luminous species have been discovered. Schistostega
pennata (syn. Gymnostomum pennatum), generally called ‘‘luminous moss,’’ is not
autoluminescent: it appears to glow emerald green or gold on cave floors, but the
glow is actually a directional reflection of dim light by a particular cell structure of
the protonema [80]. No luminous species are known from tetrapods (amphibians,
reptiles, birds, and mammals), instead they can use many other remote sensory and
communication systems; sound, odor, vibration, color vision, UV, thermal radia-
tion, ultrasonic, and even electricity.
Among the arthropods, the Myriapoda and Hexapoda contain luminous species,
but no luminous species are found in another large arthropod subgroup that
includes spiders and scorpions, the Chelicerata. Given that the larvae of the fungus
gnat, Arachnocampa, lure positively phototactic prey into their curtains of sticky
vertical threads using luminescence, it seems strange that no luminous spider has
been reported to date.
In the subphylum Myriapoda, some millipedes (Diplopoda) and centipedes
(Chilopoda) emit light, with 24 known species altogether [25]. In the subphylum
Hexapoda, luminous species are only found in elateroid beetles (Coleoptera),
fungus gnats (Diptera), and springtails (Collembola) [9, 81].
Coleoptera are the largest order in the class Insecta, containing about 350,000
species in over 160 families worldwide [22]. Luminescent species of beetles are
found in three elateroid families: Lampyridae, Phengodidae, and Elateridae [81,
82]. All species of the Lampyridae and Phengodidae (roughly 2,000 and 200
species worldwide, respectively) are luminous, and of the approximately 10,000
elaterid species worldwide, approximately 200 are luminous [83].
The Diptera (true flies) are the fourth largest order in the Insecta, containing
150 families and 150,000 species [84]. Despite their huge diversity, only a small
number of luminous species (*15 species) are known from a single family, the
Keroplatidae [9]. The bioluminescence mechanism of Arachnocampa fungus gnats
is considered to be different from that of Orfelia [85], but the details are still
unknown.
In Collembola, luminescent species have been recognized in the families
Neanuridae and Onychiuridae [4, 56], but the observation records are limited. The
author (YO) observed the luminescence of Lobella sp. (Neanuridae), in which the
specimens emitted a continuous weak light from the body after stimulation with
forceps. The function of luminescence in Lobella sp. is uncertain but it seems too
faint to deter predators or to use for intraspecific communication [9].
Recently luminescence was claimed to occur in some South American cock-
roaches (Blattodea) [86, 87], but the veracity of these reports is highly dubious [88].
It is interesting that Lepidoptera, the second largest order in Insecta [22]
apparently possess no luminous species. Most adult moths are nocturnal and some
are poisonous; thus bioluminescence would seem a suitable tool for communica-
tion by moths. However, lepidopteran insects do not seem to have evolved any
bioluminescence and instead widely use pheromones for their nocturnal commu-
nications. It seems worth noting that some nonluminous fireflies also use phero-
mones for intraspecific communication, but in contrast to the moths, the beetles are
diurnal. In Hemiptera, the fifth largest insect order [22], the elongated head of the
lantern fly Fulgora laternaria in South America had long been believed to be
luminescent, but this has now become very doubtful [89].
Luminous species of the three elateroid beetle families use the same luciferin
molecule in their luminescence mechanism, (4S)-4,5-dihydro-2-(6-hydroxy-2-
benzothiazolyl)-4-thiazolecarboxylic acid, and a homologous luciferase sharing
over 48 % amino acid identity [90]. Phylogenetic relationships among these fam-
ilies have been studied in the context of the evolution of bioluminescence [18, 91],
but are still not fully resolved. Recent molecular analysis based on the mitochon-
drial genome revealed that Lampyridae and Phengodidae are sister groups in the
cantharoid beetles [92], suggesting a single origin of their luminescence. Although
Elateridae are also closely related to the Lampyridae–Phengodidae clade [92],
phylogenetic analyses of Elateridae showed that luminous click beetles are deeply
nested within the nonluminous species, thus bioluminescence may have arisen
independently in Elateridae [93, 94]. These results indicate that bioluminescence in
beetles may have evolved once or twice [81].
Bioluminescence mechanisms of the land snail Quantula, millipedes, centi-
pedes, fungus gnats, and springtails are still unknown in detail ([9], and references
therein), but probably are independent from each other. Therefore, this author
(YO) suspects that bioluminescence has evolved at least seven times among ter-
restrial organisms, including the case of luminous fungi described hereafter.
Luminous fungi are found only in the phylum Basidiomycota (Division
Eumycota, sensu Parker [95]), that is, the taxon of fungi that produces large,
apparent fruiting bodies referred to as ‘‘mushrooms’’ (approximately 32,000 spe-
cies [96]). About 70 species of luminescent fungi have been recognized world-
wide, and they belong to three different subclades: Omphalotus, Armillaria, and
the mycenoid fungi [97]. Although all fungal luminescence is intracellular, some
species glow only in the mycelia (e.g., Armillaria species), whereas others lumi-
nesce in both mycelia and fruiting bodies (e.g., O. japonicus and M. chlorophos).
Even though the bioluminescence mechanism of the luminous fungi is not fully
understood [98], the reaction mechanism is known to be identical in all species
[99]. The function of bioluminescence in fungi has long been questioned, and
remains unconfirmed experimentally.
Terrestrial luminescent bacteria of the genus Photorhabdus adhere to the
intestine of a Heterorhabditis nematode worm: the nematode burrows into its
insect host and regurgitates Photorhabdus into the hemocoel; the bacteria grow in
the insect body and finally kill the insect. The Nematode feeds exhaustively on the
bacteria or products of bacterial degradation of the hemolymph then leaves the
cadaver in search of a new host insect. The luminescence of Photorhabdus is
observed only when it grows in the insect cadaver. The ecological functions of the
cadaver luminescence has been uncertain, but one suggestion was that it may
represent an aposematic warning to nocturnal scavenging animals or act as a lure
to attract further insect prey [100].
3.2 In the Air
Some lampyrid beetles are well known for luminescing during flight, and are
rightly commonly called fireflies. However, not all luminous beetles are capable of
flight. Some phengodid (e.g., Rhagophthalmus) and lampyrid beetles (e.g.,
Lampyris) have luminous but flightless wingless females (Fig. 3) and are com-
monly called glow-worms, whereas the males are capable of flight (winged) but
are nonluminous or only very dimly luminescent [20, 55]. Male and female adults
of some lampyrid genera are able to fly but are nonluminous, such as Lucidota,
Pyropyga, and Pristolycus [101]. The manipulation of luminescence in some
winged fireflies, such as in Luciola and Photinus, is very sophisticated: they use
species-specific flash signals to recognize conspecific males and females [51, 82,
101]. All luminous click beetles are likely capable of flight as adults, and it has
been suggested that these males and females use the light for sexual communi-
cation while flying [57, 101].
3.3 Underground: Why in Soil?
In the Oligochaeta (earthworms and potworms), luminous species have been

reported from 16 genera worldwide [9, 102]. Why do these underground dwellers
produce light? The answer remains unclear, but it is considered to play a defensive
role against predators [102]. Luminous earthworms discharge luminescent mucus
from the mouth, the anus, and/or the body wall upon mechanical stimulation [102].
These earthworms are potentially palatable: M. phosphoreus was consumed by
various potential predators, such as an earwig, a spider, a mole cricket [103], a
topminnow, a newt, and a lizard (Oba, unpublished). The New Zealand giant
luminous earthworm Octochaetus multiporus was eaten by a kiwi bird without ill
effect (VB Meyer-Rochow cited in [102], and personal communication);
M. phosphoreus captured by a centipede (Lithobiidae gen. sp.) was observed at
nighttime in nature (Fig. 11; H Yoshida, personal communication).
These observations indicate that the light of luminous earthworms may be to
startle, but not to aposematically warn predators in the darkness underground.
Sivinski and Forrest [103] fed M. phosphoreus to mole crickets, an underground-
dweller that feeds on earthworms. A mole cricket immediately captured the worm,
but dropped the worm from its grasp and drew away when the worm discharged
luminous mucus. Finally, this mole cricket attacked the worm again and consumed
it, but this observation suggests that the luminescence of earthworms may provide
the prey with some opportunity to escape from the predator.
Pupae of some lampyrid species, such as Luciola, produce underground mud
cocoons and emit a dim green light from their entire body. When disturbed, they
also emit an intense yellow light from their ventral photophore (Fig. 12). The
biochemical mechanism of the dim glow in lampyrid pupae is understood [104],
but the biological advantage of the dual colored luminescence remains unknown.
3.4 Fresh Water: Why so Rare?
Compared with the rich diversity of luminous organisms in the sea, luminous
freshwater dwellers are quite rare. Fish and crustaceans make up a large per-
centage of the list of luminous organisms [1, 105], but there are apparently no
luminescent freshwater fish and crustacean species.
The limpet Latia neritoides (Latiidae, Mollusk) found in streams of New
Zealand’s North Island is the world’s only luminous freshwater gastropod species.
Fig. 11 Luminous earthworm Microscolex phosphoreus, attacked by the lithobiid centipede at

night. Photo courtesy of Hiroshi Yoshida
Fig. 12 Luminescence of the

Japanese firefly Luciola
lateralis male pupa. The light
organ on the abdominal
segments VI and VII emits
yellow light upon stimulation.
The rest of the body
continuously emits green
light. Two independent
luciferases are involved in
this phenomenon
This small basommatophoran snail clings to the surface of submerged stones

(Fig. 13) and discharges green-yellow, brightly luminescent mucus when dis-
turbed. Meyer-Rochow and Moore [106] reported that the luminescent mucus was
released into the stream when the limpet was attacked by predators, such as fish,
Fig. 13 Luminous freshwater snail Latia neritoides discharging luminescent mucus by

stimulation at the river near Auckland, New Zealand (same as the place for Fig. 8). Photo
courtesy of So Yamashita
crayfish, or dragonfly larvae, and suggested ‘‘the predators may chase after
luminescent droplets washed away from Latia instead of attacking Latia itself’’.
Most of the lampyrid species are terrestrial for all phases of their lifecycle, but
only a few Luciola species (*10 species) are known to be aquatic during the
larval stage [107]. They adapt well to the aquatic environment where they locate
and overpower freshwater snails in order to feed on them. Most, but not all, of the
aquatic species’ larvae respire in water using tracheal gills [16, 107]. These aquatic
larvae project white glands from abdominal segments that emit a fetid smell,
suggesting that the luminescence of some aquatic larvae is also an aposematic
display to predators, such as freshwater goby and dragonfly larvae [14].
4 Marine Taxa
Although there are various terrestrial luminous organisms, they nevertheless

belong to only a few taxonomic groups. This contrasts with marine taxa, in which
luminous species are distributed across most of the large taxonomic groups, except
for diatoms, marine macro-algae, and sponges (see [2]). In total, about 80 % of the
genera containing luminous species are marine dwellers [1, 105].
4.1 Intertidal Zone
The intertidal area between the sea and land, called the littoral zone, is regularly
exposed to variable conditions: rain causes low salinity, and sunlight causes heat,
desiccation, strong UV/visible light, and high salinity. Only a few shellfish and an
earthworm are reported as luminous in this environmentally challenging zone.
Some tiny luminous gastropod snails, Angiola and Hinea (Planaxidae), can be
found under rocks in tide pools at low tide and on stony beaches [108–111]. The
luminous bivalve Pholas dactylus inhabits the shallow sublittoral zone of soft-rock
coasts, typically gneiss [4]. It is of interest that the luminous bivalves and
Fig. 14 Luminous marine

snail Angiola zonata at stony
beach of Hachijo Island,
Japan. These specimens were
found aggregated after
upturning the pictured stone
gastropod snails are found only in the harsh intertidal zone, given that there are
many thousands of related species that inhabit almost every depth of the ocean.
One possible exception is the eulimid gastropod snails Melanella luminosa, which
was found as a parasite on the scarlet sea cucumber at a depth of 10–238 m;
Marshall [112] has described its yellow glow.
In the snail Angiola zonata (syn. Angiola longispira) (Fig. 14), a patch on the
mantle (or hypobranchial gland, [109]) emits a weak blue-green light when the
animal is stimulated mechanically or exposed to high salinity ([108], S Yamashita,
personal communication). Houbrick [109] reported that all species in Angiola
appear to be nocturnal and suggested that the light in ‘‘Angiola species may have a
startling effect on predators’’. Deheyn and Wilson [111] suggested that an apo-
sematic function ‘‘is less probable considering the gregarious and cryptic behav-
iour’’ of planaxid snails. The author (YO) observed in aquaria that fry of the
intertidal teleost Kuhlia mugil consumed the soft bodies of A. zonata with no
apparent ill effect, suggesting the snail’s luminescence is not for aposematic dis-
play. In Pholas, luminescent mucus is discharged from the siphon when disturbed,
and as Harvey suggested, ‘‘the best guess is that the light masses scare away
predacious animals’’ [4], but the actual function remains uncertain [37].
The luminous earthworm Pontodrilus litoralis (syn. P. matsushimensis, P.
bermudensis) inhabits the littoral zone of sandy beaches. The function of biolu-
minescence in P. litoralis remains unclear, likewise in other luminous earthworms.
In Japan, this species is sometimes used as bait for fishing [113]. Furthermore, a
crab and goby immediately fed on living P. litoralis when introduced into aquaria,
and no apparent ill effects in the predators were observed after consuming the
luminescent earthworm, suggesting it is neither distasteful nor toxic for potential
predators. The author (YO) has observed carnivorous insects such as earwigs
(Dermaptera) and staphylinid beetles (Staphylinidae) occurring sympatrically with
P. litoralis. These observations suggest that the function of P. litoralis lumines-
cence might be to startle, but not as an aposematic display. On the other hand, as
luminous fluid is discharged only when P. litoralis specimens are severely stim-
ulated, such as being cut, squeezed, and/or beaten [9, 114], the startling hypothesis
will need to be verified.
4.2 Coastal Water
In the neritic zone, the number of luminescent species is relatively low, a reported
1–2 % of all coastal species [115]. However, each luminous species often occurs
in high density in its environment. This situation is in contrast to those in oceanic
waters where species diversity is very high, but population density of individual
species is usually low (see Sect. 4.3). Luminous species usually make up only a
small proportion of a phylum’s total number of species, but there are two
exceptions: the Cnidaria (jellyfish) and the Ctenophora (comb jelly). A large
proportion of the former phylum and nearly all of the latter are luminous [4], and
these two groups are common members of the coastal planktonic dwellers. As for
the biological function of luminescence in coastal dwellers, Morin [115] suggested
that these species primarily use fast flashes of luminescence to deter prey or
continuously glowing decoys to distract predators, giving the prey an opportunity
to escape.
Dinoflagellates (Division Chromophycota, sensu Parker [95]) are important
primary producers in coastal waters. Some of the free-living species are luminous,
and most of these are autotrophic and therefore live in shallow waters where they
can photosynthesize. The luminous dinoflagellate Noctiluca scintillans is an
obligate heterotroph, but it also lives in shallow water to feed on autotrophic
diatoms, other dinoflagellates, and even small zooplankton [37]. For the enzymatic
bioluminescence reaction, luminous dinoflagellates use open-chain tetrapyrrole
luciferin [116], which is structurally related to chlorophyll. Interestingly, luminous
krill (Euphausiidae) also use the tetrapyrrole luciferin, which is structurally very
similar to the luciferin of dinoflagellates [117, 118]. Accordingly, it is expected
that krill consume luminous dinoflagellates and chemically modify the dinofla-
gellate luciferin as a precursor to their own luciferin [119]. To date, other luminous
taxa that use dinoflagellate luciferin or its derivatives as a luminous substrate have
not been reported.
4.2.1 Cypridinid Luciferin
In ostracods, luminous species in the genera Vargula and Cypridina are coastal,
whereas species in the genus Conchoecia are midwater dwellers [120]. The two
former genera use cypridinid luciferin (also called Cypridina luciferin) as a bio-
luminescent substrate [98, 121], whereas Conchoecia use coelenterazine [98, 122].
These findings are consistent with the fact that no midwater fish are known that use
cypridinid luciferin as a bioluminescent substrate (see below).
Coastal fish with nonbacterial luminescence rely on cypridinid luciferin in
bioluminescence reactions [123, 124]. These types of luminous fish are found in
three distantly related families of the orders Perciformes and Batrachoidiformes
[123–128]: Parapriacanthus ransonneti (syn. P. beryciformes [129]) and Par-
apriacanthus dispar (Pempheridae); Pempheris ypsilychnus (Pempheridae [130]);
Fig. 15 In the marine coastal zone, cypridinid luciferin is biosynthesized by ostracods. It is

considered that some coastal fish consume the ostracods and obtain the cypridinid luciferin for
their own bioluminescence reactions. Photos courtesy of Ken-ichi Onodera (Vargula), and the
Laboratory of Marine Biology of Kochi University (Parapriacanthus and Apogon)
Apogon ellioti (syn. Jaydia ellioti) and Apogon poecilopterus (syn. Jaydia poe-
cilopterus) (Apogonidae); midshipman fish Porichthys spp. (P. notatus, myriaster,
and porosissimus; Batrachoididae); and presumably some Archamia and Rhabd-
amia species (Apogonidae).
It has been suggested that these coastal fish obtain cypridinid luciferin by
consuming Cypridina and Vargula ostracods [37]. In fact, ostracod specimens
have been discovered from the stomach of P. ransonneti ([131], Oba and Hiruta,
unpublished), and a nonluminous form of P. notatus recovered its luminescent
ability following injection of cypridinid luciferin [132]. Recently, the author (YO)
and colleagues demonstrated that cypridinid luciferin is biosynthesized from three
amino acids (Ile, Arg, Trp) in Vargula hilgendorfii (Fig. 4) and Cypridina noc-
tiluca [133–136]. These findings indicate that cypridinid luciferin produced by
coastal ostracods partly contributes to the species’ richness of luminous fish in the
coastal environment (Fig. 15). Indeed, cypridinid luciferin chemiluminesces
strongly in certain solvents, such as diglyme, dimethyl sulfoxide, and detergent-
water solution, without an enzymatic catalyst [137]. This high reactivity suggests
that cypridinid luciferin is potentially a good bioluminescent substrate by nature.
Therefore, it may not be difficult to evolve luciferase from an extant protein, if
organisms are able to obtain cypridinid luciferin by diet.
4.3 Midwater: Why so Species-Rich?
Previous research suggests that nearly all of the luminescent species known to
occur in the midwater of oceans. Beebe [138] estimated that based on a study in
Bermuda that luminous mesopelagic fish collected below a depth of 600 m rep-
resent 81 % of the families, 66 % of the species, and 97 % of all the individuals.
Calanoid copepods, krill, and Cyclothone fish represent a large part of total macro-
and microzooplanktonic marine biomass, and these taxa contain particularly many
luminous species [4].
Haddock et al. [2] listed hypotheses of why most luminescent taxa are ocean
dwellers: they suggested that the sea is favorable for the evolution of biolumi-
nescence because it is (1) a stable environment, (2) optically clear relative to other
aqueous habitats, (3) continuously dark or very dim, and (4) highly diversified
taxonomically with complex interspecific relationships. We suggest that there are
additional factors that favor the evolution or acquisition of bioluminescent capa-
bilities in marine organisms: namely ‘‘symbiotic luminescence’’ and ‘‘dietary
supply of coelenterazine’’.
4.3.1 Symbiotic Luminescence
Luminous bacteria are common in seawater, especially in temperate and coastal

oceans, but they are rarely found in freshwater or on land [139]. Accordingly,
marine animals constantly contact, ingest unintentionally, or feed on luminous
bacteria. Therefore, it is reasonable to assume that symbiotic luminescence could
have evolved independently several times in various fish and squid.
There are at least seven orders of fish and two orders of squid (orders Sepiolida
and Teuthida) that employ luminous bacteria in both coastal shallow water and
midwater [139–141] (Fig. 16).
Species of luminous bacteria found in symbiotic hosts living in shallow water
are different from those in midwater. Shallow-water fish, such as leiognathid po-
nyfish and pinecone fish, and bobtail squid Euprymna and Sepiola use Photo-
bacterium leiognathi, P. mandapamensis, Aliivibrio fischeri, A. ‘‘thorii,’’ or A.
wodanis (formerly Vibrio fischeri and V. logei [139, 142]). In contrast, midwater
species, such as chlorophthalmid and macrourid fish, predominantly use Photo-
bacterium kishitanii (formerly included in nonsymbiotic P. phosphoreum) [139–
141]. The symbiotic bacteria obtain essential nutrients from their hosts and begin
to luminesce less and less, or even die, when the host is starved [143].
Considering the advantages of bioluminescence for marine life forms, it is
surprising that only fish and squid evolved mutual symbioses with luminescent
bacteria. It has been suggested that the bioluminescence of one species of the
pelagic colonial tunicate Pyrosoma is due to bacterial symbiosis, but details are not
available [98].
Fig. 16 Luminous coastal

fish Acropoma japonicum
(Acropomatidae) harboring
the luminous bacteria
Photobacterium leiognathi
and P. mandapamensis, and
its ventral bioluminescence.
Photo courtesy of Laboratory
of Marine Biology of Kochi
University
4.3.2 Coelenterazine
Coelenterazine was originally named for the bioluminescent substrate found in

coelenterates (Cnidaria and Ctenophore), but the term is still used despite the wide
variety of marine taxa that use the same molecule for their bioluminescence
reactions [98]. Organisms that use coelenterazine or its derivatives for their bio-
luminescence are scattered at least in five phyla: the Cnidaria, Ctenophora, Mol-
lusca (e.g., the squid W. scintillans and Symplectoteuthis luminosa), Arthropoda
(e.g., ostracod Conchoecia, copepods, and the decapod shrimps), Chordata (e.g.,
myctophid lanternfish [98]), and putatively in the Echinodermata (the benthic
brittle star Amphiura filiformis [98]), Chaetognatha (the bathypelagic arrowworm
Caecosagitta macrocephala [144]), and the Radiolaria (solitary spumellarian
Thalassicolla sp. [145]; Phylum Sarcomastigophora, sensu Parker [95]). In mid-
water, fish of the family Myctophidae are very abundant, and Beebe [138] found in
study of Bermuda that approximately one-quarter of the fish species caught by net
below 600-m depth were in the Myctophidae. From the view of total specimen
quantity, the gonostomatid Cyclothone is extremely abundant in midwater [138,
146], and the involvement of coelenterazine in their luminescence has been sug-
gested [147]. On the other hand, there are no freshwater or terrestrial organisms
that use coelenterazine or its analogues in bioluminescence. This unusually
widespread and abundant recruitment of coelenterazine in marine bioluminescent
systems may be a key to understanding the species richness of marine luminescent
organisms, and pose questions as to its biosynthetic origin.
Some luminous animals that use coelenterazine in their luminescent systems are
not capable of biosynthesizing it. For example, the mysid shrimp Gnathophausia
ingens and the jellyfish Aequorea victoria lost their luminescence after a prolonged
period of a coelenterazine-free diet, but recovered luminescence when fed food
containing coelenterazine [148, 149]. These findings suggest that some luminous
organisms need a supply of coelenterazine from their diet for their luminescence
abilities. Thomson et al. [150] concluded that the luminous midwater shrimp
Systellaspis debilis is capable of coelenterazine biosynthesis on the basis of their
observation that the content of coelenterazine increased during the time in

development between newly laid eggs and just before hatching. As Shimomura
[98] mentioned, however, their analysis cannot eliminate the possibility that
coelenterazine is sequestered as an inactive form in the eggs and converted to
active coelenterazine during development; thus coelenterazine may not be bio-
synthesized de novo in the egg.
Buskey and Stearns [151] showed that the bioluminescence potential of the
marine calanoid Metridia longa was not reduced even after starvation for 3 weeks.
Furthermore, they demonstrated that the bioluminescence potential in M. longa
fully recovered within 10 h after luminescent substances were exhausted by
mechanical stimulation. As luminous copepods use coelenterazine for their
luminescence, these findings strongly suggested that M. longa can biosynthesize
coelenterazine. Recently, we demonstrated for the first time that coelenterazine is
biosynthesized in the midwater calanoid Metridia pacifica from three amino acids
(Tyr, Tyr, Phe) by feeding specimens stable isotope-labeled compounds and
analyzing the incorporation of the labeled compounds in coelenterazine by LC-
ESI-TOF/MS analysis [152].
Calanoids are one of the most abundant biomass components in the sea and are
important prey for marine carnivorous organisms [153], including luminous
‘‘coelenterazine-users,’’ such as chaetognaths, myctophid, and gonostomatid fish
[154]. Another luminous coelenterazine-user Thalassicolla is a unicellular radio-
larian, which is known to capture and ingest living copepods [155]. Unknown
copepods were found in the stomach of the myctophid lanternfish Diaphus watasei
(Oba, unpublished). Species of the genus Metridia are widely distributed in the
oceans and occur in Antarctic waters [156] as well as in the subarctic Pacific
Ocean [154], where Metridia pacifica is known as a substantial member of the
zooplankton biomass. In Toyama Bay in the southern Sea of Japan, M. pacifica
contributed 37 % (annual mean) to the total copepod biomass [157], and is one of
the major prey items for the luminous firefly squid W. scintillans (Fig. 2) [158].
Coelenterazine was also present in various nonluminous marine plankton-feeding
fish and shrimps but was not detected in benthic scavengers, such as lobsters and
crabs [98]. Taken together, it is conceivable that various marine luminous
organisms obtain coelenterazine by consuming M. pacifica and other copepods,
and for this reason, luminous organisms are abundant in the ocean, especially in
midwater (Fig. 17).
It has been known that coelenterazine exhibits chemiluminescence in various
substances, including egg yolk, BSA, and surfactants [98, 159], indicating that
coelenterazine has innate beneficial properties as a bioluminescent substrate.
Indeed, coelenterazine-type luciferases and photoproteins have been identified
from deep-sea shrimp Oplophorus, copepods, the sea pansy Renilla, and jellyfish,
but do not share significant amino acid sequence homologies [98]. This suggests
that luciferases or photoproteins using coelenterazine as a substrate or chromo-
phore have independently evolved from unrelated proteins several times in an
example of convergent evolution.
Fig. 17 In the marine midwater, coelenterazine is biosynthesized by copepods. It is considered

that various midwater organisms consume the copepods and obtain the coelenterazine for their
own bioluminescence reactions. Photos courtesy of Ken-ichi Onodera (Metridia), the Laboratory
of Marine Biology of Kochi University (Diaphus), the Uozu Aquarium (Watasenia), and Osamu
Shimomura (Aequorea)
4.4 Deep-Sea Benthos
Based on a recent study in the Bahamas, the benthic zone of the deep sea
(500–1,000 m depth) is not as species-rich in luminous organisms as the midwater
zone: less than 20 % of the collected species emitted light [160]. Johnsen et al.
[160] explain that visibility in benthic water is obstructed by particulate matter
stirred by a bottom current, which is in contrast to the optically clear midwater that
facilitates the evolution of bioluminescence [2].
In the deep-sea benthos, luminous species are found mostly in bamboo corals,
sea anemones, sea pens, sea cucumbers, and brittle stars [160]. Because it is
extremely difficult to observe natural behavior of the organisms, the functions of
their luminescence are still unclear. There are many bottom dwellers in crabs,
hermit crabs, lobsters, and isopods, but no luminous species have been reported in
these groups. An unidentified luminous sea anemone was found on the shell
inhabited by a hermit crab [160]. It is known that some sea anemones and hermit
crabs engage in a mutualistic symbiosis [161]. If the deep-sea hermit crab posi-
tively selects luminous sea anemones as a symbiont, the hermit crab should be
Fig. 18 The deep-sea

benthic rattail fish,
Coelorhynchus kamoharai.
The ventral midline glows
blue-green by harboring the
luminous bacterium
Photobacterium kishitanii
inside photophores there.
Photo courtesy of the
Laboratory of Marine
Biology of Kochi University
regarded as a luminous organism, as other taxa with luminous symbiotic bacteria

are themselves considered luminous organisms.
The rattail fish (Macrouridae) are a large family of teleosts that generally
inhabit deep-sea benthic habitats. Most of the species are luminous [162] and have
saclike photophores containing luminous bacterial cultures around the anus and
along the midline of the abdomen (Fig. 18).
The morphological features of the photophores are an important characteristic
for identifying species in the Macrouridae, but whether they use the light of their
ventral photophores for intraspecific recognition and communication in the dark is
unknown.
As the deep-sea benthos remains a largely unexplored frontier, the story of
bioluminescent life there is likely still hidden in the depths.
5 Conclusion
World-leading authorities of bioluminescence, including Newton Harvey

(1887–1959), as well as the Japanese pioneers, Sakyo Kanda (1874–1939) and
Yata Haneda (1907–1995), focused in their comprehensive works on species lists,
habitat, morphology, histology, physiology, biochemistry, biophysics, and evolu-
tion of the world’s luminous organisms, but scarcely worked on the biological
significance of the light [4, 56, 163]. This may be simply due to the difficulty of
assigning a definite biological function to the light [164]. However, in some cases
the luminescent functions of some organisms are exceptionally well identified. For
example, the flash signals of fireflies used for sexual communication [51, 54, 55],
the continuous glow of the cave glowworm Arachnocampa for prey attraction
[165], and the counter-illumination of various midwater dwellers [65] are all well
understood. Even in the case of fireflies, however, the functions of synchronous
flashing in South Asian species [166, 167], the weak luminescence in diurnal
species [55], and the glowing of eggs and pupae [104] are still not resolved. The
significance of bioluminescence has sometimes been presumed to be for self-
defense, because light emission can be produced by prodding or other forms of
physical stimulation, or the light may be for sexual communication given that
morphological variations of photophores between sexes are rather common. It is,
however, best to be wary of cursory postulations: for example, stimulation-induced
lights may be too weak to startle predators and photophore sexual dimorphism may
be involved in behaviors other than mating. Meanwhile, some luminous organisms
possess highly sophisticated light organs (equipped with lenses, color filters,
reflectors, diffusers), and are neuronally controlled [168], suggesting that the light
must have some biological significance, albeit poorly understood. Moreover, some
unique hypotheses such as ‘‘predatory counter-illumination’’ for luminous sharks
[169], and ‘‘luminescent burglar alarms’’ [115, 164] for luminous dinoflagellates
and luminous fungi ([170] and [34], respectively) may illuminate wealthiness of
bioluminescence when tested experimentally in future.
6 Perspectives
This chapter has presented an ecological and evolutionary overview of the many
divergent bioluminescent taxa found in the world. One important observation has
been the great diversity of bioluminescent substrates and enzymes that has arisen
during the evolution of the varied taxa. Moreover, the paths of convergent evo-
lution have yielded a wide variety of ecological behaviors that utilize
bioluminescence.
Although the luciferin structures of many organisms were resolved between the
1960s and 1980s, many luciferase or photoprotein genes remain unknown, such as
those of euphausiid krill Euphausia pacifica, the firefly squid W. scintillans
(Fig. 2), the midwater lanternfish of the family Myctophidae, the shallow water
fish Parapriacanthus and Porichthys, the earthworm Diplocardia longa, and the
freshwater limpet L. neritoides. Several taxa still have eluded identification of both
their luciferin and their luciferase, including the millipedes Paraspirobolus and
Motyxia, the polychaete Odontosyllis, and the brittle star Ophiopsila (summarized
in [98]). In the case of luminous fungi, enzymatic involvement in light production
is still unresolved [98, 171].
To facilitate future research of bioluminescent organisms, my group is pro-
ceeding with a DNA barcoding initiative to create a genetic reference library for
bioluminescent organisms, per Hebert et al. [172]. The database will provide a
resource for other scientists in the identification of luminous organisms with
unresolved bioluminescent systems. Given the many applications of biolumines-
cent systems using only a few well-characterized protein families, elucidating
currently unknown proteins, genes, and substrates will expand the toolkit of
molecular science and bioengineering. As there are likewise many species for
which the biological and ecological function of bioluminescence is poorly
understood or disputed in the literature, this same principle will apply to ecologists
who seek to study bioluminescent organisms.
Acknowledgments The authors thank Victor B. Meyer-Rochow (Hachijo Island Geothermal

Energy Museum and University of Oulu) for hints on the literature and for critical reading of the
manuscript; Osamu Shimomura (Institute for Advanced Research Academy of Nagoya Univer-
sity), Hiromitsu Endo (Kochi University), Naohide Nakayama (Kochi University), Ken-ichi
Onodera (Kochi University), Masashi Naito (Shizuoka University and Nagoya University), So
Yamashita (Hachijo Town Council), Osamu Inamura (Uozu Aquarium), and Hiroshi Yoshida
(Gose Industrial High School) for providing photographs.
References
1. Herring PJ (1987) Systematic distribution of bioluminescence in living organisms.

J Biolumin Chemilumin 1:147–163
2. Haddock SHD, Moline MA, Case JF (2010) Bioluminescence in the sea. Annu Rev Mar Sci
2:443–493
3. Kubodera T, Koyama Y, Mori K (2007) Observations of wild hunting behaviour and
bioluminescence of a large deep-sea, eight-armed squid, Taningia danae. Proc R Soc B
274:1029–1034
4. Harvey EN (1952) Bioluminescence. Academic Press, New York
5. Inamura O (1994) On the firefly squid (Hotaru-Ika no Hanashi). Uozu Aquarium, Uozu (in
Japanese)
6. Blest AD (1957) The function of eyespot patterns in the Lepidoptera. Behaviour
11:209–256
7. Ohba N (2004) Mystery of fireflies. Yokosuka City Mus, Yokosuka (in Japanese)
8. Costa C, Zaragoza-Caballero S (2010) Phengodidae LeConte, 1861. In: Leschen RAB,
Beutel RG, Lawrence JF (eds) Handbook of zoology, vol IV, Arthropoda: Insecta, Teilband
39, Coleoptera, Beetles, vol 2., Morphology and systematics. Walter de Gruyter, Berlin,
pp 126–135
9. Oba Y, Branham MA, Fukatsu T (2011) The terrestrial bioluminescent animals of Japan.
Zool Sci 28:771–789
10. Morin JG (1986) ‘‘Firefleas’’ of the sea: luminescent signaling in marine ostracode
crustaceans. Insect Behav Ecol 69:105–121
11. Herring PJ (1988) Copepod luminescence. Hydrobiologia 167(168):183–195
12. Underwood TJ, Tallamy DW, Pesek JD (1997) Bioluminescence in firefly larvae: A test of
the aposematic display hypothesis (Coleoptera: Lampyridae). J Insect Behav 10:365–370
13. Knight M, Glor R, Smedley SR, González A, Adler K, Eisner T (1999) Firefly toxicosis in
lizards. J Chem Ecol 25:1981–1986
14. Ohba N, Hidaka T (2002) Reflex bleeding of fireflies and prey-predator relationship. Sci
Rept Yokosuka City Mus 49:1–12 (in Japanese with English title and abstract)
15. De Cock R, Matthysen E (2003) Glow-worm larvae bioluminescence (Coleoptera:
Lampyridae) operates as an aposematic signal upon toads (Bufo bufo). Behav Ecol
14:103–108
16. Fu X, Vencl FV, Ohba N, Meyer-Rochow VB, Lei C, Zhang Z (2007) Structure and
function of the eversible glands of the aquatic firefly, Luciola leii (Coleoptera: Lampyridae).
Chemoecology 17:117–124
17. Lloyd JE (1973) Firefly parasites and predators. Coleopterists Bull 27:91–106
18. Sagegami-Oba R, Takahashi N, Oba Y (2007) The evolutionary process of bioluminescence
and aposematism in cantharoid beetles (Coleoptera: Elateroidea) inferred by the analysis of
18S ribosomal DNA. Gene 400:104–113
19. Long SM, Lewis S, Jean-Louis L, Ramos G, Richmond J, Jakob EM (2012) Firefly flashing
and jumping spider predation. Animal Behav 83:81–86
20. Branham MA, Wenzel JW (2003) The origin of photic behavior and the evolution of sexual
communication in fireflies (Coleoptera: Lampyridae). Cladistics 19:1–22
21. De Cock R, Matthysen E (1999) Aposematism and bioluminescence: experimental evidence
from glow-worm larvae (Coleoptera: Lampyridae). Evol Ecol 13:619–639
22. Grimaldi D, Engel MS (2005) Evolution of the insects. Cambridge University Press,
Cambridge
23. Sivinski J (1981) The nature and possible functions of luminescence in Coleoptera larvae.
Coleopterists Bull 35:167–179
24. Raj JS (1957) An undescribed luminous beetle larva from South India. J Bombay Natl Hist
Soc 54:788–789
25. Rosenberg J, Meyer-Rochow VB (2009) Luminescent myriapoda: a brief review. In: Meyer-
Rochow VB (ed) Bioluminescence in focus: a collection of illuminating essays. Research
Signpost, Kerala, pp 139–146
26. Marek P, Papaj D, Yeager J, Molina S, Moore W (2011) Bioluminescent aposematism in
millipedes. Cur Biol 21:R680–R681
27. Davenport D, Wootton DM, Cushing JE (1952) The biology of the Sierra luminous
millipede, Luminodesmus sequoiae, Loomis and Davenport. Biol Bull 102:100–110
28. Hudson BJ, Parsons GA (1997) Giant millipede ‘burns’ and the eye. Trans Roy Soc Trop
Med Hygiene 91:183–185
29. Houdemer ME (1926) Mote sur un Myriapode vésicant du Tonkin, Ostostigmus aculeatus
Haase. Bull Mus Hist Nat Paris 32:213–214 (in French)
30. Futuyma DJ (2005) Evolution. Sinauer, Massachusetts
31. Ohba N, Meyer-Rochow VB (2012) Insect species co-existing with the Papua New Guinea
firefly Pteroptyx effulgens share aspects of appearance and behaviour. Lampyrid 2:127–137
32. Crowson RA (1981) The biology of the Coleoptera. Academic Press, New York
33. Lev-Yadun S, Halpern M (2007) Ergot (Claviceps purpurea)—An aposematic fungus.
Symbiosis 43:105–108
34. Sivinski J (1981) Arthropods attracted to luminous fungi. Psyche 88:383–390
35. Mallefet J (2009) Echinoderm bioluminescence: where, how and why do so many
ophiuroids glow? In: Meyer-Rochow VB (ed) Bioluminescence in focus: a collection of
illuminating essays. Research Signpost, Kerala, pp 67–83
36. Grober MS (1988) Brittle-star bioluminescence functions as an aposematic signal to deter
crustacean predators. Anim Behav 36:493–501
37. Wilson T, Hastings JW (2013) Bioluminescence: living lights, lights for living. Harvard
University Press, Massachusetts
38. Abe K (1994) The light of marine fireflies. Chikuma Shobo, Tokyo (in Japanese)
39. Zarubin M, Belkin S, Ionescu M, Genin A (2012) Bacterial bioluminescence as a lure for
marine zooplankton and fish. Proc Natl Acad Sci USA 109:853–857
40. Andrews CC, Karl DM, Small LF, Fowler SW (1984) Metabolic activity and
bioluminescence of oceanic faecal pellets and sediment trap particles. Nature 307:539–541
41. Ruby EG, Morin JG (1979) Luminous enteric bacteria of marine fishes: a study of their
distribution, densities, and dispersion. Appl Environ Microbiol 38:406–411
42. Richards AM (1960) Observations on the New Zealand glow-worm Arachnocampa
luminosa (Skuse) 1890. Tran R Soc New Zealand 88:559–574
43. Broadley A, Stringer IAN (2009) Larval behaviour of the New Zealand glowworm,
Arachnocampa luminosa (Diptera: Keroplatidae), in bush and caves. In: Meyer-Rochow VB
(ed) Bioluminescence in focus: a collection of illuminating essays. Research Signpost,
Kerala, pp 325–355
44. Willis RE, White CR, Merritt DJ (2011) Using light as a lure is an efficient predatory
strategy in Arachnocampa flava, an Australian glowworm. J Comp Physiol B 181:477–486
45. Fulton BB (1941) A luminous fly larva with spider traits (Diptera, Mycetophilidae). Ann
Entomol Soc Am 34:289–302
46. Sivinski J (1982) Prey attraction by luminous larvae of the fungus gnat Orfelia fultoni. Ecol
Entomol 7:443–446
47. Matile L (1997) Phylogeny and evolution of the larval diet in the Sciaroidea (Diptera,
Bibionomorpha) since the Mesozoic. Mém Mus Natn Hist Nat 173:273–303
48. Lloyd JE (1975) Aggressive mimicry in Photuris fireflies: signal repertories by femmes
fatales. Science 187:452–453
49. Eisner T, Goetz MA, Hill DE, Smedley SR, Meinwald J (1997) Firefly ‘‘femmes fatales’’
acquire defensive steroids (lucibufagins) from their firefly prey. Proc Natl Acad Sci USA
94:9723–9728
50. Eisner T, Wiemer DF, Haynes LW, Meinwald J (1978) Lucibufagins: defensive steroids
from the fireflies Photinus ignitus and P. marginellus (Coleoptera: Lampyridae). Proc Natl
Acad Sci USA 75:905–908
51. Lewis SM, Cratsley CK (2008) Flash signal evolution, mate choice, and predation in
fireflies. Annu Rev Entomol 53:293–321
52. Redford KH (1982) Prey attraction as a possible function of bioluminescence in the larvae
of Pyrearinus termitilluminans (Coleoptera: Elateridae). Revta bras Zool S Paulo 1:31–34
53. Meyer-Rochow VB, Liddle AR (1988) Structure and function of the eyes of two species of
opilionid from New Zealand glow-worm caves (Megalopsalis tumida: Palpatores, and
Hendea myersi cavernicola: Laniatores). Proc R Soc B 233:293–319
54. Lloyd JE (1983) Bioluminescence and communication in insects. Ann Rev Entomol
28:131–160
55. Ohba N (2004) Flash communication systems of Japanese fireflies. Integ Comp Biol
44:225–233
56. Haneda Y (1985) Luminous organisms. Kouseisha-kouseikaku, Tokyo (in Japanese)
57. Stolz U, Velez S, Wood KV, Wood M, Feder JL (2003) Darwinian natural selection for
orange bioluminescent color in a Jamaican click beetle. Proc Natl Acad Sci USA
100:14955–14959
58. Hoffmann KH (1984) Environmental aspects of insect bioluminescence. In: Hoffmann KH
(ed) Environmental physiology and biochemistry of insects. Springer, Berlin, pp 225–245
59. Meyer-Rochow VB, Eguchi E (1984) Thoughts on the possible function and origin of
bioluminescence in the New Zealand glowworm Arachnocampa luminosa (Diptera:
Keroplatidae), based on electrophysiological recordings of spectral responses from the
eyes of male adults. New Zealand Entomol 8:111–119
60. Meyer-Rochow VB (1990) The New Zealand glowworm. Waitomo Caves Mus, Waitomo
Caves
61. Broadley RA (2012) Notes on pupal behaviour, eclosion, mate attraction, copulation and
predation of the New Zealand glowworm Arachnocampa luminosa (Skuse) (Diptera:
Keroplatidae), at Waitomo. N Zld Ent 35:1–9
62. Morin JG, Harrington A, Nealson K, Krieger N, Baldwin TO, Hastings JW (1975) Light for
all reasons: Versatility in the behavioral repertoire of the flashlight fish. Science 190:74–76
63. Meyer-Rochow VB (1976) Womit und warum Tiere leuchten. Selecta 10:972–974 (in
German)
64. Chakrabarty P, Davis MP, Smith WL, Berquist R, Gledhill KM, Frank LR, Sparks JS (2011)
Evolution of the light organ system in ponyfishes (Teleostei: Leiognathidae). J Morphol
272:704–721
65. Young RE (1983) Oceanic bioluminescence: an overview of general functions. Bull Mar Sci
33:829–845
66. Herring PJ (2007) Sex with the lights on? A review of bioluminescent sexual dimorphism in
the sea. J Mar Biol Ass UK 87:829–842
67. Dahlgren U (1916) Production of light by animals. J Franklin Inst 181:525–556
68. Young RE, Roper CFE (1977) Intensity regulation of bioluminescence during
countershading in living midwater animals. Fish Bull 75:239–252
69. Case JF, Warner J, Barnes AT, Lowenstine M (1977) Bioluminescence of lantern fish
(Myctophidae) in response to changes in light intensity. Nature 265:179–181
70. Widder EA (2010) Bioluminescence in the ocean: origins of biological, chemical, and
ecological diversity. Science 328:704–708
71. Hunt DM, Dulai KS, Partridge JC, Cottrill P, Bowmaker JK (2001) The molecular basis for
spectral tuning of rod visual pigments in deep-sea fish. J Exp Biol 204:3333–3344
72. Widder EA, Latz MI, Herring PJ, Case JF (1984) Far red bioluminescence from two deep-
sea fishes. Science 225:512–514
73. Douglas RH, Partridge JC, Dulai KS, Hunt DM, Mullineaux CW, Hynninen PH (1999)
Enhanced retinal longwave sensitivity using a chlorophyll-derived photosensitiser in
Malacosteus niger, a deep-sea dragon fish with far red bioluminescence. Vis Res
39:2817–2832
74. Meyer-Rochow VB, Baburina V, Smirnov S (1982) Histological observations on the eyes of
the two luminescent fishes Photoblepharon palpebratus (Boddaert) and Anomalops
katoptron (Blkr.). Zool Anz (Jena) 209:65–72
75. O’Kane DJ, Lingle WL, Porter D, Wampler JE (1990) Spectral analysis of bioluminescence
of Panellus stypticus. Mycologia 82:607–616
76. Woods WA Jr, Hendrickson H, Mason J, Lewis SM (2007) Energy and predation costs of
firefly courtship signals. Am Nat 170:702–708
77. Herring PJ (1994) Luminous fungi. Mycologist 8:181–183
78. Haneda Y (1963) Further studies on a luminous land snail, Quantula striata, in Malaya. Sci
Rept Yokosuka City Mus 8:1–9
79. Counsilman JJ, Ong PP (1988) Responses of the luminescent land snail Dyakia (Quantula)
striata to natural and artificial lights. J Ethol 6:1–8
80. Noll F (1888) Über das Leuchten der Schistostega osmundacea Schimp. Arbeiten Bot Inst
Würzburg 3:477–488 (in Germany)
81. Oba Y (2009) On the origin of beetle luminescence. In: Meyer-Rochow VB (ed)
Bioluminescence in focus: a collection of illuminating essays. Research Signpost, Kerala,
pp 277–290
82. Lloyd JE (1978) Insect bioluminescence. In: Herring PJ (ed) Bioluminescence in action.
Academic Press, New York
83. Costa C, Lawrence JF, Rosa SP (2010) Elateridae Leach, 1815. In: Leschen RAB, Beutel
RG, Lawrence JF (eds) Handbook of zoology, vol IV, Arthropoda: Insecta, Teilband 39,
Coleoptera, Beetles, vol 2., Morphology and systematics. Walter de Gruyter, Berlin,
pp 75–103
84. Yeates DK, Wiegmann BM, Courtney GW, Meier R, Lambkin C, Pape T (2007) Phylogeny
and systematics of Diptera: two decades of progress and prospects. Zootaxa 1668:565–590
85. Viviani VR, Hastings JW, Wilson T (2002) Two bioluminescent Diptera: the North
American Orfelia fultoni and the Australian Arachnocampa flava. Similar niche, different
bioluminescence systems. Photochem Photobiol 75:22–27
86. Zompro O, Fritzsche I (1999) Lucihormetica fenestrata n. gen., n. sp., the first record of
luminescence in an orthopteroid insect (Dictyoptera: Blaberidae: Blaberinae: Brachycolini).
Amazoniana 15:211–219
87. Vršanský P, Chorvát D, Fritzsche I, Hain M, Ševěík R (2012) Light-mimicking cockroaches
indicate tertiary origin of recent terrestrial luminescence. Naturwissenschaften 99:739–749
88. Merritt DJ (2013) Standards of evidence for bioluminescence in cockroaches.
Naturwissenschaften 100:697–698
89. Goemans G (2006) The Fulgoridae (Hemiptera, Fulgoromorpha) of Guatemala. In: Cano EB
(ed) Biodiversidad de Guatemala, vol 1. Pub Univ del Vall de Guatemala, Guatemala,
pp 337–344
90. Wood KV (1995) The chemical mechanism and evolutionary development of beetle
bioluminescence. Photochem Photobiol 62:662–673
91. Bocakova M, Bocak L, Hunt T, Teraväinen M, Vogler AP (2007) Molecular phylogenetics
of Elateriformia (Coleoptera): evolution of bioluminescence and neoteny. Cladistics
23:477–496
92. Timmermans MJTN, Vogler AP (2012) Phylogenetically informative rearrangements in
mitochondrial genomes of Coleoptera, and monophyly of aquatic Elateriform beetles
(Dryopoidea). Mol Phylogenet Evol 63:299–304
93. Sagegami-Oba R, Oba Y, Ôhira H (2007) Phylogenetic relationships of click beetles

(Coleoptera: Elateridae) inferred from 28S ribosomal DNA: Insights into the evolution of
bioluminescence in Elateridae. Mol Phylogenet Evol 42:410–421
94. Douglas H (2011) Phylogenetic relationships of Elateridae inferred form adult morphology,
with special reference to the position of Cardiophorinae. Zootaxa 2900:1–45
95. Parker SP (1982) Synopsis and classification of living organisms, vol. 1, 2. McGraw-Hill,
New York
96. Kirk PM, Cannon PF, Minter DM, Stalpers JA (2008) Ainsworth and Bisby’s dictionary of
the fungi, 10th edn. CAB International, Wallingford
97. Desjardin DE, Oliveira AG, Stevani CV (2008) Fungi bioluminescence revisited.
Photochem Photobiol Sci 7:170–182
98. Shimomura O (2006) Bioluminescence: chemical principles and methods. World Scientific,
Singapore
99. Oliveira AG, Desjardin DE, Perry BA, Stevani CV (2012) Evidence that a single
bioluminescent system is shared by all known bioluminescent fungal lineages. Photochem
Photobiol Sci 11:848–852
100. Waterfield NR, Ciche T, Clarke D (2009) Photorhabdus and a host of hosts. Annu Rev
Microbiol 63:557–574
101. Lloyd JE (1971) Bioluminescent communication in insects. An Rev Entomol 16:97–122
102. Rota E (2009) Lights on the ground: a historical survey of light production in the
Oligochaeta. In: Meyer-Rochow VB (ed) Bioluminescence in focus: a collection of
illuminating essays. Research Signpost, Kerala, pp 105–138
103. Sivinski J, Forrest T (1983) Luminous defense in an earthworm. Florida Entomol 66:517
104. Oba Y, Furuhashi M, Bessho M, Sagawa S, Ikeya H, Inouye S (2013) Bioluminescence of a
firefly pupa: involvement of a luciferase isotype in the dim glow of pupae and eggs in the
Japanese firefly, Luciola lateralis. Photochem Photobiol Sci 12:854–863
105. Hastings JW, Morin JG (1991) Bioluminescence. In: Prosser CL (ed) Neural and integrative
animal physiology. Wiley-Liss, New York, pp 131–170
106. Meyer-Rochow VB, Moore S (1988) Biology of Latia neritoides Gray 1850 (Gastropoda,
Pulmonata, Basommatophora): the only light-producing freshwater snail in the world. Int
Revue ges Hydrobiol 73:21–42
107. Fu X, Ballantyne L (2009) Larval respiration system and evolution in aquatic fireflies
(Coleoptera: Lampyridae: Luciolinae). In: Meyer-Rochow VB (ed) Bioluminescence in
focus: a collection of illuminating essays. Research Signpost, Kerala, pp 243–253
108. Haneda Y (1955) Luminous organisms of Japan and Far East. In: Johnson FH (ed) The
luminescence of biological systems. American Association for the Advancement of Science,
Washington DC, pp 335–385
109. Houbrick RS (1987) Anatomy, reproductive biology, and phylogeny of the Planaxidae
(Cerithiacea: Prosobranchia). Smithon Contrib Zool 445:i–iii+1–57
110. Ponder WF (1988) Bioluminescence in Hinea braziliana (Lamarck) (Gastropoda:
Planaxidae). J Moll Stud 54:361
111. Deheyn DD, Wilson NG (2011) Bioluminescent signals spatially amplified by wavelength-
specific diffusion through the shell of a marine snail. Proc R Soc B 278:2112–2121
112. Marshall BA (1997) A luminescent eulimid (Mollusca: Gastropoda) from New Zealand.
Moll Res 18:69–72
113. Yamaguchi H (1970) On the earthworm (Mimizu no Hanashi). Hokuryukan, Tokyo (in
Japanese)
114. Kanda S (1938) The luminescence of Pontodrilus matsushimensis. Rigakukai 36:1–7 (in
Japanese)
115. Morin JG (1983) Coastal bioluminescence: patterns and functions. Bull Mar Sci 33:787–817
116. Nakamura H, Kishi Y, Shimomura O, Morse D, Hastings JW (1989) Structure of
dinoflagellate luciferin and its enzymatic and nonenzymatic air-oxidation products. J Am
Chem Soc 111:7607–7611
117. Nakamura H, Musicki B, Kishi Y, Shimomura O (1988) Structure of the light emitter in krill
(Euphausia pacifica) bioluminescence. J Am Chem Soc 110:2683–2685
118. Nakamura H, Oba Y, Murai A (1993) Synthesis and absolute configuration of the ozonolysis
product of krill fluorescent compound F. Tetrahedron Lett 34:2779–2782
119. Dunlap JC, Hastings JW, Shimomura O (1980) Crossreactivity between the light-emitting
systems of distantly related organisms: novel type of light-emitting compound. Proc Natl
120. Herring PJ (1985) Bioluminescence in the Crustacea. J Crustacean Biol 5:557–573
121. Morin JG (2011) Based on a review of the data, use of the term ‘cypridinid’ solves the
Cypridina/Vargula dilemma for naming the constituents of the luminescent system of
ostracods in the family Cypridinidae. Luminescence 26:1–4
122. Oba Y, Tsuduki H, Kato S, Ojika M, Inouye S (2004) Identification of the luciferin-
luciferase system and quantification of coelenterazine by mass spectrometry in the deep-sea
luminous ostracod Conchoecia pseudodiscophora. ChemBioChem 5:1495–1499
123. Haneda Y, Johnson FH, Shimomura O (1966) The origin of luciferin in the luminous ducts
of Parapriacanthus ransonneti, Pempheris klunzingeri, and Apogon ellioti. In: Johnson FH,
Haneda Y (eds) Bioluminescence in progress. Princeton University Press, Massachusetts
124. Tsuji FI, Haneda Y, Lynch RV III, Sugiyama N (1971) Luminescence cross-reactions of
Porichthys luciferin and theories on the origin of luciferin in some shallow-water fishes.
Comp Biochem Physiol 40A:163–179
125. Haneda Y, Johnson FH, Sie EH-C (1958) Luciferin and luciferase extracts of a fish, Apogon
marginatus, and their luminescent cross-reactions with those of a crustacean, Cypridina
hilgendorfii. Biol Bull 115:336
126. Haneda Y, Johnson FH (1958) The luciferin-luciferase reaction in a fish, Parapriacanthus
beryciformis, of newly discovered luminescence. Proc Natl Acad Sci USA 44:127–129
127. Haneda Y, Tsuji FI, Sugiyama N (1969) Luminescent systems in apogonid fishes from the
Philippines. Science 165:188–190
128. Haneda Y, Tsuji FI, Sugiyama N (1969) Newly observed luminescence in apogonid fishes
from the Philippines. Sci Rept Yokosuka City Mus 15:1–9 + 2 plt
129. Tominaga Y (1963) A revision of the fishes of the family Pempheridae of Japan. J Fac Sci
Univ Tokyo, Section IV Zoology 10:269–290
130. Mooi RD, Jubb RN (1996) Descriptions of two new species of the genus Pempheris (Pisces:
Pempherididae) from Australia, with a provisional key to Australian species. Rec Australian
Mus 48:117–130
131. Johnson FH, Sugiyama N, Shimomura O, Saiga Y, Haneda Y (1961) Crystalline luciferin
from a luminescent fish, Parapriacanthus beryciformes. Proc Natl Acad Sci USA
47:486–489
132. Tsuji FI, Barnes AT, Case JF (1972) Bioluminescence in the marine teleost, Porichthys
notatus, and its induction in a non-luminous form by Cypridina (ostracod) luciferin. Nature
237:515–516
133. Oba Y, Kato S, Ojika M, Inouye S (2002) Biosynthesis of luciferin in the sea firefly,
Cypridina hilgendorfii: L-tryptophan is a component in Cypridina luciferin. Tetrahedron
Lett 43:2389–2392
134. Kato S, Oba Y, Ojika M, Inouye S (2004) Identification of the biosynthetic units of
Cypridina luciferin in Cypridina (Vargula) hilgendorfii by LC/ESI-TOF-MS. Tetrahedron
60:11427–11434
135. Kato S, Oba Y, Ojika M, Inouye S (2006) Stereoselective incorporation of isoleucine into
Cypridina luciferin in Cypridina hilgendorfii (Vargula hilgendorfii). Biosci Biotechnol
Biochem 70:1528–1532
136. Kato S, Oba Y, Ojika M, Inouye S (2007) Biosynthesis of Cypridina luciferin in Cypridina
noctiluca. Heterocycles 72:673–676
137. Goto T, Fukatsu H (1969) Cypridina bioluminescence VII. Chemiluminescence in micelle
solutions: a model system for Cypridina bioluminescence. Tetrahedron Lett 10:4299–4302
138. Beebe W (1937) Preliminary list of Bermuda deep-sea fish. Based on the collections from
fifteen hundred metre-net hauls, made in an eight-mile circle South and Nonsuch Island.
Bermuda. Zoologica NY 22:197–208
139. Dunlap PV, Urbanczyk H (2013) Luminous bacteria. In: Rosenberg E (ed) The prokaryotes:
prokaryotic physiology and biochemistry. Springer, Berlin, pp 495–528
140. Herring PJ (2002) Marine microlights: the luminous marine bacteria. Microbiol Today
29:174–176
141. Dunlap PV, Ast JC, Kimura S, Fukui A, Yoshino T, Endo H (2007) Phylogenetic analysis of
host-symbiont specificity and codivergence in bioluminescent symbioses. Cladistics
23:507–532
142. Urbanczyk H, Ast JC, Higgins MJ, Carson J, Dunlap PV (2007) Reclassification of Vibrio
fischeri, Vibrio logei, Vibrio salmonicida and Vibrio wodanis as Aliivibrio fischeri gen. nov.,
comb. nov., Aliivibrio logei comb. nov., Aliivibrio salmonicida comb. nov. and Aliivibrio
wodanis comb. nov. Int J Syst Evol Microbiol 57:2823–2829
143. Meyer-Rochow VB (1976) Loss of bioluminescence in Anomalops katoptron due to
starvation. Experientia 32:1175–1176
144. Haddock SHD, Case JF (1994) A bioluminescent chaetognath. Nature 367:225–226
145. Campbell AK, Herring PJ (1990) Imidazolopyrazine bioluminescence in copepods and
other marine organisms. Mar Biol 104:219–225
146. Miya M, Nemoto T (1986) Reproduction, growth and vertical distribution of the
mesopelagic fish Cyclothone pseudopallida (family Gonostomatidae). In: Uyeno T, Arai
R, Taniuchi T, Matsuura K (eds) Proceedings of the second international conference on the
Indo-Pacific fishes. The Ichthyological Society of Japan, Tokyo, pp 830–837
147. Mallefet J, Shimomura O (1995) Presence of coelenterazine in mesopelagic fishes from the
Strait of Messina. Mar Biol 124:381–385
148. Frank TM, Widder EA, Latz MI, Case JF (1984) Dietary maintenance of bioluminescence in
a deep-sea mysid. J Exp Biol 109:385–389
149. Haddock SHD, Rivers TJ, Robison BH (2001) Can coelenterates make coelenterazine?
Dietary requirement for luciferin in cnidarian bioluminescence. Proc Natl Acad Sci USA
98:11148–11151
150. Thomson CM, Herring PJ, Campbell AK (1995) Evidence for de novo biosynthesis of
coelenterazine in the bioluminescent midwater shrimp, Systellaspis debilis. J Mar Biol Ass
UK 75:165–171
151. Buskey EJ, Stearns DE (1991) The effects of starvation on bioluminescence potential and
egg release of the copepod Metridia longa. J Plankton Res 13:885–893
152. Oba Y, Kato S, Ojika M, Inouye S (2009) Biosynthesis of coelenterazine in the deep-sea
copepod, Metridia pacifica. Biochem Biophys Res Commun 390:684–688
153. Mauchline J (1998) The biology of calanoid copepods: advances in marine biology, vol 33.
Academic Press, San Diego
154. Padmavati G, Ikeda T, Yamaguchi A (2004) Life cycle, population structure and vertical
distribution of Metridia spp. (Copepoda: Calanoida) in the Oyashio region (NW Pacific
Ocean). Mar Ecol Prog Ser 270:181–198
155. Anderson OR (1980) Radiolaria. Springer, New York
156. Meyer-Rochow VB (1986) Luminescent Copepoda of the genus Metridia with special
reference to the Antarctic Metridia gerlachei. New Zld Antarc Rec 7:1–8
157. Hirakawa K, Imamura A (1993) Seasonal abundance and life history of Metridia pacifica
(Copepoda: Calanoida) in Toyama Bay, Southern Japan Sea. Bull Plankton Soc Japan
40:41–54
158. Hayashi S, Hirakawa K (1997) Diet composition of the firefly squid, Watasenia scintillans,
from Toyama Bay, Southern Japan Sea. Bull Japan Sea Natl Fish Res Inst 47:57–66 (in
Japanese with English title and abstract)
159. Campbell AK (2012) Darwin shines light on the evolution of bioluminescence.
Luminescence 27:447–449
160. Johnsen S, Franck TM, Haddock SHD, Widder EA, Messing CG (2012) Light and vision in
the deep-sea benthos: I. Bioluminescence at 500–1000 m depth in the Bahamian Islands.
J Exp Biol 215:3335–3343
161. Ross DM (1959) The sea anemone (Calliactis parasitica) and the hermit crab (Eupagurus
bernhardus). Nature 4693:1161–1162
162. Okamura O (1970) Studies on the macrouroid fishes of Japan: morphology, ecology and
phylogeny. Rept Usa Mar Biol Station 17:1–179 + 5 plt
163. Kanda S (1935) Fireflies (Hotaru), Nippon Hakko Seibutsu Kenkyu Kai, Tokyo (in
Japanese) (reprinted edition, 1981, Scientist Inc, Tokyo)
164. Burkenroad MD (1943) A possible function of bioluminescence. J Mar Res 2:161–164
165. Meyer-Rochow VB (2007) Glowworms: a review of Arachnocampa spp. and kin.
166. Buck J (1988) Synchronous rhythmic flashing of fireflies. Part II. Q Rev Biol 63:265–289
167. Ohba N (1999) Synchronous flashing of the firefly, Pteroptyx effulgens, in Papua New
Guinea. Sci Rept Yokosuka City Mus 46:33–40 (in Japanese with English title and abstract)
168. Anctil M, Case JF (1977) The caudal luminous organs of lanternfishes: general innervation
and ultrastructure. Am J Anat 149:1–22
169. Widder EA (1998) A predatory use of counter illumination by the squaloid shark, Isistius
brasiliensis. Env Biol Fish 53:267–273
170. Abrahams MV, Townsend LD (1993) Bioluminescence in dinoflagellates: a test of the
burglar alarm hypothesis. Ecology 74:258–260
171. Oliveira AG, Stevani CV (2009) The enzymatic nature of fungal bioluminescence.
172. Hebert PDN, Cywinska A, Ball SL, deWaard JR (2003) Biological identifications through
DNA barcodes. Proc R Soc Lond B 270:313–321
173. Branham MA (2010) Lampyridae Latreille, 1817. In: Leschen RAB, Beutel RG, Lawrence
JF (eds) Handbook of zoology, vol IV, Arthropoda: Insecta, Teilband 39, Coleoptera,
Beetles, vol 2., Morphology and systematics. Walter de Gruyter, Berlin, pp 141–149
174. Kawashima I, Lawrence JF, Branham MA (2010) Rhagophthalmidae Olivier, 1907. In:
Leschen RAB, Beutel RG, Lawrence JF (eds) Handbook of zoology, vol IV, Arthropoda:
Insecta, Teilband 39, Coleoptera, Beetles, vol 2., Morphology and systematics. Walter de
Gruyter, Berlin, pp 135–140
Biochemistry and Genetics of Bacterial
Bioluminescence
Paul Dunlap
Abstract Bacterial light production involves enzymes—luciferase, fatty acid

reductase, and flavin reductase—and substrates—reduced flavin mononucleotide
and long-chain fatty aldehyde—that are specific to bioluminescence in bacteria.
The bacterial genes coding for these enzymes, luxA and luxB for the subunits of
luciferase; luxC, luxD, and luxE for the components of the fatty acid reductase;
and luxG for flavin reductase, are found as an operon in light-emitting bacteria,
with the gene order, luxCDABEG. Over 30 species of marine and terrestrial
bacteria, which cluster phylogenetically in Aliivibrio, Photobacterium, and Vibrio
(Vibrionaceae), Shewanella (Shewanellaceae), and Photorhabdus (Enterobacte-
riaceae), carry lux operon genes. The luminescence operons of some of these
bacteria also contain genes involved in the synthesis of riboflavin, ribEBHA, and in
some species, regulatory genes luxI and luxR are associated with the lux operon. In
well-studied cases, lux genes are coordinately expressed in a population density-
responsive, self-inducing manner called quorum sensing. The evolutionary origins
and physiological function of bioluminescence in bacteria are not well understood
but are thought to relate to utilization of oxygen as a substrate in the luminescence
reaction.

Keywords Bioluminescence Bacterial luciferase Aliivibrio Photobacterium

Vibrio lux genes
Abbreviations
acyl-HSL Acyl-homoserine lactone
FMNH2 Reduced flavin mononucleotide
Kb Kilobases (thousand nucleotides)
kD Kilodaltons
P. Dunlap (&)
Department of Ecology and Evolutionary Biology, University of Michigan,
Ann Arbor, MI 48109-1048, USA
e-mail: pvdunlap@umich.edu

38 P. Dunlap
Mb Megabases (million nucleotides)

RCHO Long-chain fatty aldehyde
RCOOH Long-chain fatty acid
Contents
1 Introduction.......................................................................................................................... 38
2 Species of Luminous Bacteria ............................................................................................ 39
3 Biochemistry of Bacterial Luminescence........................................................................... 42
4 Bacterial lux Genes ............................................................................................................. 43
5 Regulation of lux Operon Expression ................................................................................ 47
6 Origin and Function of Luminescence in Bacteria ............................................................ 51
7 Outlook ................................................................................................................................ 53
References.................................................................................................................................. 53
1 Introduction
Light production by bacteria is one of several evolutionarily distinct kinds of

bioluminescence, other kinds of which are found in various terrestrial and marine
eukaryotic organisms [76, 199]. Bacterial bioluminescence has been known since
1875, when Pflüger [152] correlated the presence of bacteria in the surface slime of
a fish with luminescence [74, 159]; earlier observations of luminescence, during the
1700 and 1800s, from various sources were likely due similarly to the presence
of luminous bacteria, such as saprophytes or parasites [72, 73]. The oxygen-
dependence of bacterial luminescence, first revealed by Boyle [24], who showed
that light produced by decaying fish required air, suggests light production in
bacteria arose evolutionarily after oxygen levels began to rise through the activities
of early cyanobacteria, oxygenic phototrophs, approximately 2.4 billion years ago.
All luminous bacteria, as far as known to date, utilize the same enzymatic
reaction for light production, based on bacterial luciferase. Phylogenetic analysis
suggests that the genes coding for the bacterial luminescence enzymes arose once
evolutionarily. Currently, luminous bacteria are grouped primarily in one Gam-
maproteobacteria family, Vibrionaceae. Some luminous members of closely
related families exist, however, having apparently acquired the genes for lumi-
nescence by horizontal gene transfer. Historical perspectives on the first isolations
and analyses and early taxonomy of luminous bacteria link the study of these
bacteria to the origins of general microbiology [44, 57, 73, 159]. This chapter
Biochemistry and Genetics of Bacterial Bioluminescence 39
Fig. 1 Bacterial luminescence. Colonies of P. mandapamensis from the light organ of the
cardinalfish Siphamia tubifer (Perciformes: Apogonidae) are shown growing on a nutrient
seawater agar plate. The plate was photographed in room light (left) and (the same plate) in the
dark by the light produced by the bacteria (right). From Dunlap et al. [46]
outlines the current systematics of luminous bacteria, provides an overview of the

biochemistry and genetics of bacterial luminescence, and concludes with a dis-
cussion of the evolutionary origin and function of bacterial luminescence.
2 Species of Luminous Bacteria
Thirty or more species of bacteria have strains that make light visible to the eye
(Fig. 1) or that at least carry genes for luminescence, the lux genes (Table 1). These
bacteria, which are all Gram-negative, group phylogenetically as members of three
families of Gammaproteobacteria: Vibrionaceae (Aliivibrio, Photobacterium,
Vibrio), Enterobacteriaceae (Photorhabdus), and Shewanellaceae (Shewanella). It
should be noted, however, that most Vibrionaceae species are not luminous and
apparently lack lux genes and that only the few listed species in Enterobacteriaceae
and Shewanellaceae are known to be luminous. Also, many of the species listed
have nonluminous strains that may or may not lack lux genes [4, 19, 38, 60, 204].
The low number of luminous species, relative to the many described species in
Vibrionaceae, is consistent with loss of the lux genes over evolutionary time from
the ancestors of many lineages within the family. Furthermore, nonluminous vari-
ants apparently can arise readily through the loss of one of more core genes of the
lux operon, luxCDABEG (e.g., [204]). It should be noted here also that several
species previously classified variously as members of Photobacterium or Vibrio
(i.e., fischeri, salmonicida, logei, and wodanis) have been reclassified as members of
40 P. Dunlap
Table 1 Species and ecological sources of luminous bacteria

Species Sources Selected references
Marine
Aliivibrio
fischeri Coastal seawater, light organs of squid [22, 59, 157, 161, 163,
and fish 164, 188]
logei Coastal seawater, sediment [9, 12, 188]
salmonicida Tissue lesions of Atlantic salmon [91, 139, 188]
sifiae Coastal seawater [9, 209]
‘‘thorii’’ Light organs of squid [9, 54]
wodanis Coastal seawater, diseased farmed salmon, [9, 115, 188]
light organs of squid
Photobacterium
aquimaris Coastal seawater [208]
damselae Coastal seawater [173, 189]
kishitanii Light organs and skin of fish [6, 7]
leiognathi Coastal seawater, light organs of fish [23, 45, 65, 158]
mandapamensis Coastal seawater, light organs of fish [85, 93, 157, 191]
phosphoreum Coastal and pelagic seawater [6, 7, 25, 202]
Candidatus
Photodesmus
katoptron Light organs of Anomalops katoptron [80, 86–88, 203]
blepharon Light organs of Photobelpharon [80, 87, 88, 203]
palpebratus
Shewanella
hanedai Seawater and sediment [92]
woodyi Seawater and squid ink [117]
Vibrio
azureus Coastal seawater [207]
campbellii Coastal seawater [107, 175]
chagasii Coastal seawater, surfaces and [182, 189]
intestines of marine animals
harveyi Coastal seawater, sediment [69, 142, 157, 163, 206]
jascicida Coastal seawater [57, 193]; H. Urbanczyk
(pers. comm.)
mediterraneaa Coastal seawater [153]
orientalis Seawater, surface of shrimp [205]
owensii Coastal seawater [193]; H. Urbanczyk
(pers. comm.)
sagamiensis Coastal seawater [210]
splendidus Coastal seawater [20, 138]
vulnificus Coastal seawater, oysters [147, 189]
Brackish/Estuarine
Vibrio
cholerae Estuaries, bays, coastal seawater [93, 148, 154, 215]
Terrestrial
(continued)
Table 1 (continued)
Species Sources Selected references
Photorhabdus
asymbiotica Human skin lesions [53, 58, 99, 149, 200]
luminescens Insect larvae infected with heterorhabditid [21, 36, 58, 181]
nematodes
temperata Insect larvae infected with heterorhabditid [58, 181]
nematodes
a
Ability of this species to luminescence has not been confirmed; the single strain reported as
luminous [153] might not be available
Aliivibrio, a change that resolves long-standing confusion on the evolutionary

relationships of these bacteria with members of Vibrio and Photobacterium [9, 188].
The majority of the luminous bacteria, members of Aliivibrio, Photobacterium,
Vibrio, and Shewanella, are found in the marine environment. Luminous trains of
Vibrio cholerae can be isolated from brackish environments and freshwater as well
as from coastal seawater. Depending on the species, these bacteria occur free in
seawater and in sediments or more commonly are associated with surfaces and gut
tracts of marine animals as saprophytes and commensal symbionts (e.g., [160, 190]).
They also occur as parasites of marine animals and as highly specific mutualistic
bioluminescent light organ symbionts of many marine fish and squid. In contrast,
Photorhabdus species occur in terrestrial environments and as symbionts of ter-
restrial heterorhabditid nematodes [195]. Several of the species listed in Table 1
were described recently, and others have been recognized only recently as luminous
or as carrying lux genes. This progress suggests that future studies, especially those
employing whole genome sequence analysis (e.g., [193]), will identify many more
species and strains of bacteria that are luminous or at least carry lux genes.
Most luminous bacteria are culturable on laboratory media. Some, however, have
not yet been brought into culture despite many attempts [81]. These not-yet cultured
luminous bacteria are symbiotic in the subocular light organs of flashlight fish
(Anomalopidae) and the escal light organs of members of many families of deep-sea
anglerfish (order Lophiiformes). The inability to culture these bacteria suggests they
are obligately dependent on their host fish for nutrients necessary for reproduction or
conditions supporting their survival [81]. Early phylogenetic work placed these
bacteria in Vibrionaceae and as a species distinct from the luminous bacteria known
at the time [80, 81, 83, 84]. Recent analyses of the bacteria from light organs of two
species of anomalopid fish, using multilocus phylogenetic analysis and whole
genome sequence analysis, have identified the bacteria as members of a new Vib-
rionaceae genus, Candidatus Photodesmus, with two new species, Candidatus
Photodesmus katoptron and Candidatus Photodesmus blepharon, from Anomalops
katoptron and Photoblepharon palpebratus, respectively. The genomes of
Ca. Photodesmus katoptron and Ca. Photodesmus blepharon, approximately 1.0 and
42 P. Dunlap
1.1 Mb, respectively, are massively reduced compared to the genomes of other
members of Vibrionacaeae, which typically are 4.5–6.0 Mb, with many metabolic
and regulatory genes lost. Genome reduction and gene loss apparently account for
the inability of these bacteria to grow in laboratory culture [86–88]. For the deep-sea
anglerfish, no additional information is available at this time on the species-level
phylogenetic placement of their symbiotic luminous bacteria.
Despite much recent progress in clarifying the taxonomy of luminous bacteria
(Table 1), the ecological incidence and species-level diversity of luminous bac-
teria remain incompletely defined. One problem is that the luminescence pheno-
type can be lost; strains luminous on primary isolation often become dim or dark
in laboratory culture [3, 134, 171]. Another is that some species that grow well in
laboratory culture at room temperature, i.e., Aliivibrio logei and Shewanella
hanedai, typically produce light visible to the eye only when grown at cooler
temperatures. In some cases, i.e., luminous bacteria infecting crustaceans; [67] and
strains of Aliivibrio fischeri symbiotic with the Hawaiian sepiolid squid, Euprymna
scolopes [22], the bacteria produce a high level of light in their natural habitat but
produce little or no light when grown in laboratory culture. A further complication
is that strains of some bacteria, such V. cholerae, are known to carry lux genes but
apparently do not express them (e.g., [93, 148, 154, 215]). In addition, bacteria
identified as related to Vibrio harveyi and Vibrio cincinnatiensis carry the lux
genes but have been found to have lux gene mutations that result in a dark phe-
notype [143]. Although the phylogenetically scattered incidence of bacteria with
lux genes in Vibrionaceae presumably relates to different ecologies of the different
species, it is not obvious how having and expressing lux genes contributes to the
lifestyle of most luminous bacteria; there are no obvious ecological differences
between luminous and nonluminous species except in the case of those species that
are bioluminescent symbionts of bacterially luminous fish and squid. On-going and
future studies that examine the incidence, lux gene content, and phylogenetic
placement of luminous bacteria will undoubtedly expand understanding of the
species diversity and ecology of bacteria able to produce light.
3 Biochemistry of Bacterial Luminescence
Light emission in bacteria is catalyzed by a uniquely bacterial kind of luciferase, a

heterodimeric protein of approximately 80 kD, composed of a (40 kDa) and
b (37 kDa) subunits, with homology to long-chain alkane monooxygenases
[76, 104]. The enzyme mediates the oxidation of reduced flavin mononucleotide
(FMNH2) and a long-chain aliphatic (fatty) aldehyde (RCHO) by O2 to produce
blue-green light according to the following reaction.
luciferase
FMNH2 þ O2 þ RCHO ) FMN þ H2 O þ RCOOH þ light ð490 nmÞ
Along with bacterial luciferase, the substrates, FMNH2 and long-chain fatty
aldehyde, are specific to the bacterial luminescence reaction; bioluminescent
eukaryotes employ different chemistries and luciferases that are not homologous at
the protein or gene sequence levels to bacterial luciferase [76]. In the luminescence
reaction, binding of FMNH2 by the enzyme is followed by interaction with O2 to
form a flavin-4a-hydroperoxide. Association of this complex with aldehyde forms
a highly stable intermediate, the slow decay of which results in oxidation of the
FMNH2 and aldehyde substrates and the emission of light [76, 79]. Quantum yield
for the reaction has been estimated at 0.1–0.2 photons. The reaction is highly
specific for FMNH2, and the aldehyde substrate in vivo is likely to be tetradecanal.
FMNH2 is provided by the activity of an NADH:FMN oxidoreductase (flavin
reductase), which taps reductant from NADH generated in cellular metabolism, for
example, glycolysis and the citric acid cycle. Transfer of reductant from FMNH2
to luciferase occurs by free diffusion. Synthesis of the long-chain aldehyde is
catalyzed by a fatty-acid reductase complex composed of three polypeptides,
an NADPH-dependent acyl protein reductase (r, 54 kDa), an acyl transferase
(t, 33 kDa), and an ATP-dependent synthetase (s, 42 kDa). The complex has a
stoichiometry of r4s4t2–4, and its activity is essential for the production of light in
the absence of exogenously added aldehyde [79, 121, 183, 201]. Luciferases from
different species of luminous bacteria exhibit substantial amino acid residue and
nucleotide sequence identity [45, 122], consistent with a common evolutionary
origin of luminescence in bacteria.
4 Bacterial lux Genes
The lux operon, luxCDABEG, contains the genes necessary for light production in
bacteria (Fig. 2). The luxA and luxB genes code for the a and b subunits of
bacterial luciferase, respectively, luxC, luxD, and luxE genes, respectively, code
for the r, s, and t polypeptides of the fatty-acid reductase complex that synthesizes
and recycles aldehyde substrate for luciferase, and luxG, codes for flavin reductase
[112, 122, 141, 178, 183]. The absence of luxG from the lux operons of Photor-
habdus luminescens and newly characterized species of Ca. Photodesmus (Fig. 2)
apparently is compensated for by the activity of a flavin reductase activity coded
for by an Escherichia coli fre-like gene. Homologues of the fre-like gene are found
in various luminous bacteria [212–214].
An additional gene, luxF, which codes for a nonfluorescent flavoprotein, is
present in the lux operons of Photobacterium, between luxB and luxE (Fig. 2). The
LuxF protein might function in the luminescence system by scavenging an
inhibitory side product of the luciferase reaction [130], but it is not necessary for
light production, even in those Photobacterium species that normally carry this
gene [93]. The luxF gene apparently has been secondarily lost in Photobacterium
leiognathi [5].
44 P. Dunlap
Fig. 2 Bacterial luminescence (lux) genes. Shown are the gene content and gene order of lux
operons for those bacteria for which complete lux operon sequence data are available. Contiguous
genes of the lux operons are aligned to highlight commonalities and differences. Four distinct
types of lux operons are evident based on commonalities of gene content, organization, and
sequence similarity: (1) Aliivibrio/Shewanella type, with luxI/luxR regulatory genes; (2)
Photobacterium type, with ribEBHA genes forming a lux-rib operon; (3) Vibrio/Candidatus
Photodesmus type, without linked regulatory genes; and (4) Photorhabdus type, composed of just
five core lux genes, luxCDABE. Additional species of luminous bacteria are listed in Table 1. See
text for details and for information on accessory genes
In Photobacterium, genes involved in the synthesis of riboflavin, ribEBHA, are

part of the lux operon, forming a lux-rib operon, luxCDABFEG-ribEBHA (Fig. 2;
[8, 100, 101, 113, 176]). The absence of a transcriptional stop or other regulatory
site between the lux and rib genes indicates that these genes are coordinately
expressed from a single promoter upstream of luxC. Strains of P. phosphoreum

lack ribE, which presumably was lost in the divergence from an ancestral Pho-
tobacterium that gave rise to this species. The presence of genes for synthesis of
riboflavin as part of the lux operon might enhance light production by ensuring
coordinate synthesis of luciferase and substrates for the enzyme. The lux operon of
V. campbellii (previously classified as V. harveyi; [107]) contains ribB, coding for
3,4-dihydroxy-2-butanone 4-phosphate synthase, a key enzyme in riboflavin syn-
thesis (referred to originally as luxH; [179]). In A. fischeri, although ribB is not
part of the lux operon, its expression nonetheless is under the same regulatory
control as the lux genes [26].
The isolation of a luminous strain of V. campbellii was first reported several
years ago [175]. More recently, certain strains identified as V. harveyi, for
example, BAA-1116 (a.k.a. BB120, which has been used extensively in studies of
quorum sensing, e.g., [15]), were identified by whole genome sequence analysis as
members of V. campbellii [107]. Careful phylogenetic analysis of strains currently
referred to as V. harveyi is therefore needed to confirm their identity and the lux
operon gene organization of this species.
In Photobacterium, many strains of P. leiognathi carry two intact and appar-
ently functional lux-rib operons in their genomes [8]. The two operons, lux-rib1
and lux-rib2, are distinct in sequence and genomic location. Phylogenetic analysis
indicates that lux-rib1 and lux-rib2 are more closely related to each other than
either is to the lux and rib genes of other bacterial species [8]. These findings
indicate that lux-rib2 did not arise by a gene duplication event, and they exclude
interspecies horizontal transfer as the origin of lux-rib2 in P. leiognathi; instead,
the second operon apparently was acquired by horizontal gene transfer from a
lineage of P. leiognathi that either has gone extinct or has not yet been sampled.
The P. leiognathi lux-rib2 operon has also been found in two strains of P. man-
dapamensis, which also carry a normal P. mandapamensis lux-rib operon, and in a
strain of P. damselae, a species not previously known to be luminous [189]. In
Photobacterium aquimaris [208], the lux operon of one of two luminous strains
apparently has been replaced by horizontal acquisition of the lux operon of Pho-
tobacterium mandapamensis [192].
In addition to the presence of the ribEBHA genes as part of the lux operon of
Photobacterium, other genus-specific differences are evident in the genes upstream
of and flanking the lux operons of luminous bacteria (Fig. 2). In P. mandapam-
ensis, the lux-rib operon is preceded by lumQ and lumP, which form the lumazine
operon. The function of lumQ is not yet known, although it is thought to code for a
DNA-binding protein [109]. LumP, a 21-kDa fluorescent accessory protein
referred to as lumazine protein, functions to enhance the intensity of light emission
and to shift the emission wavelength of luciferase from blue-green (495 nm) to
blue (475–486 nm) [102, 145, 146, 151]. A lumP gene is present just upstream of
luxC in Photobacterium phosphoreum and Photobacterium aquimaris, but lumQ is
absent [192]. The LumP protein, which has been isolated from P. phosphoreum
and from a strain of P. mandapamensis (previously classified as a strain of
P. leiognathi) and also purified from P. kishitanii, contains a noncovalently bound
46 P. Dunlap
fluorophore, 6,7-dimethyl-8-ribityllumazine, the immediate biosynthetic precursor

of riboflavin [145, 165, 172]. In P. leiognathi lumP is not found, although
approximately 200 nucleotides of the P. leiognathi luxC-lumQ intergenic region
can be aligned to the P. mandapamensis lumP gene [8]. The activity of the LumP
protein apparently accounts for the blue-shifted luminescence of P. mandapam-
ensis compared to P. leiognathi, one of the diagnostic traits distinguishing these
two species [5, 102, 145, 146, 151, 93]. The genes flanking the P. leiognathi and
P. mandapamensis lux-rib operons are homologous to a single contiguous region
in nonluminous P. angustum [8, 108–111, 113].
In the examined Aliivibrio species, regulatory genes, luxI and luxR, which
control transcription of the lux operon (described below), are upstream of or flank
the luxCDABEG genes (Fig. 2). The luxI gene codes for an acyl–homoserine
lactone (acyl-HSL) synthase [166], and luxR codes for a receptor protein that
interacts with acyl-HSL to activate transcription of the lux operon [52]. In Aliiv-
ibrio fischeri, luxI is the first gene of the lux operon, and luxR, upstream of luxI, is
divergently transcribed (Fig. 2). The lux operon of Shewanella hanedai has the
same gene arrangement. This similarity, together with a high degree of lux gene
sequence identity suggests that S. hanedai acquired its lux operon by horizontal
transfer from A. fischeri or the ancestor of A. fischeri [189]. In Aliivibrio sal-
monicida, a bacterium that requires exogenous addition of aldehyde to produce a
high level of light [55], two luxR genes, homologous to A. fischeri luxR, flank the
lux operon; a luxI gene also is present, divergently transcribed from the down-
stream luxR (Fig. 2; [91, 139]). The same arrangement of luxI and luxR genes as in
A. salmonicida is present in Aliivibrio logei [119]. In contrast to A. salmonicida,
however, exogenous addition of aldehyde is not required for a high level of light
production in A. logei [119]; mutations in luxD account for the exogenous alde-
hyde requirement of A. salmonicida. Genes flanking the lux operons of other
luminous Aliivibrio species (Table 1) apparently have not yet been characterized.
An accessory protein, yellow fluorescent protein (YFP), is present in Aliivibrio
sifiae (previously referred to as A. fischeri; [9, 209] and shifts the emission
wavelength of luminescence toward yellow [11, 33, 150, 162]; the YFP gene
apparently is not linked to the lux operon.
With respect to S. hanedai and S. woodyi, comparison of genes flanking the lux
operons suggested that these species had acquired lux genes from a member of
Aliivibrio [96], a possibility confirmed through phylogenetic analysis [189]. In
Photorhabdus species, the luxCDABE genes also might have been acquired by
horizontal gene transfer, possibly from an ancestor of V. harveyi [61, 121, 123].
Phylogenetic analysis of the Photorhabdus lux genes, however, neither supports
nor contradicts horizontal acquisition of the lux genes by Ph. luminescens [189].
Certain instances of horizontal acquisition of lux genes have also been found for
Vibrio [189]. The one known luminous strain of Vibrio vulnificus, the human
pathogen [147], apparently acquired its lux genes from V. harveyi, and in
V. chagasii, a species not described as luminous [182], two luminous strains were
identified and apparently had acquired their lux genes from V. harveyi and
V. splendidus, respectively [189].
The arrangement of genes associated with luxCDABEG of the examined Vibrio

species differs substantially from that in Photobacterium and in Aliivibrio (Fig. 2).
Regulatory genes controlling transcription of the lux operon are not part of and are
not adjacent to the lux operons of those Vibrio species examined; specifically, a
luxR gene, referred to here as luxRVh which is not homologous to A. fischeri luxR,
is not physically associated with the lux operon in V. campbellii (V. harveyi). Also,
with the exception of ribB in V. campbellii, genes involved in riboflavin synthesis
in Photobacterium, ribEBHA, are not part of the lux operon in Vibrio. Conser-
vation of luxCDABEG as a unit might reflect a need for close interaction of
luciferase with the enzymes, fatty acid reductase and flavin reductase, producing
substrates for the reaction, for efficient light production. However, it is not obvious
what led to the genus-specific lux operon gene content and organization in Ali-
ivibrio, Photobacterium, and Vibrio (Fig. 2), three closely related genera of
Vibrionaceae.
5 Regulation of lux Operon Expression
The production of light consumes a substantial amount of energy, through the

synthesis and activity of the Lux proteins [41]. The retention and expression of lux
genes in many bacteria, despite this energetic cost, therefore indicates that the
activity of the luminescence system must benefit these bacteria, physiologically or
ecologically, as discussed below. Furthermore, this energetic cost presumably
accounts for the population density-responsive regulation of lux operon expression
characteristic of many luminous bacteria. Originally called autoinduction and dis-
covered through study of the pattern of luminescence and luciferase synthesis of
V. harveyi in batch culture [132, 136], this gene regulatory mechanism is now
referred to as quorum sensing to reflect its relationship with population density
[66, 70, 77, 124]. At low population density, very little luciferase is synthesized, and
consequently little light is produced, whereas at high population density, luciferase
levels are induced 100–1,000-fold and light levels increase by 103–106-fold. This
population density-responsive induction of luciferase synthesis and luminescence is
controlled in part by the production and accumulation in the cell’s local environ-
ment of small secondary metabolite signal molecules, called autoinducers, which
function via regulatory proteins to activate or derepress transcription of the lux
operon. Quorum sensing has been studied intensively in two luminous bacteria,
V. harveyi and A. fischeri. With respect to V. harveyi, a strain used extensively in
quorum-sensing research, BAA-1116, also known as BB120 (e.g., [15]), recently
was recognized through whole genome sequence analysis to be a member of
V. campbellii [107]. The information related below is therefore provisionally
ascribed to V. campbellii, pending resolution of the taxonomic status of strains
called V. harveyi that have been used in the various studies of quorum sensing. The
quorum-sensing systems of these two bacteria, V. campbellii and A. fischeri, briefly
outlined below, differ substantially at genetic and chemical levels. Despite the many
48 P. Dunlap
Fig. 3 Quorum-sensing regulatory circuitry in Vibrio campbellii (Vibrio harveyi). The expression
of lux operon, and of other quorum-sensing–regulated genes, in V. campbellii (previously classified
as V. harveyi; [107] is coordinated by three chemically distinct autoinducers, HAI-1, AI-2Vh, and
CAI-1, that modulate the phosphorylation state of luxU. The synthesis of each autoinducer is
catalyzed by a different protein, LuxM, LuxS, and CqsA, and each is recognized by a different
cytoplasmic membrane-associated two-component histidine–kinase receptor, LuxN, LuxPQ, and
CqsS, respectively. Low concentrations of the autoinducers lead to phosphorylation of LuxO and
via quorum–regulatory RNAs to the destabilization of the luxRVh transcript, thereby blocking lux
operon transcriptional activation by LuxRVh. High concentrations of the autoinducers reverse the
phosphorylation cascade, allowing formation of LuxRVh and activation of lux operon transcription.
Arrows indicate positive interactions or transcriptional activation, whereas bars indicate negative
interactions or blocking of transcription. See the text for details and references. Redrawn from [185]
differences, however, several commonalities to the two systems have been identified
[44]. Recent publications provide additional details on quorum sensing in these
bacteria as well as in nonluminous bacteria [14, 44, 155].
In V. campbellii, lux operon expression is controlled by a multicomponent
phosphorylation/dephosphorylation cascade (Fig. 3). Three chemically distinct
autoinducers are involved: 3-hydroxybutanoyl-HSL (harveyi autoinducer-1,
HAI-1), (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate (V. harveyi

autoinducer-2, AI-2Vh), and (S)-3-hydroxytridecan-4-one (cholerae autoinducer,
CAI-1) [27, 31, 90]. Synthesis of HAI-1 is dependent on LuxM [16], synthesis of
AI-2Vh is catalyzed by LuxS [167], and synthesis of CAI-1 is catalyzed by CqsA
[97, 198]. Each of these molecules is recognized by a different cytoplasmic
membrane-associated two-component histidine-kinase receptor, LuxN [16, 64],
LuxPQ [18], and CqsS [89], respectively. When concentrations of the autoinducers
are low, such as at low population density or in habitats in which the autoinducers
diffuse away rapidly from cells (i.e., in seawater), the receptor proteins function as
kinases, transferring phosphate to LuxU, a histidine-phosphotransfer protein. LuxU
then transfers the phosphate to LuxO, a DNA-binding response regulator, the
expression of which is subject to repression by LuxT [13, 17, 27, 62, 63, 106, 114,
126, 177, 197]. LuxO * P, together with sigma factor r54, then activates the
expression of genes coding for five small quorum-regulatory RNAs (Qrr), Qrr1
through Qrr5 [103, 184]. The Qrr RNAs bind and destabilize the luxRVh transcript,
blocking production of LuxRVh protein, the transcriptional activator of the lux
operon [170, 180], and thereby preventing activation of lux operon transcription.
Conversely, once autoinducer concentrations have attained high levels in the cell’s
local environment, they bind to their receptors, causing the receptors to switch from
kinases to phosphatases, leading to the dephosphorylation of LuxO. With the
resulting cessation of qrr gene transcription, a luxRVh message is produced and
translated, and LuxRVh activates lux operon transcription. Negative autoregulation
of LuxRVh adds additional complexity to this system [28, 125], as does the negative
autoregulation of LuxO, posttranscriptional control of LuxO by Qrr sRNAs [185],
and involvement of 30 :50 -cyclic AMP (cAMP) and camp receptor protein (CRP)
[29, 30, 34, 127, 137, 187]. The complexity of this regulatory system apparently
benefits V. campbellii by allowing a fine-tuning of its quorum-sensing response to
differences in the various habitats the bacterium colonizes [140, 185, 196].
In A. fischeri, quorum-sensing control of luminescence in A. fischeri involves a
population-density–dependent accumulation of the autoinducer 3-oxo-hexanoyl-
homoserine lactone (AI-1), a membrane-permeant molecule that triggers induction
of lux operon transcription when AI-1 reaches a critical concentration (Fig. 4).
Synthesis of AI-1 is catalyzed by LuxI, an acyl–homoserine lactone synthase, from
S-adenosyl methionine and acyl-HSL. The regulatory genes, luxRAf and luxI, are
directly linked to the lux operon. The luxRAf gene, which is upstream of the lux
operon and divergently transcribed from it, encodes a transcriptional activator
protein, LuxRAf, which associates with AI-1, forms a complex that binds at a site
in the lux operon promoter and facilitates the binding of RNA polymerase, thereby
activating transcription of the genes for light production, luxICDABEG. Because
luxI is a gene of the lux operon, increased transcription leads to increased pro-
duction of LuxI protein and increased synthesis of AI-1, in an autocatalytic,
positive feedback manner. The result is a rapid and strong induction of luciferase
synthesis and luminescence [48, 50–52, 94, 166, 174].
Several other regulatory factors modulate quorum-sensing in A. fischeri. These
factors include: cAMP and CRP, which activate transcription of luxRAf and
50 P. Dunlap
Fig. 4 Quorum-sensing control of luminescence in A. fischeri. The expression of the lux operon,
and of other quorum-sensing-regulated genes, in A. fischeri is mediated primarily by the
concentration of AI-1, which forms a complex with LuxRAf. Synthesis of AI-1 is dependent on
LuxI, and the AI-1/LuxRAf complex activates luxICDABEG transcription. Together with cAMP,
the CRP protein activates expression from the luxRAf promoter, increasing synthesis of LuxRAf
and potentiating the system to be induced once sufficient AI-1 has accumulated. The presence of
luxI, coding for AI-1 synthase, as part of the lux operon, leads to increased expression from the
lux operon promoter, stimulating AI-1 synthesis in an autocatalytic, positive feedback manner;
the result is a rapid and strong induction of luciferase synthesis once a threshold concentration of
AI-1 is attained. A second autoinducer, AI-2Af interacts with LuxRAf, interfering with the
interaction between AI-1 and LuxRAf. The hypothesized AI-2Af/LuxRAf complex is thought to be
transcriptionally less effective and therefore to function to delay the onset of AI-1/LuxRAf
activation of luxICDABEG transcription. See the text for details and references
thereby potentiate the cell’s response to AI-1 while repressing transcription of

luxICDABEG; negative autoregulation of luxRAf expression by LuxRAf/AI-1;
a second autoinducer, octanoyl-HSL (AI-2Af), which is synthesized by AinS and
that interacts with LuxRAf apparently interfering with AI-1 binding and thereby
delaying lux operon induction; involvement of GroEL in production of active

LuxRAf; a homologue of the V. harveyi LuxO, which functions in A. fischeri as a
repressor of luminescence apparently in a qrr-dependent manner; LitR, a protein
with substantial sequence similarity to LuxRAf and which positively regulates lux
operon expression; and an involvement of Fnr and LexA in lux operon expression
[1, 2, 35, 37, 39, 40, 42, 43, 49, 56, 68, 71, 98, 126, 128, 129, 131, 169, 186]. As in
V. campbellii, the complexity of inputs into the quorum-sensing regulatory cir-
cuitry in A. fischeri indicates both a tight integration of luminescence into the
lifestyle of the bacterium and the ability to modulate lux operon expression in
response to a variety of conditions.
6 Origin and Function of Luminescence in Bacteria
The conserved gene content and gene order of the lux operon in bacteria, lux-
CDABEG (Fig. 2), and the high levels of lux gene and Lux protein amino acid
sequence identities among luminous bacteria (e.g., [122]) indicate that all known
bacterial lux operons derive from a single common ancestor. The congruence of
phylogenies based on lux genes and other protein coding genes (and the 16S rRNA
gene) [189] indicates that bacterial luminescence arose within the Vibrionaceae
lineage and mostly likely in the ancestor that gave rise to Aliivibrio, Photobac-
terium, and Vibrio.
The homology of bacterial luciferase to long-chain alkane monooxygenases
[104] suggests that the light-emitting enzyme arose from this family of proteins,
even though none of the other enzymes in the family emit light [76]. With some
light emission, bacteria luciferase might have evolved under ecological selection,
according to the following scenario [168]. A flavoprotein catalyzing fatty acid
a-oxidation reactions with low chemiluminescent quantum yields is postulated to
have mutated under hypoxic conditions to accept FMNH2 as the flavin cofactor,
generating a fortuitously high fluorescence yield, termed ‘‘protobioluminescence,’’
via the 4a-hydroxy-FMNH product [168]. This flavin dependent, aldehyde-oxi-
dizing protoluciferase produced sufficient light, and with an appropriate emission
spectrum, to be detected by phototactic organisms. Ingestion by visually cueing
animals of particles colonized and made luminous by these early luminous bacteria
presumably enhanced their reproduction by bringing them into the animal’s
nutrient-rich digestive system, ensuring the emitter’s survival and thereby possibly
leading to selection for more intense light output [199]. It is possible that early
evolutionary steps leading to protoluciferase involved oxygen detoxification
activity that permitted early anaerobic organisms to survive an increasingly aer-
obic environment [120, 156]. An alternative hypothesis for the evolution of bac-
terial luciferase, involvement in DNA repair [32], has been refuted [194].
A single gene was hypothesized to encode bacterial protoluciferase [144].
Although a single-subunit protoluciferase, monomer or dimer, presumably would
have differed somewhat from the modern-day luciferase a-subunit and therefore
52 P. Dunlap
might have produced light, the inability of either of the extant a or b subunits alone
to produce light in vitro or in vivo [105] argues against the single-gene hypothesis.
Alternatively, bacterial protoluminescence may have arisen following a gene
duplication event that is postulated to have created luxB from luxA [10, 122, 144].
Based on amino acid sequence identities, a tandem duplication of the ancestral luxA
gene, followed by sequence divergence in the duplicated gene, is thought to have
given rise to luxB, leading to the formation of the heterodimeric luciferase present
in extant luminous bacteria. Similarly, a tandem duplication of luxB followed by
loss of approximately 300 nucleotides coding for N-terminus amino acids is thought
to have given rise to luxF in a luminescent ancestor of Photobacterium; this gene
apparently was later secondarily lost in the lineage giving rise to P. leiognathi [5,
10, 122, 144].
The association of the fatty-acid reductase genes, luxCDE, with luxA might
have predated the luxA to luxB gene duplication event. Alternatively, the presence
of ERIC sequences flanking luxA and luxB in Ph. luminescens [123] might mark an
insertion of the luxAB genes into the fatty aldehyde reductase operon during the
evolution of the bacterial luminescence system. The origins and evolution of other
luminescence genes are not well understood [144]. The evolution of the bacterial
luminescence system also involved recruitment of regulatory and other genes to
the lux operon in some species (Fig. 2).
The energetic cost of light production, involving expenditures of carbon,
nitrogen, and ATP in the synthesis of Lux proteins and in their enzymatic activities
[41] indicates that luminescence has physiological or ecological importance for
those bacteria able to express it. In the absence of selection to retain the lux genes,
this cost would lead to loss of function through mutation and gene loss, an evo-
lutionary scenario that probably accounts for the scattered incidence of luminous
strains and species in Vibrionacaeae. As outlined above, bacterial luminescence
might have arisen evolutionarily as a means of coping with oxygen. Consistent
with this possible function, luciferase, as an oxidase, might function as a secondary
respiratory chain that is active when oxygen or iron levels are too low for the
cytoplasmic membrane-associated, ATP-generating electron transport system to
operate. This activity would allow cells expressing luciferase to reoxidize reduced
coenzyme even when oxygen levels are low [75, 76, 78, 135]. Supporting this
view, growth of cytochrome-deficient luminous bacteria is dependent on induction
of luciferase, limitation for iron stimulates light production, low oxygen levels
promote the luminescence of some luminous bacteria, and luciferase synthesis can
be induced under anaerobic conditions [47, 82, 116, 118, 133]. As an alternative or
supplement to the electron transport system, the activity of luciferase in reoxi-
dizing reduced coenzyme could permit cells of luminous bacteria in low oxygen
habitats, such as in animal gut tracts, to continue to transport and metabolize
growth substrates, thereby continuing to gain energy through substrate-level
phosphorylation. Furthermore and consistent with the ecological selection scenario
above, light production presumably facilitates dissemination of luminous bacteria.
The feeding of animals on luminous particles (decaying tissues, fecal pellets, and
moribund animals infected by luminous bacteria), to which they are attracted,
would bring the bacteria into the animal’s nutrient-rich gut tract for additional
rounds of reproduction followed by dispersal [78, 135]. Recent evidence is sup-
portive of this possibility [211]. Alternatively, the function of the bacterial lux
system might be to generate a halotolerant flavodoxin, with light emission an
incidental consequence [95]. Future studies might provide additional support for
these and other proposed functions for luminescence, such as a physiological role
for luciferase activity in bioluminescent symbioses with fish and squid. Proposed
functions will be held to the standard of a demonstrated selective benefit for
luminescence, either physiological or ecological.
7 Outlook
Substantial progress has been made in the past few years in defining the taxonomy
and phylogenetic relationships of luminous bacteria and more fully characterizing
the biochemistry and genetics of bacterial luminescence. Despite this progress,
current understanding of the physiological function and ecological benefit of
luminescence in bacteria remains limited. The long-standing question, ‘‘Why do
bacteria make light?’’ remains essentially unanswered. A more detailed knowledge
of the evolutionary origins and biochemical uniqueness of bacterial luciferase
(e.g., [104]) and a more comprehensive understanding of the phylogenetic distri-
bution of lux genes through whole-genome sequence analysis (e.g., [193]) in the
context of the ecology of these bacteria (e.g., [211]) are likely to provide clues.
Particularly insightful, however, will likely be detailed comparative physiological
analysis of genetically defined mutants (e.g., [116]), an approach that addresses the
core question and provides an experimental foundation for testing specific func-
tional hypotheses.
References
1. Adar YY, Simaan M, Ulitzur S (1992) Formation of the LuxR protein in the Vibrio fischeri
lux system is controlled by HtpR through the GroESL proteins. J Bacteriol 174:7138–7143
2. Adar YY, Ulitzur S (1993) GroESL proteins facilitate binding of externally added inducer
by LuxR protein–containing E. coli cells. J Biolumin Chemilumin 8:261–266
3. Akhurst RJ (1980) Morphological and functional dimorphism in Xenorhabdus spp., bacteria
symbiotically associated with the insect pathogenic nematodes Neoaplectana and
Heterorhabditis. J Gen Microbiol 121:303–309
4. Akhurst RJ, Boemare NE (1986) A non–luminescent strain of Xenorhabdus luminescens.
J Gen Microbiol 132:1917–1922
5. Ast JC, Cleenwerck I, Engelbeen K, Urbanczyk H, Thompson FL, De Vos P, Dunlap PV,
Ast JC, Dunlap PV (2004) Phylogenetic analysis of the lux operon distinguishes two
evolutionarily distinct clades of Photobacterium leiognathi. Arch Microbiol 181:352–361
6. Ast JC, Dunlap PV (2005) Phylogenetic resolution and habitat specificity of members of the
Photobacterium phosphoreum species group. Environ Microbiol 7:1641–1654
54 P. Dunlap
7. Ast JC, Dunlap PV (2007a) Photobacterium kishitanii sp. nov., a luminous marine
bacterium symbiotic with deep–sea fish. Int J Syst Evol Microbiol 57:2073–2078
8. Ast JC, Urbanczyk H, Dunlap PV (2007) Natural merodiploidy of the lux–rib operon
of Photobacterium leiognathi from coastal waters of Honshu, Japan. J Bacteriol
189:6148–6158
9. Ast JC, Urbanczyk H, Dunlap PV (2009) Multi–gene analysis reveals previously
unrecognized phylogenetic diversity in Aliivibrio. Syst Appl Microbiol 32:379–386
10. Baldwin TO, Ziegler MM, Powers DA (1979) Covalent structure of subunits of bacterial
luciferase NH2– terminal sequence demonstrates subunit homology. Proc Natl Acad Sci
USA 76:4887–4889
11. Baldwin TO, Treat ML, Daubner SC (1990) Cloning and expression of the luxY gene from
Vibrio fischeri strain Y-1 in Escherichia coli and complete amino acid sequence of yellow
fluorescent protein. Biochemistry 29:5509–5515
12. Bang SS, Baumann P, Baumann L (1978) Phenotypic characterization of Photobacterium
logei (sp. nov.), a species related to P. fischeri. Curr Microbiol 1:285–288
13. Bassler BL (1999) How bacteria talk to each other: regulation of gene expression by quorum
sensing. Curr Opin Microbiol 2:582–587
14. Bassler B, Miller MB (2013) Quorum sensing. In: Rosenberg E, DeLong EF, Thompson F,
Lory S, Stackebrandt E (eds), The Prokaryotes (4th ed)—Prokaryotic Communities and
Ecophysiology. Springer, Berlin, pp 495–509. doi:10.1007/978-3-642-30123-0_60
15. Bassler BL, Greenberg EP, Stevens AM (1997) Cross-species induction of luminescence in
the quorum sensing bacterium Vibrio harveyi. J Bacteriol 179:4043–4045
16. Bassler BL, Wright M, Showalter RE, Silverman MR (1993) Intercellular signalling in
Vibrio harveyi, sequence and function of genes regulating expression of luminescence.
Molec Microbiol 9:773–786
17. Bassler BL, Wright M, Silverman MR (1994a) Sequence and function of LuxO, a negative
regulator of luminescence in Vibrio harveyi. Molec Microbiol 12:403–412
18. Bassler BL, Wright M, Silverman MR (1994) Multiple signalling systems controlling
expression of luminescence in Vibrio harveyi, sequence and function of genes encoding a
second sensory pathway. Molec Microbiol 13:273–286
19. Baumann P, Baumann L (1981) The marine Gram–negative eubacteria genera
Photobacterium, Beneckea, Alteromonas, Pseudomonas, and Alcaligenes. In: Starr MP,
Stolp H, Trüper HG, Balows A, Schlegel HG (eds) The prokaryotes. Springer, Berlin,
pp 1302–1331
20. Baumann P, Baumann L, Bang SS, Woolkalis MJ (1980) Reevaluation of the taxonomy of
Vibrio, Beneckea, and Photobacterium: abolition of the genus Beneckea. Curr Microbiol
4:127–132
21. Boemare NE, Akhurst RJ, Mourant RG (1993) DNA relatedness between Xenorhabdus spp.
(Enterobacteriaceae), symbiotic bacteria of Entomopathogenic nematodes, and a proposal
to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov. Int J Syst
Bacteriol 43:249–255
22. Boettcher KJ, Ruby EG (1990) Depressed light emission by symbiotic Vibrio fischeri of the
sepiolid squid Euprymna scolopes. J Bacteriol 17:3701–3706
23. Boisvert H, Chatelain R, Bassot J-M (1967) Étude d’un Photobacterium isolé de l’organe
lumineux des poissons Leiognathidae. Ann Inst Pasteur Paris 112:520–524
24. Boyle R (1668) Experiments concerning the relation between light and air in shining wood
and fish. Philos Trans 2:581–600
25. Budsberg KJ, Wimpee CF, Braddock JF (2003) Isolation and identification of
Photobacterium phosphoreum from an unexpected niche migrating salmon. Appl Environ
26. Callahan SM, Dunlap PV (2000) LuxR– and acylhomoserine– lactone–controlled non–lux
genes define a quorum–sensing regulon in Vibrio fischeri. J Bacteriol 182:2811–2822
27. Cao J-G, Meighen EA (1989) Purification and structural identification of an autoinducer for
the luminescence system of Vibrio harveyi. J Biol Chem 264:21670–21676
28. Chatterjee J, Miyamoto CM, Meighen EA (1996) Autoregulation of luxR the Vibrio harveyi
lux–operon activator functions as a repressor. Molec Microbiol 20:415–425
29. Chatterjee J, Miyamoto CM, Zouzoulas A, Lang BF, Skouris N, Meighen EA (2002) MetR
and CRP bind to the Vibrio harveyi lux promoters and regulate luminescence. Molec
30. Chen P-F, Tu S-C, Hagag N, Wu FY-H, Wu C-W (1985) Isolation and characterization of a
cyclic AMP receptor protein from luminous Vibrio harveyi cells. Arch Biochem Biophys
241:425–431
31. Chen X, Schauder S, Potier N, Dorsselaer AV, Pelczer I, Bassler BL, Hughson FM (2002)
Structural identification of a bacterial quorum–sensing signal containing boron. Nature
415:545–549
32. Czy_z A, Plata K, Wegrzyn G (2003) Stimulation of DNA repair as an evolutionary drive for
bacterial luminescence. Luminescence 18:140–144
33. Daubner SC, Astorga AM, Leisman GB, Baldwin TO (1987) Yellow light emission of
Vibrio fischeri strain Y-1: purification and characterization of the energy-accepting yellow
fluorescent protein. Proc Natl Acad Sci 84:8912–8916
34. Devine JH, Countryman C, Baldwin TO (1988) Nucleotide sequence of the luxR and luxI
genes and structure of the primary regulatory region of the lux regulon of Vibrio fischeri
ATCC 7744. Biochemistry 27:837–842
35. Dolan KM, Greenberg EP (1992) Evidence that GroEL, not r32, is involved in transcription
regulation of the Vibrio fischeri luminescence genes in Escherichia coli. J Bacteriol
174:5132–5135
36. Duchaud E, Rusniok C, Frangeul L, Buchrieser C, Givaudan A, Taourit S, Bocs S,
Boursaux-Eude C, Chandler M, Charles JF, Dassa E, Derose R, Derzelle S, Freyssinet G,
Gaudriault S, Médigue C, Lanois A, Powell K, Siguier P, Vincent R, Wingate V, Zouine M,
Glaser P, Boemare N, Danchin A, Kunst F (2003) The genome sequence of the
entomopathogenic bacterium Photorhabdus luminescens. Nat Biotechnol 21:1307–1313
37. Dunlap PV (1989) Regulation of luminescence by cyclic AMP in cya–like and crp–like
mutants of Vibrio fischeri. J Bacteriol 171:1199–1202
38. Dunlap PV, Ast JC (2005) Genomic and phylogenetic characterization of the luminous
bacteria symbiotic with the deep–sea fish Chlorophthalmus albatrossis (Aulopiformes
Chlorophthalmidae). Appl Environ Microbiol 71:930–939
39. Dunlap PV, Greenberg EP (1985) Control of Vibrio fischeri luminescence gene expression
in Escherichia coli by cyclic AMP and cyclic AMP receptor protein. J Bacteriol 164:45–50
40. Dunlap PV, Greenberg EP (1988) Control of Vibrio fischeri lux gene transcription by a
cyclic AMP receptor protein–LuxR protein regulatory circuit. J Bacteriol 170:4040–4046
41. Dunlap PV, Greenberg EP (1991) Role of intercellular chemical communication in the
Vibrio fischeri—monocentrid fish symbiosis. In: Dworkin M (ed) Microbial Cell–Cell
Interactions. American Society for Microbiology Washington, DC, pp 219–253
42. Dunlap PV, Kuo A (1992) Cell density–dependent modulation of the Vibrio fischeri
luminescence system in the absence of autoinducer and LuxR protein. J Bacteriol
174:2440–2448
43. Dunlap PV, Ray JM (1989) Requirement for autoinducer in transcriptional negative
autoregulation of the Vibrio fischeri luxR gene in Escherichia coli. J Bacteriol 171:3549–3552
44. Dunlap PV, Urbanczyk H (2013) Luminous bacteria. In: Rosenberg E, DeLong EF, Thompson
F, Lory S, Stackebrandt E (eds), The Prokaryotes (4th ed)—Prokaryotic Physiology and
Biochemistry. Springer, Berlin, pp 495–528. doi:10.1007/978-3-642-30141-4_75
45. Dunlap PV, Ast JC, Kimura S, Fukui A, Yoshino T, Endo H (2007) Phylogenetic analysis of
host–symbiont specificity and codivergence in bioluminescent symbioses. Cladistics
23:507–523
46. Dunlap PV, Gould AL, Wittenrich ML, Nakamura M (2012) Symbiosis initiation in the
bacterially luminous sea urchin cardinalfish Siphamia versicolor. J Fish Biol 81:1340–1356
56 P. Dunlap
47. Eberhard A, Hinton JP, Zuck RM (1979) Luminous bacteria synthesize luciferase
anaerobically. Arch Microbiol 121:277–282
48. Eberhard A, Burlingame AL, Eberhard C, Kenyon GL, Nealson KH, Oppenheimer NJ
(1981) Structural identification of autoinducer of Photobacterium fischeri luciferase.
Biochemistry 20:2444–2449
49. Eberhard A, Widrig CA, McBath P, Schineller JB (1986) Analogs of the autoinducer of
bioluminescence in Vibrio fischeri. Arch Microbiol 146:35–40
50. Eberhard A, Longin T, Widrig CA, Stranick SJ (1991) Synthesis of the lux gene autoinducer
in Vibrio fischeri is positively autoregulated. Arch Microbiol 155:294–297
51. Engebrecht J, Silverman M (1984) Identification of genes and gene products necessary for
bacterial bioluminescence. Proc Natl Acad Sci USA 81:4154–4158
52. Engebrecht J, Nealson K, Silverman M (1983) Bacterial bioluminescence, isolation and
genetic analysis of functions from Vibrio fischeri. Cell 32:773–781
53. Farmer JJ, Jorgensen JH, Grimont PAD, Akhurst RJ, Poinar GO, Pierce GV, Smith JA,
Carger GP, Wilson K, Hickman-Brenner FW (1989) Xenorhabdus luminescens (DNA
hybridization group 5) from human clinical specimens. J Clin Microbiol 27:1594–1600
54. Fidopiastis PM, von Boletzky S, Ruby EG (1998) A new niche for Vibrio logei, the
predominant light organ symbiont of squids in the genus Sepiola. J Bacteriol 180:59–64
55. Fidopiastis PM, Sorum H, Ruby EG (1999) Cryptic luminescence in the cold–water fish
pathogen Vibrio salmonicida. Arch Microbiol 171:205–209
56. Fidopiastis PM, Miyamoto CM, Jobling MG, Meighen EG, Ruby EG (2002) LitR, a new
transcriptional activator in Vibrio fischeri, regulates luminescence and symbiotic light organ
colonization. Molec Microbiol 45:131–143
57. Figge MJ, Robertson LA, Ast JC, Dunlap PV (2011) Historical microbiology: revival and
phylogenetic characterization of luminous bacterial cultures of M. W. Beijerinck. FEMS
Microbiol Ecol 78:463–472
58. Fischer–Le Saux M, Viallard V, Brunel B, Normand P, Boemare EN (1999) Polyphasic
classification of the genus Photorhabdus and proposal of new taxa P. luminescens subsp.
luminescens subsp. nov., P. luminescens subsp. akhurstii subsp. nov., P. luminescens subsp.
laumondii subsp. nov., P. temperata sp. nov., P. temperata subsp. temperata subsp. nov.,
and P. asymbiotica sp. nov. Int J Syst Bacteriol 49:1645–1656
59. Fitzgerald JM (1977) Classification of luminous bacteria from the light organ of the
Australian pinecone fish, Cleidopus gloriamaris. Arch Microbiol 112:153–156
60. Forst S, Nealson K (1996) Molecular biology of the symbiotic–pathogenic bacteria
Xenorhabdus spp. and Photorhabdus spp. Microbiol Rev 60:21–43
61. Forst S, Dowds B, Boemare N, Stackebrandt E (1997) Xenorhabdus and Photorhabdus spp.
Bugs that kill bugs. Ann Rev Microbiol 51:47–72
62. Freeman JA, Bassler BL (1999) A genetic analysis of the function of LuxO, a two–component
response regulator involved in quorum sensing in Vibrio harveyi. Molec Microbiol
31:665–677
63. Freeman JA, Bassler BL (1999) Sequence and function of LuxU: a two–component
phosphorelay protein that regulates quorum sensing in Vibrio harveyi. J Bacteriol
191:899–906
64. Freeman JA, Lilley BN, Bassler BL (2000) A genetic analysis of the functions of LuxN: a
two–component hybrid sensor kinase that regulates quorum sensing in Vibrio harveyi.
Molec Microbiol 35:139–149
65. Fukasawa S, Dunlap PV (1986) Identification of luminous bacteria isolated from the light
organ of the squid, Doryteuthis kensaki. J Agric Biol Chem 50:1645–1646
66. Fuqua WC, Winans SC, Greenberg EP (1994) Quorum sensing in bacteria the LuxR–LuxI
family of cell density–responsive transcriptional regulators. J Bacteriol 176:269–275
67. Giard A, Billet A (1889) Observations sur la maladie phosphorescente des Talitres et autres
crustaces. Compt Rend Biol Paris 41:593–597
68. Gilson L, Kuo A, Dunlap PV (1995) AinS and a new family of autoinducer synthesis
proteins. J Bacteriol 177:6946–6951
69. Gomez-Gil B, Soto-Rodríguez S, García-Gasca A, Roque A, Vazquez-Juarez R, Thompson

FL, Swings J (2004) Molecular identification of Vibrio harveyi–related isolates associated
with diseased aquatic organisms. Microbiology 150:1769–1777
70. Greenberg EP (1997) Quorum sensing in Gram–negative bacteria. Amer Soc Microbiol
News 63:371–377
71. Hanzelka BL, Parsek MR, Val DV, Dunlap PV, Cronan JE Jr, Greenberg EP (1999)
Acylhomoserine lactone synthase activity of the Vibrio fischeri AinS protein. J Bacteriol
181:5766–5770
72. Harvey EN (1940) Living light. Princeton University Press, Princeton
73. Harvey EN (1952) Bioluminescence. Academic Press, New York
74. Harvey EN (1957) A history of luminescence from the earliest times until 1900. American
Philosophical Society, Philadelphia
75. Hastings JW (1983) Biological diversity, chemical mechanisms, and the evolutionary
origins of bioluminescent systems. J Molec Evol 19:309–317
76. Hastings JW (2012) Bioluminescence. Cell Physiology Sourcebook. doi:10:1016/B978-0-
12-387738-3.00052-4
77. Hastings JW, Greenberg EP (1999) Quorum sensing: the explanation of a curious
phenomenon reveals a common characteristic of bacteria. J Bacteriol 181:2667–2669
78. Hastings JW, Nealson KH (1981) The symbiotic luminous bacteria. In: Starr MP, Stolp H,
Trüper HG, Balows A, Schlegel HG (eds) The prokaryotes. Springer, Berlin, pp 1332–1345
79. Hastings JW, Potrikus CJ, Gupta SC, Kurfurst M, Makemson JC (1985) Biochemistry and
physiology of bioluminescent bacteria. Adv Microb Physiol 26:235–291
80. Haygood MG (1990) Relationship of the luminous bacterial symbiont of the Caribbean
flashlight fish, Kryptophaneron alfredi (family Anomalopidae) to other luminous bacteria
based on bacterial luciferase (luxA) genes. Arch Microbiol 154:496–503
81. Haygood MG (1993) Light organ symbioses in fish. Crit Rev Microbiol 19:191–216
82. Haygood MG, Nealson KH (1985) Mechanisms of iron regulation of luminescence in Vibrio
fischeri. J Bacteriol 162:209–216
83. Haygood MG, Distel DL (1993) Bioluminescent symbionts of flashlight fish and deep–sea
anglerfish form unique lineages related to the genus Vibrio. Nature 363:154–156
84. Haygood MG, Distel DL, Herring PJ (1992) Polymerase chain reaction and 16S rRNA gene
sequences from the luminous bacterial symbionts of two deepsea anglerfish. J Marine Biol
Assoc UK 72:149–159
85. Hendrie MS, Hodgkiss W, Shewan JM (1970) The identification, taxonomy and
classification of luminous bacteria. J Gen Microbiol 64:151–169
86. Hendry TA, Dunlap PV (2011) The uncultured luminous symbiont of Anomalops katoptron
(Beryciformes: Anomalopidae) represents a new bacterial genus. Mol Phylogenet Evol
61:834–843
87. Hendry TA, Dunlap PV (2014) Phylogenetic divergence between the obligate luminous
symbionts of flashlight fishes demonstrates specificity of bacteria to host genera. Environ
Microbiol Rep. doi:10.1111/1758-2229.12135 (in press)
88. Hendry TA, deWet JR, Dunlap PV (2014) Genomic signatures of obligate host dependence in
the luminous bacterial symbiont of a vertebrate. Environ Microbiol doi:10.1111/1462-2920.
12302 (in press)
89. Henke JM, Bassler BL (2004) Three parallel quorum–sensing systems regulate gene
expression in Vibrio harveyi. J Bacteriol 186:6902–6904
90. Higgins DA, Pmianek ME, Kraml CM, Taylor RK, Semmelhack MF, Bassler BL (2007)
The major Vibrio cholerae autoinducer and its role in virulence factor production. Nature
450:883–886
91. Hjerde E, Lorentzen MS, Holden MT, Seeger K, Paulsen S, Bason N, Churcher C, Harris D,
Norbertczak H, Quail MA, Sanders S, Thurston S, Parkhill J, Willassen NP, Thomson NR
(2008) The genome sequence of the fish pathogen Aliivibrio salmonicida strain LFI1238
shows extensive evidence of gene decay. BMC Genom 9:616
58 P. Dunlap
92. Jensen MJ, Tebo BM, Baumann P, Mandel M, Nealson KH (1980) Characterization of
Alteromonas hanedai (sp. nov.), a nonfermentative luminous species of marine origin. Curr
93. Kaeding AJ, Ast JC, Pearce MM, Urbanczyk H, Kimura S, Endo H, Nakamura M, Dunlap
PV (2007) Phylogenetic diversity and co–symbiosis in the bioluminescent symbioses of
Photobacterium mandapamensis. Appl Environ Microbiol 73:3173–3182
94. Kaplan HB, Greenberg EP (1985) Diffusion of autoinducer is involved in regulation of the
Vibrio fischeri luminescence system. J Bacteriol 163:1210–1214
95. Kasai S (2006) Freshwater bioluminescence in Vibrio albensis (Vibrio cholerae biovar
albensis) NCIMB 41 is caused by a two–nucleotide deletion in luxO. J Biochem 139:471–482
96. Kasai S, Okada K, Hoshino A, Iida T, Honda T (2007) Lateral transfer of the lux gene
cluster. J Biochem Tokyo 141:231–237
97. Kelly RC, Bolitho ME, Higgins DA, Lu W, Ng WL, Jeffrey PD, Rabinowitz JD,
Semmelhack MF, Hughson FM, Bassler BL (2009) The Vibrio cholerae quorum–sensing
autoinducer CAI–1: analysis of the biosynthetic enzyme CqsA. Nat Chem Biol 5:891–895
98. Kuo A, Callahan SM, Dunlap PV (1996) Modulation of luminescence operon expression by
N–octanoyl–homoserine lactone in ainS mutants of Vibrio fischeri. J Bacteriol 178:971–976
99. Kuwata R, Yoshiga T, Yoshida M, Kondo E (2008) Mutualistic association of
Photorhabdus asymbiotica with Japanese heterorhabditid entomopathogenic nematodes.
Microbes Infect 10:734–741
100. Lee CY, Meighen EA (1992) The lux genes in Photobacterium leiognathi are closely linked
with genes corresponding in sequence to riboflavin synthesis genes. Biochem Biophys Res
Commun 186:690–697
101. Lee CY, O’Kane DJ, Meighen EA (1994) Riboflavin synthesis genes are linked with the lux
operon of Photobacterium phosphoreum. J Bacteriol 176:2100–2104
102. Lee J (1993) Lumazine protein and the excitation mechanism in bacterial bioluminescence.
Biophys Chem 48:149–158
103. Lenz DH, Mok KC, Lilley BN, Kulkarni RV, Wingreen NS, Bassler BL (2004) The small
RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi.
Cell 118:69–82
104. Li L, Liu X, Yang W, Xu W, Xu F, Wang W, Feng L, Bartlam M, Wang L, Rao Z (2008)
Crystal structure of long-chain alkane monooxygenase (LadA) in complex with coenzyme
FMN: unveiling the long-chain alkane hydroxylase. J Mol Biol 376:453–465
105. Li Z, Szittner R, Meighen EA (1993) Subunit interactions and the role of the luxA
polypeptide in controlling thermal stability and catalytic properties in recombinant
luciferase hybrids. Biochim Biophys Acta 1158:137–145
106. Lilley BN, Bassler BL (2000) Regulation of quorum sensing in Vibrio harveyi by LuxO and
sigma–54. Molec Microbiol 36:940–954
107. Lin B, Wang Z, Malanoski AP, O’Grady EA, Wimpee CF, Vuddhakul V, Alves N Jr,
Thompson FL, Gomez-Gil B, Voral GJ (2010) Comparative genomic analyses identify the
Vibrio harveyi genome sequenced strains BAA–1116 and HY01 as Vibrio campbellii.
Environ Microbiol Rep 2:81–89
108. Lin J-W, Chao Y-F, Weng S-F (1993) The lumazine protein–encoding gene in
Photobacterium leiognathi is linked to the lux operon. Gene 126:153–154
109. Lin J-W, Yu K-Y, Chao Y-F, Weng S-F (1995) The lumQ gene is linked to the lumP gene
and the lux operon in Photobacterium leiognathi. Biochem Biophys Res Commun
217:684–695
110. Lin J-W, Chao Y-F, Weng S-F (1996) Nucleotide sequence and functional analysis of the
luxE gene encoding acyl–protein synthetase of the lux operon from Photobacterium
leiognathi. Biochem Biophys Res Commun 228:764–773
111. Lin J-W, Yu K-Y, Chao Y-F, Weng S-F (1996) Regulatory region with putA gene of proline
dehydrogenase that links to the lum and lux operons in Photobacterium leiognathi. Biochem
Biophys Res Commun 219:868–875
112. Lin J-W, Chao Y-F, Weng S-F (1998) Characteristic analysis of the luxG gene encoding the
probable flavin reductase that resides in the lux operon of Photobacterium leiognathi.
Biochem Biophys Res Commun 246:446–452
113. Lin J-W, Chao Y-F, Weng S-F (2001) Riboflavin synthesis genes ribE, ribB, ribH, ribA
reside in the lux operon of Photobacterium leiognathi. Biochem Biophys Res Commun
284:587–595
114. Lin YH, Miyamoto C, Meighen EA (2000) Cloning and functional studies of a luxO
regulator LuxT from Vibrio harveyi. Biochim Biophys Acta 1494:226–235
115. Lunder T, Sørum H, Holstad G, Steigerwalt AG, Mowinckel P, Brenner DJ (2000)
Phenotypic and genotypic characterization of Vibrio viscosus sp. nov. and Vibrio wodanis
sp. nov. isolated from Atlantic salmon (Salmo salar) with ‘winter ulcer’. Int J Syst Evol
116. Makemson JC (1986) Luciferase–dependent oxygen consumption by bioluminescent
vibrios. J Bacteriol 165:461–466
117. Makemson JC, Fulayfil NR, Landry W, Van Ert LM, Wimpee CF, Widder EA, Case JF
(1997) Shewanella woodyi sp. nov., an exclusively respiratory luminous bacterium isolated
from the Alboran Sea. Int J Syst Bacteriol 47:1034–1039
118. Makemson JC, Hastings JW (1982) Iron represses bioluminescence in Vibrio harveyi. Curr
119. Manukhov IV, Khrul’nova SA, Baranova A, Zavilgelsky GB (2011) Comparative analysis
of the lux operons in Aliivibrio logei KCh1 (a Kamchatka Isolate) and Aliivibrio
salmonicida. J Bacteriol 193:3998–4001
120. McElroy WD, Seliger HH (1962) Origin and evolution of bioluminescence. In: Kasha M,
Pullman B (eds) Horizons in biochemistry. Academic Press, New York, pp 91–101
121. Meighen EA (1991) Molecular biology of bacterial bioluminescence. Microbiol Rev
55:123–142
122. Meighen EA, Dunlap PV (1993) Physiological, biochemical and genetic control of bacterial
bioluminescence. Adv Microb Physiol 34:1–67
123. Meighen EA, Szittner RB (1992) Multiple repetitive elements and organization of the lux
operons of luminescent terrestrial bacteria. J Bacteriol 174:5371–5381
124. Miller MB, Bassler BL (2001) Quorum sensing in bacteria. Annu Rev Microbiol
55:165–199
125. Miyamoto CM, Chatterjee J, Swartzman E, Szittner R, Meighen EA (1996) The role of lux
autoinducer in regulating luminescence in Vibrio harveyi control of luxR expression. Molec
126. Miyamoto CM, Dunlap PV, Ruby EG, Meighen EA (2003) LuxO controls luxR expression
in Vibrio harveyi evidence for a common regulatory mechanism in Vibrio. Molec Microbiol
48:537–548
127. Miyamoto CM, Graham AF, Meighen EA (1988) Nucleotide sequence of the luxC gene and
the upstream DNA from the bioluminescent system of Vibrio harveyi. Nucl Acids Res
16:1551–1562
128. Miyamoto CM, Lin YH, Meighen EA (2000) Control of bioluminescence in Vibrio fischeri
by the LuxO signal response regulator Molec. Microbiol 36:594–607
129. Miyashiro T, Wollenberg MS, Cao X, Oehlert D, Ruby EG (2010) A single qrr gene is
necessary and sufficient for LuxO–mediated regulation in Vibrio fischeri. Molec Microbiol
77:1556–1567
130. Moore SA, James MN (1995) Structural refinement of the non–fluorescent flavoprotein from
Photobacterium leiognathi at 160 A resolution. J Mol Biol 249:195–214
131. Müller-Breitkreutz K, Winkler UK (1993) Anaerobic expression of the Vibrio fischeri lux
regulon in E. coli is Fnr–dependent. J Biolumin Chemilumin 8:108
132. Nealson KH (1977) Autoinduction of bacterial luciferase. Occurrence, mechanism and
significance. Arch Microbiol 112:73–79
133. Nealson KH, Hastings JW (1977) Low oxygen is optimal for luciferase synthesis in some
bacteria. Ecological implications. Arch Microbiol 112:9–16
60 P. Dunlap
134. Nealson KH, Hastings JW (1979) Bacterial bioluminescence. Its control and ecological
significance. Microbiol Rev 43:496–518
135. Nealson KH, Hastings JW (1992) The luminous bacteria. In: Balows A, Trüper HG,
Dworkin M, Harder W, Schleifer K-H (eds) The prokaryotes, 2nd edn. Springer, Berlin,
pp 625–639
136. Nealson KH, Platt T, Hastings JW (1970) Cellular control of synthesis and activity of the
bacterial luminescence system. J Bacteriol 104:313–322
137. Nealson KH, Eberhard A, Hastings JW (1972) Catabolite repression of bacterial
bioluminescence, functional implications. Proc Natl Acad Sci USA 69:1073–1076
138. Nealson KH, Wimpee B, Wimpee C (1993) Identification of Vibrio splendidus as a member
of the planktonic luminous bacteria from the Persian Gulf and Kuwait region with luxA
probes. Appl Environ Microbiol 59:2684–2689
139. Nelson EJ, Tunsjø HS, Fidopiastis PM, Sørum H, Ruby EG (2007) A novel lux operon in the
cryptically bioluminescent fish pathogen Vibrio salmonicida is associated with virulence.
Appl Environ Microbiol 73:1825–1833
140. Ng W-L, Bassler BL (2009) Bacterial quorum–sensing network architectures. Ann Rev Gen
43:197–222
141. Nijvipakul S, Wongratana J, Suadee C, Entsch B, Ballou DP, Chaiyen P (2008) LuxG is a
functioning flavin reductase for bacterial luminescence. J Bacteriol 190:1531–1538
142. O’Brien CH, Sizemore RK (1979) Distribution of the luminous bacterium Beneckea harveyi
in a semitropical estuarine environment. Appl Environ Microbiol 38:933–938
143. O’Grady EA, Wimpee CF (2008) Mutations in the lux operon of natural dark mutants in the
genus Vibrio. Appl Environ Microbiol 74:61–66
144. O’Kane DJ, Prasher DC (1992) Evolutionary origins of bacterial bioluminescence. Molec
145. O’Kane DJ, Karle VA, Lee J (1985) Purification of lumazine proteins from Photobacterium
leiognathi and Photobacterium phosphoreum: bioluminescent properties. Biochemistry
24:1461–1467
146. O’Kane DJ, Woodward B, Lee J, Prasher DC (1991) Borrowed proteins in bacterial
bioluminescence. Proc Natl Acad Sci 88:1100–1104
147. Oliver JD, Roberts DM, White VK, Dry MA, Simpson LM (1986) Bioluminescence in a
strain of the human pathogenic bacterium Vibrio vulnificus. Appl Environ Microbiol
52:1209–1211
148. Palmer LM, Colwell RR (1991) Detection of luciferase gene sequence in nonluminescent
Vibrio cholerae by colony hybridization and polymerase chain reaction. Appl Environ
149. Peel MM, Alfredson DA, Gerrard JG, Davis JM, Robson JM, McDougall RJ, Scullie BL,
Akhurst RJ (1999) Isolation, identification, and molecular characterization of strains of
Photorhabdus luminescens from infected humans in Australia. J Clin Microbiol
37:3647–3653
150. Petushkov VN, Lee J (1997) Purification and characterization of flavoproteins and
cytochromes from the yellow bioluminescence marine bacterium Vibrio fischeri strain Y1.
Eur J Biochem 245:790–796
151. Petushkov VN, Ketelaars M, Gibson BG, Lee J (1996) Interaction of Photobacterium
leiognathi and Vibrio fischeri Y1 luciferases with fluorescent (antenna) proteins
bioluminescence effects of the aliphatic additive. Biochemistry 35:12086–12093
152. Pflüger E (1875) Ueber die Phosphorescenz verwesender Organismen. Arch ges Physiol
Men Tiere 11:222–263
153. Pujalte MJ, Garay E (1986) Proposal of Vibrio mediterranei sp. nov. A new marine member
of the Genus Vibrio. Int J Syst Bacteriol 36:278–281
154. Ramaiah N, Chun J, Ravel J, Straube WL, Hill RT, Colwell RR (2000) Detection of
luciferase gene sequences in non–luminescent bacteria from the Chesapeake Bay. FEMS
Microbiol Ecol 33:27–34
155. Ramsey MM, Korgaonkar AK, Whiteley M (2009) Quorum sensing in bacteria. In:
Schaechter M Encyclopedia of microbiology, 3rd edn. Academic Press, New York
156. Rees J-F, de Wergifosse B, Noiset O, Dubuisson M, Janssens B, Thompson EM (1998) The
origins of marine bioluminescence. Turning oxygen defense mechanisms into deep–sea
communication tools. J Exp Biol 201:1211–1221
157. Reichelt JL, Baumman P (1973) Taxonomy of the marine, luminous bacteria. Arch
Mikrobiol 94:283–330
158. Reichelt JL, Nealson K, Hastings JW (1977) The specificity of symbiosis pony fish and
luminescent bacteria. Arch Microbiol 112:157–161
159. Robertson LA, Figge MJ, Dunlap PV (2011) Beijerinck and the bioluminescent bacteria—
microbiological experiments in the late 19th and early 20th centuries. FEMS Microbiol Ecol
75:185–194
160. Ruby EG, Morin JG (1979) Luminous enteric bacteria of marine fish a study of their
distribution, densities, and dispersion. Appl Environ Microbiol 38:406–411
161. Ruby EG, Nealson KH (1976) Symbiotic association of Photobacterium fischeri with the
marine luminous fish Monocentris japonica, a model of symbiosis based on bacterial
studies. Biol Bull 141:574–5867
162. Ruby EG, Nealson KH (1977) A luminous bacterium that emits yellow light. Science
196:432–434
163. Ruby EG, Nealson KH (1978) Seasonal changes in the species composition of luminous
bacteria in nearshore seawater. Limnol Oceanogr 23:530–533
164. Ruby EG, Urbanowski M, Campbell J, Dunn A, Faini M, Gunsalus R, Lostroh P, Lupp C,
McCann J, Millikan D, Schaefer A, Stabb E, Stevens A, Visick K, Whistler C, Greenberg
EP (2005) Complete genome sequence of Vibrio fischeri a symbiotic bacterium with
pathogenic congeners. Proc Natl Acad Sci USA 102:3004–3009
165. Sato Y, Shimizu S, Ohtaki A, Noguchi K, Miyatake H, Dohmae N, Sasaki S, Odaka M,
Yohda M (2010) Crystal structures of the Lumazine protein from Photobacterium kishitanii
in complexes with the authentic chromophore, 6,7–dimethyl–8–(1_–D–ribityl) lumazine,
and its analogues, riboflavin and flavin mononucleotide, at high resolution. J Bacteriol
192:127–133
166. Schaefer AL, Val DL, Hanzelka BL, Cronan JE Jr, Greenberg EP (1996) Generation of cell–
to–cell signals in quorum sensing:acyl homoserine lactone synthase activity of a purified
Vibrio fischeri LuxI. Proc Natl Acad Sci USA 93:9505–9509
167. Schauder S, Shokat K, Surette MG, Bassler BL (2001) The LuxS family of bacterial
autoinducers biosynthesis of a novel quorum–sensing signal molecule. Molec Microbiol
41:463–476
168. Seliger HH (1987) The evolution of bioluminescence in bacteria. Photochem Photobiol
45:291–297
169. Shadel GS, Devine JH, Baldwin TO (1990) Control of the lux regulon of Vibrio fischeri.
J Biolumin Chemilumin 5:99–106
170. Showalter RE, Martin MO, Silverman MR (1990) Cloning and nucleotide sequence of luxR,
a regulatory gene controlling bioluminescence in Vibrio harveyi. J Bacteriol 172:2946–2954
171. Silverman M, Martin M, Engebrecht J (1989) Regulation of luminescence in marine
bacteria. In: Hopwood DA, Chater KF (eds) Genetics of bacterial diversity. Academic Press,
London, pp 71–86
172. Small ED, Koka P, Lee J (1980) Lumazine protein from the bioluminescent bacterium
Photobacterium phosphoreum. Purification and characterization. J Biol Chem 255:8804–8810
173. Smith SK, Sutton DC, Fuerst JA, Reichelt JL (1991) Evaluation of the genus Listonella and
reassignment of Listonella damsela (Love et al.) MacDonell and Colwell to the genus
Photobacterium as Photobacterium damsela comb. nov. with an emended description. Int J
Syst Bacteriol 41:529–534
174. Stevens AM, Greenberg EP (1997) Quorum sensing in Vibrio fischeri: essential elements for
activation of the luminescence genes. J Bacteriol 179:557–562
62 P. Dunlap
175. Suadee C, Nijvipakul S, Svasti J, Entsch B, Ballou DP, Chaiyen P (2007) Luciferase from
Vibrio campbellii is more thermostable and binds reduced FMN better than its homologues.
J Biochem 142:539–552
176. Sung ND, Lee CY (2004) Coregulation of lux genes and riboflavin genes in bioluminescent
bacteria of Photobacterium phosphoreum. J Microbiol 42:194–199
177. Surete MG, Miller MB, Bassler BL (1999) Quorum sensing in Escherichia coli, Salmonella
typhimurium, and Vibrio harveyi: a new family of genes responsible for autoinducer
production. Proc Natl Acad Sci USA 96:1639–1644
178. Swartzman E, Kapoor S, Graham AF, Meighen EA (1990) A new Vibrio fischeri lux gene
precedes a bidirectional termination site for the lux operon. J Bacteriol 172:6797–6802
179. Swartzman E, Miyamoto C, Graham A, Meighen EA (1990) Delineation of the
transcriptional boundaries of the lux operon of Vibrio harveyi demonstrates the presence
of two new lux genes. J Biol Chem 265:3513–3517
180. Swartzman E, Silverman M, Meighen EA (1992) The luxR gene product of Vibrio harveyi is
a transcriptional activator of the lux promoter. J Bacteriol 174:7490–7493
181. Tailliez P, Laroui C, Ginibre N, Paule A, Pagès S, Boemare N (2010) Phylogeny of
Photorhabdus and Xenorhabdus based on universally conserved protein–coding sequences
and implications for the taxonomy of these two genera. Proposal of new taxa X.
vietnamensis sp. nov., P. luminescens subsp. caribbeanensis subsp. nov., P. luminescens
subsp. hainanensis subsp. nov., P. temperata subsp. khanii subsp. nov., P. temperata subsp.
tasmaniensis subsp. nov., and the reclassification of P. luminescens subsp. thracensis as P.
temperata subsp. thracensis comb. nov. Int J Syst Evol Microbiol 60:1921–1937
182. Thompson FL, Thompson CC, Li Y, Gomez-Gil B, Vandenberghe J, Hoste B, Swings J
(2003) Vibrio kanaloae sp. nov., Vibrio pomeroyi sp. nov. and Vibrio chagasii sp. nov.,
from sea water and marine animals. Int J Syst Evol Microbiol 53:753–759
183. Tinikul R, Pitsawong W, Sucharitakul J, Nijvipakul S, Ballou DP, Chaiyen P (2013) The
transfer of reduced flavin mononucleotide from LuxG oxidoreductase to luciferase occurs
via free diffusion. Biochemistry 52:6834–6843
184. Tu KC, Bassler BL (2007) Multiple small RNAs act additively to integrate sensory
information and control quorum sensing in Vibrio harveyi. Genes Dev 21:221–233
185. Tu KC, Long T, Svenningsen SL, Wingreen NS, Bassler BL (2010) Negative feedback
loops involving small regulatory RNAs precisely control the Vibrio harveyi quorum–
sensing response. Mol Cell 37:567–579
186. Ulitzur S, Dunlap PV (1995) Regulatory circuitry controlling luminescence autoinduction in
Vibrio fischeri. Photochem Photobiol 62:625–632
187. Ulitzur S, Yashphe J (1975) An adenosine 3’,5’–monophosphate–requiring mutant of the
luminous bacteria Beneckea harveyi. Biochim Biophys Acta 404:321–328
fischeri, Vibrio logei, Vibrio salmonicida and Vibrio wodanis as Aliivibrio fischeri gen. nov.,
comb. nov., Aliivibrio logei comb. nov., Aliivibrio salmonicida comb. nov. and Aliivibrio
wodanis comb. nov. Int J Syst Evol Micro 57:2823–2829
189. Urbanczyk H, Ast JC, Kaeding AJ, Oliver JD, Dunlap PV (2008) Phylogenetic analysis of
the incidence of lux gene horizontal transfer in Vibrionaceae. J Bacteriol 190:3494–3504
190. Urbanczyk H, Ast JC, Dunlap PV (2011) Phylogeny, genomics, and symbiosis of
Photobacterium. FEMS Microbiol Rev 35:324–342
191. Urbanczyk H, Ogura Y, Hendry TA, Gould AL, Kiwaki N, Atkinson JT, Hayashi T, Dunlap
PV (2011) Genome Sequence of Photobacterium mandapamensis svers.1.1, the
bioluminescent symbiont of the cardinal fish Siphamia versicolor. J Bacteriol 193:3144–3145
192. Urbanczyk H, Furukawa T, Yamamoto Y, Dunlap PV (2012) Natural replacement of the
vertically inherited lux-rib genes of Photobacterium aquimaris by horizontally acquired
homologs. Environ Microbiol Rep 4:412–416
193. Urbanczyk H, Ogura Y, Hayashi T (2013) Taxonomic revision of Harveyi clade bacteria
(family Vibrionaceae) base don analysis of whole genome sequences. Int J Syst Evol
194. Walker EL, Bose JL, Stabb EV (2006) Photolyase confers resistance to UV light but does
not contribute to the symbiotic benefit of bioluminescence in Vibrio fischeri ES114. Appl
Environ Microbiol 72:6600–6606
195. Waterfield NR, Ciche T, Clarke D (2009) Photorhabdus and a host of hosts. Ann Rev
196. Waters CM, Bassler BL (2005) Quorum sensing cell–to–cell communication in bacteria.
Ann Rev Cell Devel Biol 21:319–346
197. Waters CM, Bassler BL (2006) The Vibrio harveyi quorum-sensing system uses shared
regulatory components to discriminate between multiple autoinducers. Genes Dev
20:2754–2767
198. Wei Y, Perez LJ, Ng WL, Semmelhack MF, Bassler BL (2011) Mechanism of Vibrio
cholerae autoinducer–1 biosynthesis. ACS Chem Biol 6:356–365
199. Widder EA (2010) Bioluminescence in the ocean origins of biological, chemical, and
ecological diversity. Science 328:704–708
200. Wilkinson P, Waterfield NR, Crossman L, Corton C, Sanchez-Contreras M, Vlisidou I,
Barron A, Bignell A, Clark L, Ormond D, Mayho M, Bason N, Smith F, Simmonds M,
Churcher C, Harris D, Thompson NR, Quail M, Parkhill J, Ffrench-Constant RH (2009)
Comparative genomics of the emerging human pathogen Photorhabdus asymbiotica with
the insect pathogen Photorhabdus luminescens. BMC Genom 10:302
201. Wilson T, Hastings JW (1998) Bioluminescence. Ann Rev Cell Devel Biol 14:197–230
202. Wimpee CF, Nadeau T-L, Nealson KH (1991) Development of species–specific
hybridization probes for marine luminous bacteria by using in vitro DNA amplification.
Appl Environ Microbiol 57:1319–1324
203. Wolfe CJ, Haygood MG (1991) Restriction fragment length polymorphism analysis reveals
high levels of genetic divergence among the light organ symbionts of flashlight fish. Biol
Bull 181:135–143
204. Wollenberg MS, Preheim SP, Polz MF, Ruby EG (2012) Polyphyly of non–bioluminescent
Vibrio fischeri sharing a lux–locus deletion. Environ Microbiol 14:655–668
205. Yang Y, Yeh LP, Cao Y, Baumann L, Baumann P, Tang JS-E, Beaman B (1983)
Characterization of marine luminous bacteria isolated off the coast of China and description
of Vibrio orientalis sp. nov. Curr Microbiol 8:95–100
206. Yetinson T, Shilo M (1979) Seasonal and geographic distribution of luminous bacteria in
the eastern Mediterranean Sea and the Gulf of Elat. Appl Environ Microbiol 37:1230–1238
207. Yoshizawa S, Wada M, Kita-Tsukamoto K, Ikemoto E, Yokota A, Kogure K (2009) Vibrio
azureus sp. nov., a luminous marine bacterium isolated from seawater. Int J Syst Evol
208. Yoshizawa S, Wada M, Kita-Tsukamoto K, Yokota A, Kogure K (2009) Photobacterium
aquimaris sp. nov., a luminous marine bacterium isolated from seawater. Int J Syst Evol
209. Yoshizawa S, Karatani H, Wada M, Yokota A, Kogure K (2010) Aliivibrio sifiae sp. nov.,
luminous marine bacteria isolated from seawater. J Gen Appl Microbiol 56:508–518
210. Yoshizawa S, Wada M, Yokota A, Kogure K (2010) Vibrio sagamiensis sp. nov., luminous
marine bacteria isolated from seawater. J Gen Appl Microbiol 56:499–507
211. Zarubin M, Belkin S, Ionescu M, Genin A (2012) Bacterial bioluminescence as a lure for
marine zooplankton and fish. Proc Natl Acad Sci USA 109:853–857
212. Zenno H, Saigo K (1994) Identification of the genes encoding NAD(P)H–flavin
oxidoreductases that are similar in sequence to Escherichiacoli Fre in four species of
luminous bacteria Photorhabdus luminescens, Vibrio fischeri, Vibrio harveyi, and Vibrio
orientalis. J Bacteriol 176:3544–3551
213. Zenno H, Inouye S, Saigo K (1992) Does the luxG gene in luminous bacteria code for an
NAD(P)H–FMN oxidoreductase? Genetics (Life Sci Adv) 11:85–91
64 P. Dunlap
214. Zenno H, Saigo K, Kanoh H, Inouye S (1994) Identification of the gene encoding the major
NAD(P)H–flavin oxidoreductase of the bioluminescent bacterium Vibrio fischeri ATCC
7744. J Bacteriol 176:3536–3543
215. Zo YG, Chokesajjawatee N, Grim C, Arakawa E, Watanabe H, Colwell RR (2009)
Diversity and seasonality of bioluminescent Vibrio cholerae populations in Chesapeake
Bay. Appl Environ Microbiol 75:135–146
Part II
Applications of Bioluminescence in
Environment and Security
Application of Enzyme Bioluminescence
in Ecology
Elena Esimbekova, Valentina Kratasyuk and Osamu Shimomura
Abstract This review examines the general principles of bioluminescent enzy-

matic toxicity bioassays and describes the applications of these methods and the
implementation in commercial biosensors. Bioluminescent enzyme system tech-
nology (BEST) has been proposed in the bacterial coupled enzyme system, wherein
NADH:FMN-oxidoreductase-luciferase substitutes for living organisms. BEST
was introduced to facilitate and accelerate the development of cost-competitive
enzymatic systems for use in biosensors for medical, environmental, and industrial
applications. For widespread use of BEST, the multicomponent reagent ‘‘Enzy-
molum’’ has been developed, which contains the bacterial luciferase, NADH:FMN-
oxidoreductase, and their substrates, co-immobilized in starch or gelatin gel.
Enzymolum is the central part of Portable Laboratory for Toxicity Detection
(PLTD), which consists of a biodetector module, a sampling module, a sample
preparation module, and a reagent module. PLTD instantly signals chemical–
biological hazards and allows us to detect a wide range of toxic substances.
Enzymolum can be integrated as a biological module into the portable biodetector–
biosensor originally constructed for personal use. Based on the example of Enzy-
molum and the algorithm for creating new enzyme biotests with tailored charac-
teristics, a new approach was demonstrated in biotechnological design and
construction. The examples of biotechnological design of various bioluminescent
methods for ecological monitoring were provided. Possible applications of enzyme
bioassays are seen in the examples for medical diagnostics, assessment of the effect
of physical load on sportsmen, analysis of food additives, and in practical courses
for higher educational institutions and schools. The advantages of enzymatic assays
are their rapidity (the period of time required does not exceed 3–5 min), high
E. Esimbekova V. Kratasyuk (&)

Institute of Biophysics SB RAS, Akademgorodok, Krasnoyarsk 660036, Russia
e-mail: ValKrat@mail.ru
E. Esimbekova V. Kratasyuk O. Shimomura
Siberian Federal University, Svobodnii Ave., 79, Krasnoyarsk 660041, Russia

DOI: 10.1007/978-3-662-43385-0_3, Springer-Verlag Berlin Heidelberg 2014
68 E. Esimbekova et al.
sensitivity, simplicity and safety of procedure, and possibility of automation of

ecological monitoring; the required luminometer is easily available.
Keywords Bioluminescence Ecological monitoring Enzymatic assay

Immobilization Integral water toxicity Luciferase
Abbreviations
ADH Alcohol dehydrogenase
BEST Bioluminescent enzyme system technology
FMN Flavin mononucleotide
FMNH2 Reduced flavin mononucleotide
L Luciferase
L+R Coupled enzyme system: NADH:FMN-oxidoreductase-luciferase
LI Luciferase index
P Induction period
PLTD Portable laboratory for toxicity detection
R NADH:FMN-oxidoreductase
RCHO Long-chain aliphatic aldehyde
RCOOH Corresponding fatty acid
TC Toxicity coefficient
Contents
1 Introduction.......................................................................................................................... 69
2 Principles of Bioluminescent Enzymatic Bioassays .......................................................... 71
2.1 The Bioassays Based on the Bacterial Coupled Enzyme System:
NADH:FMN-Oxidoreductase-Luciferase................................................................... 71
2.2 Enzymatic Reagents for Bioluminescent Analysis.................................................... 75
2.3 Biotesting Natural and Waste Waters Using the Soluble and Immobilized
Coupled Enzyme System NADH:FMN-Oxidoreductase-Luciferase ........................ 83
2.4 The Reagent ‘‘Enzymolum’’ in Toxicological Bioassay of Air ............................... 85
2.5 Signal System of Enzymatic Assays: Methodological Aspects................................ 86
2.6 How to Design New Bioluminescent Enzymatic Bioassays..................................... 87
2.7 Signal System of Enzymatic Methods for Toxicological Bioassay
of Natural and Laboratory Aquatic Ecosystems ....................................................... 91
2.8 Signal System of Enzymatic Assays for Toxicological Bioassay of Pesticides ...... 99
3 Summary ............................................................................................................................ 103
References................................................................................................................................ 105
Application of Enzyme Bioluminescence 69
1 Introduction
Present-day analytical methods based on bioluminescent reactions catalyzed by

various luciferases cover a wide range of analytical tasks from ecological moni-
toring of the environment (integral bioassays) to molecular diagnostics of various
diseases and infections (high-sensitivity and specific bioluminescent immunoas-
says and heteroduplex analyses). The prospective viability of the application of
bioluminescent methods is explained by their advantages:
• Broad range: Feasibility of constructing assays for a variety of processes and
substances.
• High sensitivity: Possibility of detecting a target substance at concentrations
down to 10-12 to 10-15 mol L-1.
• Rapidity of measurement: Bioluminescent reaction lasts milliseconds, so usu-
ally the analysis time varies within the length of period from 1 to 30 min.
• Feasibility of constructing assays for individual specific molecules.
• The ability to test a number of key physiological and biochemical processes,
for example, key metabolites such as NADH, flavins, glucose-6-phosphate, and
so on; activities of key enzymes such as NAD+ or NADH-dependent dehy-
drogenases and trypsin, among others; consumption of unique energy substrate
such as ATP; intracellular dynamics of Ca++ (a universal regulator); ribosomal
synthesis of protein molecules; genome structure and functioning; and others.
• Relative simplicity of methods.
• Availability of reagents and bioluminometers (devices for measuring light
emission intensity).
The era of bioluminescent analytical studies began with the use of luminous
bacteria enzymes, and continues to play an important role in bioluminescent
analytics. Three factors determine the prospective viability of luminous bacteria
and their enzymes in assays.
First, researchers had enough bacterial luciferase because it was possible to
obtain large amounts of biomass; luminous bacteria had long been the only cul-
tivated luminous organisms [1]. Recently, it has become possible to create
recombinant organisms by introducing the lux genes into bacteria, which has
solved the problem of getting luciferase from other luminous organisms for ana-
lytic purposes [2]. During those years when only bacterial luciferase was available,
more than 100 methods were developed using luminous bacteria and their
enzymes. Some of them are presented in the book series Methods in Enzymology
published in 1978 and 1986 [3, 4] and in the reviews by Kratasyuk and Gitelson
[5], Girotti et al. [6], and Roda et al. [7].
Second, bioluminescent assays using bacterial luciferase were characterized by
high specificity of enzyme–substrate interactions.
Finally, it is known that the energy supply of bioluminescence occurs through
the general metabolic chains of cells [8, 9]. This makes it possible to use the chains
coupling enzymes with bacterial luciferase or build such chains artificially so that
the concentration of most key metabolites (and antimetabolites) or activities of key

enzymes can be measured on the basis of luminescence.
At present, bacterial coupled enzyme systems NAD(P)H:FMN-oxidoreductase-
luciferase are actively used in bioluminescent assays for ecology, medicine,
agriculture, and other areas [7, 10]. In Reaction 1, luciferase catalyzes the oxi-
dation of long-chain aliphatic aldehydes involving reduced flavin mononucleotide;
one of the products of this reaction is a quantum of light in the blue-green spec-
trum (Reaction 1). To provide luciferase with reduced flavin mononucleotide, the
luciferase reaction is coupled with the reaction catalyzed by NAD(P)H:FMN-
oxidoreductase (Reaction 2).
Luciferase ðLÞ
ð1Þ
FMN H2 þ RCHO þ O2 ! FMN þ RCOOH þ H2 O þ hm
NADðPÞH : FMNoxidoreductase ðRÞ

ð2Þ
NADðPÞH þ FMN þ Hþ ! NADðPÞþ þFMN H2 ;
Methods based on using enzyme–substrate systems from luminous bacteria can

be nominally divided into two groups: selective analysis methods and integrated
assays such as bioluminescent toxicity assays.
Despite the diversity of bioluminescent methods used in analytical studies,
bacterial luminescence has long been the only bioluminescent system used for the
assessment of chemical pollution of various objects or total toxicity of the test
samples. Historically, the application of bacterial luminescence began with the use
of luminous bacteria. Many works have been published describing the application
of luminous bacteria for ecological monitoring [5, 6]. These methods made it
possible to determine environmental pollution by comparing the luminous inten-
sity of bacteria in the control and in the analyzed sample. As opposed to other test
objects such as paramecia, algae, crustaceans, and so on, the bioluminescent
bioassay was faster (the time of analysis didn’t exceed 20 min); however, as with
other living organisms, the essential disadvantage of this method was low accuracy
of measurement caused by the ‘‘petulance’’ of live luminous bacteria: failure to
maintain the stable state of bacterial culture during measurements and storage. The
bacteria reacted to the appearance of toxic substances either by decreasing or by
increasing the luminous intensity, which often led to ambiguous interpretation of
results. That’s why only qualified staff could work with bacteria. Because of these
shortcomings a bioassay based on luminous bacteria didn’t show very good results
in ecological laboratories. To overcome those difficulties it was decided to use the
enzymes of luminous bacteria both in soluble and immobilized states.
Since 1990, a new trend in bioluminescent analysis has been developing:
bioluminescent enzyme system technology (BEST) [11], where the bacterial
coupled enzyme system: NADH:FMN-oxidoreductase-luciferase substitutes for
living organisms in bioluminescent enzymatic toxicity bioassays. In the presence
of toxic agents, enzymes from luminous bacteria more closely reflect the toxicity
of living organisms than do the use of chemical analysis and other current methods
that cannot solve the pressing problem of how to detect, identify, and measure the
contents of the numerous chemical compounds that differ in their physicochemical
and toxic characteristics. Moreover, BEST was introduced to facilitate and
accelerate the development of cost-competitive enzymatic systems for use in
biosensors for medical, environmental, and industrial applications.
The present work examines the general principles of bioluminescent enzymatic
toxicity bioassays and describes the applications of these methods and their
implementation for commercial biosensors.
2 Principles of Bioluminescent Enzymatic Bioassays
The main principle of bacterial luciferase-based analysis methods is detection of

toxic properties in test substances and mixtures by their effect on bioluminescent
enzymatic reactions [12–15].
Bioluminescent assays are based on the phenomenon of luciferase inhibition by
the components of analyzed mixtures (Fig. 1). Many approaches and methods
have been developed for design of in vitro bioluminescent assays, based on the
bacterial coupled enzyme system: NADH:FMN-oxidoreductase-luciferase
(L + R), especially for ecological monitoring.
2.1 The Bioassays Based on the Bacterial Coupled Enzyme

System: NADH:FMN-Oxidoreductase-Luciferase
Application of enzymatic bioassays based on the bacterial coupled enzyme system

NADH:FMN-oxidoreductase-luciferase for ecological monitoring instead of living
organisms such as intact luminous bacteria, daphnia, algae, and other organisms
that can be legally used in Russia is justified by the fact that NADH:FMN-oxi-
doreductase as part of enzymatic bioassays is present in all living organisms. That
is why good correlation is expected between the effect of toxic substances on
living organisms and on the coupled enzyme system of luminous bacteria. Inte-
grated luciferase-based biotesting methods are mainly used for continuous rapid
environmental monitoring in industrial and agricultural regions, for control over
pollutant emissions of enterprises, for estimation of the efficiency of wastewater
detoxification and of the control of water and wastewater treatment facilities, as
well as for ecological risk assessment.
Bioluminescent integrated assays demonstrate changes in the bioluminescent
signal as a reaction to the appearance of large-scale classes of polluting substances
in the analyzed media. But the development of such assays depends on the
influence of pollutants and xenobiotics on bioluminescence [16–18].
Fig. 1 Scheme of luciferase biotesting [11]
Enzymatic bioluminescent bioassays are based on the classification of biolu-

minescence inhibitors and activators according to the mechanism of their influence
on fundamental physicochemical processes. The patterns of exogenous compound
influence on bioluminescent systems are classified [19, 20]. According to this
classification, there are four possible ways in which exogenous compounds act on
a bioluminescent system: (1) influence of molecules on energy transport processes
in a bioluminescent system, (2) influence of molecules on hydrogen transport
processes in a bioluminescent system, (3) influence of molecules on electron
transfer processes in a bioluminescent system, and (4) interaction of molecules
with the enzymes of a bioluminescent system. Knowing the physicochemical
basics of luminescent biotesting, it is possible to predict the analysis results and
change the sensitivity of bioassays to certain pollutant groups [17, 18, 21–23].
When enzymatic biotesting is carried out, several analysis schemes can be used
(Fig. 2). The first bioluminescent enzymatic bioassay variant is the quickest and
demonstrates good repeatability of results. The following sequence of operations is
performed: a cuvette with all the necessary components of a coupled enzyme
Fig. 2 Bioluminescent (a) I, rel. units 5÷50 µl of

testing diagram (a); modified analyzed sample
diagram of bioluminescent
testing (b) 400 Control sample
Ic
Analyzed sample
200
Iexp
100 200 300 t, s
(b) I, rel. units
400 Control sample
Ic
Analyzed sample
200
Iexp
P
100 200 300 t, s
Tmax
system of luminous bacteria, their substrates, and buffer solution are placed into a
bioluminometer, and after the maximum light emission intensity Ic (control) is
registered, from 5 to 50 lL of the test water sample or toxic substance solution are
added, and the maximum light emission intensity Iexp is registered again (Fig. 2a).
The intensity of the residual luminescence (Iexp/Ic) 100 % and the decay
constant kd are estimated. The decay constant is calculated according to the fol-
lowing formula: kd = [ln(I2/I1)]/Dt, where I1 is the peak of bioluminescence
intensity, I2 is the bioluminescence intensity at the certain moment of time after
reaching the bioluminescence maximum, and Dt is the time (minutes) needed for I1
to reach I2.
When analyzing toxicity of the water samples, the luciferase index (LI) or
toxicity coefficient (TC) are calculated according to the formulas:

LI ¼ Iexp Ic 100 %;

TC ¼ Ic Iexp Ic 100 %:
LI and TC are the residual luminescence and the degree of inhibition of the
bacterial coupled enzyme system NADH:FMN-oxidoreductase-luciferase in the
presence of analyzed sample correspondingly and TC = 100 - LI.
The criterion of toxicity is a 50 % decrease in the maximum luminescence level

of the bacterial coupled enzyme system after the analyzed sample is added, as
compared to the luminescence level in the control sample.
In the second case it is possible to achieve higher sensitivity of assays to the
toxic substances. In this variant, testing of the control sample (usually distilled
water or buffer solution) and analysis sample is performed in different cuvettes
[24]. The reaction of the bioassay is also determined by the values of TC and kd.
But in that case, it is possible to use one more parameter for toxicity analysis: the
time when the coupled enzyme system reached the luminescence maximum (Tmax;
Fig. 2b).
Application of the bacterial coupled enzyme system L + R for ecological
monitoring is justified in quite a large number of works [18, 25, 26]. Biolumi-
nescent bioassays based on the coupled enzyme system L + R were successfully
used for the analysis of water in the Yenissei River and its tributaries, drinking
water in various districts of Krasnoyarsk and Altai Territories, the salt lake Shira,
as well as in Lake Baikal [27–29].
Bioassay reaction was studied in the analysis of potable and surface waters in
the Altai region exposed to Semipalatinsk explosions and surface waters of the
Yenissei River in the radioactive waste discharge area of the Mining-Chemical
Combine (Krasnoyarsk) [26]. More than 100 samples of surface water and 170
water samples from wells and water supply systems have been analyzed. Results of
the bioluminescent assay were compared with integral tests to assess surface water
quality for its redox characteristics including hydrogen peroxide concentration,
redox-status of aquatic medium, and OH-radical formation rate and lifetime. The
assay makes possible to characterize water quality by three cleanliness classes
according to PD 53.18.24.83-89 and define pollution (chemical or biological)
causes [30]. In all parts of the Altai region the condition of surface and potable
water caused concern. Results of the bioluminescent assay were confirmed by
chemical and spectral analyses. In most samples results of bioluminescent assays
have been found to correlate with the integral assay based on redox characteristics
of the analyzed water [26].
Vetrova et al. [29] used bioluminescence bioassays based on luminous bacteria
(Photobacterium phosphopreum) and adapted coupled enzyme system
NADH:FMN-oxidoreductase-luciferase for monitoring the saline-water conditions
of Lake Shira (Khakasia, Siberia). The saltwater Lake Shira has become a very
popular health resort in southern Siberia, and the ecological problems of the lake
are currently of great concern. The differences in bioluminescence responses have
been found to be related to the salt composition and the oxidation–reduction
properties of the water. Bioluminescent kinetics parameters, which are mostly
sensitive to pollution under conditions of saline water, have been observed. The
enzymatic system in the presence of 1,4-benzoquinone are shown to be more
sensitive to redox characteristics of the salt water than in the absence of 1,4-
benzoquinone. Thus, 1,4-benzoquinone should be included in a model solution for
monitoring redox properties of the salt water. Using this technique, the results
of bioluminescence analysis are used to construct a heterogeneity map that
characterizes the spatial and temporal water quality of Lake Shira. A partial map
was based on the bioluminescence characteristics of water samples taken along the
shoreline, sampling stations in the different places, and in different depths of the
lake. The map reflects the correlation between the effect of the water samples on
bioluminescence intensity and their chemical and physical–chemical characteris-
tics. It has been demonstrated that the bioluminescence assay measurements must
be done within 2 h after the sampling time.
The advantages of enzymatic assays based on the bacterial coupled enzyme
system L + R are their rapidity (the time of analysis does not exceed 3–5 min),
high sensitivity, simple measuring procedure, possibility of automation of eco-
logical monitoring procedure, availability and safety of reagents, and a wide
market of bioluminometers [31].
They were so easy to use and convenient that they started to be applied in the
educational process, mainly in ecology practical courses [32–35].
2.2 Enzymatic Reagents for Bioluminescent Analysis
2.2.1 Ways to Obtain Stable Enzyme Preparations
Widespread use of the existing bioluminescent analytic methods is hampered by

several disadvantages, including the instability of enzyme systems during use,
limited shelf-life of enzymes–reagents for analysis, the need to control ambient
conditions (i.e., pH, temperature, etc.) and interference by substances in the test
samples, high manufacturing cost, and other factors [36].
These problems can be solved by using immobilized enzyme preparations in
bioluminescent analysis, by obtaining reagents in the microenvironment that
possess high catalytic activity and are stable for long-term storage, and making it
possible to computerize analyses as biological modules of biosensors. Immobili-
zation is the most effective and popular way among many known methods of
enzyme stabilization. Advances in obtaining highly stable immobilized enzymes
served as the basis for development of one of the foremost leading trends in
biotechnology: creation of biosensors [37].
For the last 30 years immobilization has been widely used for production of
stable reagents for bioluminescent analysis based on various bioluminescent sys-
tems: luminous bacteria, and bacterial and firefly luciferases. Many of the avail-
able immobilized reagents are successfully used in analytic measurements and in
biosensors, because they simplify the analysis procedure, sometimes enabling full
automation. At present, there are more than 40 different methods of immobilizing
luminous organisms and enzymes isolated from various organisms [38].
The area of application of these methods is extremely wide: from analytical
chemistry (analysis of NADH, FMN, ATP, and other substances), medicine
(analysis of D- and L-lactate, bile acids in blood serum, amino acids such as alanine
and phenylalanine, NADH-dependent enzymes, etc.), food industry (analysis of
the degree of bacterial content), ecological monitoring, and scientific research

dealing with cell biology and in vivo enzymatic processes.
Production of immobilized reagents for analytic purposes is one of the major
focal areas in applied enzymology. An important advantage of immobilized
enzymes is the possibility to control the enzyme stability to physical and chemical
environmental factors by way of choosing a suitable microenvironment. In an
optimal microenvironment immobilized enzymes maintain high activity in a wide
temperature range, and expansion of the range of pH optimum and ionic strength
optimum is observed.
Table 1 shows the most effective methods of bioluminescent systems immo-
bilization, and different immobilization methods are compared to reveal the
advantages and disadvantages of each of them [38]. The activity of immobilized
enzymes depends on the conditions under which the reagent was made. The
optimal microenvironment for bacterial luciferase is natural polymer gel. To make
a gel, gelatin or starch (potato, rice, or corn) can be used. By varying gel con-
centration, time, and mode of drying of immobilized enzymes it is possible to
make reagents with different enzymatic activity [39–41].
The immobilized coupled enzyme system L + R does not require special
storage conditions to save high enzymatic activity: when immobilized in starch or
gelatin gel, it maintains its activity for 2 years [42]. Moreover, immobilization in
starch and gelatin gel leads to a considerable stabilization of the coupled enzyme
system with regard to denaturation treatment: pH optimum of the enzymes
expands both to the acid and alkaline areas; high enzyme activity is maintained at
increased salt concentration; thermal stability increases [43, 44]. The highest
thermal stability is achieved in the coupled enzyme system immobilized in starch
gel [40].
Apart from reagents including NADH:FMN-oxidoreductase and luciferase,
there are reagents that include not only enzymes, but also the substrates of bac-
terial bioluminescent reaction. A patented stabilization and immobilization process
preserves up to 50 % of the enzymatic activity and produces the homogeneous
multicomponent reagent Enzymolum, which contains the bacterial luciferase,
NADH:FMN-oxidoreductase and their substrates, coimmobilized in starch or
gelatin gel [45]. The reagent is currently produced in tablet form and can be used
in the cuvette variant of a bioluminometer (Fig. 3). The other forms (e.g. on the
plane table, strips, and others) were also obtained for bioluminescent analysis.
Enzymolum can be integrated as a biological module into the portable biodetec-
tor–biosensor of original construction.
Despite the significant advantages of immobilized reagents over soluble
enzymes, the use of Enzymolum in toxicological studies was hardly possible at
first, as the sensitivity of the bioluminescent coupled enzymatic system is not
sufficient for testing pollutants at the maximum permissible concentration (MPC)
level [17]. However, there are ways of increasing its sensitivity. This could be
done by varying the conditions of testing, which is not possible for assays based on
living organisms.
Table 1 Main features of different immobilization methods [38]
Polymer carrier Relatively easy Initial activity %a Percentage activity Performancec Turnover cycles Automation
at 4 C after (days)b
Polyacryl-hydrazide Moderate 1–2 60 100 (15) G— 20 Yes
BrCN-sepharose Moderate 3–6 10–90 90 (5) G 3–5 700 Yes
BrCN-agarose Moderate 3–6 20–30 Stable (30) VG 3–5 700 Yes
Epoxy-agarose Moderate 3–6 0.1–1 70 (14) P 0.5 – No
Porous arylamino glass rods or beads Moderate 6–10 1–2 Stable (15–180) G— 700 Yes
Nylon tube reactor Complex 6–8 50–70 100 (60) G [1 900 Yes
Glass strips Easy 1–2 92–96 100 (60) G1 100 Yes
Cellulose film Easy 1–2 3–8 – G1 – Yes
Lipid films Complex 5–10 0.1–1 Stable (1–7) P— – No
Bovine serum albumin gel Easy \1 0,3–8 100 (30–60) —1 100 No
Insoluble collagen Easy 1–2 100 70 (14) 20 (240)d G— Several Yes
Starch gel Easy 10–12 100 100 (360) VG 0.1 100 Yes
Alginate gel Easy 2–10 30–60 100 (1) G— None No
Polyamide membranes Moderate 2–4 3–10 100 (180) —1 None No
a
Relative activity compared with soluble enzyme
b
Percentage of initial activity after number of days at 4 C
c
Reproducibility: G good; VG very good; P poor, or sensitivity relative to soluble enzyme set 1
d
High mechanical strength
77
Fig. 3 The multicomponent

reagent Enzymolum is a disk
of dried film, diameter
6–7 mm; dry weight
1.5 ± 0.2 mg
2.2.2 Design of the Reagent Enzymolum and the Procedure

of Toxicological Bioassay with Increased Sensitivity to Pollutants
The following methods were tested to increase the sensitivity of the reagent to
pollutants: varying the composition of the immobilized reagent; varying the ratio
of reaction mixture and test sample; varying the order of components added to the
coupled enzymatic system and test sample; introducing an additional step of
reagent incubation to the test water sample; and selecting the control solution for
biotesting [46].
Varying the Composition of the Immobilized Reagent
Information about the dependence of bioluminescence kinetic parameters on the

reaction mixture ratio, the concentration of enzymes and substrates, and enzyme
stabilizers made it possible to increase the sensitivity of the bioassays. We studied
the dependence of the sensitivity of NADH:FMN-oxidoreductase and luciferase
immobilized in starch gel with the substrates of NADH and tetradecanal to the
effect of benzoquinone and CuSO4 upon the concentration of the immobilized
reagent components. Figure 4 shows that the sensitivity of the multicomponent
reagent increased as the enzyme concentration of its content decreased. A reagent
with luciferase content 0.2 lg gave the greatest sensitivity: the residual lumines-
cence intensity was 20–30 %. Changes in the other bioluminescence parameters
(Tmax and kd), caused by varying the enzyme content in the multicomponent
reagent, were insignificant. Increasing the NADH content in the reagent also
decreased the multicomponent reagent’s sensitivity to the effect of the analyzed
pollutants. The maximum sensitivity to the effect of the analyzed substances was
observed when the NADH content in the reagent was 0.1 mM (Fig. 4).
To prevent inactivation enzymes and to maintain their activity during use and
storage, enzyme stabilizers with various mechanisms, including stabilizers of
enzyme’s SH-groups (DTT or mercaptoethanol) and BSA, were introduced to the
multicomponent reagent. Introducing enzyme-stabilizing additives to the immo-
bilized reagent increased the activity of the coupled enzymatic system L + R and
the stability of immobilized enzymes during storage, but decreased the reagent’s
sensitivity to toxic substances (Fig. 5).
(a) 100
80
Iexp/Ic,%
60
40
20
0
0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4
0.2 µg L 0.5 µg L 1µg L

[NADH], mM
(b) 80
70
60
50
Iexp/Ic,%
40
30
20
10
0
0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4
0.2 µg L 0.5 µg L 1 µg L
[NADH], mM
Fig. 4 Residual light intensity of immobilized reagent under variation of luciferase (L) and
NADH content in reagent in the presence of 50 lg L-1 benzoquinone (a) or 5 lg L-1 CuSO4
(b) [46]
It was shown previously [39] that introducing FMN into the immobilized reagent
resulted in a significant decrease in the activity of the coupled enzymatic system;
that is why FMN was not included in the reagent and FMN solution of various
concentrations was added during the analysis. When the FMN concentration in the
reaction mixture was increased, the sensitivity of the multicomponent reagent to
benzoquinone decreased and remained at the same level to CuSO4 (Fig. 6).
Thus, it is shown that by reducing the content of any component in the
immobilized reagent, it is possible to increase the reagent’s sensitivity to toxic
Fig. 5 Residual light 80

5 µg/L CuSO4
intensity of immobilized
50 µg/L benzoquinone
reagent under variation of its
content in the presence of
60
5 lg L-1 CuSO4 or
50 lg L-1 benzoquinone.
Content of stabilizers in disks
Iexp/Ic,%
was: DTT 0.1 mM, 40
mercaptoethanol 0.2 mM,
BSA 0.001 mM. Control is
the reagent without any
stabilizer [46] 20
0
control DTT mercaptoethanol BSA
Fig. 6 Residual light 100 5 µg/L CuSO4

intensity of immobilized 50 µg/L benzoquinone
reagent under variation of
FMN concentration in the 80
presence of 5 lg L-1 CuSO4
or 50 lg L-1 benzoquinone.
60
Iexp/Ic,%
FMN was injected as a

solution [46]
40
20
0
0,008 0,02 0,03 0,08
[FMN], mM
substances. This effect can be explained by the fact that the coupled enzyme
system had nonstationary conditions because the reagent contains a nonsaturating
concentration of one of the substrates (NADH). Indeed, the maximum sensitivity
of the coupled enzyme system to benzoquinone and CuSO4 was observed when the
NADH concentration was 1.3 times lower than the one corresponding to the
Michaelis constant for this substrate [40].
Varying the Order of Components Added to the Reaction Mixture
In all the experiments described above, the pollutant solution was added to the
reaction mixture after the maximum luminescence level had been reached, that is,
after the substrates and enzyme active centers had interacted and, probably,

intensity of immobilized 1 2
reagent under variation of the
order of introduction of the 100
bioassay’s components into
the reaction mixture in the 80
presence of CuSO4 or
Iexp/Ic,%
benzoquinone: 1 introduction
of analyzed water sample 60
before starting enzyme
reactions by FMN; 2 40
introduction of analyzed
water sample after light
intensity reaches its 20
maximum [46]
0
50 µg/L benzoquinone 5 µg/L CuSO4
formed enzyme–substrate complexes. The sensitivity of the coupled enzyme

system can apparently be increased by injecting the analyzed sample before the
maximum luminescence level has been reached, that is, before adding the last
reaction substrate. In this case the increase in the reagent’s sensitivity is explained
by the enhancing of competition between the substrates and the added inhibitor
toxicants.
Figure 7 shows that adding the analyzed sample before starting the reactions by
the FMN solution led to an insignificant increase in the sensitivity of the immo-
bilized coupled enzyme system to benzoquinone and CuSO4.
Incubating Enzymes in the Test Solutions
The multicomponent reagent with the control or analyzed pollutant solution was
incubated for 30–900 s, then the reaction was started with the FMN solution, and
Ic and Iexp were measured (Fig. 8).
It was shown that such additional incubation increases the sensitivity of the
immobilized reagent to copper sulfate, and the sensitivity to benzoquinone remains
at the same level. The inhibition of enzymes with the copper sulfate solution grew
proportionally to the increase of incubation time and reached its peak when the
reagent had been incubated for 120–130 s with little effect on the assay sensitivity
at 300-s incubation. A further increase of incubation time is impossible because
the activity of the immobilized reagent is significantly lowered, even when the
control solution is analyzed.
Thus, we found the conditions for biotesting aqueous solutions using the
immobilized reagent that provide the maximum sensitivity to toxic substances.

intensity of immobilized
reagent under variation of the 80
time of reagent incubation in
benzoquinone (1) or CuSO4 1
Iexp/Ic, %
60
(2) solutions [46]
40
20
2
0
0 50 100 150 200 250 300 350
Time, s
Table 2 Effects of some pollutants on bioluminescence of soluble and immobilized coupled

enzyme system NADH:FMN-oxidoreductase and luciferase [46]
Class Substance MPC, EC50, mg L-1
mg L-1
Soluble Coimmobilized L + R, C14,
L+R NADH
Quinones Benzoquinone 0.1 0.05 0.01
Naphtoquinone 0.25 0.003 0.003
Thymoquinone * 0.005 0.03
Toluquinone * 0.01 0.05
Phenols Hydroquinone 0.2 0.03 0.02
Pyrocatechin 0.1 2.2 0.002
Heavy metal CuSO4 1 0.2 0.002
salts CdSO4 0.001 310 0.08
CoCl2 0.1 40 0.1
*MPC isn’t determined
Comparing Sensitivity of Soluble and Multicomponent Immobilized Coupled

Enzyme System NADH:FMN-Oxidoreductase-Luciferase
The effect of pollutants upon the bioluminescent system was estimated according
to the parameter EC50 which is the concentration of the active substance when
bioluminescence is inhibited by 50 %. This parameter was chosen by analogy with
the generally accepted parameter EC50 which is the effective concentration of the
active substance causing a 50 % change in some vital functions of the test
organism [47]. The values of EC50 were determined for various classes of organic
pollutants using the soluble and immobilized coupled enzyme system L + R
(Table 2).
Table 2 shows that the sensitivity of the soluble coupled enzyme system L + R
to the studied pollutants was higher as compared to that of the immobilized
reagent. However, because the immobilized reagent enables measurements at the

MPC level or even lower for almost all the studied substances, it can be used for
biotesting natural and waste waters.
2.3 Biotesting Natural and Waste Waters Using the Soluble

and Immobilized Coupled Enzyme System NADH:
FMN-Oxidoreductase-Luciferase
The enzyme biotesting approach was used as a platform technology to certify

‘‘Method to measure the intensity of bioluminescence with the help of the ‘Enz-
ymolum’ reagent to detect the toxicity of drinking, natural, waste and treated waste
water’’ [24, 48]. The biotesting method of using an immobilized reagent was tested
in analyzing the wastewaters of the pulp and paper plant. The characteristics of the
samples are given in Table 3, where Sample #1 is the wastewater coming to the
department, Sample #2 is the wastewater after mechanical treatment, and Sample
#3 is the purified water from the outlet at the treatment facilities.
The criterion of toxicity was a 50 % decrease in the maximum luminescence
level of the reagent after the analyzed water sample was added, as compared to the
luminescence level in the control sample. The water’s quality was determined by
its toxicological characteristics, using the value of the biologically safe dilution,
established by Russian Federation standards, according to Table 4.
The biotesting values were analyzed after samples had been diluted with dis-
tilled water or phosphate buffer. It is common practice in biotesting to use distilled
water for diluting water samples, which makes it difficult to work with soluble
enzymes. For example, there is a high level of luminescence inhibition by the
control solution, distilled water, because of the discrepancy between the pH of
distilled water (about 5.5) and the optimum pH of the coupled enzymatic system
(about 7.0). Using 0.05 M potassium-phosphate buffer with pH 7.0 as the control
solution can lead to a misinterpretation of biotesting results. If the environment
studied is polluted with heavy metals, phosphates contained in the buffer cause
them to precipitate, which reduces the effect of heavy metals on the luminescence
of the coupled enzymatic system L + R. However, when soluble enzymes were
replaced with an immobilized reagent that had a wider pH optimum range (from
5.0 to 9.0) than had the enzyme solutions [40], no such effect was observed.
Table 5 presents the results of wastewater biotesting using the immobilized
reagent and under the Mann–Whitney U test, showing no difference between a
sample diluted by water or buffer. According to the results, samples #1 and #2
were acknowledged to be hypertoxic, and sample #3 to be toxic. In addition, it was
shown that the samples’ toxicity decreases in a row: Sample #1 [ Sample
#2 [ Sample #3, which corresponds to the changes in the chemical characteristics
of the analyzed samples. This experiment confirmed the appropriateness and
Table 3 Results of the Component Sample #1 Sample #2 Sample #3

chemical analyses of waste
and purified water samples pH 6.5 6.2 3.3
CODa, mg L-1 3,000 2,200 2,000
Dredge, mg L-1 300 74 51
NH+, mg L-1 66 53 48
Nitrites, mg L-1 \0.02 \0.02 \0.02
Nitrates, mg L-1 6.2 0.1 0.1
Waste and purified water samples from the laboratory of the
industrial waste purification department at the pulp and paper
plant where sample #1 is the wastewater, sample #2 is the
wastewater after mechanical treatment and sample #3 is the
purified water [46]
a
Chemical oxygen demand
Table 4 Toxicological Dilution factor Toxicological characteristics

characteristics of analyzed of analyzed water of analyzed water
water in accordance with its
dilution factor [46] 1 Nontoxic
2 Slightly toxic
From 3 to 10 Toxic
From 11 to 50 Highly toxic
Table 5 Results of the Test water sample Toxicity coefficient, %

toxicity bioassay of waste
water at pulp and paper plant Buffera dH2Ob
Sample #1 100 100
Sample #1, diluted 10 times 90.9 ± 7.9 90.5 ± 8.2
Sample #2 100 100
Sample #3 ±10.2 86.5 ± 8.9
a
0.05 M potassium-phosphate buffer with pH 7.0 was used as a
control sample and to prepare the analyzed water sample dilu-
tions
b
Distilled water was used as a control sample and to prepare the
analyzed water sample dilutions
efficiency of using the immobilized reagent instead of the soluble coupled enzyme
system L + R in biotests.
Another example is concerned with water bodies containing clean ‘‘reference’’
water, such as Lake Baikal. During the studies of water from Lake Baikal a
comparison was made of assays based on a soluble and immobilized coupled
enzyme system depending on the place and depth of sampling (Fig. 9). Samples
from five control points were studied.
Fig. 9 Dependencies of the Iexp/Ic, %

light emission intensity of a
50 70 90 110 130 150 170 190
soluble (lyophilized) and
0
immobilized couple enzyme
system L + R on the depth of 100
water sampling at Station 1 of
Baikal lake 200
Depth, m
300
400
500
600
The nature of luminous intensity dependence for water samples from different
depths was similar with both soluble and immobilized coupled enzyme systems.
Water samples taken from the upper (the warmest) epilimnion zone didn’t cause
intense luminescence; it must be mentioned that, depending on the location of the
sampling station, the zone was within the range of 5–50 m from the surface.
Apparently, the slight stimulation effect could be explained by the fact that the
epilimnion water, as compared to the other water body layers, contains more
oxygen due to a larger phytoplankton biomass. Water samples from the depth of
50–100 m (depending on the place of sampling) had a slight effect on the luminous
intensity with both soluble and immobilized coupled enzyme systems, which
didn’t suggest pollution, as the test readings were within the range of possible
fluctuations of enzymatic assay parameters for ‘‘normal’’ unpolluted water.
2.4 The Reagent ‘‘Enzymolum’’ in Toxicological

Bioassay of Air
Bioluminescent assays were used not only for aquatic environments, but also for
monitoring of air pollution [49]. The sensitivity of luminous bacteria and their
enzymes to air samples differing by industrial pollution degree was determined.
Air samples were collected in the clean (Akademgorodok, sample #1) and polluted
(the coal power plant area, sample #2) districts of the city of Krasnoyarsk. The air
samples were collected into liquid absorption medium (water, ethanol, or acetone).
The standard aspirating device performing 1.0 L/min was used. Chemical com-
position of the samples was analyzed with a gaseous chromatograph (Agilent
Tech. 7890A). To compare the sensitivity of assays the numbers of dilution of the
samples necessary to remove the toxic effect were considered (Table 6).
The results indicate that water is better than ethanol or acetone medium for air
sample preparation because of its sufficient capacity to absorb organic compounds,
and absence of interfering effects on bioluminescence. The sensitivity of soluble
Table 6 Comparative characteristics of bioluminescent assays used to monitor air pollution

Type of bioassay Soluble Luminous Immobilized
coupled bacteria coupled
system system
(Enzymolum)
Number of components 5 2 3
(simplicity)
Duration of assay, min 10 5–30 7
Sensitivity (number of Water 3/2000 3/700 3/16,000
dilutions), sample#1/ Ethanol 1/700 1/1 3/250
sample#2 Acetone 3/2,000 1/1 3/[2,000
Storage conditions 2 months at +15 C, 6 months at +5 C, 2 years at +4
3 years at –18 C 1 year at – to +25 C
18 C
and immobilized enzymes is 3–24 times higher than the sensitivity of bacterial-
based tests. The immobilized reagent provides the reduction of the time required to
complete the analysis (down to 7 min), easy-to-use, higher sensitivity (allowed
dilutions are up to 16,000), and the possibility to increase the volume of the sample
up to 97 % of the total. Thus there is the possibility to apply the bioluminescent
bioassays based on the immobilized reagent Enzymolum for air pollution moni-
toring [49].
2.5 Signal System of Enzymatic Assays: Methodological

Aspects
In all the existing bioassays based on living organisms, the reaction of the test
substance with the substances that are harmful for these objects is determined. In
this context the term ‘‘environmental toxicity’’ is used, although it is not always
correct. It is also considered that all living organisms, including humans, react with
the test medium in a similar way. Such a conception is based on the statement that
all living organisms have a similar structure and functions that are affected by
toxic substances. The idea that normal vital activity of all living organisms—from
amoebas to humans—is based on complex interconnected activity of many
enzymatic systems providing with such functions as respiration, digestion,
reproduction, bioluminescence, and the like, suggests that a universal test system
could be developed consisting of a number of enzymatic reactions. Indeed, it is
generally accepted that all living beings, despite the diversity of substrates
involved in metabolism, including specific ones for a given organism, have similar
basic types of reactions, and based on the activity of enzymes released under the
effect of pollutants it is possible to determine the biochemical mechanisms of these
substances affecting not only functions, but organisms on the whole.
Such a model enzymatic test system must include enzymes of different classes,
or key enzymes of metabolic processes in living organisms. In this case it will be
possible to determine which vital function of organisms will be inhibited by which
toxic substance or their mixture.
Developing such a complex model test system is possible on the basis of
bioluminescence. Using the coupling principle in bioluminescent analysis gives
almost unlimited possibilities for determining the activity of various enzymes [5]
and designing new enzymatic assays on this basis. The possible basis for devel-
opment of new bioluminescent enzymatic bioassays is the coupled enzyme reac-
tion NADH:FMN-oxidoreductase-luciferase.
The unique feature of the enzymatic analytical systems is that, due to the
coupling with bacterial luciferase, it is possible to measure the activity of more
than 100 enzymes [5] if enzyme coupling chains are created where the product of
one enzyme is the substrate of the next one. The luciferase of luminous bacteria
must be the terminal enzyme in coupling chains for such enzymes as lactic
dehydrogenase, trypsin, glucose-6-phosphate dehydrogenase, and others, making it
possible to measure the activity of many enzymes according to the luminous
intensity.
Moreover, using enzymatic reactions instead of living organisms in toxicity
bioassays makes it possible to regulate the sensitivity of enzymatic assays
depending on the objective. For example, the sensitivity of enzymatic assays to the
toxic substances may be increased by extending the coupling chain of enzymatic
reactions [50].
As a result of using several chains of coupling enzymatic reactions with bac-
terial bioluminescence, a set of enzymatic assays sensitive to different groups of
pollutants was created as a ‘‘signal system of enzymatic assays’’ [25, 51].
2.6 How to Design New Bioluminescent Enzymatic

Bioassays
To develop new bioluminescent enzymatic assays, the following methods were

suggested and used: extending the chain of enzyme coupling and using different
enzyme interaction mechanisms and types. As an example, two enzymes were
chosen: alcohol dehydrogenase (ADH) and trypsin [25]. These objects were
chosen, firstly, because they belong to different classes (oxidoreductases and
hydrolases), and secondly, because they interact differently with bacterial lucif-
erase, providing different sensitivity to the effect of toxic substances [50, 52].
Next we are going to look at the principles of designing enzymatic biolumi-
nescent assays in more detail. The easiest way to create an enzymatic bioassay is
to use direct coupling of enzymatic reactions. Such a method of assay construction
by making long enzyme coupling chains has been known for quite a long time. At
first a bioassay was developed based on the monoenzymatic reaction of luminous
(a) FMN
ADH R L
NAD + NADH FMNH 2 Light
Ethanol Acetaldehyde NAD + RCOH RCOOH
(b) Tripsin
FMN
R L
NADH FMNH 2 Light
NAD + RCOH RCOOH
Fig. 10 Examples of coupling of the enzymatic reactions
bacteria (Reaction 1). Later a coupled enzymatic bioassay was created by coupling
reaction (1) with NADH:FMN-oxidoreductase (Reaction 2) [11]. In the work of
Kudryasheva et al. [50], three enzymes were coupled with direct ADH reaction as
an example. In this case, NAD+ was added to the reaction mixture that was subject
to enzymatic reduction (Reaction 3) catalyzed by ADH, the resulting product
NADH entered Reaction (2), and then other stages of the light emission process
occurred.
ADH
ð3Þ
C2 H5 OH þ NADþ , NADH þ Hþ þ C2 H4 O
The luminous intensity during Reaction (1) is proportional to the concentration

of FMNH2 resulting from Reaction (2); the concentration of NADH depends on
the activity of ADH in Reaction (3; Fig. 10a). It should be noted that biolumi-
nescent methods used in enzyme activity measurement make it possible to couple
more than three enzymes, thus enabling development of new enzymatic assays that
can be applied for the analysis of toxicity of different samples.
Apart from direct coupling, there are other types of interaction between
enzymes that can be used for the development of enzymatic bioassays. Using the
example of reverse ADH-reaction (Reaction 3), a method was suggested for the
creation of a triple enzymatic bioassay by adding a NADH-dependent reaction to
the coupled enzyme system.
trypsin or ADH (2)

I, mV
and sample with
trypsyn or ADH (3a, 3b)
4000 1 kd background
3a
2000 kd exp
2
3b
kd control
kd exp
t, s
50 1000 1500 2000
Fig. 11 Scheme of ADH (or trypsin) activity measurement using the bioluminescence decay
constant: kd background is the decay constant for the coupled enzyme Reaction (1); kd control is the
decay constant for the triple enzyme reaction with ADH or trypsin in the absence of pollutant (2);
kdexp is the decay constant for the triple enzyme reaction with ADH or trypsin in the presence of
pollutant. 3a the pollutant inhibits trypsin or ADH activity; 3b the pollutant activates trypsin or
ADH activity
The method determined the activity of NADH-dependent dehydrogenases,

based on the measurement of the rate of reverse reaction catalyzed by dehydro-
genase, which was developed by Petushkov et al. [53]. When a constant level of
light is reached, acetic aldehyde and ADH are added to the coupled enzyme
reaction L + R. Then, based on the quasi-first-order constant of bioluminescence
recession proportional to the concentration of NADH, the activity of ADH is
calculated (Fig. 11). This method is 20 times more sensitive than spectrometric
measurements. The method is simple, one-stage, and the analysis time is 2–3 min.
Similar methods have been developed to determine the activity of a number of
other NADH-dependent dehydrogenases. All of them can be used as enzymatic
bioassays.
The third way of designing enzymatic bioassays is based on the bioluminescent
method used to measure the activity of proteolytic enzymes (Fig. 10b). It is based
on the method of measuring the activity of proteases according to the biolumi-
nescence recession constant [54]. When proteolytic enzymes are added to the
reaction mixture, hydrolysis of luciferase and NADH:FMN-oxidoreductase occurs,
causing a sharp decrease in luminous intensity (Fig. 11). Kudryasheva et al. [52]
studied the effect of anthropogenic pollutants on proteolytic enzymes using trypsin
as an example.
(a) P, s (b) P, s
40 300
30
200
20
100
10
0 0
0 0,5 1 1,5 2 0 0,05 0,1
C, mM C, µM
Fig. 12 Dependence of induction period on the concentration of 1,4-benzoquinone in the

reaction mixture, a for the coupled enzyme system L + R, and b for the triple enzyme system
ADH + L + R. Increasing the number of links in the coupling chain of enzymatic reactions
results in a tenfold increase of sensitivity to benzoquinone [50]
Thus, estimation of the effect of chemical substances on the activity of the triple
enzyme system with ADH and trypsin is based on changes in kinetic parameters of
bioluminescence (Fig. 11). The procedure for triple enzyme biotesting is as
follows.
• The maximum luminescence intensity of the coupled enzyme system L + R is
measured, and then its luminescence intensity decrease is measured for 1 min.
The decay constant (kd background) for the case of the coupled enzyme reaction is
calculated by the formula: kd background = ln(Imax/I1)/(t1 - tmax), where Imax is
the bioluminescence intensity maximum and I1 is the bioluminescence intensity
at the moment of time t1 (Fig. 11).
• 5–50-lL test sample and 5-lL ADH or trypsin solution are added to the
reaction mixture described above. Then the decay constant kd exp is measured
for the triple enzyme reaction.
• The authentic decay constant for the triple enzyme reaction is calculated by the
equation: kd = kd exp - kd background.
• For control measurement, 5-lL ADH or trypsin solution and 5–50-lL phos-
phate buffer (0.05 mol L-1, pH 6.8) or distilled water are added to the reaction
mixture described above. The control decay constant, kd control, is calculated.
• Relative activity of ADH or trypsin is calculated using the equation: A = (kd/kd
control) 9 100 %. The obtained value is proportional to the sample’s toxicity.
Thus, three types of enzyme interaction were suggested for development of a

set of enzymatic bioluminescent bioassays: direct reaction coupling, competition-
for-substrate method, and proteolytic interactions. It was demonstrated that using
different types of interactions it is possible to create enzymatic assays differing by
their sensitivity to toxic substances. Kudryasheva et al. [50] showed that increasing
the number of links in the coupling chain of enzymatic reactions results in a

tenfold increase of sensitivity to benzoquinone (Fig. 12).
The signal system of enzymatic bioassays was optimized and used for moni-
toring of natural and laboratory aquatic ecosystems and for studying the seasonal
dynamics of zooplankton nonconsumptive mortality [55], as well as for toxicity
analysis of pesticides [51] and sanitary assessment of natural polymers polyhy-
droxyalkanoates [56] and other applications.
2.7 Signal System of Enzymatic Methods for Toxicological

Bioassay of Natural and Laboratory Aquatic Ecosystems
A signal system of bioluminescent assays was developed to monitor water quality

in natural and laboratory ecosystems. It consisted of three enzymatic systems:
coupled enzyme system NADH:FMN-oxidoreductase-luciferase and triple enzyme
systems with alcohol dehydrogenase and trypsin. The results were compared with
those for luminous bacteria.
The set of bioassays was applied to a small forest pond (Siberia, Russia),
laboratory microecosystems polluted with benzoquinone and a batch culture of
blue-green algae [25].
2.7.1 Dynamics of the Pond Ecosystem and the Microecosystem

Components and the Bioassay Data
Prior to using these bioluminescent bioassays for ecological monitoring of water, it

is necessary to answer the question: are the assays too sensitive; that is, do they
respond to a natural variability of ecosystems, which is not connected to any
pollution? That is why first the response of each bioassay to the water of the pond
not subjected to heavy pollution was determined. The responses of the four bio-
assays for two extremely different parts of the ecosystem, epilimnion and hypo-
limnion, were compared [25].
The study was carried out at a small freshwater pond situated in a forest in the
vicinity of Akademgorodok (Academic Town) of the city of Krasnoyarsk (Siberia,
Russia). The pond has an oval shape, its surface area is about 3,000 m2 and its
maximum depth is 9 m. The samples were collected from epilimnion (depth about
2 m) and hypolimnion (depth about 7 ± 8 m) twice a week during three summer
months.
Water temperature in the pond had ordinary summer variations (Fig. 13). A
pronounced thermocline occurred at the depth of about 4 m in June–July and the
temperature of hypolimnion (the layer below the thermocline) was 4 ± 10 C
lower than that of the epilimnion (the layer above the thermocline). In August the
decrease of depth resulted in diminishing the difference between the epilimnion
Fig. 13 Dynamics of the pond ecosystem components: (d) epilimnion, (s) hypolimnion [25]
and hypolimnion temperature. Dissolved oxygen saturation values (Fig. 13) in the
epilimnion of the pond were significantly higher than those in the hypolimnion. In
phytoplankton, green algae Volvox aureus Ehr. and diatom Cyclotella comta (Ehr.)
Kutz. dominated. Blooms of V. aureus took place in the middle of July and in the
middle of August (Fig. 13). C. comta dominated in the middle of June, when the
water temperature was comparatively low. At the end of July and beginning of
August, a small decrease of water temperature took place (Fig. 13) and C. comta
became dominant in this period. In the hypolimnion only two samples of phyto-
plankton were taken in June and July, but the other dominant diatom and green
species then in the epilimnion, Stephanodiscus hantzschii Grun., and Closterium
peracerozum were found in the samples. Thus, the epilimnion and hypolimnion
represented separate compartments of the pond ecosystems and naturally differed
in water quality.
The variations of enzyme bioassay data were insignificant during the study
period (Fig. 14). The data for the luminous bacteria based assay varied over the
season. Water samples taken from the hypolimnion had a stronger inhibitory effect
on bacterial luminescence.
Fig. 14 Parameters of bioluminescent bioassays for the study of water from local pond: (d)
epilimnion, (s) hypolimnion. A, %: relative activity of ADH and trypsin, Ic and Iex: light
intensity in control and experimental cuvette, respectively, for NADH:FMN-oxidoreductase-
luciferase and luminous bacteria bioassays [25]
Only the bioassay based on the luminous bacteria demonstrated the significant
difference between the epilimnion and hypolimnion by the Wilcoxon test. For the
assays based on enzyme systems, the differences between the epilimnion and
hypolimnion were insignificant.
At the end of summer water samples from the pond epilimnion (depth of about
2 ± 3 m) were inoculated into three cylindrical glass experimental microecosys-
tems (MES) 25 cm in diameter and 25 cm high. They were illuminated by white
light of 2.5 W/m2 (natural intensity at the depth of sampling). Water samples from
MES were taken twice a week for 2 months. Benzoquinone was added in the last
week of study to MES 1.
The MES’s experiment consists of two parts. In the first part (lasting 42 days;
Fig. 15a) the relatively higher biomass of microalgae from the pond was
Fig. 15 Biomass of algae and the dynamics of bioassay parameters for MES: (d) MES 1
(treatment with benzoquinone on the 35th day of experiment (b), (j) MES 2, (+) MES 3. (a), %:
relative activity of ADH and trypsin, Ic and Iex: light intensity in control and experimental
cuvette, respectively, for bacteria bioassay. a and b are experiments with high and low biomasses
of microalgae, respectively [25]
inoculated into the MES 1–3 and the bioassay reactions at the high biomass were
recorded. In the second part of the experiment (Fig. 15b) MES 1–3 were reinoc-
ulated and the low biomass of microalgae was maintained in MES 1 ± 3.
In the course of the experiment microalgal species composition in the MES

were recorded as varying greatly. Throughout the experiments, green flamentous
algae Ulotrix tenerrima Kutz., Ulotrix variabilis Kutz., Spirogyra varyans (Hass.)
Kutz., and Spirogyra tenuissima (Hass.) Kutz., dominated in MES 2 and 3. In MES
1 during the first 17 days of the experiment A the dominating species was the
flamentous diatom Fragillaria virescens Ralfs., then the green flamentous algae U.
variabilis became dominant. Generally speaking, in all the MES the plankton
community of diatoms and green microalgae of quite diverse species composition
was replaced by a simpler community of flamentous algae.
Response of the bioassays ranged from inhibition of bacterial luminescence and
ADH and trypsin relative activity to stimulation of parameters of these bioassays
(Fig. 15a, b). Parameters of the bioassay based on the coupled enzyme system
remained essentially unchanged throughout the investigations (Fig. 16). Besides,
the response of this bioassay to water samples taken from different MESs was the
same (Fig. 16). There was no correlation between the response of the bioassays
and the total algae biomass.
After 35 days of experiment B, benzoquinone 10 mg 9 L-1 was added in
MES 1. Benzoquinone was taken as a xenobiotic for two reasons. First, quinones
are greatly responsible for the dissemination and destruction of the water stretches
and their inhabitants [57]. Second, Kudryasheva et al. [58, 59] have shown that the
bioluminescent system L + R is a specific assay for quinones. The concentration
chosen was close to benzoquinone content in the wastewater of the paper industry
and higher than LC50 for luminous bacteria Photobacterium phosphoreum
10 lg 9 L-1 [16]. When benzoquinone was added to MES 1, parameters of all
bioassays changed drastically. The response of the bioassay based on the coupled
system was a decrease of luminescence intensity, an increase in the time needed to
reach the maximum, and the occurrence of the induction period on the first day of
intoxication (Fig. 16). The luminous bacteria-based bioassay showed a complete
absence of light (Iex = 0) from the water from the microecosystem where the
toxicant was added (Fig. 15b). The bioassay based on ADH and trypsin showed a
decrease in the activity of the enzymes when the bioassay was performed on the
sample where benzoquinone was added (Fig. 15b).
2.7.2 Bioluminescent Control for Blooming Water
‘‘Bloom’’ in the water body can also change the response of the bioassays. It was
shown earlier that bloom of blue-green algae was the characteristic feature of the
ecosystem which indicated the distinctive rate and kinetics of the phenol bio-
degradation [60]. No bloom of blue-green algae was observed in the water body
under investigation, thus we used a laboratory culture of blue-green microalgae
Spirulina platensis as a model of a blooming pond.
Dynamics of bioassay data and growth of algae are depicted in Fig. 17. One can
see that throughout the algal growth period, the response of the bioassay based on
the coupled enzyme system varied insignificantly. It is evident from the assay data
Fig. 16 Dynamics of parameters of bioassay based on coupled enzyme system NADH:FMN-

oxidoreductase-luciferase for MES: (d) MES 1 (treatment with benzoquinone on the 35th day of
experiment (b), (j) MES 2, (+) MES 3. a and b are experiments with high and low biomasses of
microalgae, respectively, smax time of reaching the light maximum, P induction period, Ic and Iex
light intensity in control and experimental cuvette, respectively [25]
based on the triple enzyme systems that during the first days of their growth algae
release metabolites inhibiting ADH and enhancing trypsin activity. After 4 days of
algal growth, trypsin activity in the presence of the culture medium did not change.
Response of the bioassays to the algal culture medium was the same in each of the
three replicates. The bioassay based on ADH was the most sensitive. By the eighth
and ninth days, the inhibition of the triple enzyme system with ADH was almost
full (Fig. 17).
Therefore, bioluminescent assays show that their sensitivity differs by water
quality, even in the case of the unpolluted pond. The assay based on luminous
bacteria proved to be the most sensitive. The difference of water quality between
the epilimnion and hypolimnion were due to natural causes rather than by any
pollution. Hence, the result of this assay is determined by the depth at which water
Fig. 17 The growth of microalgae biomass and the bioluminescent bioassay parameters for the
study of the culture medium of S. platensis. (r) effect of culture growth medium at the beginning
of experiment, (s, h, D) three replicates, D680 optical density of S. platensis culture medium at
680 nm (the maximum of chlorophyll), A relative activity of ADH and trypsin, Ic and Iex light
intensity in control and experimental cuvette, respectively, for NADH:FMN-oxidoreductase-
luciferase bioassay [25]
samples were taken (i.e., presence of the water from deoxygenated hypolimnion);
this should be remembered when using this assay for the monitoring of water
bodies.
Changes in the response of bioassays over the period of investigation were not
related to pollution of the water body. Parameters of the bioassays did not change
dramatically even in the periods of bloom of the green algae V. aureus. Indeed, in
contrast to blue-greens, bloom of green algae is not toxic. In addition, there is no

correlation between a change in parameters of the bioassays and any of the pond
ecosystem components (pH, dissolved oxygen, NO2, NO3, NH4, etc.). From our
data we could not determine the factor responsible for variations of parameters of
the bioassays over the season. Apparently, such fluctuations in bioassay parameters
should be regarded as natural for the unpolluted water body and must be accounted
for by natural variability of water quality in the pond ecosystem.
It is evident from the experiments with the blue-green algae S. platensis that in
the course of their growth these algae release substances that have the strongest
effect on the ADH activity. Two components of the algal culture medium could
change the response of the bioassays. They are culture medium mineral salts and
metabolites released by algae as a result of their vital activity. Mineral salts are
consumed by the growing algae, so their contribution to the inhibition of the
enzyme activity decreases. The decrease of the ADH activity may take place due
to an accumulation of metabolites in the medium at the end of the exponential
growth phase.
There was no response of the coupled enzyme system, L + R, to the laboratory
culture of the blue-green algae (Fig. 17). Meanwhile, the bioassay with trypsin
responded to the algae medium at the early exponential stage of growth (the first
4 days). At this stage, the algae may release protease inhibitors into the culture
medium.
Model experiments have shown that contamination of the MES with xenobi-
otics leads to sharp changes in parameters of all the bioassays. Here again, the
most sensitive assay was that based on luminous bacteria. The most likely reason
for this must be that xenobiotics (benzoquinone) affect a number of parameters of
bacterial vital activity. They can affect respiratory and other oxidation-reduction
processes in the cell, as well as disrupt membrane structures of the cell. As a result,
the cells die, and do not produce the light.
The assay based on the coupled enzyme system is specific for quinones. If the
water body is contaminated with quinones, a number of parameters of this assay
change: P, Tmax, intensity. The induction period appeared because the NADH was
oxidized by benzoquinone. After the period of quinone reduction (P), by Reac-
tion 2, FMN can be reduced, and further stages of the bioluminescent reaction with
emission of a quantum of light become possible [59]. The ADH activity decrease
may also be explained by the sequence of the redox NADH-dependent processes
which may occur in the triple enzyme system NADH:FMN-oxidoreductase-
luciferase-alcohol dehydrogenase [50].
The decrease of trypsin-relative activity may be explained by protein modifi-
cation, for example, oxidation of the functional groups of amino acids contained in
luciferase and trypsin (SH, etc.).
Thus, it has been shown that for the unpolluted water body fluctuations in the
bioassay parameters were insignificant and resulted from natural variability of the
pond ecosystem. Parameters of the assay changed sharply when the water body
was contaminated with xenobiotics and in the case of bloom of blue-green algae. It
is necessary to emphasize that ranges of variability of bioassays, which occurred in
the unpolluted pond and unpolluted MESs, were significantly lower than the
degree of bioassay response after the addition of the pollutant (benzoquinone).
Thereby we could detect the effect of pollutants such as quinones, within the
variability of responses, caused by natural water.
Sensitivity of the assays to contaminants of polluted or ‘‘blooming’’ pond has
been shown to differ, inasmuch as the mechanisms of xenobiotic (benzoquinone)
action on the assay systems are different. Hence, the data of a single assay cannot
provide a basis for a conclusion about the presence or absence of toxic substances
in a water body. Only a set of assays, such as the one used in the present study can
be applied as an alarm system to detect an acute toxicity of aquatic ecosystems.
2.8 Signal System of Enzymatic Assays for Toxicological

Bioassay of Pesticides
Pesticides vary in their toxicity mechanism and character; for example, they can be
carcinogenic or mutagenic, or they can affect the respiratory, endocrine, immune,
or nervous systems [61, 62]. The effect of pesticides (organophosphates and
pyrethroid preparations) on the bioluminescence of the four systems has been
analyzed. The characteristics of the toxic substances analyzed are listed in Table 7.
Four bioluminescence assay systems were used in our studies (Table 8). For
each of the analyzed substances, values of EC50 and EC20 were obtained (Table 8).
They constituted 50 and 20 % of the loss of luminescence for the coupled enzyme
system and luminous bacteria, or 50 and 20 % loss of the relative enzymatic
activity for the triple enzyme systems with ADH and trypsin. The parameter EC20
was taken for the authentic determination of a toxicant’s impact on biolumines-
cence and for the comparison of the toxicant maximum permissible concentration.
The sensitivity of the coupled enzyme system to the impact of organophos-
phorous compounds differed widely. The EC20 of pirimiphos-methyl (Table 8, No.
2), for instance, was 0.9 mg 9 L-1, whereas glyphosate (Table 8, No. 3) had no
effect at all on the bioluminescence of the coupled enzyme system. It should be
noted that glyphosate is a herbicide that has low toxicity to animals. Hence, their
toxicity is in good correlation with bioluminescence inhibition.
The triple enzyme systems with ADH and trypsin are more sensitive to
organophosphorous compounds (Table 8). The sensitivity of luminous bacteria to
the impact of organophosphorous compounds is comparable to that of the coupled
enzyme system (Table 8). For example, the EC20 of pirimiphos-methyl is the same
in both cases, 0.9 mg 9 L-1.
All the studied test systems were very sensitive to pyrethroid substances in the
range 0.1–3 mg 9 L-1 (Table 7, Nos. 4–7). These insecticides, synthetic ana-
logues of natural pyrethrins, act through intestinal contact, thereby affecting the
nervous and the immune systems [63–65]. With pyrethroid, the values of EC20 are
smaller for the bacterial system than for the coupled enzymatic system. To
Table 7 Characteristics of toxic substances analyzed by bioluminescence assays [51]

Number Substance Structural formula Mr Type of Substance
1. Malathion 330.36 O
2. Pirimiphos-methyl 305.33 O
3. Glyphosate 169.07 O
4. a-Cypermethrin 416.30 P
5. Bifenthrin 422.87 P
6. Deltamethrin 505.2 P
7. k-Cyhalothrin 449.85 P
compare: with deltamethrin (Table 8, No. 6) the values of EC20 are 0.06 and
0.001 mg 9 L-1 for the coupled enzyme system and luminous bacteria, respec-
tively. This is attributed to permeability and destruction of cell membranes due to
the high lipophilicity of these substances. Sensitivity of the triple enzyme systems
to certain substances is similar to that of luminous bacteria.
Pyrethroid and organophosphorous compounds inhibited trypsin activity.
Organophosphorous compounds are known to have an effect on proteolytic
enzymes [61, 66]. They are able to inhibit esterase and proteolytic activity of both
trypsin and chemotrypsin. This may account for the inhibitory effect of organo-
phosphorous compounds observed in the triple enzyme system with trypsin.
Table 8 The influence of toxic substances on bioluminescent assay systems [51]
Number Substance Luminous bacteria Coupled enzyme system Triple enzyme system with Triple enzyme MPC PDD
photobacterium trypsin system with ADH (mg L-1) (mg kg-1)
phosphoreum
EC50 EC20 EC50 (mg L-1) EC20 EC50 (mg L-1) EC20 EC20 (mg L-1)
(mg L-1) (mg L-1) (mg L-1) (mg L-1)

1. Malathion 7 3.7 4 1.1 0.25 0.13 0.4 0.05 0.02
2. Pirimiphos- 5 0.9 3 0.9 7 3.3 11 0.01 0.01
methyl
3. Glyphosate 40 30 Not measurable [40 Not measurable [40 [40 0.02 0.1
4. a-Cypermethrin 0.5 0.31 0.7 0.3 1.61 0.16 10 0.002 0.01
5. Bifenthrin 0.15 0.09 1.25 0.3 1.17 0.11 5 0.005 –
6. Deltamethrin 0.01 0.001 0.5 0.06 1.3 0.32 0.003 0.006 0.01
7. k-Cyhalothrin 0.38 0.08 10 2.5 1.35 0.08 0.05 0.001 0.002
MPC maximum permissible concentration; PDD the person’s permissible daily dose of a toxic substance (mg kg-1 body weight)
101
Fig. 18 Relative activity (A) of ADH (1) and trypsin (2) under different concentrations of
deltamethrin. Influence of the pollutant on triple enzyme systems resulted in trypsin inhibition
and ADH activation [51]
The triple enzyme bioluminescence systems included enzymes of different

classes: ADH is an oxidoreductase and trypsin is a proteolytic enzyme. Therefore,
the sensitivities and ways of interaction of ADH and trypsin with pollutants are
different. All the toxic compounds studied, except malathion (Table 7, No. 1), had
a weak stimulatory effect on the triple enzyme system with ADH. Figure 18 shows
that deltamethrin inhibited the triple enzyme system with trypsin and activated the
triple enzyme system with ADH.
Inhibition or activation of enzymatic activity by more than 20 % is indicative of
the presence of toxic substances in the analyzed samples. Pesticides differ in their
toxicity to animals [62, 65, 67, 68]. There are potent pesticides
(LD50 \ 50 mg 9 kg-1 animal’s body weight) and pesticides of high
(50–200 mg 9 kg-1), medium (200–1,000 mg 9 kg-1) and low (more than
1,000 mg 9 kg-1) toxicity. Organophosphorous pesticides, with few exceptions, are
of high and medium toxicity, but are rather quickly inactivated in the environment.
Table 8 gives the person’s permissible daily dose (PDD) of a toxic substance
(mg 9 kg-1 body weight) and the MPCs (the established hygienic standards of the
Russian Federation: N. 1.2.1323-03). The values of EC20 for the studied biolu-
minescence systems are much higher than the MPCs. This means that the biolu-
minescence systems are not sensitive enough to detect a toxic substance at its
MPC; however, it will allow the avoidance of a false signal given by background
fluctuation of water quality, and allow authentic reaction with toxicants present in
the water with concentrations exceeding MPCs, which are indeed dangerous for
living organisms.
Considering EC20 and PDD (based on an average man’s weight of 70 kg), these
values are similar. By way of example, the PDD for malathion (Table 7, No. 1),
corrected for a man’s weight, is 1.4 mg 9 kg-1, whereas the EC20 of the coupled
enzyme system is 1.1 mg 9 L-1. This shows that sensitivity of bioluminescence
systems can be used to determine doses toxic to humans.
For pesticides, the sensitivities of the bioluminescence test systems were

compared to those determined by other bioassays [62, 67, 69–72]. In some cases,
bioluminescence bioassays are more sensitive. For example, the sensitivity of mice
to deltamethrin (Table 7, No. 6), as established by the most sensitive (for mam-
mals) hematological test, the erythrocyte micronuclei assay, is 90 mg 9 kg-1
[70], whereas the sensitivity of the bioluminescence test based on luminous bac-
teria is 0.001 mg 9 L-1. The highest sensitivity reported was that of fish used in
bioassays [62, 70]. However, analysis of that kind lacks fast response (the time
required for testing is 7 days) and is very laborious.
Thus, bioluminescence tests are sufficiently sensitive to detect compounds toxic
to humans and other mammals. The results presented can provide the basis for the
development of an alarm test bioluminescence system based on either intact
bacteria or any of several coupled enzyme systems. The described biotesting
methods are proposed for measurements in the environment presumably con-
taminated with toxic agents as a result of industrial pollution.
Inasmuch as test systems differ in their sensitivities to various toxic agents, a
deviation from the background in even one of the tests signals the presence of a
‘‘toxic factor’’ in the water. In this situation, chemical analysis is recommended.
3 Summary
It is necessity to point out that in vitro bioluminescent assay is a promising method

for analyzing the integral characteristics of various media, and its potential hasn’t
been used up yet. For example, the coupled enzyme system is used for monitoring
radiation toxicity, keeping track of the radiation effect on a microbiological and
biochemical level. The bioluminescent method of radiation toxicity monitoring in
solutions containing alpha- and beta-emitting radionuclides has been developed
[73, 74].
A new trend in using bioluminescent methods is the efficiency assessment of
detoxification by natural humic substances. This method is based on the quantita-
tive determination of the antioxidant activity of natural detoxicants, humic sub-
stances. This method makes it possible to assess their remediation properties which
are revealed by reducing the effect of toxic substances on the luminous intensity of
bacteria bioluminescent reaction in the presence of humic acids [75, 76].
Moreover, the application area of bioluminescent enzymatic assays based on
the estimation of integral toxicity is not limited by ecology. Methods based on the
use of enzymatic bioluminescent bacterial systems are applied for food product
quality. A method was suggested for detection of L- and D-lactate in beer [77]. The
effects of the mycotoxins produced by fungi of the genus Fusarium on the coupled
enzyme system: NADH:FMN-oxidoreductase-luciferase were studied [78–80]. A
possibility was demonstrated to use bioluminescent methods for safety evaluation
of food preservatives. Mei and coauthors [81] suggested detecting living bacterial
cells according to detection of cellular NADH using the coupled enzyme system:
NADH:FMN-oxidoreductase-luciferase.
Integrated bioluminescent methods are also very promising for medical
research, for example, for evaluating the gravity of endotoxicosis during treatment
in surgery and therapy. The methods are highly sensitive, rapid, and simple and
allow quantitative determination of the degree of seriousness of illness and the
estimation of the severity of a patient’s condition, disease course prediction,
estimation of the efficiency of the used detoxification methods, and determination
of the optimal operation mode of drainage facilities made of semipermeable
membranes [82–84]. Controlled perfusion of rats’ liver demonstrated a possibility
of using an integrated bioluminescent method for the intensity assessment of
pathologic oxidation processes [85]. A very interesting and promising trend in the
development of integrated bioluminescent testing is the creation of rapid analysis
for the assessment of human organism reaction to physical and mental stress.
Analysis is made by comparing the light emission intensity of the coupled enzyme
system NADH:FMN-oxidoreductase-luciferase in the presence of a person’s saliva
taken before and after a certain stress load [86]. The main advantages of the
bioluminescent method are not only its rapidity, high precision, and sensitivity, but
also noninvasiveness, because human saliva is analyzed, which reflects the func-
tional state of a person just as blood does.
Thus, the new approach of biotechnological design developed on biolumines-
cent enzymatic biosensors, methods, and reagents has been described in the
chapter. To solve the problem of how to detect, identify, and measure the contents
of the numerous chemical compounds in environmental monitoring, food product
monitoring, and medical diagnostics, we proposed Bioluminescent Enzyme Sys-
tem Technology BESTTM, wherein the bacterial coupled enzyme system:
NADH:FMN-oxidoreductase-luciferase substitutes for living organisms. BESTTM
was introduced to facilitate and accelerate the development of cost-competitive
enzymatic systems for use in biosensors.
A patented stabilization and immobilization process produces the multicom-
ponent reagent Enzymolum, which contains the bacterial luciferase, NADH:FMN-
oxidoreductase, and their substrates, coimmobilized in starch or gelatin gel. The
reagent is currently produced in tablet form and can be used only in the cuvette
variant of a bioluminometer. The other forms, for example, on the plane table,
strips, and others were also introduced for bioluminescent analysis. Enzymolum
can be integrated as a biological module into the portable biodetector–biosensor of
original construction.
Enzymolum is the central part of the Portable Laboratory for Toxicity Detection
(PLTD), which consists of a biological module, a biodetector module, a sampling
module, a sample preparation module, and a reagent module. PLTD allows us (a)
to detect a wide range of toxic substances, more than 25,000 compounds; (b) to
perform rapid screening for toxicity in emergency situations in the field and lab-
oratories; (c) to develop systems for analyzing individual compounds; (d) to
develop systems to evaluate the degree of overall toxicity; (e) to keep the high
sensitivity of reagents for many years; and (f) to perform biotesting in the presence
of high concentrations of organic substances in water. The last item suggests the
uniqueness of enzymatic bioassays, because no bioassays based on living organ-
isms give consistent results under such conditions. Living organisms use organic
substances for food and thus bioassays based on living organisms do not reflect
environmental quality correctly, resulting in the absence of accuracy and reliability
of analysis.
Prototype biosensors developed with this technology offer cost advantages,
versatility, high sensitivity (up to 10-14 mol of analyte), rapid response time (less
than 3 min), extended shelf life (up to 2 year), and flexible storage conditions (up
to +25 C).
The enzyme biotesting approach was used as a platform technology to certify
‘‘Method to measure the intensity of bioluminescence with the help of the ‘Enz-
ymolum’ reagent to detect the toxicity of drinking, natural, waste, and treated
waste water’’ [48]. The laboratory will be the principal example of a whole family
of new, portable, professional laboratories for ecological monitoring, food quality
laboratories, military departments, and other monitoring, teaching, security, and
research organizations.
Acknowledgments The work was financially supported by the Government of the Russian
Federation (Contract No 11. G34.31.0058) and Project 1762 from the Ministry of Education and
Science of Russian Federation.
References
1. Hastings J, Riley W, Massa J (1965) The purification, properties, and chemiluminescence

quantum yield of bacterial luciferase. J Biol Chem 240:1473–1481
2. Baldwin TO, Ziegler MM, Green VA et al (2000) Overexpression of bacterial luciferase and
purification from recombinant sources. In: Ziegler MM, Baldwin TO (eds) Methods in
enzymology, vol 305. Academic Press, New York, pp 135–152
3. DeLuca M (1978) Methods in enzymology. Biolumin Chemilumin 57:3–653
4. DeLuca M, McElroy WD (1986) Methods in enzymology. Biolumin Chemilumin B
133:3–649
5. Kratasyuk VA, Gitelson JI (1987) Application of luminous bacteria in bioluminescent
analysis. Uspekhi microbiologii 21:3–30
6. Girotti S, Ferri EN, Fumo MG et al (2008) Monitoring of environmental pollutants by
bioluminescent bacteria. Anal Chim Acta 608:2–29
7. Roda A, Guardigli M, Michelini E et al (2009) Bioluminescence in analytical chemistry and
in vivo imaging. Trends in Anal Chem 28:307–322
8. Hastings JW, Potrikus CJ, Gupta SC et al (1985) Biochemistry and physiology of
bioluminescent bacteria. Adv Microb Physiol 26:235–291
9. Shimomura O (2006) Bioluminescence: chemical principles and methods. World Scientific
Publishing Co. Pte. Ltd, Singapore
10. Medvedeva SE, Tyulkova NA, Kuznetsov AM et al (2009) Bioluminescent bioassays based
on luminous bacteria. J Sib Fed Univ, Biology 4:418–452
11. Kratasyuk VA (1990) Principle of luciferase biotesting. In: Proceeding of the first
international school ‘‘Biological luminescence’’, Wroclaw, Poland, 20–23 June 1989.
World Scientific Publishing Co., Singapore, pp 550–558
12. Gil TA, Belesova NP, Balayan AE et al (1988) Determination of the activity of
phenoloxidases in solution. RU Patent 1,557,521, 1 Dec 1988
13. Kratasyuk VA, Kruchinina RI, Kuznetsov AM et al (1985) The method to determine
concentration of the inhibitors of biological activity. RU Patent 1,204,639, 5 Sept 1985
14. Kratasyuk VA, Kruchinina RI, Kuznetsov AM et al (1986) The method to determine
concentration of acrylonitrile. RU Patent 1,270,658, 15 Jul 1986
15. Kratasyuk VA, Kuznetsov AM, Fish AM et al (1981) The method to determine concentration
of the inhibitors of biological activity. RU Patent 865,904, 23 Sept 1981
16. Kudryasheva NS, Kratasyuk VA, Esimbekova EN et al (1998) Development of the
bioluminescent bioindicators for analyses of environmental pollutions. Field Anal Chem
Tech 2:277–280
17. Kudryasheva N, Vetrova E, Kuznetsov A et al (2002) Bioluminescent assays: effects of
quinones and phenols. Ecotox Environ Safe 53:221–225
18. Vetrova EV, Kudryasheva NS, Kratasyuk VA (2007) Redox compounds influence on the
NAD(P)H:FMN-oxidoreductase-luciferase bioluminescent system. Photoch Photobiol Sci
6:35–40
19. Kudryasheva NS (2006) Bioluminescence and exogenous compounds. Physico-chemical
basis for bioluminescent assay. J Photochem Photobiol, B 83:77–86
20. Kudryasheva NS (2006) Nonspecific effects of exogenous compounds on bacterial
bioluminescent enzymes: fluorescence study. Curr Enzym Inhib 2:363–372
21. Kratasyuk VA, Fish AM (1980) Study of mechanism of 2,4-dinitrofluorobenzene effect on
bacterial luminescence in vitro. Biochemistry (Moscow) 45:1175–1181
22. Kratasyuk VA, Makurina VI, Kuznetsov AM et al (1991) Study of effect of sulfo-substituted
succinic acid on bacterial luminescence. Appl Biochem Micro (Moscow) 27:127–133
23. Kudryasheva NS, Esimbekova EN, Yu Kudinova I et al (2000) Effects of quinones on
NADH-dependent enzymatic bioluminescent systems. Appl Biochem Micro (Moscow)
36:409–413
24. Kratasyuk VA, Esimbekova EN (2011) Express method for biotesting of natural,
manufacturing waters and water solutions, RU Patent 2,413,771, 10 Mar 2011
25. Kratasyuk VA, Esimbekova EN, Gladyshev MI et al (2001) The use of bioluminescent
biotests for study of natural and laboratory aquatic ecosystems. Chemosphere 42:909–915
26. Kratasyuk VA, Kuznetsov AM, Rodicheva EK et al (1996) Problems and prospects of
bioluminescence assays in ecological monitoring. Sib J Ecol 5:397–403
27. Kratasyuk VA, Vetrova EV, Kudryasheva NS (1999) Bioluminescent water quality
monitoring of salt Lake Shira. Luminescence 14:193–195
28. Kudryasheva N, Shilova E, Khendogina E et al (1999) Lake Shira, a Siberian salt lake:
ecosystem, structure and functions. 3: The use of bioluminescent biotests to monitor
ecological status. Int J Salt Lake Res 8:245–251
29. Vetrova EV, Kratasyuk VA, Kudryasheva NS (2002) Bioluminescent characteristics of Shira
Lake water. Aquat Ecol 36:309–315
30. PD 53.18.24.83-89 (1990) Methods of estimation of kinetic indices of surface water quality.
Gidrometeoizdat, Moscow
31. Deryabin DG (2009) Bacterial bioluminescence: base and applied aspects. Science, Moscow
32. Gitelson JI, Kratasyuk VA (2002) Bioluminescence as an educational tool. In: Kricka LJ,
Stanley PE (eds) Bioluminescence and chemiluminescence: progress and current
applications. World scientific publishing, River Edge, pp 175–182
33. Kratasyuk VA, Gusev SM, Remmel NN et al (2007) Bioluminescence in the spaceflight and
life science training program at Kennedy space center. In: Szalay A, Hill P, Kricka L, Stanley
P (eds) Bioluminescence and chemiluminescence: chemistry, biology and applications.
World scientific publishing, San Diego, pp 257–260
34. Kratasyuk VA, Kuznetsov AM, Gitelson JI (1997) Bacterial bioluminescence in ecological
education. In: Hastings JW, Kricka ZJ, Stanley PE (eds) Bioluminescence and
chemiluminescence (molecular reporting with photons). Willey, Chichester, pp 177–180
35. Kudryashev MA, Gavrichkova OV, Kudryasheva NS et al (2002) Use of bacterial

bioluminescent bioassay by schoolchildren for ecology monitoring and relations with
human health. In: Kricka LJ, Stanley PE (eds) Bioluminescence and chemiluminescence:
progress and current applications. World Scientific Publishing, River Edge, pp 399–402
36. Kuznetsov AM, Tulkova NA, Kratasyuk VA et al (1997) The characteristics of reagents for
bioluminescent bioassays. Sib Ecol J 5:459–465
37. Turner APF (2000) Biosensors—sense and sensitivity. Science 290:1315–1317
38. Kratasyuk VA, Esimbekova EN (2003) Polymer immobilized bioluminescent systems for
biosensors and bioinvestigations. In: Arshady R (ed) Polymeric biomaterials, The PBM
Series (Introduction to Polymeric Biomaterials), vol 1. Citus Books, London, pp 301–343
39. Esimbekova EN, Kratasyuk VA, Torgashina IG (2007) Disk-shaped immobilized
multicomponent reagent for bioluminescent analyses: correlation between activity and
composition. Enzyme Microb Tech 40:343–346
40. Esimbekova EN, Torgashina IG, Kratasyuk VA (2009) Comparative study of immobilized
and soluble NADH:FMN-oxidoreductase-luciferase coupled enzyme system. Biochemistry
(Moscow) 74:695–700
41. Kratasyuk VA, Esimbekova EN (2005) Method of preparation for immobilized
multicomponent reagents for bioluminescent analysis. RU Patent 2,252,963, 27 May 2005
42. Lonshakova VI, Esimbekova EN, Kratasyuk VA (2012) Characteristics of coupled enzymatic
system of luminous bacteria co-immobilized with substrates and stabilizers into starch gel.
43. Bezrukikh AE, Esimbekova EN, Kratasyuk VA (2011) Thermoinactivation of coupled
enzyme system of luminous bacteria NADH:FMN-oxidoreductase-luciferase in gelatin. J Sib
Fed Univ, Biology 4:64–74
44. Bezrukikh AE, Esimbekova EN, Kratasyuk VA (2012) Gelatin and starch for bacterial
luciferase stabilization. Luminescence 27:114–115
45. Kratasyuk VA, Esimbekova EN (2011) Bioluminescent biomodule for analyses of various
media toxicity and method of its preparation. RU Patent 2,413,772, 10 Mar 2011
46. Esimbekova E, Kondik A, Kratasyuk V (2013) Bioluminescent enzymatic rapid assay of
water integral toxicity. Environ Monit Assess 185:5909–5916. doi:10.1007/
s10661-012-2994-1
47. Persoone G, Janssen C, De Coen W (eds) (2000) New microbiotests for routine toxicity
screening and biomonitoring. Kluwer Academic Publishers, New York
48. Sertificate N 224.0137/01.00258/2010 (2010) Method to measure the intensity of
bioluminescence with the help of the ‘‘Enzymolum’’ reagent to detect the toxicity of
drinking, natural, waste and treated waste water
49. Rimatskaya NV, Nemtseva EV, Kratasyuk VA (2012) Bioluminescent assays for monitoring
of air pollution. Luminescence 27:154
50. Kudryasheva NS, Kudinova IY, Esimbekova EN et al (1999) The influence of quinones and
phenols on the triple NAD(H)-dependent enzyme systems. Chemosphere 38:751–758
51. Vetrova E, Esimbekova E, Remmel N et al (2007) A bioluminescent signal system: detection
of chemical toxicants in water. Luminescence 22:206–214
52. Kudryasheva NS, Esimbekova EN, Remmel NN et al (2003) Effect of quinones and phenols
on the triple—enzyme bioluminescent system with protease. Luminescence 18:224–228
53. Petushkov V, Shefer L, Rodionova N et al (1987) Bioluminescent method of determination of
NAD(P)H dehydrogenase activity. Appl Biochem Biotech 23:270–274
54. Njus D, Baldwin TO, Hastings JW (1974) A sensitive assay for proteolytic enzymes using
bacterial luciferase as a substrate. Anal Biochem 61:280–287
55. Dubovskaya OP, Gladyshev MI, Esimbekova EN et al (2002) Study of possible relation
between seasonal dynamics of zooplankton nonconsumptive mortality and water toxicity in a
pond. Inland Water Biol 3:39–43
56. Shishatskaya EI, Esimbekova EN, Volova TG et al (2002) Hygienic assessment of
polyhydroxyalkanoates—natural polyethers of new generation. Gigiena Sanitaria 4:59–63
57. Stom D (1977) Influence of polyphenols and quinones on aquatic plants and their blocking of
SH-groups. Acta Hydrochim Hydrobiol 5:291–298
58. Kudryasheva NS, Kratasyuk VA, Belobrov PI (1994) Bioluminescent analysis. The action of
toxicants: physical-chemical regularities of the toxicants effects. Anal Lett 27:2931–2947
59. Kudryasheva N, Shalaeva E, Zadorozhnaya E et al (1994) Patterns of inhibition of bacterial
bioluminescence in vitro by quinones and phenols components of sewage. Biofizika
39:441–451
60. Gladyshev M, Sushchik N, Kalachova G et al (1998) The effect of algal blooms on the
disappearance of phenol in a small forest pond. Water Res 32:2769–2775
61. Galloway T, Handy R (2003) Immunotoxicity of organophosphorous pesticides.
Ecotoxicology 12:345–363
62. Hansen OCh (2004) Quantitative structure–activity relationships (QSAR) and pesticides.
From Danish ministry of the environment: environmental protection agency. Pesticides Res
94:134. http://www2.mst.dk/udgiv/publications/2004/87-7614-434-8/pdf/87-7614-435-6.pdf
63. Coles GC, Stafford KA (1999) The in vitro response of sheep scab mites to pyrethroid
insecticides. Vet Parasitol 83:327–330
64. Diel F, Horr B, Borck H et al (2003) Pyrethroid insecticides influence the signal transduction
in T helper lymphocytes from atopic and nonatopic subjects. Inflamm Res 52:154–163
65. Pauluhn J, Machemer LH (1998) Assessment of pyrethroid-induced paraesthesias:
comparison of animal model and human data. Toxicol Lett 97:361–368
66. Lundebye AK, Curtis TM, Braven J et al (1997) Effect of the organophosphorous pesticide,
dimethoate, on cardic and acetylcholinesterase (AChE) activity in the shore crab Carcinus
menaeus. Aquat Toxicol 40:23–36
67. Hernando MD, Ejerhoon M, Fernandez-Alba AR et al (2003) Combined toxicity effects of
MTBE and pesticides measured with Vibrio fishery and Daphnia magna bioassays. Water Res
37:4091–4098
68. Rahman MF, Mahboob M, Danadevi K et al (2002) Assessment of genotoxic effects of
chloropyriphos and acephate by the comet assay in mice leucocytes. Mutat Res 516:139–147
69. Datta M, Kaviraj A (2003) Acute toxicity of the synthetic pyrethroid deltamethrin to
freshwater catfish Clarias gariepinus. Bull Environ Contam Toxicol 70:296–299
70. Grisolia CK (2002) A comparison between mouse and fish micronucleus test using
cyclophosphamide, mitomycin C and various pesticides. Mutat Res 518:145–150
71. Strachan G, Preston S, Maciel H et al (2001) Use of bacterial biosensors to interpret the
toxicity and mixture toxicity of herbicides in freshwater. Water Res 35:3490–3495
72. Trajkovska S, Tosheska K, Aaron JJ et al (2005) Bioluminescence determination of enzyme
activity of firefly luciferase in the presence of pesticides. Luminescence 20:192–196
73. Kudryasheva NS, Kratasyuk VA, Rozhko TV et al (2007) Bioluminescent method for
monitoring the radiotoxicity of solutions. Patent WO 2008,036,000, 14 May 2007
74. Rozhko TV, Kudryasheva NS, Kuznetsov AM et al (2007) Effect of low-level a-radiation on
bioluminescent assay systems of various complexity. Photochem Photobiol Sci 6:67–70
75. Fedorova ES, Kudryasheva NS, Kuznetsov AM et al (2007) Bioluminescent monitoring of
detoxication processes: activity of humic substances in quinone solutions. J Photochem
Photobiol, B 88:131–136
76. Kudryasheva NS, Fedorova ES (2009) Bioluminescent assay to determine antioxidant
activity of humic substances. RU Patent 2,376,380, 20 Dec 2009
77. Girotti S, Muratori M, Fini F et al (2000) Luminescent enzymatic flow sensor for D- and L-
lactate assay in beer. Eur Food Res Technol 210:216–219
78. Kratasyuk VA, Egorova OI, Esimbekova EN et al (1998) A biological luciferase test for the
bioluminescent assay of wheat grain infection with Fusarium. Appl Biochem Micro
(Moscow) 34:622–624
79. Kratasyuk VA, L’vova LS, Egorova OI et al (1998) Effect of Fusarium mycotoxins an
bacterial bioluminescence system in vitro. Appl Biochem Micro (Moscow) 34:190–192
80. Kratasyuk VA, Plotnikova NB, L’vova LS et al (1989) Micro fungi bioluminescent assay of
grain infection. RU Patent 1,469,866, 15 Dec 1989
81. Mei C, Wang J, Lin H et al (2009) Quantitative detection of NADH by in vitro bacterial
luciferase bioluminescent. Wei Sheng Wu Xue Bao 49:1223–1228
82. Esimbekova EN, Kratasyuk VA, Abakumova VV (1999) Bioluminescent method non-
specific endotoxicosis in therapy. Luminescence 14:197–198
83. Kratasyuk VA, Kovalevskiy AN, Voevodina TV et al (1991) Determination of patients’ state
under endotoxicosis of infectious genesis. USSR Patent 1,663,548, 15 Mar 1991
84. Sovtsov SA, Kratasyuk VA (1991) Determination of endotoxicosis under surgery. USSR
Patent 1,714,512, 22 Oct 1991
85. Remmel NN, Kratasyuk VA, Maznyak OM et al (2003) Bioluminescent analysis of intensity
of pathological oxidative processes in cells of perfused rat liver after hyperthermia. B Exp
Biol Med (Moscow) 135:43–45
86. Gritsenko EV, Borodulin SV, Bytev VO et al (1996) Bioluminescent control of training
process. In: Materials of the 7th All-Russian conference on homeostasis, Krasnoyarsk, 17–22
March 1996
Detection of Organic Compounds
with Whole-Cell Bioluminescent Bioassays
Tingting Xu, Dan Close, Abby Smartt, Steven Ripp and Gary Sayler
Abstract Natural and manmade organic chemicals are widely deposited across a
diverse range of ecosystems including air, surface water, groundwater, wastewater,
soil, sediment, and marine environments. Some organic compounds, despite their
industrial values, are toxic to living organisms and pose significant health risks to
humans and wildlife. Detection and monitoring of these organic pollutants in
environmental matrices therefore is of great interest and need for remediation and
health risk assessment. Although these detections have traditionally been per-
formed using analytical chemical approaches that offer highly sensitive and spe-
cific identification of target compounds, these methods require specialized
equipment and trained operators, and fail to describe potential bioavailable effects
on living organisms. Alternatively, the integration of bioluminescent systems into
whole-cell bioreporters presents a new capacity for organic compound detection.
These bioreporters are constructed by incorporating reporter genes into catabolic
or signaling pathways that are present within living cells and emit a biolumines-
cent signal that can be detected upon exposure to target chemicals. Although
relatively less specific compared to analytical methods, bioluminescent bioassays
are more cost-effective, more rapid, can be scaled to higher throughput, and can be
T. Xu G. Sayler
Joint Institute for Biological Sciences, The University of Tennessee,
Knoxville, TN, USA
D. Close
Biosciences Division, Oak Ridge National Laboratory,
Oak Ridge, TN, USA
A. Smartt S. Ripp G. Sayler
Center for Environmental Biotechnology, The University of Tennessee,
Knoxville, TN, USA
A. Smartt S. Ripp G. Sayler (&)
Department of Microbiology, The University of Tennessee, Knoxville, TN, USA
e-mail: sayler@utk.edu

112 T. Xu et al.
designed to report not only the presence but also the bioavailability of target
substances. This chapter reviews available bacterial and eukaryotic whole-cell
bioreporters for sensing organic pollutants and their applications in a variety of
sample matrices.
Keywords Bacterial luciferase

Bioavailability
Bioreporter
Biolumines-

cence BTEX Dioxin Endocrine disruptors Environmental monitoring

Firefly luciferase Hydrocarbon PAH PCB
Abbreviations
AhR Aryl hydrocarbon receptor
AR Androgen receptor
ARE Androgen response element
ARNT AhR nuclear translocator
BPA Bisphenol-A
BTEX Benzene, toluene, ethylbenzene, and xylene
CALUX Chemical-activated luciferase expression
DDE Dichlorodiphenyldichloroethylene
DDT Dichlorodiphenyltrichloroethane
DRE Dioxin-responsive element
E2 17b-estradiol
EDC Endocrine disrupting chemical
EE2 17a-ethynylestradiol
ER Estrogen receptor
ERE Estrogen response element
GC Gas chromatography
GR Glucocorticoid receptor
HPLC High-performance liquid chromatography
MS Mass spectrometry
PAH Polycyclic aromatic hydrocarbon
PCB Polychlorinated biphenyls
PCDD Polychlorinated dibenzo-p-dioxin
PCDF Polychlorinated dibenzofuran
PMT Photomultiplier tube
PR Progesterone receptor
T3 3,30 ,5-triiodo-L-thyronine
TCA 1,1,1 trichloroethane
TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin
TCE Trichloroethylene
TR Thyroid receptor
Detection of Organic Compounds with Whole-Cell Bioluminescent Bioassays 113
Contents
1 Introduction........................................................................................................................ 113
2 Detection of Organic Compounds Using Bacterial Bioluminescent Assays .................. 114
2.1 Benzene, Toluene, Ethylbenzene, and Xylene ........................................................ 115
2.2 Polycyclic Aromatic Hydrocarbons ......................................................................... 120
2.3 Alkane Aliphatic Hydrocarbons ............................................................................... 122
2.4 Chlorinated Aliphatic Hydrocarbons........................................................................ 124
2.5 Biphenyl and Polychlorinated Biphenyls................................................................. 125
2.6 Phenol and Derivatives............................................................................................. 127
3 Evaluation of Organic Toxicant-Induced Health Risks Using Eukaryotic Cell-Based
Bioluminescent Assays...................................................................................................... 129
3.1 Bioassays for Dioxin and Dioxin-Like Compounds ............................................... 130
3.2 Bioassays for Hormonally Active Chemicals.......................................................... 135
3.3 Environmental Applications ..................................................................................... 139
4 Conclusions........................................................................................................................ 141
References................................................................................................................................ 142
1 Introduction
Synthetic organic chemistry started in the nineteenth century when, instead of

extracting urea from an animal kidney, the German chemist Friedrich Wöhler
produced it from inorganic substances, and French chemist Marcellin Berthelot
chemically synthesized fatty acids that are not found in nature [1]. Over the ensuing
200 years, chemists have subsequently learned to create an enormous number of
organic compounds, both of natural and synthetic origin, that have become
essential input materials for consumer products, agriculture, manufacturing,
pharmaceutical, and life science industries, and myriad other applications. How-
ever, despite their industrial value, some of these compounds have unfortunately
become associated with adverse health effects in humans and animals. For instance,
exposure to benzene, a naturally occurring aromatic hydrocarbon found in crude
oil, has now been linked to decreased circulating blood cell counts, leukemia, and
immunotoxicity [2], as has exposure to the toxic synthetic compound dichlorodi-
phenyltrichloroethane (DDT), which was used extensively as an insecticide until it
was banned from agricultural use worldwide because of its toxicity toward a wide
range of organisms. These compounds, like many other organic pollutants, are
persistent and prone to bioaccumulation, endowing them with a large potential to
negatively affect the well-being of living organisms.
The increasingly large scale of industrial chemical production, and its corre-
sponding increase in anthropogenic chemical consumption, has driven up a
demand for effective tools and approaches that can both detect the environmental
occurrence of these compounds as well as assess their potential biological effects
following exposure. These environmental monitoring efforts have traditionally
employed analytical methods such as high-performance liquid chromatography
114 T. Xu et al.
(HPLC) and gas chromatography coupled with mass spectrometry (GC/MS) to

detect and quantify toxic chemicals, and the ability of these analytical methods
specifically to measure chemical concentrations at high sensitivities has been
instrumental for the evaluation of the level of contamination. However, these
methods have proved to be time-consuming, expensive, and relatively difficult to
perform and, more important, are not capable of measuring bioavailability and
biological impact, two important aspects of the risk assessment paradigm.
To overcome these shortcomings, biological assays using living whole-cell
bioreporters have been developed to provide more biologically relevant informa-
tion. Bioluminescent bioreporters in particular, due to their ability to generate an
easily measurable light signal, have been well validated in the laboratory and
extensively applied in environmental monitoring. These bioreporters, being
genetically transformed to express the luciferase genes of bacterial origin (luxAB),
the full bacterial bioluminescent system (luxCDABE), or the firefly luciferase gene
(luc) as a means of light production, provide a rapid, simple, and cost-effective
complement to analytical chemical methods. As living entities, whole-cell biore-
porters act as proxies for humans and other organisms to prewarn the occurrence of
potentially toxic substances. Most environmental applications have traditionally
employed bacterial-based bioreporters for this purpose, however, eukaryotic cell-
based bioreporters are increasingly being utilized to provide more human- and
animal-relevant data. This chapter reviews the development of both bacterial and
eukaryotic cell-based bioluminescent bioreporters for sensing a broad range of
organic compounds and provides an overview of the utility and limitations of these
bioluminescent bioassays in practical applications.
2 Detection of Organic Compounds Using Bacterial

Bioluminescent Assays
Being genetically easy to manipulate and displaying rapid and robust growth,
bacteria have been extensively employed as hosts for bioreporter development.
Despite the toxic nature of organic pollutants, evolution has provided some bac-
teria with unique genetic properties that allow them to adapt to the presence of
toxic chemicals by utilizing them as carbon sources. As the generation of proteins
involved in the degradation of exposed pollutants is an energy-consuming process
and costs fitness under unexposed conditions, the catabolic process is carefully
regulated at the transcriptional level in such a way that it is only initiated upon
exposure to its corresponding chemical input. To exploit this unique feature,
bacterial bioluminescent bioreporters are constructed by transcriptionally
integrating reporter genes (luxAB, luxCDABE, or luc) within special catabolic
pathways that specifically respond to the presence of target compounds. Fortu-
nately, the diversity of catabolic capabilities and sophisticated characterization of
responsible genetic components has provided a sizable toolbox for bioreporter
construction. This section provides an overview of the genetic foundations for

bacterial bioluminescent bioreporter development and their applications toward
detection of a variety of common organic contaminants (Table 1).
2.1 Benzene, Toluene, Ethylbenzene, and Xylene
The monocyclic hydrocarbons benzene, toluene, ethylbenzene, and xylene

(BTEX) are found across various environmental matrices such as water, soil, and
other sediments due to contamination with petroleum products resulting from
gasoline spills, underground storage container leaks, runoff from manufacturing
plants, and so on. Traditionally, a sample suspected of BTEX contamination would
be subjected to a lengthy extraction process to purify the available chemicals,
followed by analytical-based testing to identify the compounds present. In an effort
to ease the process of detecting and identifying chemical pollutants, biolumines-
cent reporter strains have been developed that modulate signal production in
response to BTEX chemical exposure, indicating their presence in a sample. To
accommodate the disparate needs of detection, a variety of reporter constructs has
been developed over the years, ranging from the straightforward introduction of
bioluminescent genes into naturally BTEX-degrading organisms to track their
prevalence, to more complex introductions of both bioluminescent and
BTEX-degrading genes into specialized reporter organisms that can be tailored to
the needs of a particular investigation. Although this section focuses only on
bioluminescent BTEX reporter organisms that modulate signal in response to
compound detection, it should be noted that there is a variety of additional sources
that have evaluated BTEX presence and toxicity using constitutively biolumi-
nescent reporters as well [3–5].
Due to the prevalence of BTEX in the environment, a multitude of
bioluminescent bioreporters have been developed across a variety of different host
strains. The majority of these bioreporters function by leveraging the expression of
the TOL plasmid degradation genes that were originally identified in Pseudomonas
putida mt-2 [6]. These genes function across two distinct pathways consisting of
an upper pathway and a meta pathway. In this system, BTEX compounds are first
oxidized in the upper pathway, and then proceed to the meta-cleavage pathway
where they are further broken down before ultimately being routed to the Krebs
cycle [7–9]. Governing the expression of these pathways are two regulators, of
which the primary XylR regulator is most often utilized for bioreporter design.
This may seem counterintuitive because the xylR gene is constitutively expressed,
but its resulting protein product remains inactive until physical interaction with a
BTEX chemical imparts structural changes that permit it to bind to, and subse-
quently activate, the upper pathway promoter Pu [10]. Li et al. [11] were able to
capitalize on this interaction and construct a pTOLLUX plasmid that utilized the
xylR gene product to activate the transcription of an Aliivibrio fischeri (originally
classified as Vibrio fischeri [12]) luxCDABE gene cassette that was fused to the
Table 1 Bacterial bioluminescent bioreporter for organic contaminants and representative environmental applications
116
Reporter strain Reporter Target compound Detection limit References Environmental application
construct
E. coli DH5a (pPROBE- tubT-luxAB BTEX 0.24 lM toluene [14] Simulated aquatic oil spill [14]
LuxAB-TubT)
E. coli DH5a pGLTUR xylR-Pu-luc Benzene, Toluene, 10–20 lM toluene [13] Soil [13, 131]
Xylene 3 lM 3-xylene
P. putida TVA8 tod-luxCDABE BTEX 0.03–50 ppm BTEX [15] Soil [3]
TCE 1 lM TCE Water [18]
E. coli HMS174 (pOS25) ipbR-luxCDABE Hydrophobic 1 lM toluene [22] Soil [132]
compounds 0.1 lM naphthalene
E. coli DH5a pTOLLUX pR/xlyR-Pu- BTEX N/A [11] Soil and groundwater [11]
luxCDABE
B. sartisoli RP007 (pPROBE- phnS-luxAB PAH 0.17 lM naphthalene [14] Simulated aquatic oil spill [14]
phn-luxAB)
P. putida pPG7-JAMA21 nahR-Psal-luxAB Naphthalene 0.50 lM naphthalene vapor [133] N/Aa
P. fluorescens HK44 nahR-PnahG- PAH 12–120 lM [29] Soil [32, 131, 134]
(pUTK21) luxCDABE
E. coli DH5 (pGEc74, alkS-PalkB-luxAB 6–10-carbon 25 nM octane [37] Groundwater [20]
pJAMA7) alkane Soil [131]
E. coli alkBFG, alkJ- 5–12-carbon 10 lM octane [39] N/A
luxAB alkane
Acinetobacter baylyi ADP1 alkR-PalkM- Alkane 3 nM octane [43] Seawater [43]
luxCDABE
E. coli alkR-PalkM- Alkane 5 nM octane [45] N/A
luxAB
P. putida F1G4 sep-luxCDABE TCE 1 mM TCE [50] N/A
Methylobacterium extorquens dcmR-PdcmA- DCM 1 lM DCM [51] N/A
luxCDABE
(continued)
T. Xu et al.
Table 1 (continued)
Reporter strain Reporter Target compound Detection limit References Environmental application
construct
Ralstonia eutropha ENV307 bphR1- PCBs 0.80 lM 4-chlorobiphenyl [53] N/A
(pUTK60) luxCDABE 4.6 lM Aroclor
E. coli XL1-Blue PpcbC-luxCDABE PCBs 0.1 mM biphenyl (lowest [56] N/A
concentration tested)
E. coli XL1-Blue PpcbC-luc PCBs 0.1 mM biphenyl (lowest [56] N/A
concentration tested)
E. coli (pHYBP109) hbpR-PhbpC- OH-PCBs 1 nM 2-hydroxy-30 ,40 ,5- [58] Simulated aquatic oil spill [14]
luxAB trichlorobiphenyl Human serum [58]
P. fluorescens dmpR-Po- Phenolic 0.30 lM 2-methylphenol [63] Groundwater and semicoke-dump
OS8(pDNdmpRlux) luxCDABE compound 0.87 lM phenol leachates [63]
E. coli (pRLuc42R) luc Phenolic 0.5 lM phenol [65] N/A
compound
Acinetobacter DF4-8 mopR- Phenolic 0.03 mM phenol [67] N/A
luxCDABE compound
a
N/A not available
Detection of Organic Compounds with Whole-Cell Bioluminescent Bioassays
117
118 T. Xu et al.
native Pu promoter. When expressed in Escherichia coli DH5a, and assayed in

96-well microtiter plates, the resulting bioluminescence could be detected after a
2-h incubation with 7.5 lM toluene. However, because the XylR regulator can be
activated by a variety of BTEX chemicals, a test with known concentration toluene
spikes is required in parallel with all environmental samples. It was determined
that, under these conditions, the reporter was capable of detecting a concentration
of 168 lM of nonspecific BTEX compounds in soil, and 362 lM of nonspecific
BTEX compounds in groundwater. However, additional testing is still required to
determine which specific compound(s) are present.
In an alternative but somewhat similar approach, Willardson et al. [13]
developed a BTEX reporter strain by employing the XylR regulator to govern
expression of the firefly luciferase gene (luc). The resulting xylR-Pu-luc fusion was
housed on the pGLUTR plasmid and expressed in E. coli DH5a. The resulting
reporter was then used to detect BTEX compounds in both soil and water samples
[13]. Notably, the water samples used in these experiments were taken from near
an underground storage tank known to have leaked BTEX compounds and were
incubated directly with the reporter without preprocessing. Following this 1-h
direct incubation, the samples were treated with luciferin substrate to induce signal
generation and the total detectable BTEX concentration in the water sample was
determined to be 215 lM of toluene equivalents. Unlike the water samples, this
reporter still required soil samples to undergo an ethyl alcohol extraction to isolate
any chemical pollutants prior to exposure. However, following this extraction and
a subsequent dilution of the samples in medium, only a 1-h incubation was
required before signal generation, and under these conditions 3.44 mM toluene
equivalents could be detected. So although this method was still limited by its
inability to report specific compounds, it was capable of detecting total BTEX
compounds within 3 % of conventional detection methods, making it a powerful
tool for general BTEX detection.
In addition to reporters that utilize the TOL plasmid, a second class of reporter
organism has been developed around the toluene benzene utilization pathway (tbu)
from Ralstonia pickettii PKO1. Similar to the TOL-encoded pathway, the tbu
pathway is regulated by the TbuT protein, which activates the PtubA1 promoter in
the presence of a BTEX inducer compound. Building upon this pathway, Tecon
et al. [14] developed an E. coli-based bioreporter that harbors a pPROBE-LuxAB-
TbuT plasmid whereby the TbuT regulator acts on the PtubA1 promoter when a
BTEX inducer compound is present to transcribe the luxAB genes from A. fischeri.
Because only the luxAB genes were present, and not the full luxCDABE operon, all
assays using this reporter required the addition of the n-decanal substrate prior to
bioluminescent production. To determine its functionality under environmentally
relevant conditions, this bioreporter was used to monitor an artificial oil spill.
Using a 96-well microtiter plate assay, this bioreporter detected 4–12-lM toluene
equivalent concentrations in contaminated seawater samples that were collected as
early as 6 h after the spill. However, similar to the TOL-based systems, the
inability to differentiate between BTEX compounds limits this reporter to only
broad-spectrum compound detection.
The third class of reporters is typified by the bioluminescent P. putida TVA8

BTEX bioreporter developed by Applegate et al. [15]. This reporter, as do all
members of its class, utilizes the P. putida T-2 toluene degradation pathway (tod)
that was originally characterized by Zylstra et al. [16] and Wang et al. [17].
Similar to the previously described pathways, the tod pathway consists of a series
of genes responsible for BTEX oxidation that are under the control of a BTEX
regulatable promoter. Using this system, P. putida TVA8 was constructed by
transposon insertion of the bioluminescent luxCDABE gene cassette downstream
of the todX and todR genes for toluene recognition and transcriptional activation
and validated in wastewater samples for its ability to detect BTEX compounds
[18]. During the experiment, P. putida TVA8 was inoculated with a 1:10 ratio of
wastewater and sampled every 30 min for 8 h to observe bioluminescence. Unlike
the assays of Stiner and Halverson [19], no additional substrate was required
because this construct contained the full luxCDABE cassette. Under these condi-
tions, bioluminescence was detectable within the first 30 min [18] and significant
bioluminescent responses were observed following treatment with 23 distinct
compounds. Although this again highlights the inability of most BTEX bioreporter
organisms to differentiate individual BTEX compounds from one another, it also
shows their utility as detection systems for organic pollutants in general.
Following this successful validation, the same reporter was later utilized to test
groundwater located beneath an airfield site [20], where it successfully differen-
tiated between contaminated and remediated sites as confirmed by analytical
detection methods.
The final pathway that has been used for BTEX bioreporter construction is the
isopropylbenzene (ipb) pathway. This pathway, which was originally identified in
P. putida RE204 [21], consists of a regulatory protein (encoded by ipbR), an
operator/promoter ipbo/p, and the isopropylbenzene dioxygenase gene (ipbA). To
develop a BTEX bioreporter using this system, Selifonova and Eaton [22] trans-
formed an E. coli HMS174 strain with a plasmid containing the ipb genes fused
with the lux gene cassette of A. fischeri. This construct was then used to detect
aromatic compounds in hydrocarbon mixtures (jet fuel, diesel fuel, and creosote)
and for the direct detection of hydrocarbons extracted from sediments. It was
demonstrated that the reporter could detect a concentration as low as 0.01 ppm in
creosote hydrocarbon mixtures. Because the sediments contained a mixture of
hydrocarbons in varying quantities, light detection was measured from dilutions of
the total extracted hydrocarbons to determine if bioluminescent production
increased corresponding to the increase of hydrocarbons. It was demonstrated that
the reporter did produce bioluminescence corresponding to the sediment extraction
dilutions and could detect hydrocarbons in a dilution as low as 1:500. However,
because this reporter is not specific to BTEX compounds, the amount of light
produced may not indicate the true concentration of BTEX compounds in mixed
samples.
As a whole, it can be said that the varied classes of BTEX reporters have
successfully met their goal of providing a faster and lower cost method for the
detection of BTEX compounds. However, the major caveat for these reporters is
120 T. Xu et al.
their inability to differentiate individual BTEX compounds. Because each

bioreporter was constructed to detect a number of compounds that are under the
BTEX classification, pinpointing which contaminant(s) are present in the sample is
not currently possible. Therefore, in order to determine the exact chemical
contaminant further analysis of a sample still needs to be done. Despite this
drawback, these reporters provide a simple and efficient method for the rapid
screening of multiple categories of environmental samples, making them a
valuable first-line analysis tool for large-scale monitoring projects.
2.2 Polycyclic Aromatic Hydrocarbons
Polycyclic aromatic hydrocarbons (PAHs) are compounds consisting of two or

more fused benzene rings. Due to repeated spills and the seepage of petroleum
products, they have become some of the most common soil and water contami-
nants and can be found around the globe. There is a pressing need for the iden-
tification and remediation of PAHs because they pose significant human health
risks such as heart disease, cancers, and kidney and liver damage [23]. In order to
meet this need, PAH-detecting bioreporters have been developed that utilize either
the naphthalene, phenanthrene, or isopropylbenzene degradation pathways for
control of their bioluminescent signals.
By far, the majority of the PAH bioreporters exploit the naphthalene degra-
dation pathway in order to modulate their bioluminescent signal. This pathway
consists of two separate systems, an upper pathway (nahABCDEF) that degrades
naphthalene to salicylate, and a lower pathway that then degrades the salicylate to
acetaldehyde and pyruvate [24]. A single regulatory gene, nahR, governs the
expression of these genes in response to naphthalene, making it an ideal target for
bioreporter design. Burlage et al. [25] were the first to make use of this pathway
when they constructed P. putida RB1351. This strain contains the luxCDABE gene
cassette under the control of the upper NAH promoter, Pnah, allowing biolumi-
nescent expression to be modulated in response to naphthalene bioavailability.
This reporter strain has been extensively investigated by Dorn et al., who refer to
the strain as P. putida RB1353 [26–28]. From these studies, it has been concluded
that the lux expression can be altered from a 1 °C temperature change and a
change in pH of 0.2 [27], the reporter can successfully be integrated into a fiber-
optic detection system for monitoring microbial activity in porous media in real-
time [28], and that the fiber-optic detection system can be used for the realtime
in situ monitoring of bioactive zone formation and dynamics [26].
The most widely known naphthalene pathway-based reporter, however, is likely
P. fluorescens HK44, which has become one of the most evaluated microbial
bioreporters ever to be developed. Similar to P. putida RB1351, HK44 expresses
the upper naphthalene pathway (nahABCDEF) and the regulator gene nahR,
however, in the lower pathway the nahG gene is fused to the luxCDABE cassette
from A. fischeri [29]. Using this set-up King et al. [29] demonstrated a
bioluminescent response to naphthalene at concentrations as low as 1.56 lM after

15 min of exposure in a chemostat culture. More important, however, it was also
shown in this study that the bioluminescent signal generated by HK44 was capable
of responding to naphthalene in a dose-responsive manner, allowing for realtime
detection and monitoring. Throughout its widespread use, HK44 has since been
tested against a variety of organic compounds and has been applied to monitor
PAH occurrence in various environmental matrices (recently reviewed by Trögl
et al. [30]). Although the majority of applications using strain HK44 have been
performed on extracts of water, soil, and sediment samples, Valdman and Gutz
[31] recently demonstrated the utility of agar gel-immobilized HK44 reporter cells
for the detection of naphthalene and related compounds in the vapor phase. Var-
ious concentrations of naphthalene vapors were flowed into sampling tubes and
bioluminescence was monitored in a luminometer. The limit of detection under
this experimental design was determined to be 20 nM naphthalene, and a linear
relationship between naphthalene concentration and bioluminescent response was
obtained between the concentrations of 50 and 260 nM.
Because of the characteristics and popularity of the P. fluorescens HK44
bioreporter, it was selected as the model organism for a first of its kind multiyear
controlled field release study. In 1996 P. fluorescens HK44 was released in a
controlled environmental test site to monitor the long-term ability of a genetically
modified organism to detect and degrade naphthalene. Over time, environmental
naphthalene was detected in two ways, either through the detection of naphthalene
vapors by HK44 biosensor modules interfaced with fiber-optic cables [32], or
through direct interaction of HK44 bioreporter cells with naphthalene in the soil
via observation of the resultant bioluminescent signal using a photomultiplier tube
(PMT) [32]. The continued detection of HK44 throughout the 2-year study proved
its ability to persist in the environment, and bioluminescent detection on-site
demonstrated its utility as a continuous reporter for naphthalene bioavailability
[32, 33].
It is important to remember, however, that despite the widespread use of
P. fluorescens HK44 and its related naphthalene-based sensor organisms, other
methods for PAH bioreporter construction have been employed. Among these has
been the use of the phenanthrene degradative genes from the Burkholderia sp.
strain RP007, which was first described by Laurie and Lloyd-Jones in 1999 [34].
Tecon et al. [126] exploited this operon for the construction of a bioreporter for the
detection of naphthalene, phenanthrene, and related PAH compounds. The resul-
tant reporter strain, B. sartisoli RP007, harnessed the regulatory genes phnR and
phnS from the phenanthrene pathway to regulate expression of the luxAB genes.
Under this design, when a PAH compound interacts with the PhnR protein it
causes the downstream activation of the phnS promoter, which then allows tran-
scription of the A. fischeri luxAB genes. Similar to other reporters that only contain
the luxAB genes, this reporter is limited in that the substrate n-decanal must be
added for the production of light concurrent with naphthalene exposure. However,
when n-decanal is supplied, the reporter demonstrated a minimal detection limit of
0.17 lM after a 3-h incubation. When exposed to an artificial oil spill,
122 T. Xu et al.
bioluminescence was detected above the minimal detection limit after 3 h, and
continued to produce a response after 5 days, demonstrating a longevity of signal
that can be crucial for environmental monitoring applications.
2.3 Alkane Aliphatic Hydrocarbons
Alkanes are saturated hydrocarbon structures that can be deposited environmen-

tally from a wide variety of sources. Most commonly, however, they originate
from the natural seepage of crude oil deposits or from anthropogenic releases of
fuel products and industrial lubricants. Due to their highly hydrophobic nature,
they are not acutely bioavailable, and therefore have traditionally been difficult to
detect. Historically, the predominant method for alkane detection by microbial
bioreporters has been through expression of the regulatory region of the alkane-
responsive alk operon from Pseudomonas oleovorans. In 1973 it was discovered
that the genes responsible for octane degradation in P. oleovorans were located on
a plasmid and could be transferred between organisms [35]. Although it was
known at this time that the degradation function encoded by these genes was
inducible in the presence of alkanes, it took another 15 years before the genetic
structure of the operon was fully identified [36]. It was then a further 9 years
before it was first exploited as a sensing component for bioreporter development
[37]. The alk operon consists of two distinct sections, with the first encoding three
genes for alkane catabolism (alkBFG) and second encoding a regulatory compo-
nent (alkS) that can activate transcription in the presence of 6- to 10-carbon
alkanes [38].
Sticher et al. [37] were able to remove the alkS regulatory component and
coexpress it along with a fusion of the inducible alkB promoter and the Vibrio
harveyi luxAB genes. This effectively governed the transcription of the luxAB
genes in response to alkane presence, although it required an exogenous
application of decanal to serve as the substrate for the luxAB luciferase in order to
elicit a bioluminescent response. Following a survey of decanal concentrations, it
was determined that the assay conditions had to be amended with 2 mM decanal to
ensure that the results were indicative solely of the induction of the luxAB genes by
alkanes, and not limited by a lack of substrate for the resultant bioluminescent
reaction. Under laboratory conditions, this decanal-supplemented assay was
capable of detecting 5- to 10-carbon chain length alkanes with a response time of
1–2 h. The minimum detection level for octane was determined to be 24.5 nM,
however, induction at this level resulted in only a 1.4-fold increase in light
production. It is also important to note that the assay could be inhibited by the
presence of alicyclic hydrocarbons, aromatic hydrocarbons, alkylbenzenes, or
biphenyls, which limited its use in environmental applications. Despite this
handicap, however, the assay was used to monitor diesel-oil–contaminated
groundwater samples, but was limited to reporting in octane equivalents inasmuch
as it is not specific for individual hydrocarbon species. Similarly, because of the
complex nature of diesel oil, a coassay was required to determine the level of
inhibition caused by nonalkane chemicals, which then allowed a corrected octane
equivalency to be determined. Although this assay format was not ideal, it rep-
resented the first time that a bioluminescent microbial assay was used to monitor
for environmental alkane contamination, and provided a valuable first step toward
the development of improved sensor moieties.
Building upon this system, Minak-Bernero et al. [39] were able to take further
advantage of the remaining P. oleovorans alk operon genes and develop an alkane
sensor that did not require the exogenous addition of decanal in order to produce a
bioluminescent signal. To accomplish this, they constitutively co-expressed the
alkBFG alkane catabolism genes, the alkJ alcohol dehydrogenase gene, and the
luxAB genes. Under this system, the alkBFG gene products performed their native
function of reducing the target alkanes to alcohols, the alkJ gene product then
converted those alcohols into aldehydes, and the LuxAB luciferase proteins then
used the resultant aldehydes as substrates for the generation of a bioluminescent
signal. The ultimate result was a bioluminescent microbial bioreporter that could
respond to the presence of alkanes within seconds after exposure to produce a
detectable signal. This sensor was approximately as sensitive as the decanal-
dependent sensor developed previously [37], giving a linear response to octane in
the 10- to 200-lM range [39]. Additionally, because the P. oleovorans alk gene
products were capable of modifying 5- to 12-carbon chain length primary alcohols
and aldehydes [40], and the LuxAB luciferase protein could accept 6-carbon and
longer chain length aldehydes [41], this sensor was theoretically capable of
sensing any alkane between pentane and dodecane. However, because the system
constitutively expressed all of the alk and lux genes, the sensor would report the
detection of any bioavailable pathway intermediate products indiscriminately. This
made it impossible to differentiate alkanes, alcohols, or aldehydes in a given
sample. So although detection had become increasingly autonomous, the
specificity of the system was reduced.
In 2010 there was a renewed interest in the detection of alkane hydrocarbons in
seawater due to the highly publicized Deepwater Horizon oil spill in the Gulf of
Mexico [42]. This spurred renewed testing with the available reporter strains and
demonstrated that, although the detection characteristics were similar to those
obtained in groundwater samples [37], the reporters could be inhibited by salt
during in situ analysis with laboratory-contaminated seawater [14], limiting their
use under environmental conditions. To overcome this deficiency, Zhang et al.
[43] developed a bioluminescent reporter using the native alk operon system in
Acinetobacter baylyi ADP1 rather than expressing a modified version of the
P. oleovorans alk operon in E. coli as had been done previously. This reporter was
constructed using homologous recombination to introduce the luxCDABE genes
from Photorhabdus luminescens in place of the alkM alkane hydroxylase gene in
the A. baylyi ADP1 chromosome, placing them under the control of the naturally
alkane-inducible AlkR regulator protein. Although the detection limit was higher
than that of P. oleovorans-based E. coli reporter strains [37], the A. baylyi ADP1
reporter strain was capable of detecting alkanes between 7- and 36-carbons in
124 T. Xu et al.
length, giving it a significantly enhanced detection profile [43]. A. baylyi

ADP1 was also demonstrated to be significantly more tolerant to seawater than
E. coli, which allowed the reporter to function without any interference from salt
contamination. Another advantage of this reporter is that A. baylyi ADP1 was
shown to adhere to the oil-water interface and emulsify the oil into small droplets
by forming a single layer of cells around the droplet surface. This effectively
presented an increased sample concentration for the reporter to detect, and aided in
decreasing response time to 0.5 h. Testing with alternative contaminants such as
salicylate and toluene did not show any significant induction of bioluminescent
signal [44], suggesting that the A. baylyi ADP1 reporter may be more specific than
previous P. oleovorans-based versions as well. One year later, similar inroads for
the detection of longer chain alkanes were made by Kumari et al. [45], who
expressed the luxAB genes under the control of the alk operon from Alcanivorax
borkumensis SK2 in E. coli. However, they ultimately chose to focus on the
development of an EGFP (enhanced green fluorescent protein)-based fluorescent
reporter strain in place of the luxAB-expressing strain that required the addition of
decanal to produce a bioluminescent signal.
2.4 Chlorinated Aliphatic Hydrocarbons
Chlorinated aliphatic hydrocarbons have been widely used throughout the

chemical industry for decades. Through mishandling and environmental releases
over this time frame, they have become widespread in soils and groundwater [46],
and because of their widespread deposition and toxic nature, they now pose a
significant risk to both human and environmental health [47, 48]. For these rea-
sons, there is an increasing interest in the development of bioreporters that are
capable of determining the location and bioavailability of these toxic compounds
in order to direct remediation efforts for their disposal.
Classically, bioreporter-based chlorinated aliphatic hydrocarbon detection
mechanisms have been established around the tod operon. This operon consists of
three genes responsible for oxidation of toluene to cis-toluene dihydrodiol under
the control of a regulatable promoter that is upregulated in response to increasing
toluene concentrations [16]. Applegate et al. [15] were able to leverage this action
by replacing the downstream tod genes with a complete luxCDABE operon to
develop a P. putida strain (TVA8) capable of responding to challenges with either
toluene or the chlorinated aliphatic hydrocarbon trichloroethylene (TCE) by
production of a bioluminescent signal [49]. Using this reporter, it was possible to
detect TCE at a lower detection limit between 1 and 5 lM and an upper limit of
230 lM, although the results of the analysis could be easily skewed in the pres-
ence of contaminating toluene. Despite this detriment, the P. putida TVA8 bio-
reporter was successfully deployed under environmental conditions, where it was
able to detect TCE and 1,1,1 trichloroethane (TCA) in contaminated groundwater
samples as confirmed by analytical analysis [20], further demonstrating its utility.
More recently, a second P. putida operon has been discovered that can be used
as an alternative to the traditional tod-based approach. This operon, the sep operon,
consists of three efflux pump-encoding genes that are regulated in response to a
variety of common chemical solvents [50]. When the luxCDABE operon was
cloned in place of the upstream sepA gene, the result was a strain that modulated
bioluminescent activity in response to TCE availability. This strain was still
susceptible to interference by the same contaminant chemicals as the TVA8 strain
[15], however, it did present investigators with another tool for the realtime
detection and monitoring of a wide range of halogenated solvents and chlorinated
aliphatic hydrocarbons.
Since the time the tod and sep-based reporter systems were first developed, a
more specific reporter has emerged that can sense and respond to the presence of
the chlorinated aliphatic hydrocarbon dichloromethane (DCM). The selectivity of
this reporter is due to its utilization of the dcm operon from Methylobacterium
extorquens DM4, which is able to grow on DCM as a sole carbon source. The dcm
operon consists of the genes dcmAR, with the dcmA gene upregulated in the
presence of DCM and the dcmR gene encoding a contrasting negative regulatory
element. By cloning the luxCDABE genes under control of the dcm promoter, it
was possible to elicit a bioluminescent response from aerosolized DCM at a range
between 12 lM and 1.2 mM. Induction of the bioluminescent signal could be
observed at 1 h posttreatment at the 1.2-mM level, but increased to 2.3 h at the
12-lM level. In the liquid phase, the reporter could detect DCM between a range
of 1.2 lM–12 mM, and the induction time was relatively decreased compared to
aerosolized samples, requiring only 0.5 h at the 12-mM concentration. Regardless
of the medium used (aerosol or liquid) there was a correlation between biolumi-
nescent output and DCM concentration at an R2 value of 0.99 [51]. In contrast to
the nonspecific reaction of tod- and sep-based systems, this level of specificity and
dose–response kinetics highlights what can be achieved by modulating the
selectivity of the upstream regulatory element that is used for bioreporter
generation.
2.5 Biphenyl and Polychlorinated Biphenyls
PCBs represent some of the most widely distributed and persistent environmental
contaminants due to their resistance to physical, chemical, and biological degra-
dation. It is because of this exceptional stability that PCBs found extensive use as
dielectric and coolant fluids in transformers, capacitors, and electric motors.
Evidence of their consequent discharge can be found in nearly all environmental
ecosystems, including water, sediments, soils, and air, and their tendency to bio-
accumulate in living organisms magnifies their presence throughout the food chain
as well. Microbiologically, there are a handful of known bacterial species that can
utilize biphenyl as a sole source of carbon and energy, predominantly via oxidative
degradation mediated by the biphenyl gene cluster (bph) [52]. Layton et al. [53]
126 T. Xu et al.
were the first to exploit the bph pathway for PCB bioluminescent biosensing by
linking the bph R1 regulatory region to a plasmid-localized luxCDABE gene
cassette that was inserted into Ralstonia eutropha to create the bioreporter
ENV307(pUTK60). Validation was performed against biphenyl, 2-, 3-, and
4-chlorobiphenyl, and an Aroclor PCB mixture. However, due to the poor aqueous
solubility of PCBs, a surfactant was added to the test samples to promote increased
bioavailability. Minimum detection limits ranged from 0.80 lM for 4-chlorobi-
phenyl to 4.6 lM for Aroclor in a 96-well microtiter plate assay over a 6-h
incubation period. The need to add surfactants to PCB samples then drove this
group to create an improved toxicity-based bioassay because, in the standard
Microtox test, the toxicity of the surfactants toward the A. fischeri reporters
interferes with the toxicity profile of the PCB compounds [54]. To circumvent this
limitation, Layton et al. [55] developed their toxicity assay using indigenous
wastewater microorganisms displaying surfactant resistance (Stenotrophomonas
sp. and Alcaligenes eutrophus) that were engineered to bioluminesce constitutively
via plasmid insertion of a luxCDABE gene cassette. Results showed these two
strains to be 400 times more resistant than A. fischeri to the commonly used
surfactant polyoxyethylene 10 lauryl ether, signifying their potential practicality in
PCB and other compound toxicity bioassays that require the addition of
surfactants.
Pseudomonas sp. DJ-12 expresses a meta-cleavage dioxygenase via the
pcbABCD operon that enables degradation of select biphenyl compounds. Park
et al. [56] created plasmid-based gene fusions between the pcbC promoter and
luxCDABE and luc to create two bioluminescent bioreporters in E. coli host cells.
Bioassays performed in 96-well microtiter plates over 30-min exposure periods
indicated responsiveness to biphenyl compounds between the 0.1 and 1-mM
exposure concentrations analyzed. Testing at lower concentrations to establish true
detection limits still needs to be performed, as well as compound specificity
studies, but these bioreporters demonstrate a potential addition to the inventory of
biphenyl-responsive bioreporters.
In bacteria and higher organisms, PCBs are biotransformed by cytochrome
P-450 monooxygenases and metabolized to hydroxylated PCBs (OH-PCBs). Cer-
tain bacteria are able to use hydroxybiphenyls as sole carbon and energy sources
via mediation of the hbp gene cluster under regulatory control of the hbpR gene
[57]. Recognizing this, Turner et al. [58] linked the hbpR gene from Pseudomonas
azelaica to the luxAB genes on a plasmid-based (pHYBP109) system that was
inserted into E. coli to create a bioluminescent reporter for OH-PCBs. The biore-
porter was tested against 27 OH-PCBs with dose-dependent responses successfully
obtained with limits of detection in the range from 10-5 to 10-9 M in 4-h incu-
bation assays followed by the addition of the n-decanal substrate. The bioreporter
was also used by Tecon et al. [14] in a luxAB-based multibioreporter assay to
monitor for oil spill constituents in aquatic ecosystems, subsequently allowing for
the simultaneous detection of biphenyls, short-chain linear alkanes, and
monoaromatic and polyaromatic compounds within a 3-h assay. Validation of the
bioreporter was also applied diagnostically in human serum samples spiked with
an individual OH-PCB (2-hydroxy-30 ,40 -dichlorobiphenyl) as well as a mixture of

10 OH-PCBs, with demonstrated detection limits as low as 5 9 10-8 M within a
4-h bioassay time frame [58].
With a goal of expanding the bioreporter’s chemical detection portfolio, Tropel
et al. [59] then subjected this hbpR promoter/operator region to site-directed
mutagenesis to modify its recognition specificity. Using this approach, they suc-
cessfully created an hbpR-luxAB P. azelaica bioreporter that was capable of
responding to both m-xylene and 2-hydroxybiphenyl. This ability to combine
different regulatory pathways within a single bioreporter to enable biosensing
across different chemical classes facilitates simplified multitargeted bioassays for
expanded environmental monitoring.
2.6 Phenol and Derivatives
Phenols and their derivatives serve as some of the most common environmental
pollutants in soil, water, and air. Deposition occurs through both natural events
(i.e., decomposition of organic material, forest fires, and atmospheric degradation
of benzene) and industrial activities where it is produced in massively high vol-
umes (approximately 7 billion kg per year) as an important precursor component
of plastics, epoxies, explosives, detergents, herbicides, and pharmaceutical drugs.
Under such large-scale manufacturing demands, environmental impacts
(especially in relation to wastewater discharges) are well recognized, with 12
phenolic compounds registered on the US Environmental Protection Agency’s list
of priority pollutants [phenol, 2-chlorophenol, 2,4-dichlorophenol, 2,4-dimethyl-
phenol, 2,4-dinitrophenol, 4-nitrophenol, 2-nitrophenol, pentachlorophenol,
2,3,4,6-tetrachlorophenol, 2,4,6-trichlorophenol, 4-chloro-3-methylphenol (syno-
nym 4-chlor-m-cresol), and 2-methyl-4,6-dinitrophenol (synonym 4,6-dinitro-o-
cresol)]. Accordingly, microorganisms have evolved to utilize these phenols,
which provides an inroad for the development of bioreporter assays based on their
genetic pathways to detect and monitor these phenolic compounds [60]. Over two
decades ago, Shingler et al. [61] isolated a Pseudomonas strain, CF600, capable of
using specific phenols and derivatives as sole sources of carbon. Elucidation of its
genetic pathway for doing so described the now well-understood dmp operon [62]
that later became the platform for the luxCDABE-based bioluminescent bioreporter
P. fluorescens OS8(pDNdmpRlux) [63]. Reporter OS8(pDNdmpRlux) demon-
strated a response portfolio to a variety of phenols, including 2-, 3-, and 4-
methylphenol (synonyms o-, m-, and p-cresol), 2,3-, 2,4-, 3,4-, and 2,6-dimeth-
ylphenol, resorcinol, and 5-methylresorcinol, with maximum detection limits
achieved under 2-methylphenol (0.30 lM) and phenol (0.87 lM) exposures of a 4-
h duration. As is customary with these types of bioreporters, specific phenols
cannot be individually identified and the bioluminescent signal rather represents
the total phenolic content in its bioavailable form. When applied to natural, mixed
contaminant groundwater and semicoke dump leachates containing primarily
128 T. Xu et al.
phenol and methylated phenols, the bioreporter successfully bioindicated phenol

bioavailability in nine of the ten samples analyzed, with this single negative
sample hypothesized to contain phenolic constituents in a non bioavailable form
[63]. Building upon these results, Wise and Kuske [64] devised a second gener-
ation bioreporter using mutated versions of the regulatory DmpR region of the dmp
operon to increase the range of phenolics detected. This new reporter construct
extended the range of detectable compounds to include 2-chlorophenol, 2,
4-dichlorophenol, 4-chloro-3-methylphenol, and 2- and 4-nitrophenol. Similarly,
Gupta et al. [65] performed a more defined DmpR mutation approach and linked
gene expression to firefly luciferase within an E. coli host cell to create a biolu-
minescent bioreporter (pRLuc42R) capable of detecting phenol at a lower limit of
0.50 lM within a 3-h assay time frame, however, its ability to detect phenolic
compounds in realworld samples has yet to be tested.
Microbes belonging to the genus Acinetobacter also utilize phenol as a sole
carbon source, and have thus also been transformed into bioluminescent biore-
porters. Their genetic architecture consists of a mop operon wherein the MopR
regulator activates phenol hydroxylase expression upon binding with phenolic
compounds such as phenol, 3-chlorophenol, and 2- and 3-methylphenol [66].
Abd-El-Haleem et al. [67] developed the Acinetobacter bioreporter DF4-8 via
linkage of this regulatory activity to the luxCDABE gene cassette to create a
bioluminescent bioreporter capable of detecting phenol at a limit of detection of
0.03 mM within an approximate 4-h assay. When exposed to slurries of aged soils
obtained from a phenol-contaminated industrial site, the bioreporter elicited a
bioluminescent signal within approximately 6 h. This group then further devel-
oped a luxCDABE-based constitutively bioluminescent Acinetobacter bioreporter
(DF4/PUTK2) for chemical toxicity assessment and showed its decreasing levels
of bioluminescence in response to several phenolic compounds ranging from 50 to
500 ppm (EC50 = 333 ppm) [68]. They also immobilized the bioreporters in
calcium alginate and demonstrated an 8-week storage capacity at 4 °C in a 96-well
microtiter plate format, suggesting application toward a prepackaged, off-the-shelf
sensor platform. A larger panel of bioreporters for monitoring phenol-related
toxicity was developed by Wiles et al. [69] using four wastewater Pseudomonas
isolates engineered to carry a chromosomally integrated luxCDABE cassette.
When exposed to natural wastewater effluent samples in a 96-well microtiter plate
format under a 5-min incubation period, this panel effectively bioindicated phe-
nolic concentration shifts in concentrations ranging from approximately 10 to
800 ppm (EC50 = 454–757 ppm). In both this study and the previously described
Acinetobacter reporter study, toxicity profiles were compared against the standard
Microtox assay, where A. fischeri is used as the sensor microorganism [70–72],
and in both cases Microtox performed less reliably. The advantage of using
indigenous Pseudomonads or Acinetobacter strains in these studies is their natural
robustness to the wastewater environment undergoing testing, whereas A. fischeri,
being native to the marine environment, is less ecologically adapted and
oftentimes responds less efficiently or requires additional sample preparation steps
to adjust its performance efficiency [73].
3 Evaluation of Organic Toxicant-Induced Health Risks

Using Eukaryotic Cell-Based Bioluminescent Assays
In addition to bacteria, eukaryotic cells, including the lower eukaryotic organism

Saccharomyces cerevisiae, and cultured mammalian cell lines have been
increasingly employed to serve as hosts for bioluminescent reporter assays against
organic toxicants. However, unlike in bacterial bioassays where catabolic
pathways for the biodegradation of target substances are exploited for reporter
development, eukaryotic cells generally are not able to utilize such toxic organic
compounds as carbon sources or lack exhaustively characterized catabolic path-
ways altogether. These deficiencies have been overcome as research on cell
biology and toxicology have revealed that many essential receptor-mediated sig-
naling transduction pathways for normal cellular functions can be disrupted by the
binding of exogenous organic toxicants to endogenous receptors [74], which thus
allows reporter development using similar techniques. Under these strategies, the
characterization of key receptors and transcriptional response elements in these
pathways forms the foundation for eukaryotic-cell–based bioluminescent biore-
porters, which are commonly created by fusing bioluminescent reporter genes to
the response element of a target pathway so that bioluminescent expression is
transcriptionally modulated through chemical-receptor binding. This design
requires coexpression of cognate receptors that are either endogenously present in
some cell lines or, in the case of nonexpressing cell lines and S. cerevisiae, can be
co-introduced with the reporter construct. This means that, because reporter gene
expression is integrated into endogenous toxicity pathways, eukaryotic whole-cell
bioreporters essentially measure chemical-triggered biological effects, making
them ideal tools for providing pathway-specific risk assessment information. This
is particularly important during environmental contaminant evaluation where
multiple pollutants with a range of toxicological effects often coexist throughout a
contaminated site. Because it is common for structurally different chemicals to
exert similar toxic effects or for the same compound to induce multiple pathways
(i.e., some PCBs have been shown to have both dioxin-like and estrogenic activity
[75, 76]), eukaryotic cell-based bioassays can therefore serve as valuable tools for
the estimation of potential harmful effects that may not be detectable using
traditional analytical means.
Currently, the major use of these eukaryotic cell-based reporters has been for
the detection of organic contaminants responsible for dioxin-like (Table 2) or
endocrine-disrupting activities (Table 3). This section summarizes a variety of
bioluminescent-based bioassays that have been developed for the detection of
these two activities. Due to their extensive use in the literature, it is impractical
and unnecessary to list all of the applications, however, a focus is drawn on
highlighting the predominant application areas with recent examples to provide an
overview of the usefulness and limitations of these unique reporter systems.
130 T. Xu et al.
Table 2 Eukaryotic cell-based bioluminescent bioreporters for dioxin-like chemicals and recent
environmental applications
Host cell Reporter construct Detection EC50 References Recent
(organism) (reporter name) limit (TCDD) (TCDD) environmental
application
S. cerevisiae hAhR and hARNT, 1 nM 4.8 nM [91] Sewage sludge
DRE(95)-luc [135]
Sediment [91]
HepG2 DRE(94)-luc 1 pM 0.35 nM [82] Solid municipal
(human) waste [136]
H4IIE (rat) DRE(94)-luc 0.5 fM 10 pM [83] River water [137]
(H4IIE.Luc) Sediment [137]
Hepa1 DRE(94)-luc 0.1–1 pM 30 pM [80] Storm water [138]
(mouse) (H1L1.1c2) Seawater [92]
RTH-149 DRE(94)-luc 4 pM 64 pM [139] Seawater [140]
(rainbow (RTL 2.0)
trout)
RHEK-1 DRE(94)-luc 10 pM 200 pM [141] N/Aa
(human) (HKY1.7)
Hepa1 DRE(920)-luc 0.01 pM 10–16 pM [142] N/A
(mouse) (H1L7.5c3)
a
N/A not available
3.1 Bioassays for Dioxin and Dioxin-Like Compounds
In their most basic form, dioxins are any compound containing a heterocyclic
6-membered ring consisting of 2 oxygen atoms and 4 alternative atoms. Practi-
cally, dioxins are persistent pollutants that can bioaccumulate over time, leading to
increased health risks for organisms of higher trophic levels, with the classic
example being 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD is the most
toxic of the known dioxins, and is thus used as a representative model, with the
relative toxicity of other chemicals expressed in toxic equivalency factors [77].
Collectively, chemicals that inflict toxic effects similar to TCDD are classified as
dioxin-like, and can lead to hepatotoxicity, embryotoxicity, teratogenicity,
immunotoxicity, dermal toxicity, carcinogenesis, or lethality [78, 79]. Dioxin-like
activities have been found in various groups of organic compounds, including
polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans
(PCDFs), some PCBs, and PAHs.
The mechanism of action for dioxin-regulated gene expression begins when the
chemical binds to the aryl hydrocarbon receptor (AhR) in the cytosol. This
chemical–AhR complex then translocates into the nucleus and forms a dimer with
an AhR nuclear translocator (ARNT) protein. The dioxin:AhR:ARNT complex
then binds to specific DNA sequences called dioxin-responsive elements (DREs),
which results in the activation of the adjacent responsive gene(s) [80]. Because the
resulting increase in gene expression is directly proportional to the toxicity of the
Table 3 Eukaryotic cell-based bioluminescent bioreporters for endocrine-disrupting chemicals and recent environmental applications
Host cell Reporter construct (reporter name) Detection limit EC50 References Recent environmental application
(organism)
Estrogenic and antiestrogenic
S. cerevisiae hERa, ERE(92)-luxCDABE (BLYES) 45 pM E2 0.24 nM E2 [118] Freshwater [143]
Drinking water [143, 144]
S. cerevisiae hERa, ERE(92)-luc (BMAEREluc/ 30 pM E2 0.5 nM E2 [116] Wastewater [145]
ERa)
S. cerevisiae hERb, ERE-luc 0.1 nM E2 0.5 nM E2 [116] N/Aa
(BMAEREluc/ERb)
MCF-7 ERE-luc (MVLN) 1 pM 20 pM [98, 99] River water [137]
(human) Sediment [137]
Wastewater [146]
MCF-7 ERE-luc (MELN) 1 pM E2 5 pM E2 [102] Wastewater [125, 147]
(human) Freshwater [148, 149]
Sediment [148, 149]
T-47D (human) ERE(93)-luc (T-47D ER-CALUX) 0.5 pM E2 6 pM E2 [104] Wastewater [150, 151]
Freshwater [151, 152]
Sediment [151]
T-47D (human) ERE(93)-luc (T-47D-KBluc) 1 pM E2 10 pM E2 [105] Wastewater [126, 153, 154]
Freshwater [153]
HeLa (human) hERa, ERE-luc (HELNa) 1 pM E2 5 pM E2 [102] Drinking water [155]
HeLa (human) hERb, ERE-luc (HELNb) 1 pM E2 10 pM E2 [102] N/A
U2-OS (human) hERa, ERE(93)-luc (ERa-CALUX) 0.8 pM E2 20 pM E2 [113] Indoor dust [156]
Detection of Organic Compounds with Whole-Cell Bioluminescent Bioassays
Drinking water, wastewater, and

freshwater [157]
Androgenic and anti-androgenic
S. cerevisiae hAR, ARE(94)-luxCDABE (BLYAS) 2.5 nM DHT 9.7 nM DHT [119] N/A
(continued)
131
Table 3 (continued)
132
Host cell Reporter construct (reporter name) Detection limit EC50 References Recent environmental application
(organism)
S. cerevisiae hAR, ARE(92)-luc (BMAAREluc/AR) 50 pM testosterone 10 nM [116, 117] Sediment [137]
testosterone
0.5 nM DHT 5.5 M DHT
T-47D (human) ARE(92)-luc (AR-LUX) 46 pM methyltrieno-lone 86 pM [107] Freshwater [158]
methyltrieno-
lone
MDA-MB-453 MMTV-luc (final reporter known as 0.1 nM DHT [108, 126] Wastewater [126, 159]
(human) MDA-kb20
O2-US (human) hAR, HRE (hormone response 3.6 pM DHT 0.13 nM DHT [113] Drinking water, wastewater, and
element)-luc freshwater [157]
Glucocorticoid receptor (GR) agonist and antagonist
MDA-MB-453 MMTV-luc (final reporter known as 10 nM dexamethasone N/A [108] Wastewater [159]
(human) MDA-kb2)
O2-US hGR, HRE(93)-luc (final reporter 0.2 pM dexamethasone 0.37 nM [113] Wastewater [160]
known as GR-CALUX) dexamethasone
Thyroid receptor (TR) agonist and antagonist
PC12 r(rat) Avian TRa1, TRE(9 4)-luc (PC-DR- 30 pM 3,3’,5-triiodo-L- 0.18 nM T3 [161] Wastewater, drinking water, and
LUC) thyronine (T3) surface water [162]
Progesterone receptor (PR) agonist and antagonist
O2-US hPR, HRE(93)-luc (PR-CALUX) 1.3 pM Org2058 0.09 nM Org2058 [113] Indoor dust [156]
Drinking water, wastewater, and
freshwater [157]
a
N/A not available
T. Xu et al.
binding chemical [81], this process provides an excellent platform for the devel-
opment of regulatable bioluminescent bioreporter-based dioxin detection
strategies.
The first dioxin-responsive bioluminescent reporter to be developed was used
by Postlind et al. [82] to track expression of the CYP1A1 and CYP1A2 genes of the
human cytochrome P450 gene family. This was accomplished by cloning the 50
flanking region from either the CYP1A1 or CYP1A2 genes upstream of a luc gene
in a human expression vector. These vectors were then introduced to a human
hepatoma (HepG2) cell line and challenged with TCDD and other compounds.
Under transient transfection conditions where cells were exposed to TCDD for
24 h the day after plasmid introduction, reporters expressing the CYP1A2 50
flanking sequences displayed a detection minimum of 0.01 nM TCDD, whereas
those expressing CYP1A1 50 sequences were detectable down to a concentration of
0.001 nM. Each was capable of responding to TCDD treatment in a dose–response
fashion, however, although the signal maximum for the CYP1A2-based reporter
was 10-fold over control upon treatment with 10-nM TCDD, the CYP1A1-based
reporter reached its signal maximum at 65-fold over control upon 100-nM treat-
ment. This gave the CYP1A1-based reporter both a larger range of detection, as
well as a greater signal intensity throughout that range. Commensurate with its
lackluster performance compared to CYP1A1, the CYP1A2-based reporter con-
struct was not able to function at all under stable expression conditions. Whereas
the CYP1A1-based reporter was capable of stable expression and could detect
10-nM TCDD at 0.5 h postexposure, with increasing reporter activity positively
correlating with exposure time up to 24 h. The EC50 for TCDD detection by this
reporter was determined to be 0.35 nM, making it the first functionally useful
dioxin-detecting human cell line.
Building upon this expression strategy, Garrison et al. [80] used a construct
consisting of a mouse mammary tumor virus promoter mediated luc gene under the
control of a 484 base pair 50 upstream mouse Cyp1a1 gene sequence that contained
4 DREs to generate a range of bioluminescent dioxin-responsive bioreporter cell
lines. When treated with 1 nM TCDD for 24 h the day after plasmid introduction,
all of these lines were capable of responding to TCDD challenge with a
corresponding bioluminescent output. This demonstrated that the mouse DREs
could be successfully recognized by the dioxin:AhR:ARNT complex across a wide
range of species, and therefore that the assay could be performed in specific cell
types to determine species-specific bioavailability of dioxins and dioxin-like
compounds. Bolstered by these findings, a second set of stably transfected dioxin-
responsive bioluminescent bioreporter cell lines were developed. Of these cell
lines, the mouse H1L1.1c2 line (Hepa1-derived) was chosen as a model for
characterization because it had the greatest level of induction and was found to
respond reliably to TCDD treatment in a dose–response fashion (although this
dose–response relationship was also reported for the human HepG2-derived
HG2L1.1c3 cell line, no data were presented to support this claim at the time). The
model H1L1.1c2 reporter cell line displayed a minimal detection limit
between 0.1 and 1.0 pM of TCDD, with a maximal induction of 80-fold over
134 T. Xu et al.
control at 1.0 nM, providing an ED50 of 0.02 nM. This is less than the previous
value of 0.35 nM established by Postlind et al. [82] with their human HepG2 cell
line, and represented a lower maximal detection limit (1.0 vs. 100 nM) as well.
Also unlike the Postlind study, Garrison et al. [80] were not able to detect a
bioluminescent signal until 2 h posttreatment, and reached a maximum induction
at 4 h posttreatment following application of 1.0-nM TCDD. However, despite
being mostly in line with the Postlind study, the Garrison study remains notable for
its demonstration of bio-reporter function across a wide variety of cell lines, and
for demonstrating that the inducing chemicals did not act as competitive substrates
for the luciferase enzyme, providing a significant advantage for the bioluminescent
assay over older, more traditional chemical-based assays.
Following up these successful demonstrations against specific chemicals in
laboratory settings, Murk et al. [83] transported the system to a rat hepatoma
H4IIE cell line and rebranded its use as a chemical-activated luciferase expression
(CALUX) assay. Using this new cell line they were able to demonstrate TCDD
detection down to 0.5 fM, with a maximum detection limit between 100 pM and
1.0 nM, and an EC50 of 10 pM. Similar to the earlier experiments [80, 82], they
were also able to demonstrate a dose-response relationship between TCDD and
bioluminescent signal, and did not discover any significant substrate inhibition.
What solidified the CALUX assay as the predominant method for dioxin-like
compound detection, however, was the successful demonstration of its use with
environmental samples and its ability to mimic the results of existing, more com-
plex and more costly in vivo assay results [83]. Murk and colleagues demonstrated
that the luc-expressing H4IIE reporter cells could be exposed to extracted sediment
and water samples to determine toxic equivalency factors rapidly and inexpen-
sively. Although it was ultimately determined that unpurified sediment samples
could become toxic to the H4IIE-luc cells, it is primarily highlighted that purifi-
cation was not required for water samples, which significantly improved the speed
at which they could be assayed. Perhaps more important, however, was the
validation of the CALUX assay against the in vitro zebrafish early life stage assay.
Although the presence of heavy metals led to a poor correlation between the two
assays when performed directly in unprocessed environmental water samples, this
correlation significantly improved following sample extraction. When compared
side by side under laboratory controlled conditions, the EC50 as determined by the
zebrafish early life stage assay was found to be 21 pM, whereas the EC50 of the
CALUX assay was 10 pM. Moreover, the working range for the CALUX assay was
improved compared to the zebrafish early life stage assay, and reduced both the
time and cost involved in its performance.
Through its low cost, lack of substrate inhibition, ability to be adapted for
high-throughput, and ability to function in disparate cellular backgrounds, the
CALUX assay has become the predominant method for assaying dioxin and dioxin-
like chemicals using mammalian cells. Since its early demonstrations as a tool for
laboratory-based chemical toxicity testing and environmental pollutant screening, it
has been used for a wide variety of applications including veterinary [84], food
product testing [85–88], and human clinical sample analysis [89, 90]. And as the
applications for the CALUX assay have expanded, so have the organisms that have
been employed for its use. Although still predominantly performed in mammalian
cell lines, recently the assay has been reconstituted in yeast by co-expressing the
genes for AhR and ARNT with a DRE-mediated luc luciferase. This switch away
from human cells and into the microbial eukaryote S. cerevisiae was done with the
hope that it would provide a more robust and simplified expression system that
could improve deployability and reduce costs. It was found that the yeast-based
system could respond to TCDD treatment in a dose–response fashion, with a
minimum detection time of 3.5 h and an EC50 value similar to that of the early
human cellular reporters at 4.8 nM. Inasmuch as this assay was validated against the
classical H4IIE-luc cell line-based CALUX assay and found to be in good agree-
ment [91], it has since been used as a first stage in vivo screen for dioxin-like
chemical load detection in composted sewage sludge [91], proving its worth as an
alternative means of detection when human cell lines cannot be used.
As a final note, with the use of CALUX assays continuing to proliferate, it is
important to recognize that the results of the assay can vary from lab to lab, and
even from run to run [92]. In a validation study, Besselink et al. [93] found 14.6
and 26.1 % intralaboratory assay reliability levels for pure compounds and whole
matrix, respectively, and 6.5 and 27.9 % interlaboratory assay reliability levels for
pure compounds and whole matrix, respectively. Therefore, in light of these dis-
crepancies, it is important to review the sample cleanup methods, the effects of the
solvents used during extraction and testing, any known interaction with synergistic
or antagonistic compounds used, the cell line utilized, and the analysis methods
employed before comparing results between multiple sources in order to determine
the validity of the comparison [92]. Despite these caveats, however, the CALUX
assay remains the predominant method for bioluminescent screening of
compounds eliciting dioxin-like activities.
3.2 Bioassays for Hormonally Active Chemicals
The vertebrate endocrine system consists of a group of signaling molecules

collectively called hormones, which act through binding to their corresponding
nuclear receptors in order to transcriptionally modulate expression of genes
involved in different stages of an animal lifecycle including development, growth,
and reproduction. Due to the critical roles of hormones, any interference with the
endocrine system may lead to various adverse health effects in humans and
wildlife [74], and a variety of organic compounds has been discovered that are
considered as endocrine disruptors because of their ability to mimic or repress the
function of these natural hormones in vivo [94, 95]. The presence of these
chemicals in plastics, pesticides, herbicides, household products, cosmetics, and
pharmaceuticals, and their wide use and discharge into the environment through
anthropogenic activity and industrial waste has made the detection of endocrine
disruptive activity an increasingly large concern.
136 T. Xu et al.
The major mechanism of action for endocrine disrupting chemicals (EDCs) is

to modulate the transcriptional activity of hormone receptors, which includes (but
is not limited to) estrogen receptors (ER—with two isoforms ERa and ERb),
androgen receptors (AR), glucocorticoid receptors (GR), progesterone receptors
(PR), and thyroid receptors (TR). Because of this mechanism of action, biolumi-
nescent bioreporters for EDCs are constructed similar to the dioxin bioreporters by
conditionally expressing a reporter gene (either luc or luxCDABE) under the
control of the corresponding response element. Therefore, detection of organic
compounds using these bioassays is activity—rather than structure-oriented.
Cell-based reporter gene assays for EDCs including bioluminescent bioassays are
routinely reviewed in the literature (for recent examples see [96, 97]) and are
summarized here in Table 3.
Depending on the source of the target receptors, these bioreporters can be
divided into two types, those that exploit endogenous receptors and those that
require manual receptor cointroduction. For those that exploit endogenous recep-
tors, the ER-positive human breast cancer cell lines MCF-7 and T-47D are the
model platforms for bioassays of estrogenic and antiestrogenic activities. The first
stable bioluminescent bioreporter for ER agonists and antagonists in mammalian
cells was the MCF-7-derived MVLN reporter cell line developed by Pons et al. [98]
and subsequently shown to be responsive to both estrogenic and antiestrogenic
substances [99]. In these reporter cells, luc gene expression was placed under the
control of the estrogen response element (ERE) derived from the 50 flanking region
of the Xenopus Vitellogenin A2 gene. Due to the limited techniques available at that
time, the original tests with these cells were performed in a now rarely used 6-well
plate format. Nevertheless, under these conditions treatment with the natural
estrogen 17b-estradiol (E2) exhibited an EC50 of *20 pM after a 24-h exposure
period. However, as technology has improved, this assay has been modernized, and
is now commonly carried out in a more standard 96-well plate format and with a
48-h exposure [100, 101]. Concurrent with this modernization has been the intro-
duction of additional bioreporter cell lines that function in a similar manner. For
instance, building upon the work of Pons et al. [98], Balaguer et al. [102] later
developed a similar MCF-7-derived MELN reporter cell line, in which luc
expression was linked to an ERE and a b-Globin promoter. After 16 h of incuba-
tion, the MELN cell line was able to detect E2 concentrations as low as *1 pM
with an EC50 value of *5 pM when performed in a 24-well plate. Other chemicals
including a nonylphenol mixture, 4n-nonylphenol, 2,40 -dichlorodiphenyldichloro-
ethylene (2,40 -DDE), and 4,40 -DDE (both of which are DDT breakdown products)
also tested positive for estrogenic activity under the same assay conditions. The
reproducibility and lab-to-lab variation of the MELN assay against a battery of
compounds with known estrogenic or antiestrogenic activity was recently tested in
a higher throughput 96-well plate format [103], and this study revealed that,
although the individual laboratories maintained mean intralaboratory coefficients of
variations of either 32.1 or 56.8 % for their EC50 values, both labs produced similar
rankings of estrogenic or antiestrogenic potency of most of the chemicals,
highlighting the utility of this assay for comparative applications.
Moving away from the MCF-7 cell line, Legler et al. [104] developed a T-47D
cell-based ER-CALUX reporter cell line for detection of ER agonists and antag-
onists. Using a minimal TATA box promoter and three tandem repeats of ERE,
this cell line (T-47D ER-CALUX) displayed very low background biolumines-
cence in solvent controls and a maximum induction of approximately 100- and
76-fold compared to unexposed background following a 24-h exposure to 30 pM
E2 in 24- and 96-well plate formats, respectively. This bioassay was also capable
of detecting E2 down to *0.5 pM and is the most sensitive among reported
estrogen-specific assays. A similar T-47D-KBluc reporter developed by Wilson
et al. [105] has a comparable EC50 value for E2 (10 pM in KBluc vs. 6 pM in
EREtata-luc) but possesses a larger dynamic detection range from 1 pM to 100
nM. However, despite these similarities, it is worth noting that controversial
results were obtained in a follow-up study comparing the in vitro T-47D
ER-CALUX assay with an in vivo transgenic zebrafish assay expressing the same
reporter construct [106]. Despite the synthetic estrogen 17a-ethynylestradiol (EE2)
testing 100 times more potent than E2 in the transgenic zebrafish assay, it showed
equal estrogen agonistic activity compared to E2 in the cell-based in vitro assay.
One possible explanation for these differences is that the binding affinities of test
compounds may be different when interacting with ERs that have originated from
different species, which would explain the poor performance of human cell-based
assays to predict toxicokinetics in zebrafish models.
Under similar development strategies, bioluminescent bioreporters screening for
AR agonists and antagonists have been generated as well. These reporters take
advantage of AR-positive cell lines, such as T-47D and the human breast cancer cell
line MDA-MB-453 in order to supply the receptors needed for successful activation
of their chosen bioluminescent expression systems. Blankvoort et al. [107] were the
first to develop a stable T-47D AR-LUX cell line for androgenic and antiandrogenic
effects by using a rat probasin promoter-derived ARE-mediated luc reporter con-
struct. This reporter cell line was shown to be capable of detecting methyltrienolone
down to 46 pM after 24 h of exposure and detecting the environmentally relevant
antiandrogenic compounds 4,40 -DDE and (RS)-3-(3,5-dichlorophenyl)-5-methyl-5-
vinyloxazolidine-2,4-dione (vinclozoline) as well. However, because of the coex-
pression of other hormone receptors (such as ERa, ERb, and PR) in the T-47D
cellular background, there remains a possibility that nonspecific responses may have
been detected. To overcome this issue, Wilson et al. [108] developed a bioassay for
chemicals mimicking/blocking androgen and glucocorticoid activities utilizing the
MDA-MB-453 cell line, which expressed high levels of AR and GR but showed
undetectable or very low levels of alternative receptors [109, 110]. The resulting
reporter cell line, named MDA-kb2, expressed the luc gene under the regulation of
an AR- and GR-responsive mouse mammary tumor virus (MMTV) promoter and,
because of the characteristic low-level expression of competing hormone receptors,
provided a significant decrease in background activity that led to an increased
signal-to-noise detection ratio.
This decrease in alternative receptor expression resulting from the use of the
MDA-kb2 cell line helped to reduce cross-talk between different pathways [111],
138 T. Xu et al.
but was not the only means for accomplishing this goal. With the hope of providing
a more receptor-specific bioassay, several cell lines with little to undetectable levels
of nontarget receptors have been utilized through the introduction of specific
receptors that are not natively expressed. Examples of this approach include the use
of human cervical cancer cell line HeLa and osteosarcoma cell line U2-OS as
parental cells for reporter development. In addition to the MELN reporter cell line,
the HELNa and HELNb bioreporters were developed by Balaguer et al. [102] by
cointroducing ERa and ERb, respectively. Although the E2-generated biolumi-
nescent response of these cell lines is similar to that observed in MELN assays, it
should be noted that TCDD can elicit an antiestrogenic response in HeLa cell-based
assays compared to its demonstrated estrogenic activity in MELN assays. This
differential behavior highlights an example of the effect of pathway cross-talk (in
this case between the AhR- and ER-mediated pathways), which must always be
accounted for during data interpretation. To reduce the prevalence of this cross-talk,
a panel of CALUX bioassays has subsequently been developed for selective
detection of chemicals interacting with ERa, ERb, AR, GR, and PR using the U2-
OS cell line, which demonstrates little or no natural activity of any of these
receptors [112–114]. These reporter cell lines were generated by cointroducing a
vector that conferred constitutive receptor expression, and a second vector that
permitted target receptor-mediated expression of the luc gene. Using this approach,
the ERa- and AR-CALUX bioassays were also shown to be well correlated with
other animal-based assays (R2 value of 0.46 and 0.87 for AR and ERa assays,
respectively), making them useful tools for predicting potential in vivo activities
with a reduced chance of cross-talk–based interference [115].
For similar reasons to those listed above, as the techniques for genetic
expression continue to improve, there has been an increased interest in using the
lower eukaryotic organism S. cerevisiae as a platform for hormonally active
chemical screening. Due to their lack of human hormone receptor expression, fast
and robust growth, and relatively simplified genetic manipulation techniques,
yeast-based bioreporters can now be constructed using a stepwise transformation
of a recombinant human hormone receptor (e.g., hERa, hERb, and hAR) of
interest and a receptor-responsive reporter gene. Several luc-based yeast biore-
porters have been developed for the rapid profiling of estrogenic and androgenic
potentials, and have demonstrated a lower detection limit for E2 and dihydrotes-
tosterone of 30 and 50 pM, respectively [116, 117]. Despite this reduced sensi-
tivity compared to mammalian cell-line–based assays, the yeast-based assays were
capable of reporting relative potency of test chemicals more rapidly, with only a
2.5-h incubation time.
In particular, two yeast bioassays, BLYES [118] and BLYAS [119], which have
been developed to detect estrogenic and androgenic activity, respectively, stand
out from the other bioreporters mentioned above with respect to their choice of
bioluminescent reporter genes. Instead of using the luc reporter gene, each of these
utilizes a bioluminescent end point resulting from expression of the luxCDABE
genes. The switch to the lux system eliminates the need for exogenous luciferin
addition and/or cell lysis and permits autonomous bioluminescent signal
generation and detection. This allows the assay to proceed more rapidly, and with
near realtime signaling. Bioluminescent signal detection can occur as early as 1 h
after exposure to 2.8 nM E2 in the BLYES assay [118], which allowed the BLYES
and BLYAS assays to be used to evaluate the toxicity and potential endo-
crine-disrupting activities of a battery of 68 chemicals quickly and efficiently in a
cost-effective manner [120].
Compared to bioreporters relying on endogenous receptor-mediated signaling,
the test responses generated using recombinant yeast bioreporters and mammalian
bioreporters with manually introduced receptors are less likely to be subject to
nonspecific interactions, which may provide improved mechanistic insight.
However, it still remains to be seen if an enhanced prediction of in vivo effects
might be achieved using bioassays without exogenous manipulation of the
signaling receptors. This creates a potential tradeoff that will need to be evaluated
on a case-by-case basis.
3.3 Environmental Applications
The discharge of chemicals through anthropogenic activity and industrial waste in

the environment urges a careful assessment of ecologically relevant compounds
for their potential toxic effects. One particular phenolic compound of recent
emerging importance is bisphenol-A (BPA). BPA is used extensively in the pro-
duction of polycarbonate plastics and is widely and controversially implicated in
causing negative health effects due to its biological action as an endocrine dis-
ruptor [121]. Its presence in drinking and wastewaters has become particularly
relevant, along with other mid- to long-chain alkylphenols and alkylphenol eth-
oxylates that have been suggested to exhibit similar properties. For these reasons, a
number of bioluminescent reporter systems using both mammalian cell lines and
yeast have recently been employed to characterize its endocrine-disrupting
activity. Michelini et al. [117] used a yeast-based hAR/ARE-luc bioassay to
demonstrate the antiandrogenic potency of BPA with a half maximal inhibitory
concentration (IC50) of 5 lM against 10 nM testosterone. BPA’s estrogenic
potential was later supported by a number of different estrogen assays, yielding
EC50 values of 2.8 and 0.8 lM using the BLYES [118] and T47D ER-CALUX
[104] assays, respectively. In addition, several widely used industrial compounds,
such as polyfluorinated iodine alkanes, nonylphenol isomers, and phthalates have
also been evaluated by the MVLN and H4IIE-luc reporter assay for their estro-
genic and dioxin-like potentials [101, 122–124]. These uses, although by no means
exhaustive, highlight the utility of these eukaryotic cell-based bioluminescent
bioassays to offer a high-throughput and relatively inexpensive route for profiling
the potential toxicities of the ever-expanding number of chemicals that are rou-
tinely being used and released to the environment, providing valuable preliminary
data for assessing the deleterious effects of their exposure.
140 T. Xu et al.
The continuing release of organic compounds through urban and industrial

wastewater has also raised concerns regarding the efficiency of its associated
treatment processes, as any residual toxic chemical present in the treated effluent is
directly discharged into surface water and can thus affect downstream aquatic
ecosystems, and in some cases, drinking water supplies. Of particular interest has
been the generation of disinfection by-products from oxidative chemical treatment
and their potential as endocrine disruptors. To help elucidate this issue, several
bioluminescent bioassays have been applied to determine the endocrine-disrupting
potencies of industrial wastewater before and after ozonation treatment. For
example, Schiliro et al. [125] utilized the MELN assay along with another non-
bioluminescent cell-based proliferation assay to measure the E2 equivalents of pre-
and postozone treated wastewater from a textile industrial wastewater treatment
plant. The MELN bioluminescent bioassay estimated an average of 15.34
(±13.00) pM and 9.29 (±9.10) pM E2 equivalents in pre- and post-ozonation
samples, respectively. Although they did note some discrepancy between the
measured E2 equivalent values between the MELN assay and the proliferation
assay (8.62 (±6.16) pM preozonation and 2.64 (±2.13) pM postozonation), they
pointed out that both assays identified a comparable degree of reduction in
estrogenic potentials as a result of the ozonation process. Furthermore, with
respect to the possible generation of more toxic by-products during ozonation of
naphthenic acid, a primary organic constituent of the wastewater produced during
the hot water extraction of bitumen from oil sands in surface mining operations,
He et al. [126] compared the endocrine-disrupting activity between untreated and
ozone-treated oil sands process-affected water. This study demonstrated that an-
tiandrogenic activity was reduced in ozone-treated water compared to untreated
water through the use of a MDA-kb2 bioreporter assay. However, unlike in the
study of Schiliro et al., and in disagreement with the general idea that ozone
disinfection is an effective means to reduce estrogenicity [127], it was reported that
the estrogenic potential of the oil sands wastewater was not affected by ozonation
when evaluated with the T47D-KBluc bioreporter assay. It is therefore worth
noting that wastewater treatment efficiency varies on a case-by-case basis, and that
bioluminescent bioassays should therefore be used as a rapid preliminary
screening method before applying comprehensive chemical analyses.
Last but not least, to help better understand the potential risks of chemical
exposure, eukaryotic cell-based bioluminescent bioassays are increasingly being
utilized in combination with chemical analysis to survey ecosystems affected by the
discharge of toxic chemicals. Traditionally the pollutant composition would be
fully characterized instrumentally, however, bioluminescent bioassays now provide
a rapid and cost-effective means of simultaneously assessing potential biological
effects as well. In addition, with an increased understanding that additive and
possible synergistic effects of complex mixtures could contribute to the overall
environmental impact [128], there is an understanding that chemical analysis alone
may not generate sufficient information for risk assessment. Similarly, unidentified
hormonal activity may be overlooked by instrumental analysis, which only looks
for known targets. This was demonstrated by Fenet et al. [129] when they linked
the concentrations of alkylphenols quantified by GC/MS with their contribution

toward the total estrogenic activity in environmental samples using the MELN
reporter assay. This study recognized varying degrees of correlation between
chemically determined concentrations and total estrogenicity on a sample-by-
sample basis, demonstrating that, although alkylphenols of GC/MS-determined
concentrations could explain a large part of the estrogenic potency in the studied
sediment samples, their abundance only provided little to a very low contribution
toward the overall observed estrogenic effects in the water samples. These findings
have since been repeated, suggesting the presence of other unintended or unknown
estrogenic contaminants in studied sites [100]. However, despite their utility to
assess biological effects rapidly, it is critical to acknowledge that these biolumi-
nescent assays are not capable of identifying the causative agents, and therefore
should not be used as a stand-alone technique for environmental evaluation. A
comprehensive assessment of organic pollution requires thorough chemical and
toxicological analyses and is often time-consuming. Therefore, the major role of
cell-based bioluminescent assays should be to serve as a rapid and economical
initial screening tool to prealert samples eliciting positive responses for further
investigations and to reduce expense and labor on samples with negative responses.
4 Conclusions
Equipped with a bioluminescent reporter system, living whole-cell bioreporters are

capable of sensing the presence of detrimental organic contaminants and internally
transforming that input cue into an output signal in the form of light for easy
detection. Compared to chemical analysis using costly instrumentations and
complicated protocols, bioluminescent bioreporter-based assays are inexpensive,
easy to perform, and capable of rapid and high-throughput detection. Bioreporters
are often criticized for their compromised specificity, but it is important to note
that they are not intended to replace analytical methods for the identification of the
exact composition of a contaminated sample. Unlike analytical approaches, which
can define structures and measure concentrations, whole-cell bioreporters are
designed to survey biological potentials such as the biodegradation and toxicity
potentials that are measured by catabolism-based bacterial reporters and
toxicology-based eukaryotic reporters, respectively. With the major concerns of
environmental monitoring being contamination evaluation and risk assessment, the
most suitable application of bioluminescent bioassays is for the rapid prescreening
of large numbers of samples to prioritize them for further in-depth examinations in
combination with other analyses (including analytical methods) to provide bio-
logically relevant data for comprehensive risk assessments. Realizing that
eukaryotic-based bioreporters provide more human- centric biologically relevant
information than the bacterial-based bioreporters, there is greater motivation
toward their application in establishing toxicokinetic profiles for improved
142 T. Xu et al.
surveillance and modeling of human/animal health impacts. This includes both the
lower eukaryotic yeast and mammalian cell lines that almost exclusively rely upon
firefly luciferase as a signaling element, although newer versions of ‘‘humanized’’
bacterial luciferase capable of being expressed under eukaryotic genetic controls
without the necessary addition of a light-activating substrate are becoming
available for higher throughput, more data intensive, realtime chemical toxicity
profiling [130]. As the inventory of bioluminescent bioreporters expands toward
more chemical targets with greater specificity, sensitivity, and human relevance, it
is clear that the bioreporter’s role as an environmental sentinel is here to stay.
References
1. Yeh BJ, Lim WA (2007) Synthetic biology: lessons from the history of synthetic organic
chemistry. Nat Chem Biol 3:521–525
2. Snyder R (2000) Overview of the toxicology of benzene. J Toxicol Env Health-Pt A
61:339–346
3. Dawson JJC, Iroegbu CO, Maciel H, Paton GI (2008) Application of luminescent biosensors
for monitoring the degradation and toxicity of BTEX compounds in soils. J Appl Microbiol
104:141–151
4. Girotti S, Bolelli L, Roda A, Gentilomi G, Musiani M (2002) Improved detection of toxic
chemicals using bioluminescent bacteria. Anal Chim Acta 471:113–120
5. Xu T, Close DM, Sayler GS, Ripp SA (2013) Genetically modified whole-cell bioreporters
for environmental assessment. Ecol Indic 28:125–141
6. Williams PA, Murray K (1974) Metabolism of benzoate and methylbenzoates by
Pseudomonas putida mt-2: evidence for existance of a TOL plasmid. J Bacteriol
120:416–423
7. Franklin FCH, Bagdasarian M, Bagdasarian MM, Timmis KN (1981) Molecular and
functional analysis of the TOL Plasmid pWWO from Pseudomonas putida and cloning of
genes for the enitre regulated aromatic ring meta-cleavage pathway. Proc Natl Acad Sci
USA 78:7458–7462
8. Worsey MJ, Franklin FCH, Williams PA (1978) Regulation of degradative pathway
enzymes coded for by TOL plasmid pWWO from Pseudomonas putida mt-2. J Bacteriol
134:757–764
9. Worsey MJ, Williams PA (1975) Metabolism of toluene and xylenes by Pseudomonas
putida mt-2: evidence for a new function of TOL plasmid. J Bacteriol 124:7–13
10. Ramos JL, Marques S, Timmis KN (1997) Transcriptional control of the Pseudomonas tol
plasmid catabolic operons is achieved through an interplay of host factors and plasmid-
encoded regulators. An Rev Microbiol 51:341–373
11. Li Y-F, Li F-Y, Ho C-L, Liao VH-C (2008) Construction and comparison of fluorescence
and bioluminescence bacterial biosensors for the detection of bioavailable toluene and
related compounds. Environ Pollut 152:123–129
fischeri, Vibrio logei, Vibrio salmonicida and Vibrio wodanis as Aliivibrio fischeri gen.
nov., comb. nov., Aliivibrio logei comb. nov., Aliivibrio salmonicida comb. nov and
Aliivibrio wodanis comb. nov. Int J Syst Evol Microbiol 57:2823–2829
13. Willardson BM, Wilkins JF, Rand TA, Schupp JM, Hill KK, Keim P, Jackson PJ (1998)
Development and testing of a bacterial biosensor for toluene-based environmental
contaminants. Appl Environ Microbiol 64:1006–1012
14. Tecon R, Beggah S, Czechowska K, Sentchilo V, Chronopoulou PM, McGenity TJ, van der
Meer JR (2010) Development of a multistrain bacterial bioreporter platform for the
monitoring of hydrocarbon contaminants in marine environments. Environ Sci Technol
44:1049–1055
15. Applegate BM, Kehrmeyer SR, Sayler GS (1998) A chromosomally based tod-luxCDABE
whole-cell reporter for benzene, toluene, ethybenzene, and xylene (BTEX) sensing. Appl
16. Zylstra G, McCombie W, Gibson D, Finette B (1988) Toluene degradation by Pseudomonas
putida F1: genetic organization of the tod operon. Appl Environ Microbiol 54:1498–1503
17. Wang Y, Rawlings M, Gibson DT, Labbe D, Bergeron H, Brousseau R, Lau PCK (1995)
Identification of a membrane protein and a truncated LysR type regulator associated with
the toluene degradation pathway in Pseudomonas putida F1. Mol Gen Genet 246:570–579
18. Kuncova G, Pazlarova J, Hlavata A, Ripp S, Sayler GS (2011) Bioluminescent bioreporter
Pseudomonas putida TVA8 as a detector of water pollution. Operational conditions and
selectivity of free cells sensor. Ecol Indic 11:882–887
19. Stiner L, Halverson LJ (2002) Development and characterization of a green fluorescent
protein-based bacterial biosensor for bioavailable toluene and related compounds. Appl
20. Bhattacharyya J, Read D, Amos S, Dooley S, Killham K, Paton GI (2005) Biosensor-based
diagnostics of contaminated groundwater: assessment and remediation strategy. Environ
Pollut 134:485–492
21. Eaton RW, Timmis KN (1986) Characterization of a plasmid-specified pathway for
catabolism of isopropylbenzne in Pseudomonas putida RE204. J Bacteriol 168:123–131
22. Selifonova OV, Eaton RW (1996) Use of an ipb-lux fusion to study regulation of the
isopropylbenzene catabolism operon of Pseudomonas putida RE204 and to detect
hydrophobic pollutants in the environment. Appl Environ Microbiol 62:778–783
23. Mumtaz MM, George JD, Gold KW, Cibulas W, Derosa CT (1996) ATSDR evaluation of
health effects of chemicals. 4. Polycyclic aromatic hydrocarbons (PAHs): understanding a
complex problem. Toxicol Ind Health 12:742–971
24. Grund AD, Gunsalus IC (1983) Cloning of genes for naphthalene metabolism in
Pseudomonas putida. J Bacteriol 156:89–94
25. Burlage RS, Sayler GS, Larimer F (1990) Monitoring of naphthalene catabolism by
bioluminescence with nah-lux transcriptional fusions. J Bacteriol 172:4749–4757
26. Dorn JG, Brusseau ML, Maier RM (2005) Real-time, in situ monitoring of bioactive zone
dynamics in heterogeneous systems. Environ Sci Technol 39:8898–8905
27. Dorn JG, Frye RJ, Maier RM (2003) Effect of temperature, pH, and initial cell number on
luxCDABE and nah gene expression during naphthalene and salicylate catabolism in the
bioreporter organism Pseudomonas putida RB1353. Appl Environ Microbiol 69:2209–2216
28. Dorn JG, Mahal MK, Brusseau ML, Maier RM (2004) Employing a novel fiber optic
detection system to monitor the dynamics of in situ lux bioreporter activity in porous media:
system performance update. Anal Chim Acta 525:63–74
29. King JMH, Digrazia PM, Applegate B, Burlage R, Sanseverino J, Dunbar P, Larimer F,
Sayler GS (1990) Rapid, sensitive bioluminescent reporter technology for naphthalene
exposure and biodegradation. Science 249:778–781
30. Trogl J, Chauhan A, Ripp S, Layton AC, Kuncova G, Sayler GS (2012) Pseudomonas
fluorescens HK44: lessons learned from a model whole-cell bioreporter with a broad
application history. Sensors 12:1544–1571
31. Valdman E, Gutz IGR (2008) Bioluminescent sensor for naphthalene in air: Cell
immobilization and evaluation with a dynamic standard atmosphere generator. Sens
Actuator B-Chem 133:656–663
32. Ripp S, Nivens DE, Ahn Y, Werner C, Jarrell J, Easter JP, Cox CD, Burlage RS, Sayler GS
(2000) Controlled field release of a bioluminescent genetically engineered microorganism
for bioremediation process monitoring and control. Environ Sci Technol 34:846–853
144 T. Xu et al.
33. Sayler GS, Ripp S (2000) Field applications of genetically engineered microorganisms for
bioremediation processes. Curr Opin Biotechnol 11:286–289
34. Laurie AD, Lloyd-Jones G (1999) The phn genes of Burkholderia sp. strain RP007
constitute a divergent gene cluster for polycyclic aromatic hydrocarbon catabolism.
J Bacteriol 181:531–540
35. Chakrabarty A, Chou G, Gunsalus I (1973) Genetic regulation of octane dissimilation
plasmid in Pseudomonas. Proc Natl Acad Sci USA 70:1137–1140
36. Eggink G, Engel H, Meijer W, Otten J, Kingma J, Witholt B (1988) Alkane utilization in
Pseudomonas oleovorans. Structure and function of the regulatory locus alkR. J Biol Chem
263:13400–13405
37. Sticher P, Jaspers MCM, Stemmler K, Harms H, Zehnder AJB, van der Meer JR (1997)
Development and characterization of a whole-cell bioluminescent sensor for bioavailable
middle-chain alkanes in contaminated groundwater samples. Appl Environ Microbiol
63:4053–4060
38. Owen DJ, Eggink G, Hauer B, Kok M, McBeth DL, Yang YL, Shapiro JA (1984) Physical
structure, genetic content and expression of the alkBAC operon. Mol Gen Genet
197:373–383
39. Minak-Bernero V, Bare RE, Haith CE, Grossman MJ (2004) Detection of alkanes, alcohols,
and aldehydes using bioluminescence. Biotechnol Bioeng 87:170–177
40. Bosetti A, van Beilen JB, Preusting H, Lageveen RG, Witholt B (1992) Production of
primary aliphatic alcohols with a recombinant Pseudomonas strain, encoding the alkane
hydroxylase enzyme system. Enzyme Microb Technol 14:702–708
41. Francisco W, Abu-Soud H, Baldwin T, Raushel F (1993) Interaction of aldehyde substrate
and inhibitors to bacterial luciferase. J Biol Chem 268:24734–24741
42. Atlas RM, Hazen TC (2011) Oil biodegradation and bioremediation: a tale of the two worst
spills in U.S. history. Environ Sci Technol 45:6709–6715
43. Zhang D, He Y, Wang Y, Wang H, Wu L, Aries E, Huang WE (2012) Whole-cell bacterial
bioreporter for actively searching and sensing of alkanes and oil spills. Microb Biotechnol
5:87–97
44. Zhang DY, Fakhrullin RF, Ozmen M, Wang H, Wang J, Paunov VN, Li GH, Huang WE
(2011) Functionalization of whole-cell bacterial reporters with magnetic nanoparticles.
Microb Biotechnol 4:89–97
45. Kumari R, Tecon R, Beggah S, Rutler R, Arey JS, van der Meer JR (2011) Development of
bioreporter assays for the detection of bioavailability of long-chain alkanes based on the
marine bacterium Alcanivorax borkumensis strain SK2. Environ Microbiol 13:2808–2819
46. van Hylckama Vlieg JET, Janssen DB (2001) Formation and detoxification of reactive
intermediates in the metabolism of chlorinated ethenes. J Biotechnol 85:81–102
47. Arcangeli J-P, Arvin E (1997) Modeling of the cometabolic biodegradation of
trichloroethylene by toluene-oxidizing bacteria in a biofilm system. Environ Sci Technol
31:3044–3052
48. Sponza DT (2003) Toxicity and treatability of carbontetrachloride and tetrachloroethylene
in anaerobic batch cultures. Int Biodeterior Biodegrad 51:119–127
49. Shingleton JT, Applegate BM, Nagel AC, Bienkowski PR, Sayler GS (1998) Induction of
the tod operon by trichloroethylene in Pseudomonas putida TVA8. Appl Environ Microbiol
64:5049–5052
50. Phoenix P, Keane A, Patel A, Bergeron H, Ghoshal S, Lau P (2003) Characterization of a
new solvent-responsive gene locus in Pseudomonas putida F1 and its functionalization as a
versatile biosensor. Environ Microbiol 5:1309–1327
51. Lopes N, Hawkins SA, Jegier P, Menn F-M, Sayler GS, Ripp S (2012) Detection of
dichloromethane with a bioluminescent (lux) bacterial bioreporter. J Ind Microbiol
Biotechnol 39:45–53
52. Furukawa K, Fujihara H (2008) Microbial degradation of polychlorinated biphenyls:
Biochemical and molecular features. J Biosci Bioeng 105:433–449
53. Layton AC, Muccini M, Ghosh MM, Sayler GS (1998) Construction of a bioluminescent
reporter strain to detect polychlorinated biphenyls. Appl Environ Microbiol 64:5023–5026
54. Bradley C, Berube PR (2008) Characterization of anionic surfactant-induced toxicity in a
primary effluent. J Environ Eng Sci 7:63–70
55. Layton AC, Gregory B, Schultz TW, Sayler GS (1999) Validation of genetically engineered
bioluminescent surfactant resistant bacteria as toxicity assessment tools. Ecotox Environ
Safe 43:222–228
56. Park SH, Lee K, Chae JC, Kim CK (2004) Construction of transformant reporters carrying
fused genes using pcbC promoter of Pseudomonas sp DJ-12 for detection of aromatic
pollutants. Environ Monit Assess 92:241–251
57. Jaspers MCM, Suske WA, Schmid A, Goslings DAM, Kohler HPE, van der Meer JR (2000)
HbpR, a new member of the XylR/DmpR subclass within the NtrC family of bacterial
transcriptional activators, regulates expression of 2-hydroxybiphenyl metabolism in
Pseudomonas azelaica HBP1. J Bacteriol 182:405–417
58. Turner K, Xu S, Pasini P, Deo S, Bachas L, Daunert S (2007) Hydroxylated polychlorinated
biphenyl detection based on a genetically engineered bioluminescent whole-cell sensing
system. Anal Chem 79:5740–5745
59. Tropel D, Bahler A, Globig K, van der Meer JR (2004) Design of new promoters and of a
dual-bioreporter based on cross-activation by the two regulatory proteins XylR and HbpR.
60. Krastanov A, Alexieva Z, Yemendzhiev H (2013) Microbial degradation of phenol and
phenolic derivatives. Eng Life Sci 13:76–87
61. Shingler V, Franklin FCH, Tsuda M, Holroyd D, Bagdasarian M (1989) Molecular analysis
of a plasmid-encoded phenol hydroxylase from Pseudomonas CF600. J Gen Microbiol
135:1083–1092
62. Shingler V, Bartilson M, Moore T (1993) Cloning and nucleotide-sequencing of the gene
encoding the positive regulator (DmpR) of the phenol catabolic pathway encoded by
PVI150 and identification of DmpR as a member of the NtrC family of transcriptional
activators. J Bacteriol 175:1596–1604
63. Leedjarv A, Ivask A, Virta M, Kahru A (2006) Analysis of bioavailable phenols from
natural samples by recombinant luminescent bacterial sensors. Chemosphere 64:1910–1919
64. Wise AA, Kuske CR (2000) Generation of novel bacterial regulatory proteins that detect
priority pollutant phenols. Appl Environ Microbiol 66:163–169
65. Gupta S, Saxena M, Saini N, Mahmooduzzafar, Kumar R, Kumar A (2012) An effective
strategy for a whole-cell biosensor based on putative effector interaction site of the
regulatory DmpR protein. PLoS ONE 7: e43527
66. Ehrt S, Schirmer F, Hillen W (1995) Genetic organization, nucleotide sequence and
regulation of expression of genes encoding phenol hydroxylase and catechol 1,2-
dioxygenase in Acinetobacter calcoaceticus NCIB8250. Mol Microbiol 18:13–20
67. Abd-El-Haleem D, Ripp S, Scott C, Sayler GS (2002) A luxCDABE-based bioluminescent
bioreporter for the detection of phenol. J Ind Microbiol Biotechnol 29:233–237
68. Zaki S, Abd-El-Haleem D, Abulhamd A, Elbery H, AbuElreesh G (2008) Influence of
phenolics on the sensitivity of free and immobilized bioluminescent Acinetobacter
bacterium. Microbiol Res 163:277–285
69. Wiles S, Whiteley AS, Philp JC, Bailey MJ (2003) Development of bespoke bioluminescent
reporters with the potential for in situ deployment within a phenolic-remediating wastewater
treatment system. J Microbiol Methods 55:667–677
70. Ghosh SK, Doctor PB (1992) Toxicity screening of phenol using Microtox. Environ Toxicol
Water Quality 7:157–163
71. Ismailov AD, Pogosyan SI, Mitrofanova TI, Egorov NS, Netrusov AI (2000) Bacterial
bioluminescence inhibition by chlorophenols. Appl Biochem Microbiol 36:404–408
72. Kudryasheva N, Vetrova E, Kuznetsov A, Kratasyuk V, Stom D (2002) Bioluminescence
assays: Effects of quinones and phenols. Ecotox Environ Safe 53:221–225
146 T. Xu et al.
73. Berglind R, Leffler P, Sjostrom M (2010) Interactions between pH, potassium, calcium,
bromide, and phenol and their effects on the bioluminescence of Vibrio fischeri. J Toxicol
Env Health-Pt A 73:1102–1112
74. Diamanti-Kandarakis E, Bourguignon JP, Giudice LC, Hauser R, Prins GS, Soto AM,
Zoeller RT, Gore AC (2009) Endocrine-disrupting chemicals: an endocrine society
scientific statement. Endocr Rev 30:293–342
75. Alcock RE, Behnisch PA, Jones KC, Hagenmaier H (1998) Dioxin-like PCBs in the
environment—human exposure and the significance of sources. Chemosphere
37:1457–1472
76. Soto AM, Sonnenschein C, Chung KL, Fernandez MF, Olea N, Serrano FO (1995) The E-
SCREEN assay as a tool to identify estrogens—an update on estrogenic environmental
pollutants. Environ Health Perspect 103:113–122
77. Van den Berg M, Birnbaum L, Bosveld A, Brunström B, Cook P, Feeley M, Giesy JP,
Hanberg A, Hasegawa R, Kennedy SW (1998) Toxic equivalency factors (TEFs) for PCBs,
PCDDs, PCDFs for humans and wildlife. Environ Health Perspect 106:775–792
78. Ahlborg UG, Brouwer A, Fingerhut MA, Jacobson JL, Jacobson SW, Kennedy SW, Kettrup
AA, Koeman JH, Poiger H, Rappe C (1992) Impact of polychlorinated dibenzo-p-dioxins,
dibenzofurans, and biphenyls on human and environmental health, with special emphasis on
application of the toxic equivalency factor concept. Environ Toxicol Pharmacol
228:179–199
79. Peterson RE, Theobald HM, Kimmel GL (1993) Developmental and reproductive toxicity
of dioxins and related compounds: cross-species comparisons. Crit Rev Toxicol 23:283–335
80. Garrison P, Tullis K, Aarts J, Brouwer A, Giesy J, Denison M (1996) Species-specific
recombinant cell lines as bioassay systems for the detection of 2,3,7,8-tetrachlorodibenzo-p-
dioxin-like chemicals. Toxicol Sci 30:194–203
81. Safe SH (1995) Modulation of gene expression and endocrine response pathways by 2,3,7,8-
tetrachlorodibenzo-p-dioxin and related compounds. Pharmacol Ther 67:247–281
82. Postlind H, Vu T, Tukey R, Quattrochi LC (1993) Response of human CYP1-luciferase
plasmids to 2,3,7,8-tetrachlorodibenzo-p-dioxin and polycyclic aromatic hydrocarbons.
Toxicol Appl Pharmacol 118:255–262
83. Murk AJ, Legler J, Denison MS, Giesy JP, vandeGuchte C, Brouwer A (1996) Chemical-
activated luciferase gene expression (CALUX): a novel in vitro bioassay for Ah receptor
active compounds in sediments and pore water. Fundam Appl Toxicol 33:149–160
84. Murk AJ, Leonards PEG, Bulder AS, Jonas AS, Rozemeijer MJC, Denison MS, Koeman
JH, Brouwer A (1997) The CALUX (chemical-activated luciferase expression) assay
adapted and validated for measuring TCDD equivalents in blood plasma. Environ Toxicol
Chem 16:1583–1589
85. Bovee TFH, Hoogenboom LAP, Hamers ARM, Traag WA, Zuidema T, Aarts J, Brouwer A,
Kuiper HA (1998) Validation and use of the CALUX-bioassay for the determination of
dioxins and PCBs in bovine milk. Food Addit Contam 15:863–875
86. Cederberg T, Laier P, Vinggaard AM (2002) Screening of food samples for dioxin levels:
comparison of GC-MS determination with the CALUX bioassay. Organohalogen Compd
58:409–412
87. Tsutsumi T, Amakura Y, Nakamura M, Brown DJ, Clark GC, Sasaki K, Toyoda M, Maitani
T (2003) Validation of the CALUX bioassay for the screening of PCDD/Fs and dioxin-like
PCBs in retail fish. Analyst 128:486–492
88. Van Overmeire I, Carbonnelle S, Van Loco J, Roos P, Brown D, Chu M, Clark G, Goeyens
L (2002) Validation of the CALUX bioassay: quantitative screening approach.
Organohalogen Compd 58:353–356
89. Pauwels A, Cenijn PH, Schepens P, Brouwer A (2000) Comparison of chemical-activated
luciferase gene expression bioassay and gas chromatography for PCB determination in
human serum and follicular fluid. Environ Health Perspect 108:553–557
90. Van Wouwe N, Windal I, Vanderperren H, Eppe G, Xhrouet C, Massart A-C, Debacker N,
Sasse A, Baeyens W, De Pauw E (2004) Validation of the CALUX bioassay for PCDD/F
analyses in human blood plasma and comparison with GC-HRMS. Talanta 63:1157–1167
91. Leskinen P, Hilscherova K, Sidlova T, Kiviranta H, Pessala P, Salo S, Verta M, Virta M
(2008) Detecting AhR ligands in sediments using bioluminescent reporter yeast. Biosens
Bioelectron 23:1850–1855
92. Windal I, Denison MS, Birnbaum LS, Van Wouwe N, Baeyens W, Goeyens L (2005)
Chemically activated luciferase gene expression (CALUX) cell bioassay analysis for the
estimation of dioxin-like activity: critical parameters of the CALUX procedure that impact
assay results. Environ Sci Technol 39:7357–7364
93. Besselink HT, Schipper C, Klamer H, Leonards P, Verhaar H, Felzel E, Murk AJ, Thain J,
Hosoe K, Schoeters G, Legler J, Brouwer B (2004) Intra- and interlaboratory calibration of
Ò
the DR CALUX bioassay for the analysis of dioxins and dioxin-like chemicals in
sediments. Environ Toxicol Chem 23:2781–2789
94. Colborn T, vom Saal FS, Soto AM (1993) Developmental effects of endocrine-disrupting
chemicals in wildlife and humans. Environ Health Perspect 101:378–384
95. Kavlock RJ, Daston GP, DeRosa C, FennerCrisp P, Gray LE, Kaattari S, Lucier G, Luster
M, Mac MJ, Maczka C, Miller R, Moore J, Rolland R, Scott G, Sheehan DM, Sinks T,
Tilson HA (1996) Research needs for the risk assessment of health and environmental
effects of endocrine disruptors: a report of the US EPA-sponsored workshop. Environ
Health Perspect 104:715–740
96. Eltzov E, Kushmaro A, Marks RS (2009) Biosensors for endocrine disruptors. In: Shaw I
(eds) Endocrine-disrupting chemicals in food. Woodhead Publishing in Food Science
Technology and Nutrition, pp 183–208
97. Svobodova K, Cajthaml T (2010) New in vitro reporter gene bioassays for screening of
hormonal active compounds in the environment. Appl Microbiol Biotechnol 88:839–847
98. Pons M, Gagne D, Nicolas JC, Mehtali M (1990) A new cellular model of response to
estrogens: a bioluminescent test to characterize (anti)estrogen molecules. Biotechniques
9:450
99. Demirpence E, Duchesne MJ, Badia E, Gagne D, Pons M (1993) MVLN cells—a
bioluminescent MCF-7-derived cell line to study the modulation of estrogenic activity.
J Steroid Biochem Mol Biol 46:355–364
100. Shue MF, Chen FA, Chen TC (2010) Total estrogenic activity and nonylphenol
concentration in the Donggang River. Taiwan. Environ Monit Assess 168:91–101
101. Wang C, Wang T, Liu W, Ruan T, Zhou QF, Liu JY, Zhang AQ, Zhao B, Jiang GB (2012)
The in vitro estrogenic activities of polyfluorinated iodine alkanes. Environ Health Perspect
120:119–125
102. Balaguer P, Francois F, Comunale F, Fenet H, Boussioux AM, Pons M, Nicolas JC, Casellas
C (1999) Reporter cell lines to study the estrogenic effects of xenoestrogens. Sci Total
Environ 233:47–56
103. Witters H, Freyberger A, Smits K, Vangenechten C, Lofink W, Weimer M, Bremer S, Ahr
PHJ, Berckmans P (2010) The assessment of estrogenic or anti-estrogenic activity of
chemicals by the human stably transfected estrogen sensitive MELN cell line: results of test
performance and transferability. Reprod Toxicol 30:60–72
104. Legler J, van den Brink CE, Brouwer A, Murk AJ, van der Saag PT, Vethaak AD, van der
Burg B (1999) Development of a stably transfected estrogen receptor-mediated luciferase
reporter gene assay in the human T47D breast cancer cell line. Toxicol Sci 48:55–66
105. Wilson VS, Bobseine K, Gray LE (2004) Development and characterization of a cell line
that stably expresses an estrogen-responsive luciferase reporter for the detection of estrogen
receptor agonist and antagonists. Toxicol Sci 81:69–77
106. Legler J, Zeinstra LM, Schuitemaker F, Lanser PH, Bogerd J, Brouwer A, Vethaak AD, De
Voogt P, Murk AJ, Van der Burg B (2002) Comparison of in vivo and in vitro reporter gene
assays for short-term screening of estrogenic activity. Environ Sci Technol 36:4410–4415
148 T. Xu et al.
107. Blankvoort BMG, de Groene EM, van Meeteren-Kreikamp AP, Witkamp RF, Rodenburg
RJT, Aarts J (2001) Development of an androgen reporter gene assay (AR-LUX) utilizing a
human cell line with an endogenously regulated androgen receptor. Anal Biochem
298:93–102
108. Wilson VS, Bobseine K, Lambright CR, Gray LE (2002) A novel cell line, MDA-kb2, that
stably expresses an androgen- and glucocorticoid-responsive reporter for the detection of
hormone receptor agonists and antagonists. Toxicol Sci 66:69–81
109. Hall RE, Tilley WD, McPhaul MJ, Sutherland RL (1992) Regulation of androgen receptor-
gene expression by steroids and retinoic acid in human breast-cancer cells. Int J Cancer
52:778–784
110. Vladusic EA, Hornby AE, Guerra-Vladusic FK, Lakins J, Lupu R (2000) Expression and
regulation of estrogen receptor beta in human breast tumors and cell lines. Oncol Rep
7:157–167
111. Aranda A, Pascual A (2001) Nuclear hormone receptors and gene expression. Physiol Rev
81:1269–1304
112. Quaedackers ME, Van den Brink CE, Wissink S, Schreurs R, Gustafsson JA, Van der Saag
PT, Van der Burg B (2001) 4-hydroxytamoxifen trans-represses nuclear factor-kappa B
activity in human osteoblastic U2-OS cells through estrogen receptor (ER)alpha, and not
through ER beta. Endocrinology 142:1156–1166
113. Sonneveld E, Jansen HJ, Riteco JAC, Brouwer A, van der Burg B (2005) Development of
androgen- and estrogen-responsive bioassays, members of a panel of human cell line-based
highly selective steroid-responsive bioassays. Toxicol Sci 83:136–148
114. van der Burg B, Schreurs R, van der Linden S, Seinen W, Brouwer A, Sonneveld E (2008)
Endocrine effects of polycyclic musks: do we smell a rat? Int J Androl 31:188–193
115. Sonneveld E, Riteco JAC, Jansen HJ, Pieterse B, Brouwer A, Schoonen WG, van der Burg
B (2006) Comparison of in vitro and in vivo screening models for androgenic and estrogenic
activities. Toxicol Sci 89:173–187
116. Leskinen P, Michelini E, Picard D, Karp M, Virta M (2005) Bioluminescent yeast assays for
detecting estrogenic and androgenic activity in different matrices. Chemosphere 61:259–266
117. Michelini E, Leskinen P, Virta M, Karp M, Roda A (2005) A new recombinant cell-based
bioluminescent assay for sensitive androgen-like compound detection. Biosens Bioelectron
20:2261–2267
118. Sanseverino J, Gupta RK, Layton AC, Patterson SS, Ripp SA, Saidak L, Simpson ML,
Schultz TW, Sayler GS (2005) Use of Saccharomyces cerevisiae BLYES expressing
bacterial bioluminescence for rapid, sensitive detection of estrogenic compounds. Appl
119. Eldridge ML, Sanseverino J, Layton AC, Easter JP, Schultz TW, Sayler GS (2007)
Saccharomyces cerevisiae BLYAS, a new bioluminescent bioreporter for detection of
androgenic compounds. Appl Environ Microbiol 73:6012–6018
120. Sanseverino J, Eldridge ML, Layton AC, Easter JP, Yarbrough J, Schultz TW, Sayler GS
(2009) Screening of potentially hormonally active chemicals using bioluminescent yeast
bioreporters. Toxicol Sci 107:122–134
121. Vandenberg LN, Maffini MV, Sonnenschein C, Rubin BS, Soto AM (2009) Bisphenol-A
and the great divide: A review of controversies in the field of endocrine disruption. Endocr
Rev 30:75–95
122. Bonefeld-Jorgensen EC, Long MH, Hofmeister MV, Vinggaard AM (2007) Endocrine-
disrupting potential of bisphenol A, bisphenol A dimethacrylate, 4-n-nonylphenol, and
4-n-octylphenol in vitro: new data and a brief review. Environ Health Perspect 115:69–76
123. Mankidy R, Wiseman S, Ma H, Giesy JP (2013) Biological impact of phthalates. Toxicol
Lett 217:50–58
124. Preuss TG, Gurer-Orhan H, Meerman J, Ratte HT (2010) Some nonylphenol isomers show
antiestrogenic potency in the MVLN cell assay. Toxicol in Vitro 24:129–134
125. Schiliro T, Porfido A, Spina F, Varese GC, Gilli G (2012) Oestrogenic activity of a textile
industrial wastewater treatment plant effluent evaluated by the E-screen test and MELN
gene-reporter luciferase assay. Sci Total Environ 432:389–395
126. He YH, Wiseman SB, Hecker M, Zhang XW, Wang N, Perez LA, Jones PD, El-Din MG,
Martin JW, Giesy JP (2011) Effect of ozonation on the estrogenicity and androgenicity of
oil sands process-affected water. Environ Sci Technol 45:6268–6274
127. Pereira RO, Postigo C, de Alda ML, Daniel LA, Barcelo D (2011) Removal of estrogens
through water disinfection processes and formation of by-products. Chemosphere
82:789–799
128. Kortenkamp A (2007) Ten years of mixing cocktails: a review of combination effects of
endocrine-disrupting chemicals. Environ Health Perspect 115:98–105
129. Fenet H, Gomez E, Pillon A, Rosain D, Nicolas JC, Casellas C, Balaguer P (2003)
Estrogenic activity in water and sediments of a French river: contribution of alkylphenols.
Arch Environ Contam Toxicol 44:1–6
130. Close DM, Patterson SS, Ripp SA, Baek SJ, Sanseverino J, Sayler GS (2010) Autonomous
bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian
cell line. PLoS ONE 5:e12441
131. Bundy JG, Campbell CD, Paton GI (2001) Comparison of response of six different
luminescent bacterial bioassays to bioremediation of five contrasting oils. J Environ Monit
3:404–410
132. Diplock EE, Mardlin DP, Killham KS, Paton GI (2009) Predicting bioremediation of
hydrocarbons: Laboratory to field scale. Environ Pollut 157:1831–1840
133. Werlen C, Jaspers MCM, van der Meer JR (2004) Measurement of biologically available
naphthalene in gas and aqueous phases by use of a Pseudomonas putida biosensor. Appl
134. Paton GI, Reid BJ, Sempled KT (2009) Application of a luminescence-based biosensor for
assessing naphthalene biodegradation in soils from a manufactured gas plant. Environ Pollut
157:1643–1648
135. Kapanen A, Vikman M, Rajasärkkä J, Virta M, Itävaara M (2013) Biotests for
environmental quality assessment of composted sewage sludge. Waste Manag
33:1451–1460
136. Sakai S, Takigami H (2003) Integrated biomonitoring of dioxin-like compounds for waste
management and environment. Ind Health 41:205–214
137. Hilscherova K, Dusek L, Sidlova T, Jalova V, Cupr P, Giesy JP, Nehyba S, Jarkovsky J,
Klanova J, Holoubek I (2010) Seasonally and regionally determined indication potential of
bioassays in contaminated river sediments. Environ Toxicol Chem 29:522–534
138. Kanematsu M, Hayashi A, Denison MS, Young TM (2009) Characterization and potential
environmental risks of leachate from shredded rubber mulches. Chemosphere 76:952–958
139. Richter CA, Tieber VL, Denison MS, Giesy JP (1997) An in vitro rainbow trout cell
bioassay for aryl hydrocarbon receptor-mediated toxins. Environ Toxicol Chem 16:543–550
140. Hahn ME (2002) Biomarkers and bioassays for detecting dioxin-like compounds in the
marine environment. Sci Total Environ 289:49–69
141. Yang J-H, Lee H-G, Park K-Y (2008) Development of human dermal epithelial cell-based
bioassay for the dioxins. Chemosphere 72:1188–1192
142. He G, Tsutsumi T, Zhao B, Baston DS, Zhao J, Heath-Pagliuso S, Denison MS (2011)
Third-generation Ah receptor–responsive luciferase reporter plasmids: Amplification of
dioxin-responsive elements dramatically increases CALUX bioassay sensitivity and
responsiveness. Toxicol Sci 123:511–522
143. Bergamasco AMD, Eldridge M, Sanseverino J, Sodre FF, Montagner CC, Pescara IC,
Jardim WF, Umbuzeiro GD (2011) Bioluminescent yeast estrogen assay (BLYES) as a
sensitive tool to monitor surface and drinking water for estrogenicity. J Environ Monit
13:3288–3293
150 T. Xu et al.
144. Jardim WF, Montagner CC, Pescara IC, Umbuzeiro GA, Bergamasco AMD, Eldridge ML,
Sodre FF (2012) An integrated approach to evaluate emerging contaminants in drinking
water. Sep Purif Technol 84:3–8
145. Salste L, Leskinen P, Virta M, Kronberg L (2007) Determination of estrogens and
estrogenic activity in wastewater effluent by chemical analysis and the bioluminescent yeast
assay. Sci Total Environ 378:343–351
146. Furuichi T, Kannan K, Suzuki K, Tanaka S, Giesy JP, Masunaga S (2006) Occurrence of
estrogenic compounds in and removal by a swine farm waste treatment plant. Environ Sci
Technol 40:7896–7902
147. Mahjoub O, Escande A, Rosain D, Casellas C, Gomez E, Fenet H (2011) Estrogen-like and
dioxin-like organic contaminants in reclaimed wastewater: transfer to irrigated soil and
groundwater. Water Sci Technol 63:1657–1662
148. David A, Gomez E, Ait-Aissa S, Rosain D, Casellas C, Fenet H (2010) Impact of urban
wastewater discharges on the sediments of a small Mediterranean river and associated
coastal environment: Assessment of estrogenic and dioxin-like activities. Arch Environ
Contam Toxicol 58:562–575
149. Mnif W, Zidi I, Hassine AIH, Gomez E, Bartegi A, Roig B, Balaguer P (2012) Monitoring
endocrine disrupter compounds in the Tunisian Hamdoun River using in vitro bioassays.
Soil Sediment Contam 21:815–830
150. Maletz S, Floehr T, Beier S, Klumper C, Brouwer A, Behnisch P, Higley E, Giesy JP,
Hecker M, Gebhardt W, Linnemann V, Pinnekamp J, Hollert H (2013) In vitro
characterization of the effectiveness of enhanced sewage treatment processes to eliminate
endocrine activity of hospital effluents. Water Res 47:1545–1557
151. Vethaak AD, Lahr J, Schrap SM, Belfroid AC, Rijs GBJ, Gerritsen A, de Boer J, Bulder AS,
Grinwis GCM, Kuiper RV, Legler J, Murk TAJ, Peijnenburg W, Verhaar HJM, de Voogt P
(2005) An integrated assessment of estrogenic contamination and biological effects in the
aquatic environment of The Netherlands. Chemosphere 59:511–524
152. Houtman CJ, Booij P, van der Valk KM, van Bodegom PM, van den Ende F, Gerritsen
AAM, Lamoree MH, Legler J, Brouwer A (2007) Biomonitoring of estrogenic exposure and
identification of responsible compounds in bream from Dutch surface waters. Environ
Toxicol Chem 26:898–907
153. Leusch FDL, De Jager C, Levi Y, Lim R, Puijker L, Sacher F, Tremblay LA, Wilson VS,
Chapman HF (2010) Comparison of five in vitro bioassays to measure estrogenic activity in
environmental waters. Environ Sci Technol 44:3853–3860
154. Wehmas LC, Cavallin JE, Durhan EJ, Kahl MD, Martinovic D, Mayasich J, Tuominen T,
Villeneuve DL, Ankley GT (2011) Screening complex effluents for estrogenic activity with
the T47D-KBluc cell bioassay: assay optimization and comparison with in vivo responses in
fish. Environ Toxicol Chem 30:439–445
155. Maggioni S, Balaguer P, Chiozzotto C, Benfenati E (2013) Screening of endocrine-
disrupting phenols, herbicides, steroid estrogens, and estrogenicity in drinking water from
the waterworks of 35 Italian cities and from PET-bottled mineral water. Environ Sci Pollut
Res 20:1649–1660
156. Suzuki G, Tue NM, Malarvannan G, Sudaryanto A, Takahashi S, Tanabe S, Sakai S,
Brouwer A, Uramaru N, Kitamura S, Taldgami H (2013) Similarities in the endocrine-
disrupting potencies of indoor dust and flame retardants by using human osteosarcoma
(U2OS) cell-based reporter gene assays. Environ Sci Technol 47:2898–2908
157. Van der Linden SC, Heringa MB, Man HY, Sonneveld E, Puijker LM, Brouwer A, Van der
Burg B (2008) Detection of multiple hormonal activities in wastewater effluents and surface
water, using a panel of steroid receptor CALUX bioassays. Environ Sci Technol
42:5814–5820
158. Blankvoort BMG, Rodenburg RJT, Murk AJ, Koeman JH, Schilt R, Aarts J (2005)
Androgenic activity in surface water samples detected using the AR-LUX assay: indications
for mixture effects. Environ Toxicol Pharmacol 19:263–272
159. Bellet V, Hernandez-Raquet G, Dagnino S, Seree L, Pardon P, Bancon-Montiny C, Fenet H,

Creusot N, Ait-Aissa S, Cavailles V, Budzinski H, Antignac JP, Balaguer P (2012)
Occurrence of androgens in sewage treatment plants influents is associated with antagonist
activities on other steroid receptors. Water Res 46:1912–1922
160. Schriks M, van Leerdam JA, van der Linden SC, van der Burg B, van Wezel AP, de Voogt P
(2010) High-resolution mass spectrometric identification and quantification of
glucocorticoid compounds in various wastewaters in The Netherlands. Environ Sci
Technol 44:4766–4774
161. Jugan ML, Levy-Bimbot M, Pomerance M, Tamisier-Karolak S, Blondeau JP, Levi Y
(2007) A new bioluminescent cellular assay to measure the transcriptional effects of
chemicals that modulate the alpha-1 thyroid hormone receptor. Toxicol in Vitro
21:1197–1205
162. Jugan ML, Oziol L, Bimbot M, Huteau V, Tamisier-Karolak S, Blondeau JP, Levi Y (2009)
In vitro assessment of thyroid and estrogenic endocrine disruptors in wastewater treatment
plants, rivers and drinking water supplies in the greater Paris area (France). Sci Total
Environ 407:3579–3587
Part III
Applications of Bioluminescence
in Agriculture and Bioprocess
Detection of Bacteria with Bioluminescent
Reporter Bacteriophage
Jochen Klumpp and Martin J. Loessner
Abstract Bacteriophages are viruses that exclusively infect bacteria. They are
ideally suited for the development of highly specific diagnostic assay systems.
Bioluminescent reporter bacteriophages are designed and constructed by integra-
tion of a luciferase gene in the virus genome. Relying on the host specificity of the
phage, the system enables rapid, sensitive, and specific detection of bacterial
pathogens. A bioluminescent reporter phage assay is superior to any other
molecular detection method, because gene expression and light emission are
dependent on an active metabolism of the bacterial cell, and only viable cells will
yield a signal. In this chapter we introduce the concept of creating reporter phages,
discuss their advantages and disadvantages, and illustrate the advances made in
developing such systems for different Gram-negative and Gram-positive patho-
gens. The application of bioluminescent reporter phages for the detection of
foodborne pathogens is emphasized.
Keywords Reporter bacteriophage Luciferase Pathogen detection Food-

borne pathogens
Abbreviations
d Day(s)
g Gram(s)
h Hour(s)
L Liter(s)
min Minute(s)
ml Milliliter(s)
mol Mole(s)
s Second(s)
J. Klumpp M. J. Loessner (&)

Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstrasse 7,
8092 Zurich, Switzerland
e-mail: martin.loessner@ethz.ch

156 J. Klumpp and M. J. Loessner
Contents
1 Introduction........................................................................................................................ 156
2 Bioluminescence as Reporter System............................................................................... 158
3 Principles of Reporter Bacteriophage Construction and Use .......................................... 159
4 Specific Bioluminescent Reporter Phages........................................................................ 162
4.1 Antibiotic Resistance ................................................................................................ 166
4.2 Combination of Bioluminescent Reporter Phages with Other Technologies......... 166
5 Summary, Conclusions, and Outlook ............................................................................... 167
References................................................................................................................................ 167
1 Introduction
Bacteriophages are viruses that solely infect bacteria and rely on the host
metabolism for reproduction, making them perfect intracellular parasites. They
represent the most abundant biological entities on earth and are a major driving
force for bacterial evolution [1, 2]. Most bacteriophages belong to the order
Caudovirales, the tailed bacteriophages, with the three families Siphoviridae,
Myoviridae, and Podoviridae. Bacteriophages propagate themselves by adsorption
to a host bacteria cell, penetration of the cell wall, and subsequent injection of the
phage DNA or RNA. The phage can follow a temperate lifestyle, which is char-
acterized by the integration of the phage genome into the host chromosome and
coreplication with the bacterial chromosome. In contrast, a lytic lifestyle results in
reprogramming of the host cell by the invading DNA, resulting in production of
new virus progeny and subsequence cell lysis. Temperate phages (in the integrated
state called ‘‘prophage’’) can excise their genome from the host chromosome under
adverse physiological conditions and enter the lytic cycle, thereby eventually
killing the host. In both cases, phage DNA is transduced into the host cell during
the initial infection, a basic step and prerequisite for development of any reporter
bacteriophage.
Bacteriophages have been widely used for therapeutic purposes and for diag-
nostics of pathogenic bacteria for many years. Phage therapy has been one of the
pillars of eastern Europe and Soviet Union healthcare systems but the concept was
completely abandoned in the Western world due to the invention of antibiotics [3,
4]. The current crisis caused by antibiotic-resistant bacteria prompted a reconsid-
eration of bacteriophage therapy, and sparked a new area of research using these
viruses. In addition to application in medical settings, bacteriophages are becoming
increasingly popular for biocontrol of pathogenic bacteria in food. Multiple studies
demonstrate the feasibility of the approach in controlling Salmonella, Campylo-
bacter, Listeria, and many other pathogens in a variety of foods, as well as in
livestock [5–9]. Also, enzymatic components of the phage, such as endolysins, can
be used to control infections and remove unwanted pathogens [10, 11].
Detection of Bacteria with Bioluminescent Reporter Bacteriophage 157
Bacteriophages or their components also exhibit great potential for develop-

ment of diagnostic assays targeted at bacterial pathogens. One especially
intriguing method of using phages to detect bacteria are so-called ‘‘reporter pha-
ges.’’ Here, the phage is genetically modified to carry and transduce a reporter
gene, which can be a fluorescent or luminescent marker or any other detectable
gene product, which is expressed upon infection of the bacterial host. Such
reporter phages have been developed on the basis of firefly luciferase [12], bac-
terial luciferase [13], beta-galactosidase [14, 15], green fluorescent protein [16,
17], and ice nucleation protein [18]. Monitoring the product or substrate converted
by the action of the reporter enzyme enables realtime detection of low numbers of
viable bacteria.
Recent large outbreaks of foodborne pathogens, such as the EHEC contamina-
tion of sprouts in Germany in 2011 or the occurrence of Listeria monocytogenes on
cantaloupe melons in the United States in 2012, prompted a quest for simple,
specific, and sensitive detection methods for the food industry. Current detection
methods for bacterial pathogens from foods still mostly rely on the conventional
enrichment and plating methods, which suffer from a number of drawbacks, such as
long incubation times (24–48 h, depending on the type of enrichment needed),
error-prone procedures, costly reagents, and low specificity. A number of molecular
methods for pathogen detection have been developed in recent years, such as PCR
or other nucleic acid amplification-based assays, molecular probes, or various
immunoassays. Although massively reducing the time-to-result and (in some cases)
the detection limit, some assays lack specificity, and cross-reactivity in immuno-
assays may be problematic. Others, such as PCR-based assays or DNA hybrid-
ization technologies offer quick and highly specific results, but are unable to
distinguish between live and dead cells and often require cost-intensive equipment
and expert staff. Moreover, these procedures are often hampered by the complexity
of the sample matrix and uneven distribution of the pathogen in the test sample [19].
Reporter bacteriophages offer a sensitive and efficient alternative, featuring a
number of advantages: they are highly specific, robust, and easy to handle, and
detect only live cells. Bioluminescent reporter bacteriophages especially require
very little processing time until sample testing. Although a suitable substrate and
instrument for signal recording is required, the assay is generally inexpensive, and
trained staff is not needed to perform the analysis. Furthermore, most foods are
unlikely to feature a background activity that would mask or yield false-positive
signals in bioluminescence measurements. Last, most bacteriophages can be
manufactured with ease in large quantities, rendering the production of the
detection agent very cost-efficient. Attempts to replace the luminometer machine
by inexpensive Polaroid films or off-the-shelf digital cameras showed great
potential in further simplifying the assay procedures [20, 21].
In this chapter, we provide a summary of the current knowledge on biolumi-
nescent (and other) reporter bacteriophages, and their application for diagnostics
of bacteria from food and other environments.
2 Bioluminescence as Reporter System
Bioluminescence is mostly mediated by the luciferase enzyme, and the genes

encoding the components of this system can be found in different organisms. Most
widely used are the luxA and luxB genes from Vibrio fischeri and Vibrio harveyi
(Lux), as well as the firefly luciferase (Luc) from Photinus pyralis. Both catalyze a
different light-emitting reaction (see below). The luxA and luxB genes encode the
two subunits for the heterodimeric bacterial luciferase enzyme. In some organisms,
the enzyme works best if expressed from a fused luxAB gene, instead of individual
genes [22–24]. The other components encoded on the lux operon (luxCDE) are
necessary for the conversion of medium-chain fatty acid to the aldehyde substrate
to be oxidized by the luciferase. Because the substrate can easily be supplied
exogenously for reporter phage assays, the luxCDE genes are dispensable.
Attempts have been made to clone the full luxABCDEI operon into a phage,
although with mixed results [25, 26]. The luxI and luxR genes encode a quorum-
sensing system, in which LuxI generates an acyl-homoserine-lacton autoinducer
that interacts with the LuxR protein and amplifies the light-emitting reaction [27].
The bacterial luciferase enzyme catalyzes the oxidation of flavin mononucle-
otide (FMNH2) and a long-chain aldehyde to water, FMH, and a carboxylic acid,
thereby emitting light.
FMNH2 þ O2 þ R COH ! FMN þ H2 O þ R COOH þ Light ð490 nmÞ
Equation 1. Light-emitting reaction catalyzed by the bacterial luciferase

In contrast, the insect (firefly) luciferase catalyzes a two-step reaction with an
unstable intermediate based on the substrate luciferin, a carboxylic acid present in
many firefly species.
1: Luciferin þ ATP ! Luciferyl adenylate þ Pyrophosphate

2: Lyciferyl adenlyate þ O2 ! AMP þ Oxyluciferin þ Light ð560 nmÞ
Equation 2. Light-emitting reaction catalyzed by the firefly luciferase

The luciferase is engineered to be encoded by the reporter bacteriophage under
control of a promoter, which should be highly activated upon infection of the
bacterial host cell. ATP and FMNH2 are provided by the metabolism of the host
cells. The substrates (aldehyde or Luciferin) have to be supplied exogenously. A
significant difference exists between the accessibility of the substrates: whereas the
aldehyde used by the bacterial luciferase readily diffuses through bacterial cell
walls and membranes, the Luciferin does not. As a consequence, target bacterial
cells have to be lysed either by letting the phage complete its lifecycle or by
external action for the insect luciferase to gain access to its substrate. Both
enzymes are reasonably active at 30 °C, but exhibit temperature-dependence.
3 Principles of Reporter Bacteriophage Construction

and Use
The construction of the bioluminescent reporter bacteriophage follows one unique

principle: the reporter genes are integrated in the phage genome, but due to a lack
of an own metabolism, the phage particles of course remain ‘‘dark.’’ Only when
the phage infects a suitable host and the DNA transduced into the bacterium, gene
expression can occur, which results in active reporter protein and light-emitting
bacteria.
One intrinsic property of the phage is essential for the development of biolu-
minescent reporter phages: the approach harnesses the host specificity of bacte-
riophages, which is limited to genus or species level and sometimes even serovar
or strain level. Ideally, a reporter phage should be able to target all relevant
members of a pathogen group, at the desired discrimination level (species, serovar,
strain), but no related organisms. In some cases, this is difficult to achieve, for
example, in Salmonella enterica with its more than 2,500 serovars. In other cases,
however, a narrow host range is desirable, such as when discriminating between
pathovars of Pseudomonas [28]. The choice of bacteriophage is quite essential for
the success of the assay. Both virulent and temperate bacteriophages are, in
general, suited for the task. However, a temperate phage often displays a very
narrow and limited host range, and might not be the ideal choice for detection of
all members of a species. Also, prophages confer homoimmunity to similar tem-
perate phages, which might prevent gene expression from reporter phage genomes.
Also, bacteria might become resistant to phage infection by various mecha-
nisms, such as receptor mutation, CRISPR systems, restriction-modification sys-
tems, or abortive infection, and thus generate a false-negative signal. Although the
likelihood for resistance development is low (especially in a food production
setting, where potentially resistant bacteria are removed from the production chain
with the food and not reintroduced), the possibility needs to be addressed. Bac-
teriophage resistance may be associated with a high fitness cost and therefore
could be expected to be lost quickly when the selective pressure (phage) is
removed [29]. Moreover, bacteriophage resistance occurs in subfractions of a
bacterial population, whereas the rest of the population is still sensitive to infection
and produces a detectable light signal when infected with a luciferase phage.
Another prerequisite for the construction of a reporter phage is that the phage
genome is amenable to genetic manipulation. Several different approaches can be
used for constructing a reporter phage. Besides transposition and homologous
recombination, direct cloning is a possibility with smaller genomes of temperate
phages [30]. Direct cloning requires the availability of a genetic manipulation
system for the host bacterium. A genetically stable insertion of luxAB into a phage
genome under control of a strong promoter should not negatively affect host range
or infectivity or ability to multiply of the phage. An important consideration is
where to place the reporter genes. In some cases, it may be possible to replace
nonessential genetic regions by the reporter system, although identification of such
regions is not an easy task. Phage genes are usually densely packed on the gen-
omes, often featuring coding capacities of more than 90 % [31–33]. Moreover,
insertion of large gene fragments into noncoding regions might be difficult to
achieve. Despite the recent advances in sequencing technologies and an increasing
number of phage genomes available, the function of most genes in phage genomes
is not clear [34, 35]. Determining a nonessential gene or a noncoding region in
which a reporter gene may be inserted is therefore not trivial. This fact also
represents the major difficulty in transposition-based methods for generating a
reporter phage: random insertion likely results in disruption of functions required
for infection and multiplication.
A further restriction to the more straightforward design and construction of
reporter phages is that direct cloning is rarely possible. In the best case, chro-
mosomally integrated temperate phages (prophages) will be amenable to genetic
manipulation in the same way as the host chromosome. However, this depends on
the availability of suitable tools for manipulating this organism. In contract,
genetic manipulation of virulent bacteriophage chromosomes is inherently much
more difficult, because the phage DNA is only accessible for modification during
the infection process. The method of choice is homologous recombination and
double cross-over between the mutation locus placed on a plasmid and the phage
DNA, which will result in mutated DNA incorporated into new phage particles.
Because the frequency of successful recombination is very low, the challenge is to
isolate the recombinant virus from the background of wild-type phages. However,
the desired phage can be relatively conveniently selected from the background by
screening for ‘‘bioluminescent plaques’’ formed on infected host cells (in the
presence of the substrate for the luciferase enzyme).
The reporter gene should be placed under control of a strong promoter, active
upon infection of the bacterial host. Naturally, a phage late-gene promoter would
be best suited for this task. Alternatively, nonphage promoters to enhance suffi-
cient levels of reporter gene expression in certain host bacteria could also be used.
The physical structure of the phage genome plays a major role in determining
the correct placement of a reporter gene. Phage genomes must be circularized or
primed by proteins for efficient, loss-free DNA replication in the host cell.
Therefore, most phage genomes feature overlapping single-stranded ends or ter-
minal redundancies to enable circularization upon infection [36], the latter of
which may be extensive [37]. Any genetic manipulation, in particular addition of
sequence, must take into account that phage capsids provide limited space, and
packaging signals must not be destroyed by gene insertion. This is well illustrated
by construction of Listeria reporter phage A511::luxAB. The 2.2 kb luxAB gene
was inserted into the 30 -region of the major capsid protein gene, which features a
strong promoter and is highly expressed during phage replication [38]. At the time
this work was done in 1995, no complete genome sequence and no information
about the A511 genome structure was available. Only 12 years later, the complete
genome sequence of A511 and the 3.125 bp fixed terminal redundancy of its
chromosome were resolved [37]. These findings explained why it was possible to
insert the 2.2 bp luxAB fusion into the A511 genome. However, it is still unclear
how the A511 terminal redundancy is generated and maintained [37]. In another
example, Kuhn et al. reported severe difficulties in obtaining a transposon-mutated
Felix O1 Salmonella reporter phage, which was attributed to the phage not being
able to accommodate additional genetic information. Eventually, essential genetic
information had to be deleted and supplied in trans to enable recombinant reporter
phage generation [25].
A very important advantage of the detection of bacteria using bioluminescent
phages is the fact that bacteriophages rely on the host cell metabolism for gene
expression and protein synthesis, and the bioluminescence reaction also requires
substrates such as FMNH2 originating from an active metabolism. Consequently,
luciferase and bioluminescence cannot be produced outside a viable host cells. The
reporter phage assay is therefore superior to PCR or any other molecular method,
which cannot distinguish between live and dead cells.
The theoretical detection limit of a reporter phage assay is a single viable cell.
However, this is greatly dependent on the reporter system and detector, and spe-
cific characteristics of the phage, such as its multiplication rate and requirements.
The matrix from which bacteria are isolated, enriched, and detected also plays an
important role. Clearly, there is a significantly better performance of the assay in
broth or buffer than directly in a food matrix [14, 29, 39]. The food surface
provides hiding niches for bacterial target cells, which absorb the majority of
phage particles, or inactivate them by indigenous substances. Also, free diffusion
of the phage particle is essential for successful binding to the target host, a con-
dition not given in most food materials [40]. In most cases, a pre-enrichment step
is strictly required for detection of bacterial cells, with a reasonable lower limit of
10 or 100 cells per ml or g food being detectable [14, 29, 39]. With longer pre-
enrichment, this limit may be lowered down to one cfu/ml/g or less [39], which is
well within the regulatory limits for most foods and pathogens.
Some drawbacks of bioluminescent reporter phage assays should also be
mentioned here, such as the lack of thermostability of the LuxAB fusion from
Vibrio, which is progressively inactivated at temperatures of 30 °C and above,
limiting its use to bacteria that can grow at or below this temperature (the psy-
chotrophic pathogen Listeria represents an ideal case). Also, emission of biolu-
minescence from infected bacteria is transient, and the time window for
measurement is shortly after injection of the aldehyde substrate. This is caused by
the limited supply of FNMH2 in infected cells, which is rapidly depleted by the
bioluminescence reaction, resulting in a short burst of light emission of typically
less than 20 s [15, 38]. Increasing the measurement time will therefore not result in
a lower detection limit, that is, a better signal-to-noise ratio [15].
Furthermore, the use of relatively simple single-tube luminometers is not easily
applicable in large-scale screening of food materials. Bioluminescence measure-
ment from phage-infected bacteria in small liquid samples is much better suited to
multiplates and semiautomated luminescence readers with substrate injection
systems. However, such systems may be relatively expensive, similar to any other
modern high-throughput screening method. Yet, reporter bacteriophages may also
be integrated into lab-on-a-chip approaches. Bacteriophages can be immobilized
on membranes and other solid supports [19, 41–43], and the detection system
could potentially be miniaturized. Hazbon et al. reported simplification of the
detection of the light-emitting reaction from Mycobacterium reporter phages by
using inexpensive Polaroid film, and achieved equal sensitivity compared to a
luminometer, albeit requiring a longer detection time [20].
Finally, the lack of a suitable bacteriophage for the target organism, or diffi-
culties in genetic manipulation might present major hurdles for development of
other or additional bioluminescent phages.
4 Specific Bioluminescent Reporter Phages
This chapter provides an overview of bioluminescent reporter bacteriophages

constructed for specific organisms in the past, but also discusses applications for
the detection of pathogens in food and hospital settings.
Early attempts to construct recombinant bacteriophages featuring a reporter
molecule for bacterial detection (not necessarily a bioluminescent marker) were
undertaken by Castilho et al., who cloned a promoterless b-galactosidase gene into
bacteriophage Mu. When host cells were infected, a random insertion of the lacZ
gene in the host chromosome could occur, and if the insertion happened down-
stream of a host promoter, b-galactosidase was produced and resulted in a color
reaction with an exogenous substrate, enabling cell detection [44].
A few years later, Ulitzur and coworkers modified phage lambda by introducing
the bacterial luciferase genes into the bacteriophage genomic DNA, and created
the first luciferase reporter phage described, termed L28 [13]. The resulting bio-
luminescent reporter bacteriophage could detect as little as 10 viable Escherichia
coli cells approximately 100 min after infection [13, 45].
Salmonella was the target for several attempts to construct useful reporter
phages. As one of the major foodborne pathogens with millions of cases world-
wide and a very large projected economic loss, screening foods for Salmonella
contamination is essential for food producers. Different luciferase phages were
constructed, based on phages P1, P22, and Felix O1 [25, 30, 46]. Chen and
Griffiths reported Salmonella detection in eggs, and could detect all of the 51
tested Salmonella isolates with no false-positives using a three-phage cocktail. The
test detected 10 cfu/ml of Salmonella from broth with 6 h pre-enrichment, and
within 24 h directly from contaminated whole eggs [47]. Another P22-based
reporter phage was evaluated by Stewart and Turpin et al. in two independent
studies and it was able to detect as low as 100 Salmonella cells [48, 49]. Thouand
et al. report a P22::luxAB assay for detection of Salmonella in poultry samples,
with detection limits of 102–104 cfu/ml, depending on the matrix used [50].
However, P22 is a narrow host-range phage, and thus not suited for general Sal-
monella diagnostics. In 2002, Kuhn and coworkers described another reporter
phage based on the broad host-range Salmonella phage Felix-O1. Despite multiple
attempts, the authors could not insert the luxAB genes by transposon mutagenesis
[25], likely because of the terminally redundant invariable genome ends of this
phage (Klumpp and Marti, unpublished results). The Felix-O1 reporter was
eventually made by replacement of three genes with a luxAB cassette, and in trans
supply of one of the essential genes. However, the reporter phage performed
poorly in strains other than the propagation host [25]. One interesting property of
this specific construct is the fact that it is genetically locked; that is, it can only
reproduce in the complemented laboratory host strain. Therefore, it is unable to
reproduce and spread into the environment, a fact that might help address general
concerns regarding use of GMO reporter phages.
In 1991, Kodikara et al. developed a reporter phage for near-online detection of
enterobacteria. For proof-of-concept, an abattoir environment was used, and meat-
processing surfaces were sampled. The authors reported a 104 cfu/g detection limit
for a 1 h quick test, and could significantly reduce the limit to 10 cfu using a 4 h
sample pre-enrichment [51].
E. coli, namely serotype O157:H7 was also used as a target for reporter phage
detection. The phage was based upon the temperate virus UV10, and featured
promoterless bacterial luxAB genes that were inserted by transposon mutagenesis
on a 3.6-kb fragment [52]. The authors claimed that UV10 was able to detect
O157:H7 after only 1 h of infection, and could detect 64 % of screened E. coli
O157:H7 isolates. Use of a temperate phage is probably the reason for the
insufficient range of detectable isolates, and construction of further reporter phages
was suggested [52].
In 2008, Ripp and coworkers published the construction of an E. coli O157:H7
reporter based on phage PP01 [26]. Earlier, the same phage was engineered as a
fluorescent reporter phage by Oda et al., to present a GFP molecule on its capsid.
The phage enabled specific tagging of target bacteria, which could be seen under
the fluorescence microscope [17]. Ripp et al. engineered the phage to encode the
luxI/R quorum sensing, and a luxCDABE bioluminescence operon. The reporter
phage is described to respond specifically and autonomously to the presence of
E. coli O157:H7. The presence of the LuxI and R proteins serves for signal
amplification. LuxI produces an autoinducer N3–(oxohexanoyl-) L-homoserine
lactone (OHHL), which activates LuxR, which in turn upregulates the lux gene
transcription. The LuxCDE gene products catalyze synthesis of the luciferase
aldehyde substrate, avoiding the need for external substrate addition during
detection [26]. The phage featured a detection limit of 103 cfu/ml from pure
cultures. To push this value down to 1 cfu/ml, an enrichment step was introduced,
resulting in a total assay time of approximately 10–12 h. In apple juice, the
detection limit for E. coli O157:H7 could was 100 cfu/ml, and in water it was
1 cfu/ml. In contrast, the limit was a massive 106 cfu/g from ground beef, possibly
due to a high background bioluminescence because of the intrinsic presence of the
OHHL autoinducer in beef [26]. This points to a major drawback of signal au-
toamplification: the presence of an inducer-like substance can trigger a light-
emitting reaction and might produce false-positive results.
Arguably one of the first reporter phages developed for Gram-positive patho-
gens and successfully applied to foods was Listeria phage A511:luxAB [38].
Listeria is the causative agent of the rare but severe infection termed listeriosis
[53], and is almost exclusively transmitted via contaminated foods [54, 55].
Culture-based detection methods for Listeria take 4–6 days, which is quite prob-
lematic considering the short shelf-life of most ready-to-eat foods and delicates-
sen. Phage A511 is a virulent myovirus of Listeria and can infect the majority of
Listeria strains [56]. It later became a model organism for the bacteriophage
subfamily Spounavirinae, which comprises many related phages of potential
biotechnological interest [37, 57]. The reporter phage was developed with the goal
to reduce detection time and limits. Current regulations demand the absence of
Listeria from 25 g of food for certain types of food, which is still difficult to
confirm even by the most modern molecular detection methods. In this work, a
luxAB gene cassette from Vibrio harveyi was inserted downstream of the major
capsid gene of A511, and expression is driven by the dedicated and strong Pcps
promoter, resulting in high expression levels. Recombination into the wild-type
phage was achieved by plasmid-based double-crossover during phage infection
and genome replication [38]. Mutant phages could be identified and isolated using
the luciferase activity, and could be calculated to have occurred at a relatively high
frequency of approximately 1:50,000. Maximum signal intensity is approximately
100–140 min postinfection of Listeria target cells [38].
In a follow-up study, the efficacy of A511:luxAB was evaluated in artificially
and naturally contaminated foods [39]. The phage enabled highly sensitive
detection of Listeria contamination in 55 out of 348 tested field samples, with no
false-positives. A pre-enrichment step of 20 h was used to achieve best results,
which is superior to the 4–6 days required for the standard plating method.
Depending on the type of food, detection of 0.1 cell/g could be achieved, whereas
in other, more complex types of food, such as minced meat, positive diagnostics of
10 cells/g food were possible [39]. It should be noted that A511 is not species
specific, and can infect and yield signals with other Listeria species. This is not
necessarily a drawback, inasmuch as any Listeria contamination is considered
undesirable and a marker of poor hygiene or contaminated raw products [58].
More recently, the same concept was used to modify A511 with a different
reporter system. The celB glycosidase from Pyrococcus furiosus features extreme
heat stability—in contrast to the original luxAB system, which is instable above
35 °C—and sufficient pH stability. The enzyme is most active at 102–106 °C and
pH 5–5.5, a major advantage compared to the temperature-sensitive luciferase
enzyme [15]. Because of its versatile activity, featuring both b-galactosidase and
b-glycosidase activity, a wide range of chromogenic, fluorescent, or chemilumi-
nescent substrates can be used for pathogen detection. The system has been proven
to be suitable for detection of Listeria from the food matrix, with detection limits
as low as 10 cells. The assay can be fully automated to 96-well plate format [15].
One advantage of the celB reporter phage is that detection does not need to be
carried out in a specific time window as with luciferase activity, but the color
conversion of a cromogenic substrate is an end point assay and can be determined
any time after incubation.
Earlier attempts to construct bioluminescent reporter phages have focused on

clinically relevant pathogens such as Mycobacterium tuberculosis. These attempts
were driven by the slow-growing nature of these bacteria and the resulting long
time required for detection. The luciferase reporter phages developed for this
species used firefly luciferase and were based on phages TM4, L5, and D29 [12,
59, 60]. Both D29 and L5 exhibit a relatively broad host range, infecting M.
tuberculosis, M. smegmatis, and M. bovis. TM4 infects M. avium, M. tuberculosis,
M. smegmatis, and M. ulcerans. In 1995, Sarkis and coworkers presented the L5
luciferase reporter phage for Mycobacterium smegmatis [12]. The temperate
bacteriophage carries the firefly luciferase genes inserted into a tRNA gene region.
This is in contrast to previous attempts at generating Mycobacterium-specific
reporter phages, which utilized phage TM4 or D29 and a shuttle plasmid strategy
[59, 60]. The latter experiments resulted in a reporter system with relatively poor
detection capacity of 104–105 cells [12]. However, this approach is somewhat
different from using a lytic phage, as the system actually depends on formation of
lysogens, which then constitutively express the lux genes, and light output
increases for many hours after infection [12]. The drawback of using the firefly
luciferase is that because of the poor diffusion properties of the substrate, bacterial
target cells must actually be lysed in order to generate the light signal.
Several reports have followed up on the detection of mycobacteria by bacte-
riophages. In recent approaches, bacteriophages are utilized to deliver a GFP or
other fluorophor to mycobacterial cells, which is then used to monitor them by
fluorescence microscopy or flow cytometry [61]. A study conducted in Mexico
revealed a 76 % detection rate of M. tuberculosis from 523 sputum samples using
luciferase reporter phage. In a later study, the same authors report detection of the
target bacteria in 94 % of 84 contaminated sputum samples [62, 63]. Luciferase
reporter phages and ‘‘fluoromycobacteriophages’’ have also been used for detec-
tion of drug-resistant isolates of Mycobacterium tuberculosis [60, 64] (see below
for an extended discussion of this specific application.).
Not surprisingly, bioluminescent bacteriophages were also designed to target
potential biosafety-relevant human pathogens, such as Bacillus anthracis and
Yersinia pestis [65]. For B. anthracis, a reporter phage was developed based on the
temperate, but broad host-range Wb phage, which infects all nonencapsulated B.
anthracis strains [66]. Most Bacillus ACT-group phages infect all three organisms
with similar efficiency, and are thus not useful for the above purpose. In contrast,
codetection of B. thuringiensis and B. anthracis would not be considered prob-
lematic for detection in foods. The Vibrio harveyi luxA and luxb were inserted into
the dispensable wp40/wp41 locus, under control of an optimized Bacillus promoter
and featuring a suitable ribosome binding site [66]. The assay yields a quick
response time (first light signal only 16 min following infection), and a detection
limit as low as 1,600 cfu/ml of vegetative cells. Spores may be triggered to ger-
minate and could be detected after as low as 60 min [66]. However, use of a
temperate phage has the massive drawback of not necessarily lysing the host cell.
Immunity to infection can easily occur, resulting in an absence of light signal even
though the target organism is present.
The same authors also produced a reporter phage for Yersinia pestis [67], which
(in an improved version) could potentially also be used to assess antibiotic sus-
ceptibility of this pathogen. Yersinia typing phage UA1122 was equipped with the
bacterial luxAB genes in a noncoding region, and the ability of the phage to
generate a drug-concentration-dependent signal was harnessed [68].
Plant pathogenic bacteria also represent targets for the development of reporter
bacteriophages, as they cause significant losses in agriculture, and early-warning
and screening systems are needed. Schofield et al. report the development of phage
PBSPCA1::luxAB, which specifically targets Pseudomonas cannabina pv. alisal-
ensis, the causative agent of bacterial blight of crucifers [28]. The Vibrio harveyi
luxAB genes were inserted into an phoH homologue in the phage genome, under
control of a strong bacterial promoter [28]. The detection limit was 260 cfu/ml, at
approximately 120 min after infection [28, 69].
4.1 Antibiotic Resistance
A simple but intriguing concept for testing of antibiotic susceptibility of bacterial

isolates was proposed by Ulitzur in 1987 [13], and further developed with reporter
bacteriophages for Mycobacteria. If the host cell is treated with an antibiotic to
which it is sensitive, phage multiplication cannot continue, gene expression of
reporter genes from the phage genome is attenuated or abolished, and cells emit
less or no light. Ulitzur et al. confirmed this hypothesis by providing antibiotic-
response curves for different antibiotic substances on E. coli infected by reporter
phage k L28 [13]. In a more recent study, Schofield et al. transferred this concept
to a reporter phage for Yersinia pestis [68]. The bioluminescent signal generated
by A1122::luxAB was fitness-dependent and directly linked to the drug concen-
tration used for challenge. The phage-based antibiotic sensitivity test performed
similar to the CLSI microdilution method, but yielded results after 60–120 min
instead of 48 h [68]. This concept has been further exploited by reporter phages for
Mycobacterium [60, 62, 70]. Riska et al. could detect the resistance to fast-acting
drugs such as Rifampicin and slow-acting drugs such as ethambutol or ciprofl-
oxacine within hours or 2–3 days, respectively [21]. Similar results were also
obtained with myocbacterial fluorescence reporter phages [64].
4.2 Combination of Bioluminescent Reporter Phages

with Other Technologies
The detection limit of bioluminescent reporter phages can be further and signifi-
cantly improved if immunomagnetic separation technologies are used in pre-
enrichment of target bacteria. Especially suitable seems the use of cell-wall
binding domains of bacteriophage endolysins coated on paramagnetic beads for
fast and highly efficient enrichment of pathogens [71, 72]. Pre-enriching the target
bacteria with magnetic separation, followed by detection with bioluminescent
reporter phages could lower the detection limit by several orders of magnitude, and
provide ultrafast diagnostics of viable bacteria only (Kretzer and Loessner
unpublished data). The methods may be automated and performed in a high-
throughput setting. Favrin et al. used classical immunomagnetic separation for this
purpose, and were able to detect as low as 3 cfu Salmonella enteritidis per 25 g of
food [73].
5 Summary, Conclusions, and Outlook
The use of bioluminescent bacteriophages for detection of foodborne pathogens

offers a number of unique advantages. The system allows for rapid and highly
specific detection of selected bacterial agents, and requires a minimum of equip-
ment and training to use. Bioluminescent reporter bacteriophages can detect only
viable bacteria, as the reporter gene expression relies on an active host metabo-
lism. The assays are fast, highly specific, and generally inexpensive. Despite these
clear advantages, the reporter bacteriophage technology is not as widely accepted
or used as one would imagine. One potential drawback of the reporter phage
construction is the requirement of a sufficiently specific but broad host-range
bacteriophage. If none is available, it must be isolated from the environment, often
a laborious and lengthy process, and not always crowned by success. Especially in
closely related organisms such as Salmonella, Shigella, and E. coli, phages specific
for one but not the other organisms are difficult to find [74]. It may also be
challenging to achieve appropriate genetic manipulation of the selected phages
without affecting the phage lifecycle or ability to infect its host. However, with the
increased availability of genome sequences of bacteriophages and bacterial hosts
due to novel DNA sequencing technologies, the further development of reporter
bacteriophage platforms might gain momentum, especially if difficult-to-detect
bacterial species are targeted. Last, but not least, coupling of efficient enrichment
and immobilization technologies with bioluminescent bacteriophage assays pro-
vides the microbiologist with powerful diagnostic tools for specific detection of
bacterial pathogens.
References
1. Hendrix RW (2003) Bacteriophage genomics. Curr Opin Microbiol 6:506–511

2. Hendrix RW, Hatfull GF, Smith MC (2003) Bacteriophages with tails: chasing their origins
and evolution. Res Microbiol 154:253–257
3. Sulakvelidze A, Alavidze Z, Morris JG Jr (2001) Bacteriophage therapy. Antimicrob Agents
Chemother 45:649–659
4. Summers WC (2001) Bacteriophage therapy. Annu Rev Microbiol 55:437–451
5. Greer GG (2005) Bacteriophage control of foodborne bacteria. J Food Prot 68:1102–1111

6. Hooton SP, Atterbury RJ, Connerton IF (2011) Application of a bacteriophage cocktail to
reduce Salmonella Typhimurium U288 contamination on pig skin. Int J Food Microbiol
151(2):157–163
7. Carvalho CM, Gannon BW, Halfhide DE, Santos SB, Hayes CM, Roe JM, Azeredo J (2010)
The in vivo efficacy of two administration routes of a phage cocktail to reduce numbers of
Campylobacter coli and Campylobacter jejuni in chickens. BMC Microbiol 10:232
8. Connerton PL, Timms AR, Connerton IF (2011) Campylobacter bacteriophages and
bacteriophage therapy. J Appl Microbiol 111:255–265
9. Guenther S, Herzig O, Fieseler L, Klumpp J, Loessner MJ (2012) Biocontrol of Salmonella
Typhimurium in RTE foods with the virulent bacteriophage FO1-E2. Int J Food Microbiol
154:66–72
10. Loessner MJ (2005) Bacteriophage endolysins—current state of research and applications.
Curr Opin Microbiol 8:480–487
11. Fischetti VA (2010) Bacteriophage endolysins: a novel anti-infective to control Gram-
positive pathogens. Int J Med Microbiol 300:357–362
12. Sarkis GJ, Jacobs WR Jr, Hatfull GF (1995) L5 luciferase reporter mycobacteriophages: a
sensitive tool for the detection and assay of live mycobacteria. Mol Microbiol 15:1055–1067
13. Ulitzur S, Kuhn J (1987) Introduction of lux genes into bacteria, a new approach for specific
determination of bacteria and their antibiotic susceptibility. In: Schlomerich J, Andreesen R,
Kapp A, Ernst M (eds) Bioluminescence and Chemiluminescence: New Perspectives. Wiley,
New York, pp 463–472
14. Goodridge L, Griffiths M (2002) Reporter bacteriophage assays as a mean to detect foodborne
pathogenic bacteria. Food Res Int 35:863–870
15. Hagens S, de Wouters T, Vollenweider P, Loessner MJ (2011) Reporter bacteriophage
A511:celB transduces a hyperthermostable glycosidase from Pyrococcus furiosus for rapid
and simple detection of viable Listeria cells. Bacteriophage 1:143–151
16. Funatsu T, Taniyama T, Tajima T, Tadakuma H, Namiki H (2002) Rapid and sensitive
detection method of a bacterium by using a GFP reporter phage. Microbiol Immunol
46:365–369
17. Oda M, Morita M, Unno H, Tanji Y (2004) Rapid detection of Escherichia coli O157:H7 by
using green fluorescent protein-labeled PP01 bacteriophage. Appl Environ Microbiol
70:527–534
18. Wolber PK, Green RL (1990) Detection of bacteria by transduction of ice nucleation genes.
Trends Biotechnol 8:276–279
19. Minikh O, Tolba M, Brovko LY, Griffiths MW (2010) Bacteriophage-based biosorbents
coupled with bioluminescent ATP assay for rapid concentration and detection of Escherichia
coli. J Microbiol Methods 82:177–183
20. Hazbon MH, Guarin N, Ferro BE, Rodriguez AL, Labrada LA, Tovar R, Riska PF, Jacobs
WR Jr (2003) Photographic and luminometric detection of luciferase reporter phages for drug
susceptibility testing of clinical Mycobacterium tuberculosis isolates. J Clin Microbiol
41:4865–4869
21. Riska PF, Jacobs WR Jr (1998) The use of luciferase-reporter phage for antibiotic-
susceptibility testing of mycobacteria. Methods Mol Biol 101:431–455
22. Boylan MO, Pelletier J, Dhepagnon S, Trudel S, Sonenberg N, Meighen EA (1989)
Construction of a fused LuxAB gene by site-directed mutagenesis. J Biolumin Chemilumin
4:310–316
23. Escher A, O’Kane DJ, Lee J, Szalay AA (1989) Bacterial luciferase alpha beta fusion protein
is fully active as a monomer and highly sensitive in vivo to elevated temperature. Proc Natl
24. Olsson O, Escher A, Sandberg G, Schell J, Koncz C, Szalay AA (1989) Engineering of
monomeric bacterial luciferases by fusion of luxA and luxB genes in Vibrio harveyi. Gene
81:335–347
25. Kuhn J, Suissa M, Wyse J, Cohen I, Weiser I, Reznick S, Lubinsky-Mink S, Stewart G,

Ulitzur S (2002) Detection of bacteria using foreign DNA: the development of a
bacteriophage reagent for Salmonella. Int J Food Microbiol 74:229–238
26. Ripp S, Jegier P, Johnson CM, Brigati JR, Sayler GS (2008) Bacteriophage-amplified
bioluminescent sensing of Escherichia coli O157:H7. Anal Bioanal Chem 391:507–514
27. Ripp S, Jegier P, Birmele M, Johnson CM, Daumer KA, Garland JL, Sayler GS (2006)
Linking bacteriophage infection to quorum sensing signalling and bioluminescent bioreporter
monitoring for direct detection of bacterial agents. J Appl Microbiol 100:488–499
28. Schofield DA, Bull CT, Rubio I, Wechter WP, Westwater C, Molineux IJ (2012)
Development of an engineered bioluminescent reporter phage for detection of bacterial
blight of crucifers. Appl Environ Microbiol 78:3592–3598
29. Capparelli R, Nocerino N, Lanzetta R, Silipo A, Amoresano A, Giangrande C, Becker K,
Blaiotta G, Evidente A, Cimmino A, Iannaccone M, Parlato M, Medaglia C, Roperto S,
Roperto F, Ramunno L, Iannelli D (2010) Bacteriophage-resistant Staphylococcus aureus
mutant confers broad immunity against staphylococcal infection in mice. PLoS ONE
5:e11720
30. Ulitzur S, Kuhn J (2000) Construction of lux bacteriophages and the determination of specific
bacteria and their antibiotic sensitivities. Methods Enzymol 305:543–557
31. Dorscht J, Klumpp J, Bielmann R, Schmelcher M, Born Y, Zimmer M, Calendar R, Loessner
MJ (2009) Comparative genome analysis of Listeria bacteriophages reveals extensive
mosaicism, programmed translational frameshifting, and a novel prophage insertion site.
32. Kilcher S, Loessner MJ, Klumpp J (2010) Brochothrix thermosphacta bacteriophages feature
heterogeneous and highly mosaic genomes and utilize unique prophage insertion sites.
33. Schmuki MM, Erne D, Loessner MJ, Klumpp J (2012) Bacteriophage P70: Unique
morphology and unrelatedness to other Listeria bacteriophages. J Virol 86:13099–13102
34. Klumpp J, Fouts DE, Sozhamannan S (2012) Next generation sequencing technologies and
the changing landscape of phage genomics. Bacteriophage 2:190–199
35. Klumpp J, Fouts DE, Sozhamannan S (2013) Bacteriophage functional genomics and its role
in bacterial pathogen detection. Brief Funct Genomic 12(4): 354–365
36. Casjens S, Gilcrease EB (2009) Determining dna packaging stragety by analysis of the
termini of the chromosomes in tailed-bacteriophage virions. In: Clokie MRJ, Kropinski A
(eds) Bacteriophages—Methods and protocols. vol 2: molecular and applied aspects. Humana
Press, New York, pp 91–111
37. Klumpp J, Dorscht J, Lurz R, Bielmann R, Wieland M, Zimmer M, Calendar R, Loessner MJ
(2008) The terminally redundant, nonpermuted genome of Listeria bacteriophage A511: a
Model for the SPO1-like myoviruses of gram-positive bacteria. J Bacteriol 190:5753–5765
38. Loessner MJ, Rees CE, Stewart GS, Scherer S (1996) Construction of luciferase reporter
bacteriophage A511:luxAB for rapid and sensitive detection of viable Listeria cells. Appl
39. Loessner MJ, Rudolf M, Scherer S (1997) Evaluation of luciferase reporter bacteriophage
A511:luxAB for detection of Listeria monocytogenes in contaminated foods. Appl Environ
40. Hagens S, Loessner MJ (2010) Bacteriophage for biocontrol of foodborne pathogens:
calculations and considerations. Curr Pharma Biotechnol 11:58–68
41. Anany H, Chen W, Pelton R, Griffiths MW (2011) Biocontrol of Listeria monocytogenes and
Escherichia coli O157:H7 in meat by using phages immobilized on modified cellulose
membranes. Appl Environ Microbiol 77:6379–6387
42. Naidoo R, Singh A, Arya SK, Beadle B, Glass N, Tanha J, Szymanski CM, Evoy S (2012)
Surface-immobilization of chromatographically purified bacteriophages for the optimized
capture of bacteria. Bacteriophage 2:15–24
43. Arya SK, Singh A, Naidoo R, Wu P, McDermott MT, Evoy S (2011) Chemically
immobilized T4-bacteriophage for specific Escherichia coli detection using surface plasmon
resonance. The Analyst 136:486–492
44. Castilho BA, Olfson P, Casadaban MJ (1984) Plasmid insertion mutagenesis and lac gene
fusion with mini-mu bacteriophage transposons. J Bacteriol 158:488–495
45. Ulitzur S, Kuhn J (1989) Detection and/or identification of microorganisms i a test sample
using bioluminescence or other exogenous genetically introduced marker, Patent C12N15/52,
06/739,957 USPTO
46. Kuhn J, Suissa M, Chiswell D, Azriel A, Berman B, Shahar D, Reznick S, Sharf R, Wyse J,
Bar-On T, Cohen I, Giles R, Weiser I, Lubinsky-Mink S, Ulitzur S (2002) A bacteriophage
reagent for Salmonella: molecular studies on Felix 01. Int J Food Microbiol 74:217–227
47. Chen J, Griffiths M (1996) Salmonella detection in egg using Lux + bacteriophages. J Food
Prot 59:908–914
48. Stewart G, Smith T, Denyer S (1989) Genetic engineering for bioluminescent bacteria. Food
Sci Technol Today 3: 19-22
49. Turpin P, Maycroft KA, Bedford J, Rowlands CL (1993) A rapid luminescent-phage based
MPN method for the enumeration of Salmonella typhimurium in environmental samples. Lett
Appl Microbiol 16: 24-27
50. Thouand G, Vachon P, Liu S, Dayre M, Griffiths MW (2008) Optimization and validation of
a simple method using P22:luxAB bacteriophage for rapid detection of Salmonella enterica
serotypes A, B, and D in poultry samples. J Food Prot 71:380–385
51. Kodikara CP, Crew HH, Stewart GS (1991) Near on-line detection of enteric bacteria using
lux recombinant bacteriophage. FEMS Microbiol Lett 83:261–266
52. Waddell TE, Poppe C (2000) Construction of mini-Tn10luxABcam/Ptac-ATS and its use for
developing a bacteriophage that transduces bioluminescence to Escherichia coli O157:H7.
FEMS Microbiol Lett 182:285–289
53. Vazquez-Boland JA, Kuhn M, Berche P, Chakraborty T, Dominguez-Bernal G, Goebel W,
Gonzalez-Zorn B, Wehland J, Kreft J (2001) Listeria pathogenesis and molecular virulence
determinants. Clin Microbiol Rev 14:584–640
54. Farber JM, Peterkin PI (1991) Listeria monocytogenes, a food-borne pathogen. Microbiol
Rev 55:476–511
55. McLauchlin J, Mitchell RT, Smerdon WJ, Jewell K (2004) Listeria monocytogenes and
listeriosis: a review of hazard characterisation for use in microbiological risk assessment of
foods. Int J Food Microbiol 92:15–33
56. Loessner MJ, Busse M (1990) Bacteriophage typing of Listeria species. Appl Environ
57. Klumpp J, Lavigne R, Loessner MJ, Ackermann HW (2010) The SPO1-related
bacteriophages. Arch Virol 155:1547–1561
58. Hagens S, Loessner MJ (2007) Luciferase Reporter Bacteriophages. In: Marks RS, Cullen
DC, Karube I, Lowe CR, Weetall HH (eds) Handbook of Biosensors and Biochips. Wiley,
Hoboken
59. Pearson RE, Jurgensen S, Sarkis GJ, Hatfull GF, Jacobs WR Jr (1996) Construction of D29
shuttle phasmids and luciferase reporter phages for detection of mycobacteria. Gene
183:129–136
60. Jacobs WR Jr, Barletta RG, Udani R, Chan J, Kalkut G, Sosne G, Kieser T, Sarkis GJ, Hatfull
GF, Bloom BR (1993) Rapid assessment of drug susceptibilities of Mycobacterium
tuberculosis by means of luciferase reporter phages. Science 260:819–822
61. Piuri M, Jacobs WR Jr, Hatfull GF (2009) Fluoromycobacteriophages for rapid, specific, and
sensitive antibiotic susceptibility testing of Mycobacterium tuberculosis. PLoS ONE 4:e4870
62. Banaiee N, Bobadilla-Del-Valle M, Bardarov S Jr, Riska PF, Small PM, Ponce-De-Leon A,
Jacobs WR Jr, Hatfull GF, Sifuentes-Osornio J (2001) Luciferase reporter
mycobacteriophages for detection, identification, and antibiotic susceptibility testing of
Mycobacterium tuberculosis in Mexico. J Clin Microbiol 39:3883–3888
63. Banaiee N, Bobadilla-del-Valle M, Riska PF, Bardarov S Jr, Small PM, Ponce-de-Leon A,
Jacobs WR Jr, Hatfull GF, Sifuentes-Osornio J (2003) Rapid identification and susceptibility
testing of Mycobacterium tuberculosis from MGIT cultures with luciferase reporter
mycobacteriophages. J Med Microbiol 52:557–561
64. Rondon L, Piuri M, Jacobs WR Jr, de Waard J, Hatfull GF, Takiff HE (2011) Evaluation of
fluoromycobacteriophages for detecting drug resistance in Mycobacterium tuberculosis.
J Clin Microbiol 49:1838–1842
65. Schofield DA, Molineux IJ, Westwater C (2011) ‘Bioluminescent’ reporter phage for the
detection of category A bacterial pathogens. J Vis Exp 53:e2740
66. Schofield DA, Westwater C (2009) Phage-mediated bioluminescent detection of Bacillus
anthracis. J Appl Microbiol 107:1468–1478
67. Schofield DA, Molineux IJ, Westwater C (2009) Diagnostic bioluminescent phage for
detection of Yersinia pestis. J Clin Microbiol 47:3887–3894
68. Schofield DA, Molineux IJ, Westwater C (2012) Rapid identification and antibiotic
susceptibility testing of Yersinia pestis using bioluminescent reporter phage. J Microbiol
Methods 90:80–82
69. Schofield D, Bull CT, Rubio I, Wechter WP, Westwater C, Molineux IJ (2013) ‘‘Light-
tagged’’ bacteriophage as a diagnostic tool for the detection of phytopathogens.
Bioengineered 4:50–54
70. Carriere C, Riska PF, Zimhony O, Kriakov J, Bardarov S, Burns J, Chan J, Jacobs WR Jr
(1997) Conditionally replicating luciferase reporter phages: improved sensitivity for rapid
detection and assessment of drug susceptibility of Mycobacterium tuberculosis. J Clin
71. Schmelcher M, Shabarova T, Eugster MR, Eichenseher F, Tchang VS, Banz M, Loessner MJ
(2010) Rapid multiplex detection and differentiation of Listeria cells by use of fluorescent
phage endolysin cell wall binding domains. Appl Environ Microbiol 76:5745–5756
72. Kretzer JW, Lehmann R, Schmelcher M, Banz M, Kim KP, Korn C, Loessner MJ (2007) Use
of high-affinity cell wall-binding domains of bacteriophage endolysins for immobilization
and separation of bacterial cells. Appl Environ Microbiol 73:1992–2000
73. Favrin SJ, Jassim SA, Griffiths MW (2003) Application of a novel immunomagnetic
separation-bacteriophage assay for the detection of Salmonella enteritidis and Escherichia
coli O157:H7 in food. Int J Food Microbiol 85:63–71
74. Marti R, Zurfluh K, Hagens S, Pianezzi J, Klumpp J, Loessner MJ (2013) Long tail fibers of
the novel broad host range T-even bacteriophage S16 specifically recognize Salmonella
OmpC. Mol Microbiol 87:818–834
Part IV
Applications of Bioluminescence
in Health
for Medical Diagnostics
Ludmila A. Frank and Vasilisa V. Krasitskaya
Abstract Nowadays luciferases are effectively used as analytical instruments in a

great variety of research fields. Of special interest are the studies dealing with
elaboration of novel analytical systems for the purposes of medical diagnostics.
The ever-expanding spectrum of clinically important analytes accounts for the
increasing demand for new techniques for their detection. In this chapter we have
made an attempt to summarize the results on applications of luciferases as
reporters in binding assays including immunoassay, nucleic acid hybridization
assay, and so on. The data over the last 15 years have been analyzed and clearly
show that luciferase-based assays, due to extremely high sensitivity, low cost, and
the lack of need for skilled personnel, hold much promise for clinical diagnostics.
Keywords Bioluminescence
Ca2+-regulated photoprotein Diagnostics

Immunoassay Luciferase Nucleic acid hybridization assay
Abbreviations
Ab Antibody
Ag Antigen
ATP Adenosine-50 -triphosphate
BRET Bioluminescence resonance energy transfer
CMV Cytomegalovirus
ELISA Enzyme-linked immunosorbent assay
EYFP Yellow fluorescent protein
GFP Green fluorescent protein
hCG Human chorionic gonadotropin
L. A. Frank (&) V. V. Krasitskaya

Photobiology Laboratory, Institute of Biophysics, Russian Academy of Sciences,
Siberian Branch, Krasnoyarsk 660036, Russia
e-mail: lfrank@yandex.ru
L. A. Frank
Siberian Federal University, Svobodnii Avenue 79, Krasnoyarsk 660041, Russia

176 L. A. Frank and V. V. Krasitskaya
hFSH Follicle stimulating gonadotropic hormone

hGH Human growth hormone
hGLuc Highly bright Gaussia luciferase
hLH Luteinizing gonadotropic hormone (lutropin)
JAK2 Janus kinase 2
LIPS Luciferase immunoprecipitation system
LNA Locked nucleic acid
MBL Mannose-binding lectin
miRNA Micro RNA
mRNA Messenger RNA
MTB Mycobacterium tuberculosis
NASBA Nucleic acid sequence-based amplification
PAP Prostatic acid phosphatase
PCR Polymerase chain reaction
PEXT Primer extension
PSA Prostate specific antigen
RLuc Renilla luciferase
RT-PCR Reverse transcription polymerase chain reaction
SNP Single nucleotide polymorphism
TLR4 Toll-like receptor 4
TSH Thyroid stimulation hormone (thyrotropin)
Contents
1 Introduction........................................................................................................................ 176
2 Immunoassay: State-of-the-Art ......................................................................................... 177
2.1 Luciferases as Labels for Immunoassay .................................................................. 179
3 Nucleic Acid Hybridization Assay Based on Bioluminescent Reporters ....................... 183
3.1 Bioluminescent Assay of Infectious Agents............................................................ 185
3.2 Bioluminescent Assay in Oncology Diagnostics..................................................... 187
4 Multianalytical Bioluminescent Assay ............................................................................. 189
5 Bioluminescent Binding Assay in Homogeneous Format ............................................... 192
6 Summary, Conclusions, and Outlook ............................................................................... 193
References................................................................................................................................ 194
1 Introduction
At present, among bioluminescent systems the best-known are those derived from
fireflies, marine bacteria, coelenterates, and copepods. ‘‘Best-known’’ here implies
availability of cDNAs coding corresponding light-emitting proteins and affordable
Application of Enzyme Bioluminescence for Medical Diagnostics 177
recombinant proteins, chemically synthesized substrates, information on 3D

structure, and mechanism of light-producing reaction. A high quantum yield of the
reactions and a high sensitivity of modern photometers make possible the detection
of luciferases down to the attomole. The foregoing provides the basis for a variety
of analytical applications of luciferases: from environment monitoring to detection
of different events in living cells. In this chapter, we focus on bioluminescence
application in in vitro binding assays that play an increasingly important part in
medical diagnostics. The data presented demonstrate a successful use of luciferase
reporters in immunoassay, nucleic acid hybridization assay, simultaneous detec-
tion of several targets in one sample, and carried out in solid-phase and homo-
geneous formats.
2 Immunoassay: State-of-the-Art
Immunoassay is a diverse group of analytical techniques used throughout clinical

laboratories. Since 1959 when immunoassay principles were expounded by Yalow
and Berson there has been an exponential growth in the range of applications and
in the number of novel and ingenious assay designs. Regardless of the application
and underlying technology the assay involves four components: the antigen (Ag)
to be detected, the antibody (Ab) used for detection, the method to separate the
antigen–antibody complex (Ag–Ab) from unbound reactants (in the case of het-
erogeneous assay), and the method for Ag–Ab complex detection. The most
popular and simple to understand type of immunoassay is the immunometric
(sandwich) solid-phase design (Fig. 1a). In this case, the surface is activated with
antibody (Ab) that captures test antigen from the sample and the antibody Ab
(tracer), specific to another part of the antigen and labeled with a signal generation
molecule or isotope. After incubation resulting in Ab–Ag–Ab complex formation
the unbound labeled antibody is washed away. The final stage of the assay
involves the measurement of signal level, which in this type of analysis is pro-
portional to antigen concentration in the sample.
When the target analyte is small molecules (certain hormones, drugs, etc.), the
type of immunoassay applied is different. In this case, only one antibody and the
target analyte labeled with a suitable signal generation material are used as the key
reagents. The sample analyte competes with the labeled one for binding with
antibody on the surface (competitive immunoassay). In this type of assay the signal
level is indirectly proportional to antigen concentration in the sample (Fig. 1b).
Of less use is the so-called homogeneous immunoassay that does not require a
separation stage for unbound labeled antibody. The signal is generated by the
tracer only on binding with analyte. Under specific design of the assay the complex
formation quenches the signal completely.
A wide variety of immunoassay designs and formats has been developed over
the years (see [1]). In general the efficacy of any immunoassay depends on two
Fig. 1 Scheme for: a sandwich-type and b competitive immunoassays, and c basic antibody
structure (human IgG) demonstrating pairs of identical heavy (blue) and light (yellow) chains.
Both pairs contain variable domens, VH (dark blue) and VL (dark yellow), responsible for antigen
binding
factors: the efficiency of the immunocomplex formation and the ability of the
detection system to register this complex with high sensitivity. Complex formation
is provided by antibodies’ specificity and binding affinity to the antigen. Anti-
bodies are proteins, which are produced in animals by immunological response to
the presence of a foreign substance (Fig. 1c schematically presents a basic anti-
body structure). Antibodies are a crucial component of immunoassay performance
due to their ability to bind to an extremely wide range of natural and man-made
molecules, cells, and viruses; exceptional specificity for the analyte that enables us
to assay it in complex biological media (sera, urine, etc.); and the strength of
binding, providing the formation of a strong noncovalent complex Ag–Ab that
survives at processing and signal generation.
The immunocomplex detection system (signal generation plus measuring tool)
accounts for immunoassay sensitivity. The prime requirement for the one is the
detection of the label above background noise. A wide range of labels and their
nature-dependent detection systems are applied in immunological assay.
Radiolabels are the first to be used but the problems dealing with radioactive wastes
and inherent instability of radiolabeled reagents have stimulated the development
of nonradioactive labels. The enzymes catalyzing chemical reactions producing

visual signals (e.g., color or light) are now used more frequently as compared to
other labels. Enzymes may be detected at very low concentration because a single
enzyme molecule catalyzes many reactions without being consumed. Thus the
signal and consequently the assay sensitivity increase by several orders in contrast to
a label that produces just one signal event. To become a tracer, the enzyme is
covalently linked to antibody or antigen (depending on the assay format). The other
conjugation method involves a molecular biology technique in which a gene
encoding enzyme and gene encoding biospecific polypeptide (antigen, antibody,
etc.) are joined in one frame. Translation of this fusion gene yields a single
polypeptide with properties of both biospecific and reporter modules. The main
shortcoming of enzyme-based assay is that enzymes and substrates may be unstable
and require special conditions to maintain activity (pH, temperature, cofactors,
absence of inhibitors, etc.). To suit as a label, any enzyme should fulfill some more
conditions: availability, stability to chemical modification and under storage, sim-
plicity of use, and lack of toxicity. The search for enzymes meeting the above
requirements is being continued. Nowadays luciferases are the point of interest in
this view because a high quantum yield of bioluminescent reaction and capacities of
modern photometers make possible their detection down to the attomole. So they are
excellent reporters for a great variety of analytical applications. The perspectives of
application of the presently known luciferases as labels for immunoassay are further
considered.
2.1 Luciferases as Labels for Immunoassay
Bacterial luciferases are relatively big (around 80 kDa) heterodimeric proteins.

They catalyze long-chain aldehyde oxidation and need reduced flavin, which can
be provided by NADH:FMN-oxydoreductase. The system is too complicated to be
applied as a reporter in immunoassay.
Firefly luciferases are single-chain polypeptides with a molecular mass around
60 kDa, catalyzing ATP-dependent luciferin oxidation. The bioluminescent reac-
tion demonstrates the highest quantum yield (48 %, [2]) among those known
today. Therefore as little as 0.02 pg of the protein (subattomolar level!) can be
measured. Luciferases’ cDNAs from some fireflies (e.g., Photinus pyralis, Luciola
mingrelica) were cloned; the recombinant analogues were obtained and are now
commercially available products. The enzymes and their genetically modified
improved analogues have been applied as genetic reporters in molecular biology
and for ATP detection in microbiology. But due to firefly luciferase instability to
chemical conjugation with Ab or Ag, its application as a label in immunoassay
looked unreasonable. Nevertheless the firefly luciferase-based labels were obtained
using gene-fusing technology. The luciferase gene was fused with DNA, coding
biotin-acceptor polypeptide and expressed in Escherichia coli cells. A chimeric
protein fully retained luciferase activity and also was efficiently biotinylated
Fig. 2 Biotin-streptavidin/avidin system (a), the strongest noncovalent biological interaction

known (kd * 10-15 M) and one of the most widely used affinity pairs in molecular,
immunological, and cellular assays. Biotin (B) is introduced into the molecule of interest and into
reporter, and streptavidin (Stavi) with four biotin-binding sites serves as a strong and specific
bridge between these molecules (b). Sometimes, conjugates streptavidin-reporter are used (c)
in vivo. Using biotinylated luciferase in combination with streptavidin and bio-

tinylated antibodies, the sensitive immunoassays of hCG, human growth hormone
(hGH), TSH, PSA, and staphylococcal protein A have been developed [3–5]. The
principle of how the biotin–streptavidin system works is shown in Fig. 2.
Another technique applies the auxiliary label enzyme, acetate kinase or pyru-
vate phosphate dikinase [6]. These enzymes being stable to chemical modifications
yield ATP as one of the reaction products. The auxiliary enzyme conjugated with
antibody forms the immunocomplex with a target and its activity is determined by
measuring the amount of produced ATP using intact firefly luciferase.
A big group of luciferases responsible for bioluminescence of different marine
animals (soft corals Renilla reniformis and Renilla muelleri, copepods Metridia
longa and Gaussia princeps, jellyfish Aequorea victoria, hydroid polyp Obelia
longissima etc.) catalyzes oxidation of the same substrate molecule, coelenter-
azine. (One may see its structure in Fig. 3.) The cDNAs of all the luciferases listed
were cloned, their recombinant analogues obtained and are now commercially
available, as well as coelenterazine and its chemical derivatives.
Renilla luciferases (RLuc) are single-chain polypeptides with a molecular mass
around 36 kDa. With genetic modifications applied, their analogues with essen-
tially improved properties regarding thermal stability, light output, or color were
obtained [7, 8], thus making them more suitable as reporters for any kind of assay.
Nevertheless the essential loss of RLuc activity chemically conjugated with the
other molecules is observed [9]. A highly sensitive bioluminescent immunoassay
Fig. 3 Luciferase immunoprecipitation system. Immunocomplex antibody to tumor-associated

protein: fusion of this protein-RLuc is captured by magnetic beads, covered with protein A/G.
The bioluminescent signal arises with substrate (coelenterazine) addition. Protein A/G:
recombinant fusion protein that combines IgG (Fc fragment) binding domains of proteins A
and G
was developed using the luciferase genetically fused with antigene or antibody.
Figure 3 displays the luciferase immunoprecipitation system (LIPS), developed by
Burbelo and coauthors to detect antibodies responsible for tumor-associated
proteins [10], and a variety of infectious agents [11–13]. Briefly, this approach
involves: (a) construction of antigen–RLuc fusion and expression of the fusion
protein in mammalian COS cells; (b) incubation of RLuc–antigen fusion and
analyzed sera sample to bind antibody of interest; (c) immobilization of the
complex RLuc–antigen fusion–antibody on protein A/G beads, and (d) quantita-
tion of antigen-specific antibody by adding coelenterazine and measuring light
production. The most important point of this technology is the mammalian
production of fusion protein to provide correct folding and specific posttransla-
tional modifications of the antigenic domain, which are impossible under bacterial
expression. Of course it makes the analysis more expensive, although in some
crucial cases it is justifiable by high sensitivity.
Fusion proteins RLuc–antibody have mostly been engineered for ex vivo or
in vivo analytical applications. For these constructions, the variable regions
VL and VH chains of immunoglobulin (Fig. 1c) being minimal engineered anti-
body fragments, capable of efficient target binding, are usually used as luciferase
partners. Under bacterial expression, periplasmic secretion of chimeric protein is
usually employed in order to provide antibody domain affinity. As shown for
anticarcinoembryonic antigen antibody–RLuc fusion proteins [14], bacterial
expression allowed purification of small amounts of proteins, with only about half
of them recovered intact, whereas under mammalian expression the recovery was
greater, and owing to the lack of proteases, the protein purified remained stable for
several months when stored at 4 °C.
Luciferases from M. longa and G. princeps are single-chain polypeptides
(20–24 kDa) that contain 10 Cys residues, organizing several disulfide bonds. The
bacterial expression gives mostly unfolded proteins and the obtaining of an active
protein requires a special refolding procedure or expression in eukaryotic cells.
These luciferases are unstable to chemicals, too: Metridia luciferase chemically

conjugated with biotin loses almost 70 % of bioluminescent activity [15, 16].
Gaussia luciferase is the smallest native luciferase yet discovered (19.9 kDa). Its
biotinylated active derivative was obtained using gene-fusing technology such as
biotinylated firefly luciferase and applied to detect a biotinylated model DNA
fragment through streptavidin bridge [17].
Luciferases from marine ostracods Vargula hilgendorfii and Cypridina
noctiluca are highly homologous single-chain glycosylated polypeptides (around
62 kDa) containing 34 Cys residues. They are very stable (50-h half-life time at
37 °C) possibly due to the formation of several Cys–Cys bridges. Recently the
recombinant Cypridina luciferase expressed in yeast was chemically conjugated
with small haptenes: biotin or prostaglandin E2 with a loss of activity exceeding
70 % [18–20]. Despite this, these derivatives displayed high sensitivity in inter-
feron alpha and prostaglandin E2 immunoassay.
Both types of marine luciferases from copepods and ostracods are secreted
proteins and, in accord with numerous publications, are successfully used as
secreted reporter enzymes for continuous in vivo and ex vivo monitoring without
cell destruction, whereas their application in in vitro analytical systems is a rare
case.
Luciferases of a peculiar kind are Ca2+-regulated photoproteins responsible for
bioluminescence of a number of marine coelenterates such as jellyfish A. victoria
and Clytia gregaria, and hydroid polyps O. longissima and Obelia geniculata,
among others.
Ca2+-regulated photoproteins are stable complexes of apophotoprotein (single-
chain polypeptide with a molecular mass around 20 kDa) and the preoxidized
substrate molecule peroxycoelenterazine, which is strongly but noncovalently
immobilized in the protein hydrophobic cavity [21–23]. The primary structures of
photoproteins reveal high homology and all have three Ca2+-binding sites.
Upon Ca2+ addition, proteins undergo conformational changes resulting in coel-
enterazine decarboxylation. The products of the reaction are CO2, coelenteramide,
and a flash of blue light. In contrast to the luciferase reaction, photoprotein bio-
luminescence does not depend on oxygen or substrate concentration. Biolumines-
cence is detected in a luminometer allowing injection of calcium solution and
simultaneous measurement of light. The cDNAs of the proteins were cloned,
expressed in bacterial cells both in a soluble form and as inclusion bodies, and
isolated with a high yield. Recombinant apoproteins were effectively activated with
a synthetic coelenterazine under calcium-free conditions in the presence of O2 and a
reducing reagent producing photoprotein of high activity (close to that of a natural
protein) without folding problems. Simple technology of expression and purifica-
tion provides 15–20 mg recombinant photoprotein of high purity and activity per
1 g of bacterial cells. Regarding estimating photoproteins as labels, several obvious
advantages of those should be taken into account: availability of recombinant
proteins; high quantum yield of the reaction and virtual absence of the background
signal making the photoprotein detection down to an attomole; practically
unlimited linear range of bioluminescence; the simplicity of the reaction trigger;
tolerance to chemical modifications, and conjugation with the other molecules. The
conjugates in solution as well as in frozen and lyophilized states were found to be
stable and well-stored. A great number of publications described photoprotein-
based immunoassay of different analytes of clinical interest: hormones, interleu-
kins, oncomarkers, and the like, and infections (see [24]). The assays were carried
out in different formats (sandwich, competitive), and performed in solid-phase,
homogeneous, and even in flow injection variants. The papers consider investiga-
tions of model samples as well as sera, saliva, and mucous of patients and exper-
imental animals. Clinical trials began in 1994. At that time aequorin-based
thyrotropin (TSH) assay (developed and marketed by SeaLite Inc., USA) was tested
in the Pathology and Laboratory Medicine Departments of Emory University and
Veterans Administration Hospital (Atlanta, USA) [25]. The authors measured
serum TSH in 153 euthyroid individuals with thyroidal illnesses (primary hypo- and
hyperthyroids, thyroid cancer, etc.) applying bioluminescent methods and com-
pared the results with the data obtained by commercially available Nichols, ACS-
180 and TOSOH methods. The aequorin-based method was found to have the
required performance characteristics (e.g., the functional sensitivity of 0.017 mIU/
L, which is higher than in the cases of ACS-180 and TOSOH assays) and is ‘‘clearly
qualified as a last, third-generation TSH assay.’’
From the above presented data it appears that the application of classical
luciferases as reporters in immunoassay is complicated by their instability to
chemical conjugation with biospecific molecules (recognition elements) or by the
problems of genetically fused proteins’ folding under their bacterial expression. By
contrast, Ca2+-regulated photoproteins are free of these shortcomings and this
makes them perfect in vitro reporting molecules. The immunoassay on their basis
is fast and simple and, what is most important, highly sensitive in detecting the
target.
3 Nucleic Acid Hybridization Assay Based

on Bioluminescent Reporters
Nucleic acid hybridization is based on the ability of individual single-stranded

nucleic acid molecules to form double-stranded molecules (i.e., to hybridize to
each other) in accordance with complementarity of bases. Standard nucleic acid
hybridization assays involve the formation of heteroduplexes between labeled
single-stranded nucleic acid probes and complementary sequences within a target
nucleic acid. In recent years, bioluminescent proteins have found rapidly
expanding application as effective labels in DNA/RNA assays, in clinical diag-
nostics as well. The polymerase chain reaction (PCR) has proved to be of great
value in diagnostic research. Its ability to amplify specific nucleic acid sequences
several millionfold has facilitated detection of a small number of DNA copies. For
some diagnoses it is necessary to establish the presence or absence of the sequence
of interest (diseases associated with infectious agents, gene deletions, SNP, etc.).
But more often, quantitative PCR analysis is important for evaluation of therapy
effectiveness, detection of gene expression through reverse transcriptase-poly-
merase chain reaction (RT-PCR), and so on.
Many strategies have been developed to quantitate PCR products. The tradi-
tional approach of calculation is visualization by DNA band density after sepa-
ration on agarose gel, or scintillation counting of radiolabeled products. The
obvious shortcomings of these techniques—low sensitivity, bulkiness to perform,
or necessity to use radioactive materials—hamper implementation of these assays
in routine settings. The assay improvement was achieved by using automated
capillary electrophoresis and laser-induced fluorescence [26]. The need for
electrophoresis was avoided by using magnetic beads coated with streptavidin to
capture biotinylated PCR fragments [27]. After hybridization with a hapten-
labeled probe, these beads were analyzed either by flow cytometry or by immu-
noenzymatic assay based on chemiluminescent, fluorescent, or colorimetric labels.
Quantitative realtime PCR assay allows continuous monitoring of accumulation of
PCR products during the amplification reaction. This provides identification of the
cycle of near-logarithmic PCR product generation (threshold cycle) and, by
inference, the relative quantification of the template DNA present at the start of the
reaction. Because the amplification products are monitored in realtime as being
formed cycle by cycle, no postamplification handling is required. The absolute
quantification is performed according to either an internal standard coamplified
with the sample DNA, or to an external standard curve obtained by parallel
amplification of serial known concentrations of a reference DNA sequence.
However, realtime PCR has certain disadvantages such as the high cost of the
equipment and requirements for high technical skills. Despite the use of high
temperatures, nonspecific primer annealing may occur during PCR, and errors will
be exponentially amplified through multiple cycles. In order to discriminate these
products and to obtain a higher level of specificity, a hybridization step should be
included in assays.
Several methods have been developed for quantitating PCR products using a
microplate format. The tag can be attached to the DNA during the PCR reaction or
via a labeled probe. The PCR product is immobilized on a surface using biotin–
streptavidin, digoxigenin–antidigoxigenin, direct chemical conjugation, or other
methods. The DNA is then detected with a colorimetric, fluorescent, or lumines-
cent tag. The obvious advantages of microplate assays are low cost, fewer steps,
and the possibility to handle a large number of samples simultaneously. However,
in the desirable exponential PCR reaction phase, the concentration of a product is
often too low to be detected by standard methods. This technical hurdle may be
overcome using sensitive detection methods.
In recent years, bioluminescence has found rapidly expanding application in
DNA/RNA assays. With no excitation required, bioluminometric assay offers
higher detectability, wider linear range, and much simpler instrumentation than
fluorometric methods. A variety of hybridization assays has been published, most
of them describing the use of Ca2+-regulated photoproteins as reporters. The first
reports describing the use of recombinant aequorin as a reporter molecule in a
Fig. 4 Bioluminometric
nucleic acid hybridization
assays with recombinant
aequorin as a reporter
proposed by a Xiao [28] and
Siddigi [30], and b Galvan
[29]. D, digoxigenin, Aeq,
aequorin
bioluminometric nucleic acid hybridization assay appeared in 1996 [28–30]

(Fig. 4). By now photoproteins are applied as reporters in the assay of infectious
agents; quantitation and detection of gene expression during infectious disease;
detection of cancer markers, microRNAs, and SNP; quantification of the allele
burden at oncogenic somatic point mutation. Some examples for illustration are
considered below.
3.1 Bioluminescent Assay of Infectious Agents
Early detection and recognition of pathogens is extremely important for effective

treatment and prevention of epidemic diseases. The aequorin-based hybridization
assays are well suited to detect clinically important agents, specifically when
pathogens are either difficult to isolate or propagate. In the case of tuberculosis,
conventional plating techniques require approximately 4 weeks of cell growth in
an approved high biosafety facility prior to enumeration of colon-forming units
(CFU). A reverse transcription-PCR (RT-PCR) assay was developed by Actor for
rapid identification of Mycobacterium tuberculosis (MTB) infection in tissues
from murine models. Tissue samples were collected, reverse transcribed, and
amplified in the presence of biotinylated 16 s rRNA MTB specific primer
sequences. Amplicons captured on streptavidin-coated wells were coupled with a
digoxigenin labeled probe, and detected using an aequorin-conjugated antidigox-
igenin antibody. The assay allowed detection of the infection within 24 h after
tissue was obtained [31]. In a similar system, malaria pathogen following infection
of mice with Plasmodium berghei was detected within blood cells after a one-day
exposure [31].
The extremely high sensitivity of pathogen detection using bioluminescence

has been highlighted in an assay of cytomegalovirus (CMV) within patient serum
samples [31]. In this case an oligonucleotide probe was directly labeled with
aequorin. As few as 4 copies of CMV genome template were detected in less than
1 h.
Guenthner and Hart developed a quantitative competitive PCR assay of the
human immunodeficiency virus Type 1 (HIV-1) DNA or RNA. The test is based
on the presence of a competitive internal standard containing an internal 80-bp
deletion of HIV-1 gag target sequence. Using a primer pair in which one primer is
biotinylated, PCR amplicons are bound to a streptavidin-coated microplate,
denatured, and probed with a digoxigenin-labeled wild-type or internal-standard
probe. The hybridized probes are detected with antidigoxigenin antibody–aequorin
conjugates. Results indicated high sensitivity with a quantifiable range of
100–10,000 copies of HIV gag [32].
Song et al. have developed a nucleic acid sequence-based amplification
(NASBA) assay for amplification of Chlamydia trachomatis 16S rRNA and
coupled it with a microtiter plate bioluminescent assay employing aequorin for
detection and quantitation. NASBA products were hybridized with a biotinylated
capture probe and fluorescein-labeled detection probe. The capture probe was
immobilized on a streptavidin-coated plate. The hybrids were detected by an
aequorin antifluorescein conjugate. This assay can detect 1,000 molecules of 16S
rRNA providing a sensitivity equal to or greater than one genomic equivalent; it is
quantitative through four logs of bacterial load and may be useful for large-scale
epidemiological studies aimed at understanding the relationship between variables
such as antigen load, serotype, and transmission rates [33]. In a similar study,
Coombes et al. have developed a NASBA-based bioluminescent assay of
Chlamidia pneumonia RNA, which may be applied toward elucidation of the role
of differentially expressed chlamydial genes during infection and disease pathol-
ogy. They used NASBA amplification techniques coupled with bioluminescent
readout and were able to quantitate ompA RNA over nearly an 8-log linear range,
beginning at approximately 100 ompA RNA molecules. In addition, the sensitivity
of detection was tenfold greater than Northern blotting and detection using
chemiluminescent-labeled probes [34].
Doleman and colleagues developed a bioluminescence DNA hybridization
assay to detect the most deadly species of malaria, namely, Plasmodium falcipa-
rum. Of great interest is that the assay does not require PCR amplification of the
sample and is based on the competition between the target DNA (analyte) and the
biotinylated DNA for hybridization (probe) on the microplate, which is detected
by the streptavidin–aequorin conjugate signal. This bioluminescence hybridization
assay demonstrated a detection limit of 3 pg/lL and was employed to detect the
target DNA in standard and spiked human serum samples. This assay is potentially
suitable for multiplex analysis, even in laboratories that lack sophisticated
equipment [35].
3.2 Bioluminescent Assay in Oncology Diagnostics
Molecular diagnostics is an essential analytical tool in oncology. It implies

diagnosis of cancer of inherited type, identification and diagnosis of molecular
biomarkers that aid in early detection, and prediction of responses to therapies,
tests for cancer risk, and so on. Many methods have been developed for cancer
diagnostics including the ones with bioluminescent readout.
Galvan and coauthors developed a bioluminescence hybridization assay com-
bined with reverse transcriptase polymerase chain reaction to detect mRNA for
prostate-specific antigen (PSA) [29]. Because metastatic prostate cancer calls for a
therapeutic intervention that differs from that applied to organ-confined disease,
the detection of circulating PSA-expressing cells becomes a unique test for correct
disease staging. The authors used aequorin as a reporter molecule for a microtiter
well-based hybridization assay (Fig. 4b). PSA mRNA from a single cell, in the
presence of one million cells that do not express PSA, was detected, with a signal-
to-background ratio being 2.5. Typical variation coefficients obtained were 6 %.
The configuration of the proposed assay does not require labeling of PCR products
during amplification. The amplified DNA is determined by simultaneous hybrid-
ization with capture and detection probes. The applicability of the assay is not
limited to PCR products but extends to any target DNA for which two specific
oligonucleotide probes can be synthesized.
The need for early molecular markers and their detection methods in cancer
diagnosis is tremendous. Recent studies revealed that miRNA mutation or
misexpression correlate either positively or negatively with various human can-
cers. miRNAs are considered as useful early diagnostic and prognostic cancer
markers, candidates for therapeutic intervention, and targets for basic biomedical
research. However, methods for highly sensitive and rapid detection of miRNA
directly from cells that would serve as a suitable diagnostics platform are lacking.
Cissell et al. have developed an assay for detection of microRNA, miR21 in breast
cancer cells using Renilla luciferase as a label [36]. The miR21 was linked to
several cancers. The levels of miR21, for instance, were found to be elevated in
breast, liver, ovarian, pancreatic, and brain tumors compared to corresponding
normal tissues [37, 38]. Here, RLuc was conjugated to an oligonucleotide probe
complementary to the miR21 sequence. A competitive assay was set up between
miR21 from the sample and RLuc-labeled miR21 probe for binding with the
biotinylated anti-miR21 oligonucleotide probe (Fig. 5). The duplexes obtained
were immobilized on the streptavidin-activated surface. The signal was measured
after the addition of RLuc substrate coelenterazine and was correlated with the
amount of free miR21 in the sample. The hybridization assay was developed in a
microplate format with a total assay time of 1.5 h and without the need for sample
PCR-amplification, thus making it more suitable for application in clinical diag-
nostics. The optimized assay allowed a detection limit of 1 fmol. The method is
applicable for sensitive, accurate, and precise measurement of miR21 in vitro and
ex vivo.
Fig. 5 Scheme for

bioluminescence-based
hybridization assay
developed for detection of
miR21 [36]
The need for detection of minority mutation (i.e., a few mutants within a high
excess of wild-type alleles) arises frequently in the field of cancer genetics.
Routine tumor biopsies often consist of an inhomogeneous mixture of stromal cells
plus tumor cells encompassing a wide range of genetic profiles and mutations.
Quantification of the mutant allele burden (percentage of the mutant allele) is
critical for diagnosis, monitoring of therapy, and detection of minimal residual
disease. Detection of minority mutation is often important in mitochondrial disease
and mutation/polymorphism screening of pooled DNA from many individuals.
With point mutations, the challenge is to quantify the mutant allele while dis-
criminating from a large excess of the normal allele that differs in a single base-
pair. Tsiakalou et al. reported the first bioluminometric assay for quantification of
the allele burden and its application to JAK2 V617F somatic point mutation [39].
This mutation is known to be strongly associated with the myeloproliferative
disorders polycythemia vera and essential thrombocythemia [40]. The proposed
method is performed in microtiter wells and involves a single PCR for amplifi-
cation of both alleles, followed by primer extension (PEXT) reactions with allele-
specific primers (three cycles). The products are captured in microtiter wells and
detected by oligo(dT)-conjugated photoprotein aequorin. The authors
demonstrated that the luminescence signal from the mutant allele is linearly related
to the allele burden. As low as 0.85 % of the mutant allele can be detected and the
linearity extends up to 100 %. The assay is complete within 50 min after the
amplification step. The method can be applied to a large number of reported
somatic mutations where quantification of the allele burden is required.
Iliadi et al. developed two bioluminescent methods that enable absolute
quantification of the allele, with this mutation as an example [41]. The first method
exploits the ability of a nonextendable locked nucleic acid (LNA) effectively to
inhibit the PCR-amplification of the normal allele while the amplification of the
mutant allele remains unaffected. The second method employs allele-specific PCR
primers, thereby allowing the amplification of the corresponding allele only. In
both assays, absolute quantification of the mutant allele is achieved through
coamplification of the recombinant DNA internal standard (DNA competitor). The
amplified products from the target and internal standard are quantified by a
hybridization assay performed at microtiter wells and exploiting the advantages of
the aequorin reporter. The ratio of luminescence values for target DNA and DNA
competitor linearly relate to the number of JAK2 V617F allele copies initially
present in the sample. The methods allow absolute quantification of less than 300
copies of the mutant allele even in samples containing less than 1 % of the mutant
allele. The novelty and advantages of the proposed method are: (a) the concept of
competitive PCR is exploited for the absolute quantification of the mutant and
normal alleles in single-point mutations using properly designed recombinant
DNA internal standards; (b) the combination of the above principle with a
high-throughput and highly sensitive bioluminometric assay; (c) the considerably
lower cost of instrumentation and reagents compared to other methods, such as
realtime PCR and BEAMing technology; and (d) incorporation of LNA probes in
the competitive PCR simplifies the methods because it does not require two
allele-specific primers.
Bioluminescence-based systems for detection of nucleic acids appeared to be a
powerful and flexible analytical tool for accurate and rapid assay. In the case of
photoproteins, sufficient assay sensitivity allows the detection of amplified product
before the linear relationship of the target to product is lost, as well as the direct
detection of low copies of unamplified target. The use of such analytical systems
considerably improves the assays in terms of simplicity and costs.
4 Multianalytical Bioluminescent Assay
The multianalyte approach allows intensification of the assay procedure especially

when the proper diagnostics require simultaneous detection of several analytes in
one sample. Both immuno- and molecular assays of the kind were developed using
either different bioluminescent reporters or luciferases in tandem with the other
reporters.
Ito et al. [42] developed a highly sensitive and rapid immunoassay of two
antigens involving aequorin and firefly luciferase labels. The authors detected two
couples of antigens: prostatic acid phosphatase (PAP) with PSA or PSA with
alphafetoprotein (AFP). As tracers, chemical conjugate aequorin–antidigoxigenin
Fab fragment and biotinylated in vivo firefly luciferase were applied. A Ca2+
injection triggered flash-type aequorin bioluminescence and then the mixture of
luciferin, ATP and Mg2+ was placed into the wells to initiate luciferase biolumi-
nescence. With the developed technique applied, PAP, PSA, and AFP were
detected in standard and then in clinical sera. The values obtained from the
developed simultaneous assay were in good correlation with those derived from
conventional methods.
Two kinds of biotinylated in vivo firefly Luciola lateralis luciferases with
different bioluminescent spectra maxima—559 nm and 607 nm—were applied for
simultaneous assay of pepsinogen I and pepsinogen II [43]. To divide signals,
several optic filters were applied, but the high signals’ overlap and long way to
form immunocomplexes made the assay rather bulky.
Fig. 6 Dual analyte single-well bioluminescence assay based on photoprotein obelin variants
with substantially altered bioluminescence spectra and kinetics. a Bioluminescence of mixture of
obelins transmitted through filter I (fast violet signal) and filter II (slow green signal); dashed
line, time for filter replacement. Upper inset, bioluminescence spectra of obelins (colored lines)
and optical filter transmission (black lines). r.l.u., relative light units. b Scheme for simultaneous
solid-phase immunoassay of two targets
With site-directed mutagenesis applied, photoprotein obelin mutants with

substantially altered bioluminescence spectral and kinetic characteristics were
obtained: W92F, H22E emitting fast (kd = 0.6 s-1) violet signal (kmax = 387 nm)
and Y138F with slow (kd = 6.1 s-1) greenish light (kmax = 498 nm) with small
spectral overlapping [44]. Using those as reporters, a dual analyte single-well
bioluminescence assay was developed based on the spectral and time signal res-
olution (Fig. 6). The approach was successfully applied to simultaneous immu-
noassay of: (a) total and IgG-bound prolactins [45]; (b) gonadotropic hormones,
luteinizing (lutropin or hLH) and follicle stimulating (hFSH); (c) gene allelic
variants at SNP genotyping of the human F5 gene encoding Factor V Leiden
polymorphism 1691 G ? A (R506Q) [46]. Many clinical samples were investi-
gated and the obtained results were in good agreement with those obtained by
traditional techniques.
Tannous et al. proposed dual analyte bio/chemiluminometric method for
simultaneous genotyping of IVS-1-110 locus of the human globin gene in a single
microtiter well [47]. They used aequorin and alkaline phosphatase as reporters
with consequent triggering of their bio/chemiluminescence reaction. The principle
of the approach is illustrated in Fig. 7. Genomic DNA, isolated from whole blood,
was first subjected to polymerase chain reaction using primers flanking the
polymorphic site. A single oligonucleotide-ligation reaction employing two allele-
specific probes, labeled with biotin and digoxigenin, and a common probe carrying
a characteristic tail was then performed. When the probes perfectly matched their
target sequence, they were joined covalently by ligase, whereas a mismatch at the
junction inhibited ligation. The ligation products were captured in a microtiter well
through hybridization of the tail with an immobilized complementary oligonu-
cleotide and were detected by adding a mixture of the streptavidin–aequorin
complex and antidigoxigenin-alkaline phosphatase conjugate. The aequorin was
Fig. 7 Principle of
bioluminometric SNP
genotyping by
oligonucleotide ligation
reaction. N probe is specific
for normal allele, M probe for
mutant allele, and C probe is a
common probe. BSA, bovine
serum albumin; ALP, alkaline
phosphatase
measured first by adding Ca2+, then the wells were washed and the chemilumi-
nescence of bound alkaline phosphatase was measured after incubation with
substrate. The ratio of the obtained luminescence signals gives the genotype of
each sample.
The same dual-analyte bio/chemiluminometric assay but for detection of PEXT
reaction products was applied by Konstantou et al. for two SNPs of human
mannose-binding lectin (MBL2) gene (-550 and -221) and one SNP of
cytochrome P450 gene CYP2D6 (CYP2D6*3) [48]. MBL2 is a key component of
the innate immune system, and its deficiency is associated with the increased
susceptibility to various infections and autoimmune disorders. The CYP2D6 is
important because it is involved in the metabolism of many commonly prescribed
drugs [49]. PCR-amplified DNA fragments that span the SNP of interest are
subjected to two PEXT reactions using normal and mutant primers in the presence
of digoxigenin-dUTP and biotin-dUTP. The primers perfectly complementary with
the target DNA are extended by DNA polymerase thus forming digoxigenin-
labeled or biotin-labeled products that are then mixed and analyzed by dual bio/
chemiluminometric assay. The proposed method provides good discrimination
between the two alleles. Patient genotypes showed 100 % concordance with direct
DNA sequencing data.
Elenis et al. developed a method of simultaneous genotyping of two common
SNPs within the toll-like receptor 4 (TLR4) gene, that is, A896G and C1196T [50].
The method consists of a single PCR of the region spanning both polymorphic
sites, followed by a quadruple PEXT reaction in a single tube. Biotinylated
nucleotide is incorporated into extended primers. All four products are captured on
streptavidin-coated microtiter wells and detected with a combination of four
reporters, aequorin, and alkaline phosphatase, b-galactosidase, and horseradish
peroxidase. Bio/chemiluminescence of each reporter was measured sequentially
after washing the wells and incubation with a corresponding substrate. For each
SNP, 46 individuals were genotyped. The accuracy of this method was confirmed
by sequencing. The proposed quadruple-allele bio/chemiluminometric approach

provides an accurate, simple, rapid, and reproducible method for high-sample–
throughput SNP genotyping.
5 Bioluminescent Binding Assay in Homogeneous Format
Homogeneous format of any assay simplifies detection by circumventing immo-

bilization, incubation, and washing steps that are required in both ELISA and DNA
hybridization assays. The detection in this case is performed due to emergence (or
quenching) of the signal during the formation of specific complexes. This kind of
format is essential for high-throughput assay in vivo and ex vivo, for discovering
and investigation of macromolecules’ interactions, protein activity, and the like.
(See for reference, the exhaustive review [51]). The subject is considered in this
volume in another chapter. Nevertheless preliminary investigations on the
development of the assay design are carried out on simple in vitro examples and
moreover there are some communications on homogeneous assays in real
physiological samples. Here we just briefly dwell on some principal points.
There are two general approaches for homogeneous bioluminescent binding assay
based on BRET phenomenon or on split-protein reassembly technology (Fig. 8).
BRET is a naturally occurring phenomenon resulting from radiationless energy
transfer between a bioluminescent donor and fluorescent acceptor [52, 53]. The
efficiency of the process is known to depend on overlapping of donor luminescence
and acceptor absorption spectra and on the distance between the proteins. The last
one must be around 10 nm and is determined by the presence and effectiveness of
molecules’ biospecific interactions including antigen–antibody, ligand–acceptor,
and complementary oligonucleotides. The BRET-based immunoassay principle is
illustrated by hen egg lysozyme analysis [54] (Fig. 8a). The pair of Renilla luciferase
(donor) yellow fluorescent protein (EYFP, acceptor) was applied as a reporter in a
noncompetitive homogeneous immunoassay based on antigen-dependent reassoci-
ation of antibody variable domains. Two himeric proteins, an antibody heavy-chain
variable fragment, luciferase (VH-RLuc) and antibody light-chain variable fragment,
yellow fluorescent protein (VL-EYFP) emit correspondingly blue bioluminescent
(max = 475 nm) and yellow fluorescent (max = 525 nm) signals. The addition of
antigen induces dimerization of the two proteins and tethering of RLuc and EYFP. As
a result an effect of energy transfer between bioluminescent donor (RLuc) and
fluorescent acceptor (EYFP) arises and the additional yellow peak is observed in the
blue-centered luciferase’s spectrum. The luminescence ratio of 525/475 nm depends
on the antigen concentration and is used for its evaluation. A more effective pair for
BRET-based assay was developed recently, with a highly bright Gaussia luciferase
variant (hGLuc) as a donor and highly photostable red fluorescent protein tdTomato
as an acceptor [55]. The hGLuc is more suitable inasmuch as its emission
(max = 470 nm) is far from that of tdTomato (max = 580 nm) and overlaps well
with the excitation of tdTomato (max = 554 nm). The pair gave rather persistent
Fig. 8 Two general

approaches for homogeneous
bioluminescent binding
assay: a based on BRET
phenomenon and b on
split-protein reassembly
technology. RLuc, Renilla
luciferase; EYFP, yellow
fluorescent protein
signals for different buffers and selected pHs, and is insensitive to complicated
sample matrices such as serum. The pair has a large luminescence spectra separation
(110 nm) and provides high assay sensitivity. Instead of GFP the quantum dots are
used as energy acceptors in nucleic acid hybridization assays [56, 57] or synthetic
fluorochromes such as Cy3 or Cy3.5 [58].
In split-protein analytical systems, two polypeptides serve as biospecific rec-
ognition elements attached to the fragmented reporter. The protein domains
interact with the desired target resulting in a ternary complex that drives the
reassembly of the split-protein reporter recovering its activity [59] (Fig. 8b). For
successful creation of a split reporter, a few criteria must be met: each reporter
fragment by itself should not exhibit any activity, the affinity of fragments in the
absence of the target should be negligible, and the reassembled split-protein must
provide an easily measurable readout. This strategy has been successfully used in
reassembling several luciferases as reporters, from R. reniformis [60], firefly [61],
G. princeps [62], and photoprotein aequorin [63]. Here are only several examples
but in reality, the range of biological activities and processes that can be monitored
with a bioluminescence readout using split-protein reassembly both in in vitro and
in vivo settings is unprecedented.
6 Summary, Conclusions, and Outlook
Bioluminescent proteins have been intensively used as high sensitive reporters in

all kinds of binding assays including immunoassay, protein–protein and protein–
ligand assays, and nucleic acid hybridization assays. They are distinctly divided
into two groups: luciferases and Ca2+-regulated photoproteins. Photoproteins are

resistant to chemical modifications and this makes the synthesis of highly active
conjugates with different biospecific molecules to be applied as the labels possible.
By the very nature of photoproteins, the bioluminescent signal of these labels does
not depend on oxygen and substrate and practically always correlates with the
label concentration linearly. Luciferases, by contrast, are not stable and the labels
based on those are usually obtained by genetic fusing. The problems with proper
folding of the fused proteins under bacterial expression require the involvement of
eukaryotic expressing cells (mammalians, insects) that are rather expensive in
terms of routine application. It should be noted, however, that the advantage of
luciferase-based labels is the ability to increase the assay sensitivity by accumu-
lating the signal at the substrate excess. In principle, luciferases seem more suit-
able reporters for studies ex vivo and in vivo. As to Ca2+-regulated photoproteins,
they are also used for in vivo investigations as unexcelled probes in tracing
intracellular calcium fluxes.
The proteins with altered properties (stability, luminescence spectra, signal
kinetics, etc.) were obtained with the help of site-directed mutagenesis. The novel
variants obtained provide the development of multianalytical systems aimed at
simultaneous detection of several targets in one sample. This approach benefits the
assay in terms of price and time, and excludes the mistakes of separate
measurements.
A high sensitivity of bioluminescent labels promotes miniaturization of the
assay, that is, elaboration of microfluid and nanochip technologies and portable
light-detection devices. The fastest response of these labels provides the fastest
detection of analyte which is very important for cases of urgent diagnostics. Owing
to portability, low cost, and the lack of need for skilled personnel, such analytical
systems hold much promise for applications in clinical diagnostics.
References
1. Wild D (2005) The immunoassay handbook, 3rd edn. Elsevier, Oxford

2. Ando Y, Niwa K, Yamada N, Enomoto T, Irie T, Kubota H, Ohmiya Y, Akiyama H (2008)
Firefly bioluminescence quantum yield and color change by pH-sensitive green emission. Nat
Photonics 2:44–47
3. Tatsumi H, Fukuda S, Kikuchi M, Koyama Y (1996) Construction of biotinylated firefly
luciferases using biotin acceptor peptides. Anal Biochem 243:176–180
4. Seto Y, Iba T, Abe K (2001) Development of ultra-high sensitivity bioluminescent enzyme
immunoassay for prostate-specific antigen (PSA) using firefly luciferase. Luminescence
16:285–290
5. Minekawa T, Ohkuma H, Abe K, Maekawa H, Arakawa H (2009) Development of ultra-high
sensitivity bioluminescent enzyme immunoassay for hepatitis B virus surface antigen using
firefly luciferase. Luminescence 24:394–399
6. Maeda M (2003) New label enzymes for bioluminescent enzyme immunoassay. J Pharm
Biomed Anal 30:1725–1734
7. Loening AM, Fenn TD, Wu AM, Gambhir SS (2006) Consensus guided mutagenesis of
Renilla luciferase yields enhanced stability and light output. Prot Eng Des Sel 19(9):391–400
8. Stepanyuk GA, Unch J, Malikova NP, Markova SV, John Lee J, Vysotski ES (2010)
Coelenterazine-v ligated to Ca2+-triggered coelenterazine-binding protein is a stable and
efficient substrate of the red-shifted mutant of Renilla muelleri luciferase. Anal Bioanal Chem
398:1809–1817
9. Krasitskaya VV, Burakova LP, Pyshnaya IA, Frank LA (2012) Bioluminescent reporters for
identification of gene allelic variants. Rus J Bioorg Chem 38(3):298–305
10. Burbelo PD, Goldman R, Mattson TL (2005) A simplified immunoprecipitation method for
quantitatively measuring antibody responses in clinical sera samples by using mammalian-
produced Renilla luciferase-antigen fusion proteins. BMC Biotechnol 5(22):692–699
11. Burbelo PD, Ching KH, Mattson TL et al (2007) Rapid antibody quantification and
generation of whole proteome antibody response profiles using LIPS (luciferase
immunoprecipitation systems). Biochem Biophys Res Commun 352:889–895
12. Ramanathan R, Burbelo P, Groot S et al (2008) A luciferase immunoprecipitation systems
assay enhances the sensitivity and specificity of diagnosis of Strongyloides stercoralis
infection. J Infect Dis 198:444–451
13. Burbelo PD, Issa AT, Ching KH, Cohen JI, Iadarola MJ, Marques A (2010) Rapid, simple,
quantitative and highly sensitive antibody detection for lyme disease. Clin Vaccine Immunol
17(6):904–909
14. Venisnik KM, Olafsen T, Loening AM, Iyer M, Gambhir SS, Wu AM (2006) Bifunctional
antibody-Renilla luciferase fusion protein for in vivo optical detection of tumors. Protein Eng
Des Sel 19(10):453–460
15. Markova SV, Golz S, Frank LA, Kalthof B, Vysotski ES (2004) Cloning and expression of
cDNA for a luciferase from the marine copepod Metridia longa. J Biol Chem
279(5):3212–3217
16. Borisova VV, Frank LA, Markova SV, Burakova LP, Vysotski ES (2008) Recombinant
Metridia luciferase isoforms: expression, refolding and applicability for in vitro assay.
17. Verhaegen M, Christopoulos TK (2002) Overexpression, purification and analytical
application of a bioluminescent reporter for DNA hybridization. Anal Chem 74:4378–4385
18. Nakajima Y, Kobayashi K, Yamagishi K, Enomoto T, Ohmiya Y (2004) cDNA cloning and
characterization of a secreted luciferase from the luminous japanese ostracod, Cypridina
noctiluca. Biosci Biotechnol Biochem 68:565–570
19. Wu C, Kawasaki K, Ogawa Y, Yoshida Y, Ohgiya S, Ohmiya Y (2007) Preparation of
biotinylated Cypridina luciferase and its use in bioluminescent enzyme immunoassay. Anal
Chem 79:1634–1638
20. Wu C, Irie S, Yamamoto S, Ohmiya Y (2009) A bioluminescent enzyme immunoassay for
prostaglandin E2 using Cypridina luciferase. Luminescence 24:131–133
21. Liu ZJ, Vysotski ES, Chen CJ, Rose JP, Lee J, Wang BC (2000) Structure of the Ca2+-
regulated photoprotein obelin at 1.7 Å resolution determined directly from its sulfur
substructure. Protein Sci 9:2085–2093
22. Head JF, Inouy S, Teranishi K, Shimomura O (2000) The crystal structure of the photoprotein
aequorin at 2.3 Å resolution. Nature 405:372–376
23. Markova SV, Vysotski ES, Blinks JR, Burakova LP, Wang BC, Lee J (2002) Obelin from the
bioluminescent marine hydroid Obelia geniculata: cloning, expression, and comparison of
some properties with those of other Ca2+-regulated photoproteins. Biochemistry
41:2227–2236
24. Frank LA (2010) Ca2+-regulated photoproteins: effective immunoassay reporters. Sensors
10:11287–11300
25. Sgoutas DS, Tuten TE, Verras AA, Love A, Barton EG (1995) AquaLite bioluminescence
assay of thyrotropin in serum evaluated. Clin Chem 41:1637–1643
26. Fasco MJ, Treanor CP, Spivack S, Figge HL, Kaminsky LS (1995) Quantitative RNA-
polymerase chain reaction-DNA analysis by capillary electrophoresis and laser-induced
fluorescence. Anal Biochem 224:140–147
27. Vlieger AM, Medenblik AM, van Gijlswijk RP, Tanke HJ, van der Ploeg M, Gratama JW,
Raap AK (1992) Quantitation of polymerase chain reaction products by hybridization-based
assays with fluorescent, colorimetric, or chemiluminescent detection. Anal Biochem 205:1–7
28. Xiao L, Chumfu Y, Nelson CO (1996) Quantitation of RT-PCR amplified cytokine mRNA by
aequorin-based bioluminescence immunoassay. J Immunol Methods 199:139–147
29. Galvan B, Christopoulos TK (1996) Bioluminescence hybridization assay using recombinant
aequorin. Application to the detection of prostate-specific antigen mRNA. Anal Chem
68:3545–3550
30. Siddigi AM, Jennings VM, Kidd MR, Actor JK, Hunter RL (1996) Evaluation of
electrochemiluminescence and bioluminescence-based assays for quantitating specific
DNA. J Clin Lab Anal 10:423–431
31. Actor JK (2000) Bioluminescent quantitation and detection of gene expression during
infectious disease. Comb Chem High Throughput Screen 3(4):277–288
32. Guenthner PC, Hart CE (1998) Quantitative, competitive PCR assay for HIV-1 using a
microplate-based detection system. Biotechniques 24(5):810–816
33. Song X, Coombes BK, Mahony JB (2000) Quantitation of Chlamidia trachomatis 16S rRNA
using NASBA amplification and bioluminescent microtiter plate assay. Comb Chem High
Throughput Screen 3(4):303–313
34. Coombes BK, Mahony JB (2000) Nucleic acid sequence based amplification (NASBA) of
Chlamydia pneumoniae major outer membrane protein (ompA) mRNA with bioluminescent
detection. Comb Chem High Throughput Screen 3(4):315–327
35. Doleman L, Davies L, Rowe L, Moschou EA, Deo S, Daunert S (2007) Bioluminescence
DNA hybridization assay for Plasmodium falciparum based on the photoprotein aequorin.
Anal Chem 79:4149–4153
36. Cissell KA, Rahimi Y, Shrestha S (2008) Bioluminescence-based detection of microRNA,
miR21 in breast cancer cells. Anal Chem 80:2319–2325
37. Zhu S, Si ML, Wu H, Mo YY (2007) MicroRNA-21 targets the tumor suppressor gene
tropomyosin 1 (TPM1). J Biol Chem 282:14328–14336
38. Si ML, Zhu S, Wu H, Lu Z, Wu F, Mo YY (2007) miR-21-mediated tumor growth. Oncogene
26:2799–2803
39. Tsiakalou V, Petropoulou M, Ioannou PC (2009) Bioluminometric assay for relative
quantification of mutant allele burden: application to the oncogenic somatic point mutation
JAK2 V617F. Anal Chem 81:8596–8602
40. Levine RL, Wadleigh M, Cools J, Ebert BL, Wernig G, Huntly BJ, Boggon TJ, Wlodarska I,
Clark JJ, Moore S, Adelsperger J, Koo S, Lee JC, Gabriel S, Mercher T, D’Andrea A,
Fröhling S, Döhner K, Marynen P, Vandenberghe P, Mesa RA, Tefferi A, Griffin JD, Eck MJ,
Sellers WR, Meyerson M, Golub TR, Lee SJ, Gilliland DG (2005) Activating mutation in the
tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid
metaplasia with myelofibrosis. Cancer Cell 7(4):387–397
41. Iliadi A, Petropoulou M, Ioannou PC, Christopoulos TK, Anagnostopoulos NI, Kanavakis E,
Traeger-Synodinos J (2011) Absolute quantification of the alleles in somatic point mutations
by bioluminometric methods based on competitive polymerase chain reaction in the presence
of a locked nucleic acid blocker or an allele-specific primer. Anal Chem 83:6545–6551
42. Ito K, Nishimura W, Maeda M, Gomi K, Inouye S, Arakawa H (2007) Highly sensitive and
rapid tandem bioluminescent immunoassay using aequorin labeled Fab fragment and
biotinylated firefly luciferase. Anal Chem Acta 58:245–251
43. Ohkuma H, Abe K, Kosaka Y, Maeda M (2000) Detection of luciferase having two kinds of
luminescent colour based on optical filter procedure: application to an enzyme immunoassay.
44. Frank LA, Borisova VV, Markova SV, Malikova NP, Stepanyuk GA, Vysotski ES (2008)
Violet and greenish photoprotein obelin mutants for reporter applications in dual-color assay.
Anal Bioanal Chem 391:2891–2896
45. Kudryavtsev AN, Krasitskaya VV, Petunin AI, Burakov AY, Frank LA (2012) Simultaneous
bioluminescent immunoassay of serum total and IgG-bound prolactins. Anal Chem
84:3119–3124
46. Krasitskaya VV, Kudryavtsev AN, Shimomura O, Frank LA (2013) Obelin mutants as
reporters in bioluminescent dual-analyte binding assay. Anal Methods 5:636–640
47. Tannous BA, Verhaegen M, Christopoulos TK, Kourakli A (2003) Combined flash- and
glow-type chemiluminescent reactions for high-throughput genotyping of biallelic
polymorphisms. Anal Biochem 320:266–272
48. Konstantou J, Ioannou PC, Christopoulos TK (2007) Genotyping of single nucleotide
polymorphisms by primer extension reaction and dual-analyte bio/chemiluminometric assay.
Anal Bioanal Chem 388:1747–1754
49. Daly AK (2003) Pharmacogenetics of the major polymorphic metabolizing enzymes. Fundam
Clin Pharmacol 17:27–41
50. Elenis DS, Ioannou PC, Christopoulos TK (2009) Quadruple-allele chemiluminometric assay
for simultaneous genotyping of two single-nucleotide polymorphisms. Analyst 134:725–730
51. Ozava T, Yoshimura H, Kim SB (2013) Advances in fluorescence and bioluminescence
imaging. Anal Chem 85:590–609
52. Xia Z, Rao J (2009) Biosensing and imaging based on bioluminescence resonance energy
transfer. Curr Opin Biotechnol 20:37–44
53. Roda A, Guardigli M, Michelini E, Mirasoli M (2009) Nanobioanalytical luminescence:
Förster-type energy transfer methods. Anal Bioanal Chem 393:109–123
54. Arai R, Nakagawa H, Tsumoto K, Mahoney W, Kumagai I, Ueda H, Nagamune T (2001)
Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence
resonance energy transfer. Anal Biochem 289:77–81
55. Li F, Yu J, Zhang Z, Cui Z, Wang D, Wei H, Zhang XE (2013) Use of hGluc/tdTomato pair
for sensitive BRET sensing of protease with high solution media tolerance. Talanta
109:141–146
56. Cissel KA, Campbell S, Deo SK (2008) Rapid, single-step nucleic acid detection. Anal
Bioanal Chem 391:2577–2581
57. Kumar M, Zhang D, Broyles D, Deo SK (2011) A rapid, sensitive and selective
bioluminescence resonance energy transfer (BRET)-based nucleic acid sensing system.
Biosens Bioelectron 30:133–139
58. Yamakawa Y, Ueda H, Kitayama A, Nakamune T (2002) Rapid homogeneous immunoassay
of peptides based on bioluminescence resonance energy transfer from firefly luciferase.
J Biosci Bioenq 93(6):537–542
59. Shekhman SS, Ghosh I (2011) Split-protein systems: beyond binary protein-protein
interaction. Curr Opin Chem Biol 15(6):789–797
60. Mie M, Thuy NPB, Kobatake E (2012) Development of a homogeneous immunoassay
system using protein A fusion fragmented Renilla luciferase. Analyst 137:1085–1089
61. Ohmuro-Matsuyama Y, Chung C-I, Ueda H (2013) Demonstration of protein-fragment
complementation assay using purified firefly luciferase fragment. BMC Biotechnol 13:31–39
62. Kim SB, Takenaka Y, Torimura M (2011) A bioluminescent probe for salivary cortisol.
Bioconjug Chem. 22(9):1835–1841
63. Scott SD, Hamorsky KT, Ensor CM (2011) Cyclic AMP receptor protein-aequorin molecular
switch for cyclic AMP. Bioconjug Chem 22(3):475–481
Index
Note: Page numbers followed by ‘‘f’’ and ‘‘t’’ indicate figures and tables respectively
0–9 Alcohol dehydrogenase (ADH), 87, 90, 95, 96,

1,4-benzoquinone, 74, 78, 79, 82t, 91, 93, 95, 98, 99, 101t
98, 99 activity measurement, scheme of, 89f
dependence of induction period on con- activity under different concentration of
centration of, 90f deltamethrin, 102f
under variation of biomass of algae and, 94f
FMN concentration, 80f in coupling of enzymatic reactions, 88f
luciferase concentration, 79f in pond water study, 93f
order of bioassay components, 81f Aliivibrio, 23, 39, 40t, 41, 47, 51
time of reagent incubation, 82f regulatory genes, 46
17a-ethynylestradiol (EE2) testing, 137 Alkane aliphatic hydrocarbons, 122–124
2-hydroxy-3’,4’-dichlorobiphenyl, 127 in seawater, 123
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), Alkanes, 122, 123, 124, 126, 139
130, 133, 134, 135, 138 Alpha–emitting radionuclides, 103
2,4’-dichlorodiphenyldichloroethylene (2,4’- Antigen–antibody complex (Ag–Ab), 177, 192
DDE), 136 Aposematism (warning display), 4, 5, 7–8
(2S,4S)-2-methyl-2,3,3,4-tetra- in Lampyridae, 7
hydroxytetrahydrofuran borate, 49 luminescence
3-hydroxybutanoyl-HSL, 48–49 in centipedes, 8
3-oxo-hexanoyl-homoserine lactone, 49 in millipedes, 8, 8f
3’:5’-cyclic AMP (cAMP), 49 in marine organisms, 8–9
4a-hydroxy-FMNH product, 51 in mushrooms, 8
4n-nonylphenol, 136 in poisonous butterflies, 8
(4S)-4,5-dihydro-2-(6-hydroxy-2-benzothiaz- Aryl hydrocarbon receptor (AhR), 130
olyl)-4-thiazolecarboxylic acid, 15 Autoinducers, 47, 48–49, 163
6,7-dimethyl-8-ribityllumazine, 46 Automated capillary electrophoresis, 184
A B
Abortive infection, 159 Bacterial bioluminescent assays, detection of
Acetate kinase, 180 organic compounds using, 114–115
Acinetobacter baylyi ADP1, 123, 124 benzene, toluene, ethylbenzene, and xylene
Acinetobacter bioreporter (BTEX), 115–120. See also BTEX
DF4/PUTK2, 128 bioreporters
DF4-8, 128 alkane aliphatic hydrocarbons, 122–124
Acyl-homoserine lactone (acyl-HSL) synthase, biphenyls, 125–127
46 chlorinated aliphatic hydrocarbons,
AhR nuclear translocator (ARNT) gene/pro- 124–125
tein, 130, 134, 135 phenol and derivatives, 127–128

DOI: 10.1007/978-3-662-43385-0, Ó Springer-Verlag Berlin Heidelberg 2014
200 Index
Bacterial bioluminescent assays, detection of in prey attraction, 10–12

organic compounds using (cont.) searchlight, 13
polychlorinated biphenyls, 125–127 night-vision illumination, 13
polycyclic aromatic hydrocarbons, self-defense, 4–10
120–122. See also PAH bioreporters aposematism, 4, 7–8
Bacterial luciferase, 42, 157 Batesian mimicry, 5
bioluminescent system, 70 photophores, 5, 6
shortcomings of, 70 Bioluminescent assays
enzyme-substrate interactions, 69 application areas, 75–76
luciferase system, 70 enzymatic bioassays. See Enzymatic
NAD(P)H:FMN-oxidoreductase bioluminescent bioassays
system, 70 enzyme incubation, 81–83
Bacterial luminescence existing methods, disadvantages of, 75
biochemistry of, 42–43 immobilized enzymes, 76
lux genes (see lux genes) varying the composition of, 78–80
origin and function of, 51–53 of infectious agents, 185–186
fatty-acid reductase genes, 52 NADH:FMN-oxidoreductase-luciferase
flavin cofactor, 51 (see NADH:FMN-oxidoreductase-
growth dependence actors, 52 luciferase)
halotolerant flavodoxin, 53 in oncology diagnostics, 187–189
species of, 39–42 reaction mixture, varying order
ecological incidence and diversity, 42 of components, 80–81
and ecological sources of, 40–41t Bioluminescent binding assay, in
not-yet cultured species, 41–42 homogeneous format, 192–193,
Bacterial lux genes. See lux genes 193f
Bacteriophages, 156, 157, 159, 160, 161–162, Bioluminescent enzyme system technology
164, 165, 166 (BEST), 70
bioluminescent, 167 Bioluminescent organisms
Batesian mimicry, 5, 8 deep-sea benthos, 26–27
BEAMing technology, 189 defensive reactions, 4–5
Beta-emitting radionuclides, 103 diversity of, 5f
Beta-galactosidase, 157 marine taxa, 19
Bioluminescence coastal water, 21–22
application viability of, 69 intertidal zone, 19–20
assays, reagents for (see Bioluminescent in midwater, 23–26
assays) occurrence, 4
by bacteria, 38 prey attraction, hypotheses, 10
colonies of P. mandapamensis, 39f terrestrial taxa, 14
luciferase, 42 in air, 16–17
origin and function of (see under Bac- in freshwater, rarity, 17–19
terial luminescence) on land, 14–16
oxygen-dependence of, 38 in underground, 17
species of (see under Bacterial Bioluminescent plaques, 160
luminescence) Bioluminometric SNP genotyping, 191f
cDNA coding, 176 Biotin-dUTP, 191
kinetics parameters, 74 Biotin–streptavidin conjugation, 184
as reporter system, 158 and avidin system, 180f
Bioluminescence, function of Biphenyls, 125–127
camouflage, 12–13 gene cluster (bph), 125, 126
functionless bioluminescence, 14 Bisphenol-A (BPA), 139
intraspecific communication, 12 Blooming water, bioluminescent control for,
light organ lure, 10 95–99
attraction of spore-dispersing organ- based on coupled enzyme system, 96f
isms, 11 contamination of MES, 98
Index 201
growth of microalgae biomass and, 97f D

sensitivity of assays to contamination, 99 Deep-sea benthos, 26–27
BLYAS, 138, 139 Dichlorodiphenyltrichloroethane (DDT), 113
BLYES, 138, 139 Digoxigenin–antidigoxigenin conjugation,
BRET (bioluminescence resonance energy 184, 186, 189, 191
transfer) phenomenon, 192, 193f Digoxigenin-dUTP, 191
BSA (bovine serum albumin), 29, 78 Dioxin-responsive elements (DREs), 130
BTEX (benzene, toluene, ethylbenzene, and bioluminescent reporter, 133
xylene), 115–120. See also BTEX Direct chemical conjugation, 184
bioreporters Direct reaction coupling, 90
BTEX bioreporters, 115–117 DNA barcoding, 28
firefly luciferase gene (luc), 118 DNA hybridization assays, 186, 192
isopropylbenzene (ipb) pathway, 119 for Plasmodium falciparum (malaria), 186
for organic contaminants and DNA hybridization technologies, 157
representative environmental DNA/RNA assays, 183
applications, 116–117t DTT (dithiothreitol), 78, 80f
P. putida TVA8 BTEX bioreporter, 119 Dual analyte single-well bioluminescence
T-2 toluene degradation (tod) pathway, assay, 190, 190f
119
in toluene benzene utilization (tbu) path-
way, 118 E
and TOL-encoded pathway, 118 Ecological monitoring, 70, 71, 76, 91, 105
EGFP (enhanced green fluorescent protein)-
based fluorescent reporter strain,
C 124
Calanoids, 23, 25 ELISA (enzyme-linked immunosorbent
Ca2+-regulated photoproteins, 182, 183, 184, assay), 192
194 Endocrine-disrupting chemicals (EDCs), 136
Camouflage, 12–13 eukaryotic cell-based bioluminescent
Camp receptor protein (CRP), 49 assays for, 131–132t
celB glycosidase, 164 Endolysins, 156, 166
Chemical-activated luciferase expression Enterobacteriaceae, 39
(CALUX) assay, 134, 135 Environmental toxicity, 86
H4IIE-luc cell line-based, 134, 135 Enzymatic bioluminescent bioassays. See also
variation in results, 135 Bioluminescent assays
Chemically synthesized substrates, 177 designing new assays, 87–91
Chlorinated aliphatic hydrocarbons, 124–125 coupling enzymes, example of, 88f
1,1,1 trichloroethane (TCA), 124 enzyme interaction, 90
dichloromethane (DCM), 125 light emission process, 88
trichloroethylene (TCE), 124 enzyme activity measurement
Coastal ostracod (Vargula hilgendorfii), 7f NADH-dependent dehydrogenases, 89
Coelenterazine, 4, 21, 23, 24–25, 180, 181, proteolytic enzymes, 88f, 89
182 for pesticides, 99–103
biosynthesized by copepods, 26f influence of toxic substance on, 101t
Competition-for-substrate method, 90 pond water analysis, parameters of, 93f
Competitive immunoassay, 177, 178f principles of, 71–73
Counter-illumination, 4, 12 biotesting diagram, 73f
hypothesis, 13 coupled enzyme system (see
in marine mesopelagic dwellers, 13 NADH:FMN-oxidoreductase-
CRISPR systems, 159 luciferase)
Cypridinid luciferin, 21–22, 22f exogenous compounds on, 72
Cytochrome P450 gene CYP2D6 luciferase biotesting, 72, 72f
(CYP2D6*3), 191 modified diagram, 73f
Cytomegalovirus (CMV), assay of, 186 single system of, 86–87
202 Index
for toxicological bioassay, 91–95, 99–103 H

Enzymes, 179 Harvey, Newton, 27
auxiliary label enzyme, 180 HELNa bioreporters, 138
immobilized, 75, 76, 86 HELNb bioreporters, 138
incubating in test solution, 81–83 High-performance liquid chromatography
of luminous bacteria, 69, 70, 72 (HPLC), 113–114
proteolytic, 89, 100 Homogeneous immunoassay, 177, 192
test system, 87 Human HepG2-derived HG2L1.1c3 cell line,
Enzymolum, 76, 78f, 104 133, 134
design of, 78 Human immunodeficiency virus
in toxicological bioassay of air, 85–86 Type 1 (HIV-1), 186
comparative characteristics of, 86t Human mannose-binding lectin (MBL2) gene,
Escherichia coli cells, 162, 167, 179 191
DH5a, 118 Humanized bacterial luciferase, 142
fre-like gene, 43 Hydroxylated PCBs (OH-PCBs), 126
serotype O157:H7, 163
Estrogen response elements (EREs), 136
Eukaryotic cell-based bioluminescent assays I
dioxin-like activities, 130t Ice nucleation protein, 157
endocrine-disrupting activities, 131–132t Immobilization
enzymes in bioluminescent assays
coupled enzyme system, 76
F effective methods of, 76, 77t
Felix-O1 reporter, 162, 163 reagents
Fireflies, 5, 8, 11, 16, 27, 179 in benzoquinone, 80f, 82f
Firefly luciferase, 75, 114, 118, 128, 142, 157, in CuSO4, 80f, 81, 82f
179, 182, 189 enzyme-stabilizing additives in, 78
luc genes, 118, 158, 165 FMN in, 79, 80f
Firefly luciferin, 4 variation of luciferase, 79f
Firefly mimics, 8 variation of NADH content, 79f, 80
Firefly squid (Watasenia scintillans), 5, 6f, 21 varying the composition of, 78–79
Fishing-lines, 10 Immunoassay, 177–179
Flavin mononucleotide (FMN), 70, 79, 81, 81f, homogeneous, 177, 192
98 luciferase as labels, 179–183
reduced FMN (FMNH2), 42, 43, 51, 88, Immunocomplex detection system, 178
88f, 158, 161 In vitro binding assays, 177
Fluoromycobacteriophages, 165 Information on 3D structure, 177
Fungus gnats (Arachnocampa luminosa), 10, Integrated bioluminescent methods, 104. See
11, 11f, 15, 16 also NADH:FMN-oxidoreductase-
luciferase
Isopropylbenzene (ipb)
G pathway, 119, 120
Gas chromatography coupled with mass spec-
trometry (GC/MS), 114
Glow-worms, 16 J
Gram-negative pathogens, 39 JAK2 (Janus kinase 2) V617F somatic
Gram-positive pathogens, 163 point mutation, 188, 189
Green fluorescent protein (GFP), 157, 163, Japanese firefly (Luciola lateralis), 18f
165, 193 Japanese fireworm (Odontosyllis), 9, 10f
Groundwater monitoring
diesel-oil-contaminated, 122
TCE and 1,1,1 trichloroethane (TCA) K
contaminated, 124 Krebs cycle, 115
Index 203
L M
Laser-induced fluorescence, 184 Mann-Whitney U test, 83
Light emission reaction MCF-7 cell line
catalyzed by bacterial luciferase, 158 b-Globin promoter, 136
catalyzed by firefly luciferase, 158 -derived MELN reporter cell line, 136
Light production. See Bioluminescence -derived MVLN reporter cell line, 136
Light-emitting proteins, 176 MDA-kb2 cell line, 137
Light-producing reaction, 177 bioreporter assay, 140
Listeria, 156, 161, 164 MELN assay, 136, 138, 140, 141
Listeria reporter phage A511::luxAB, 160, 161 reporter cell line, 136, 138
Listeriosis, 164 Mercaptoethanol, 78, 80f
Littoral zone, 19 Microecosystem (MES) components, 91–95
luminous earthworms in, 20 experiments, 93
Locked nucleic acid (LNA), 188, 189 biomass of algae, 94f
Luciferase dynamics of bioassay parameters for,
biotesting, 72–73 94f
scheme, 72f Midwater hatchetfish (Sternoptyx pseudobscu-
biotin-streptavidin/avidin system, 180f ra), 13f
as immunoassay labels, 179–183 Misdirection (or distraction/obstruction of
bacterial luciferases, 179 sight), 4
biotinylated luciferase, 180 Mouse mammary tumor virus (MMTV) pro-
firefly luciferases, 179 moter, 137
immunoprecipitation system (see Lucifer- Multianalytical bioluminescent assay,
ase immunoprecipitation system 189–192
(LIPS)) Mushrooms, 8, 16
luciferase index (LI), 73 attraction of spore-dispersing organisms,
single-chain polypeptides, 181 11
homologous glycosylated, 182 Mycobacterium reporter phages, 162, 165, 166
Luciferase immunoprecipitation system Mycobacterium tuberculosis (MTB) infection,
(LIPS), 181, 181f 185
Luciferin, 4, 15, 21, 28, 118, 138, 179, 189
cypridinid luciferin, 21–22, 22f
substrate luciferin, 158 N
Luminescent secretion clouds, 7 N3-(oxohexanoyl-) L-homoserine lactone
Luminous species (OHHL), 163
brittle star (Ophiopsila), 9f NADH:FMN-oxidoreductase-luciferase, 70,
coastal fish (Acropoma japonicum), 24f 71–72, 76
earthworm (Microscolex phosphoreus), 18f advantages of, 75
freshwater snail (Latia neritoides), 19f in Lake Shira water analysis, 74
marine snail (Angiola zonata), 20f in pond water MES, 91–95
millipede (Paraspirobolus lucifugus), 8, 8f in single system of enzymatic assays, 87
mushroom (Mycena chlorophos), 8, 9f Natural water biotesting
lux genes, 39, 41, 43–47 reference water, Lake Baikal, 84
gene content and gene order of, 44f samples
luxCDABEG, 39, 43 characteristics, 83, 84t
luxF, 43 light emission intensity dependency on,
lux-rib operons, 44, 45 85f
genus-specific differences, 45 toxicity bioassay of, 84t
nonfluorescent flavoprotein coding, 43 toxicological characteristics of, 84t
lux operon expression, regulation of, 47–51 using immobilized coupled enzyme sys-
population density-responsive regulation, tem, 83–85
47 using soluble enzyme system, 83–85
luxRVh (lux operon in V. harveyi), 47 Nucleic acid hybridization assay, 185f
204 Index
based on bioluminescent reporters, toxicological bioassay with increased sen-

183–185 sitivity to, 78–79
with recombinant aequorin as reporter, in Yenissei River water, 74
185f Polychlorinated biphenyls (PCBs), 125–127,
Nucleic acid sequence-based amplification 130
(NASBA) assay, 186 Polychlorinated dibenzofurans (PCDFs), 130
for Chlamidia pneumonia RNA, 186 Polychlorinated dibenzo-p-dioxins (PCDDs),
130
Polycyclic aromatic hydrocarbon (PAHs),
O 120–122, 130. See also PAH
Off-the-shelf digital cameras, 157 bioreporters
Organic toxicant-induced health risks, 129 Polymerase chain reaction (PCR), 183, 184
using eukaryotic cell-based bioluminescent PCR-based assays, 157
assays Pond ecosystem, 91–95
advantage of, 140 bioluminescent bioassays for, 93f
bioassays for dioxin and dioxin-like biomass of algae, 94f
compounds, 130, 130t, 133–135 dynamics of, 92f
for endocrine-disrupting chemicals, Portable Laboratory for Toxicity Detection
131–132t, 136 (PLTD), 104
for environmental applications, Primer extension (PEXT) reactions, 188, 191
139–141 pRLuc42R bioreporter, 128
for hormonally active chemicals, Prophage, 156
135–139 Prostatic acid phosphatase (PAP) with PSA,
Organophosphorous pesticides, 99, 100, 102 189
Proteolytic interactions, 90
Protobioluminescence, 51
P Prototype biosensors, 105
PAH bioreporters, 120–122 PSA with alphafetoprotein (AFP), 189
environmental naphthalene detection, 121 Pseudomonas fluorescens OS8(pDNdmpRlux),
P. fluorescens HK44, 120, 121 127
phenanthrene degradative genes, 121 Pseudomonas oleovorans alk operon genes,
Pepsinogen I assay, 189 122, 123
Pepsinogen II assay, 189 Pseudomonas strain, CF600, 127
Pesticides, toxicological bioassay of, 99–103 pTOLLUX plasmid, 115
characteristics of toxic substances, 100t Pyrethroid, 99, 100
inhibition of trypsin activity, 100 Pyruvate phosphate dikinase, 180
sensitivity of coupled enzyme, 99
triple enzyme systems, 99, 102f
Phengodid beetle (Rhagophthalmus ohbai), 5, Q
6f, 7 Quantitative realtime PCR assay, 184
Phenol and derivatives, 127–128 Quorum sensing, 45, 47
Photobacterium, 39, 40t, 51, 52 in A. fischeri, 47
colonies of P. mandapamensis, 39f 3’:5’-cyclic AMP (cAMP), 49
Photorhabdus, 39, 41t control of luminescence in, 50f
Polaroid films, 157 octanoyl-HSL (AI-2Af), 50–51
Pollutants, 71, 74 regulatory factors in, 49–50
anthropogenic pollutants studies, 89 in Vibrio campbellii (V. harveyi), 47, 48
on bioluminescence of soluble enzyme phosphorylation/dephosphorylation
system, 82t cascade, 49f
on immobilized coupled enzyme system,
82t
in Lake Shira water, 74 R
maximum permissible concentration Realtime PCR, 184, 189
(MPC), 76 Receptor mutation, 159
Index 205
Recombinant proteins, 176–177 T

Reduced flavin mononucleotide (FMNH2), 42, T-47D ER-CALUX, 137
43 Terrestrial luminous organisms, 14
NADH:FMN oxidoreductase, 43 Thyrotropin (TSH) assay, 183
Renilla luciferases (RLuc), 180 third-generation assay, 183
bioluminescence-based hybridization Toll-like receptor 4 (TLR4) gene, 191
assay, 188f Toluene benzene utilization (tbu) pathway,
for miR21 in breast cancer cells, 187 118
Reporter bacteriophages, 157 Toluene degradation (tod) pathway, 119
advantage of, 161 Toxicity coefficient (TC), 73, 74
antibiotic resistance, 166 and luciferase index (LI), 73, 74
in combination with other technologies, Toxicological bioassay
166–167 pond ecosystem, 91–95
construction and use, 159–162 signal system of enzymatic assays for
drawbacks of, 161–162 pesticides, 99–103
on foodborne pathogens, 167 Trypsin, 69, 87, 90, 91, 95, 100, 102
specific bioluminescent phages, 162–166 in microalgae biomass, 97f, 98
on biosafety-relevant human pathogens, in pond water analysis, 93f
165 scheme of activity, 89f
on clinically relevant pathogens, 165 triple enzyme systems, 96, 99
plant pathogenic bacteria, 166
Restriction-modification systems, 159
Reverse transcriptase-polymerase chain reac- U
tion (RT-PCR) assay, 184 U2-OS cell line, 138
for Mycobacterium tuberculosis (MTB)
infection, 185
for prostate-specific antigen (PSA), 187 V
(RS)-3-(3,5-dichlorophenyl)-5-methyl-5-vi- Vertebrate endocrine system, 135
nyloxazolidine-2,4-dione (vinclozo- Vibrio, 39, 40t, 51
line), 137 Vibrio harveyi luxAB genes, 122
Russian Federation standards, 83 Vibrionaceae, 38, 39
S W
(S)-3-hydroxytridecan-4-one (cholerae autoin- Waste water biotesting
ducer, CAI-1), 49 reference water, Lake Baikal, 84
Sakyo Kanda, 27 samples
Salmonella, 162, 167 characteristics, 83, 84t
Salmonella reporter phage Felix O1, 161 light emission intensity dependency on,
Sandwich-type immunoassay, 178f 85f
Shewanella, 39, 40t, 41, 42, 46 toxicity bioassay of, 84t
Shewanellaceae, 39 toxicological characteristics of, 84t
Shigella, 167 using immobilized coupled enzyme sys-
Single-tube luminometers, 161 tem, 83–85
Split-protein reassembly technology, 192 using soluble enzyme system, 83–85
Spore-feeding fungus gnat (Keroplatus nippo-
nicus), 11, 11f
Startling (or deterring), 4 X
click beetles, 6 Xenobiotics, 71
phengodid, 6 contamination of MES, 98
Symbiotic luminescence, 23–24 Xenopus Vitellogenin A2 gene, 136
206 Index
Y Z
Yata Haneda, 27 Zebrafish assay, 134, 137
Yeast-based assays, 138, 139
hAR/ARE-luc bioassay, 139
Yellow fluorescent protein (YFP), 46, 192
energy transfer (EYFP), 192
Yenissei River water analysis, 74
Yersinia pestis, 166

Bioluminescence Fundamentals and Applications in Biotechnology - 2014

Uploaded by

Copyright:

Available Formats

Bioluminescence Fundamentals and Applications in Biotechnology - 2014

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Bioluminescence Fundamentals and Applications in Biotechnology - 2014

Uploaded by

Copyright:

Available Formats

Advances in Biochemical Engineering/Biotechnology 144

Series Editor: T. Scheper

For further volumes:

Volumes are organized topically and provide a comprehensive discussion of

In references, Advances in Biochemical Engineering/Biotechnology is abbreviated

Dan Close Elena Esimbekova Paul Dunlap Ludmila A. Frank

Nanyang Technological University

ISSN 0724-6145 ISSN 1616-8542 (electronic)

Library of Congress Control Number: 2014943751

Ó Springer-Verlag Berlin Heidelberg 2014

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

Part I Fundamentals of Bioluminescence

Eco-Evo Bioluminescence on Land and in the Sea . . . . . . . . . . . . . . . 3

Biochemistry and Genetics of Bacterial Bioluminescence . . . . . . . . . . 37

Part II Applications of Bioluminescence in Environment

Application of Enzyme Bioluminescence in Ecology . . . . . . . . . . . . . . 67

Detection of Organic Compounds with Whole-Cell Bioluminescent

Part III Applications of Bioluminescence in Agriculture

Detection of Bacteria with Bioluminescent Reporter Bacteriophage . . . 155

Part IV Applications of Bioluminescence in Health

Application of Enzyme Bioluminescence for Medical Diagnostics . . . . 175

Yuichi Oba and Darrin T. Schultz

Abstract This review discusses the evolution of bioluminescence organisms that

Y. Oba (&) D. T. Schultz

G. Thouand and R. Marks (eds.), Bioluminescence: Fundamentals and Applications 3

3.2 In the Air..................................................................................................................... 16

Bioluminescent organisms occur in a wide variety of habitats, from tropical jun-

Fig. 4 Coastal ostracod

Luminescent secretion clouds discharged by agitated bioluminescent organisms

Fig. 5 Luminous millipede Paraspirobolus lucifugus collected in Okinawa, Japan. Blue-green

The function of the luminescence in millipedes and centipedes has been

brittle star Ophiopsila (Ophiuroidea, Echinodermata) [35]. Found in shallow reefs,

2.2 Light Organ Lure

Fig. 8 Luminescence of the carnivorous fungus gnat Arachnocampa luminosa on a riverbank

Fig. 9 Luminescence of spore-feeding fungus gnat Keroplatus nipponicus on Hachijo Island

2.3 Intraspecific Communication

Many organisms use bioluminescence to camouflage themselves in the open from

Species in three genera of deep-sea dragonfish, Malacosteus, Aristostomias, and

2.6 Functionless Bioluminescence

Because of its accessibility to observers, bioluminescence in terrestrial habitats has

Terrestrial luminous organisms comprise arthropods and fungi. One exception is

3.2 In the Air

3.3 Underground: Why in Soil?

In the Oligochaeta (earthworms and potworms), luminous species have been

3.4 Fresh Water: Why so Rare?

Fig. 11 Luminous earthworm Microscolex phosphoreus, attacked by the lithobiid centipede at

Fig. 12 Luminescence of the

This small basommatophoran snail clings to the surface of submerged stones

Fig. 13 Luminous freshwater snail Latia neritoides discharging luminescent mucus by

Although there are various terrestrial luminous organisms, they nevertheless

4.1 Intertidal Zone

Fig. 14 Luminous marine

4.2 Coastal Water

4.2.1 Cypridinid Luciferin

Fig. 15 In the marine coastal zone, cypridinid luciferin is biosynthesized by ostracods. It is

4.3 Midwater: Why so Species-Rich?

4.3.1 Symbiotic Luminescence

Luminous bacteria are common in seawater, especially in temperate and coastal