ORIGINAL RESEARCH ARTICLE
published: 14 December 2012
doi: 10.3389/fmicb.2012.00423
Widespread occurrence of two carbon fixation pathways in
tubeworm endosymbionts: lessons from hydrothermal
vent associated tubeworms from the Mediterranean Sea
Vera Thiel 1 *†‡ , Michael Hügler 2 ‡ , Martina Blümel 1 † , Heike I. Baumann 1 , Andrea Gärtner 1 ,
Rolf Schmaljohann 1 , Harald Strauss 3 , Dieter Garbe-Schönberg 4 , Sven Petersen 1 , Dominique A. Cowart 5 ,
Charles R. Fisher 5 and Johannes F. Imhoff 1
1
GEOMAR, Helmholtz Centre for Ocean Research Kiel, Kiel, Germany
Water Technology Center Karlsruhe, Karlsruhe, Germany
3
Institut für Geologie und Paläontologie, Westfälische Wilhelms-Universität Münster, Münster, Germany
4
Institut für Geowissenschaften, Christian – Albrechts – Universität Kiel, Kiel, Germany
5
Mueller Laboratory, Department of Biology, The Pennsylvania State University, University Park, PA, USA
2
Edited by:
Andreas Teske, University of North
Carolina at Chapel Hill, USA
Reviewed by:
John Stolz, Duquesne University, USA
James F. Holden, University of
Massachusetts Amherst, USA
*Correspondence:
Vera Thiel , Biochemistry and
Molecular Biology, The Pennsylvania
State University, 231 South Frear,
University Park, PA 16802, USA.
e-mail: vut1@psu.edu
†
Present address:
Vera Thiel , Department of
Biochemistry and Molecular Biology,
The Pennsylvania State University,
University Park, USA;
Martina Blümel , Christian Albrechts
Universität Kiel, Institut für
Pflanzenzüchtung, Kiel, Germany.
‡
Vera Thiel and Michael Hügler have
contributed equally to this work.
Vestimentiferan tubeworms (siboglinid polychetes) of the genus Lamellibrachia are common members of cold seep faunal communities and have also been found at sedimented
hydrothermal vent sites in the Pacific. As they lack a digestive system, they are nourished
by chemoautotrophic bacterial endosymbionts growing in a specialized tissue called the
trophosome. Here we present the results of investigations of tubeworms and endosymbionts from a shallow hydrothermal vent field in the Western Mediterranean Sea. The
tubeworms, which are the first reported vent-associated tubeworms outside the Pacific,
are identified as Lamellibrachia anaximandri using mitochondrial ribosomal and cytochrome
oxidase I (COI) gene sequences. They harbor a single gammaproteobacterial endosymbiont. Carbon isotopic data, as well as the analysis of genes involved in carbon and sulfur
metabolism indicate a sulfide-oxidizing chemoautotrophic endosymbiont. The detection
of a hydrogenase gene fragment suggests the potential for hydrogen oxidation as alternative energy source. Surprisingly, the endosymbiont harbors genes for two different
carbon fixation pathways, the Calvin-Benson-Bassham (CBB) cycle as well as the reductive tricarboxylic acid (rTCA) cycle, as has been reported for the endosymbiont of the vent
tubeworm Riftia pachyptila. In addition to RubisCO genes we detected ATP citrate lyase
(ACL – the key enzyme of the rTCA cycle) type II gene sequences using newly designed
primer sets. Comparative investigations with additional tubeworm species (Lamellibrachia
luymesi, Lamellibrachia sp. 1, Lamellibrachia sp. 2, Escarpia laminata, Seepiophila jonesi )
from multiple cold seep sites in the Gulf of Mexico revealed the presence of acl genes
in these species as well. Thus, our study suggests that the presence of two different
carbon fixation pathways, the CBB cycle and the rTCA cycle, is not restricted to the Riftia
endosymbiont, but rather might be common in vestimentiferan tubeworm endosymbionts,
regardless of the habitat.
Keywords: hydrothermal vent, vestimentiferan tubeworm, carbon fixation, endosymbiont, acl gene, cbbM gene,
Lamellibrachia, Mediterranean Sea
INTRODUCTION
Vestimentiferan tubeworms are often dominant members of
chemosynthetic communities present at reduced environments
such as hydrothermal vents and cold seeps (Vrijenhoek, 2010).
So far, hydrothermal vent-associated tubeworms have not been
found outside the Pacific. In contrast, seep-associated tubeworms
have been found in the Gulf of Mexico (GoM), the Mediterranean
Sea, and the margins of the Atlantic Ocean (Cordes et al., 2009;
Vrijenhoek, 2010).
The Mediterranean Sea is the world’s largest enclosed sea, and
represents a hot spot of biodiversity with a considerable number
of endemic species (Myers et al., 2000). Its only connection to the
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Atlantic Ocean is the narrow and shallow Strait of Gibraltar, which
is the sole route for exchange of propagules between these two
water bodies. The only vestimentiferan tubeworms documented to
date in the Mediterranean Sea belong to the genus Lamellibrachia
and specimens from several Mediterranean mud volcanoes were
recently described as the new species Lamellibrachia anaximandri
(Southward et al., 2011). The genus Lamellibrachia has a worldwide distribution, and occurs in several types of chemosynthetic
environments from the shallow to the deep-sea (e.g., Kojima et al.,
2002). Within the Mediterranean Sea, Lamellibrachia spp. have
been discovered in the vicinity of mud volcanoes in the Alboran Sea at 572 m depth (Hilário et al., 2011), from several mud
December 2012 | Volume 3 | Article 423 | 1
Thiel et al.
volcanoes in the Eastern Mediterranean Sea at a depth of about
3,000 m (Olu-Le Roy et al., 2004; Bayon et al., 2009; Duperron
et al., 2009; Southward et al., 2011) and also from two sunken ship
wrecks in the Eastern and Western Mediterranean (Hughes and
Crawford, 2008; Gambi et al., 2011; Figure 1).
Hydrocarbon seep communities in the GoM were among the
first seep communities to be discovered, are extensively studied,
and have a high diversity of tubeworm species (Kennicutt et al.,
1985; Miglietta et al., 2010). The Louisiana Slope in the northern
GoM area extends from the continental shelf to the salt deformation edge of the Sigsbee Escarpment, and ranges from about
300 to 3,000 m in depth. This area is home to at least six known
morphospecies of vestimentiferan tubeworms (Miglietta et al.,
2010), including the most commonly studied seep tubeworms,
Lamellibrachi luymesi (van der Land and Nørrevang, 1975), and
Seepiophila jonesi (Gardiner et al., 2001).
In contrast to the well-known hydrothermal vent tubeworm
Riftia pachyptila, which inhabits hard substrate in hot sulfidic environments, members of the genus Lamellibrachia live in sedimented
areas and are most common in cold seep environments. Seep habitats are generally much less dynamic than vent habitats and may be
stable for centuries (Fisher et al., 1997). Compared to vent environments, emanating seep fluids are cooler, often enriched in methane
and concentrations of dissolved sulfide may be quite low (Southward et al., 2011). Lamellibrachia tubeworms can obtain sulfide
from the underlying sediments using the buried, permeable posterior region of the tube termed the“root”(Julian et al., 1999; Freytag
et al., 2001). Since Lamellibrachia, like other siboglinid polychetes,
lack a digestive tract, they are dependent on their endosymbionts
for nutrition. Sulfide is transported via hemoglobin molecules in
Carbon fixation in tubeworm endosymbionts
the blood to the trophosome, a large organ that harbors dense
populations of gammaproteobacterial endosymbionts (reviewed
by Childress and Fisher, 1992). These endosymbionts oxidize the
sulfide to obtain energy and reducing power for autotrophic carbon fixation. A portion of the synthesized organic matter serves in
turn as energy source for the host tubeworm (Bright et al., 2000;
Stewart and Cavanaugh, 2006). Lamellibrachia spp. are not only
found at cold seeps, but also at sediment covered hydrothermal
sites, e.g., Lamellibrachia barhami along the Juan de Fuca Ridge
(Juniper et al., 1992), Lamellibrachia columna near hydrothermal
vents in the Lau Basin in the southwest Pacific (Southward, 1991)
and Lamellibrachia satsuma at hydrothermal sites off southern
Japan (Miake et al., 2006). Even though the so-called “vent” and
“seep” tubeworm genera are clearly specialized for their preferred
in situ conditions, they have been found at the same site, sometimes
occurring only meters apart, e.g., the seep tubeworm L. barhami
and the vent species Ridgeia piscesae at Middle Valley in 2,400 m
depth in the northeast Pacific Ocean (McMullin et al., 2003).
Vestimentiferan endosymbionts form a monophyletic cluster within the gammaproteobacteria. They have been shown to
cover very large geographic ranges, with nearly identical 16S
rRNA in hosts separated by thousands of kilometers. Within
the endosymbiont cluster four different groups (one “vent”group, three “seep”-groups) are distinguishable. So-called “vent”
endosymbionts appear to be specific for vent vestimentiferan hosts
(e.g., Riftia, Tevnia, Ridgeia, and Oasisia), while three different 16S
rRNA gene clusters (groups 1–3), possibly representing different
strains, were found only in “seep” vestimentiferans (Nelson and
Fisher, 2000; McMullin et al., 2003). Site depth has been postulated
to be a factor in defining which of the three endosymbiont strains
FIGURE 1 | Map with locations of vestimentiferan tubeworms of the genus Lamellibrachia described from previous studies (black circles) and this
study (red circle) within the Mediterranean Sea.
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December 2012 | Volume 3 | Article 423 | 2
Thiel et al.
is found in a particular “seep” host, with “group 3” occurring only
in shallow water host specimens (McMullin et al., 2003).
The best-studied tubeworm endosymbiont is Candidatus
Endoriftia persephone, the gammaproteobacterial endosymbiont
of the vent-associated tubeworm Riftia pachyptila. Its metabolic
capacities have been subject of detailed metagenomic, proteomic,
and enzymatic studies (Felbeck, 1981; Felbeck et al., 1981; Markert
et al., 2007, 2011; Robidart et al., 2008; Gardebrecht et al., 2012).
Candidatus Endoriftia persephone is a sulfide-oxidizing chemoautotroph. Sulfide is oxidized to sulfate via sulfite and adenosine
phosphosulfate (APS). The enzymes involved in the so-called
APS pathway are dissimilatory sulfite reductase (DsrAB, working
in reverse as sulfide oxidase), APS reductase (AprAB), and ATP
sulfurylase (Markert et al., 2007, 2011). Quite surprising is the
presence of two alternative carbon fixation pathways in the Riftia
endosymbiont, the Calvin–Benson–Bassham (CBB) cycle as well
as the reductive tricarboxylic acid (rTCA) cycle (Felbeck, 1981;
Markert et al., 2007). Both pathways show unique features. The
CBB cycle seems more energy-efficient due to modified enzyme
equipment (Markert et al., 2011; Gardebrecht et al., 2012; Kleiner
et al., 2012) while the rTCA cycle harbors a novel type of ATP
citrate lyase (Hügler and Sievert, 2011). In contrast to the Riftia
endosymbiont there are no genomic, proteomic or metabolomic
studies of the endosymbiont(s) of Lamellibrachia spp.
This study reports the recovery of vestimentiferan tubeworms
from the Palinuro volcanic complex, a submarine volcano in
the Tyrrhenian Sea (Western Mediterranean Sea), north of Sicily
(Figure 1). The Palinuro complex is part of the active Aeolian
Island Arc and consists of several volcanic edifices aligned over
a strike length of 55 km (Petersen et al., 2008; Passaro et al.,
2010). The volcanic complex is up to 25 km wide at its base and
its shallowest portion rises from 3,000 m to a water depth of
less than 100 m. Iron and manganese-bearing precipitates were
first documented at Palinuro by Kidd and Ármannson (1979)
Carbon fixation in tubeworm endosymbionts
providing the first evidence for hydrothermal activity in the area.
Hydrothermal sulfides were described by Minniti and Bonavia
(1984) and Puchelt and Laschek (1987) within sediment sampled
from the most westerly summit of Palinuro. The discovery of living
vestimentiferan tubeworm colonies on top of the main volcanic
edifices in this western summit in 2006 as well as temperatures
of up to 60˚C in sediment cores recovered from the seafloor indicated that active hydrothermal venting was taking place at the
time although black smoker style venting has not been observed
(Petersen et al., 2008; Monecke et al., 2009). Two colonies of these
tubeworms were sampled in spring 2011.
We describe the results from detailed analyses of the Palinuro
tubeworms and their endosymbionts, which are the first reported
vent-associated tubeworms outside the Pacific Ocean. For comparison, several seep tubeworm species from the GoM were also
analyzed (Figure 2), providing deeper insights into the geographic
dispersal, phylogeny, and metabolic potential of tubeworms and
their endosymbionts.
MATERIALS AND METHODS
SAMPLING SITE, SAMPLE COLLECTION, AND PROCESSING OF
PALINURO TUBEWORMS
Vestimentiferan tubeworm specimens were retrieved from two different colonies termed colony #1 and #2 on the western summit
of the Palinuro volcanic complex (Mediterranean Sea, 39˚32.44′ N,
14˚42.38′ E, depth: 630 m) during the Pos412 cruise of R/V Poseidon in spring 2011. Sampling was conducted using a Mohawktype remotely operated vehicle (ROV) supplied by Oceaneering
Inc. (Aberdeen, UK) fitted with a robotic arm. Locations of the
tubeworm collections are given in Table 1.
The ROV was also equipped with a fluid sampling system (Kiel
in situ pumping system, KIPS, Garbe-Schönberg et al., 2006) capable of acquiring four 550 mL water samples per dive with in situ
filtration. Parallel to the sampling nozzle was a temperature probe
FIGURE 2 | Map of sampling locations for tubeworm specimens in the Gulf of Mexico included in this study. Scale is in degrees longitude; 1˚ = 111.12 km.
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December 2012 | Volume 3 | Article 423 | 3
Thiel et al.
Carbon fixation in tubeworm endosymbionts
Table 1 | Position of tubeworm colonies sampled during R/V
POSEIDON cruise Pos412 at Palinuro volcanic complex, Tyrrhenian
Sea and temperatures measured within colonies.
Tubeworm
Station
Geographical
Depth
Temperature
colony no.
no.
position
(m)
(˚C)
634
Top: 14.2
Center: 19.4
630
Top: 15.6
Center: 14.7
(TMS; ROV)
Colony #1
231-1
39˚32.45′ N/
014˚42.41′ E
Base: 15.9
Colony #2
241-3
39˚32.427′ N/
014˚42.384′ E
Base: 14.2
attached to a data logger. Fluids and temperatures around the
colonies were sampled using KIPS and the temperature probe by
maneuvering the ROV’s robotic arm into the fluid in close proximity to the living tubeworms. Live tubeworms were sampled using
the ROV’s robotic arm. Immediately after tubeworm sampling, the
dive was terminated, and the ROV was recovered. Upon recovery,
the tubeworms obtained from colony #1 were put into sterile Petri
dishes using sterile tweezers followed by dissection with a sterile
scalpel. The animal was then separated from the tube; subsamples
were recovered from vestimentum (host tissue free of symbionts)
and trophosome (endosymbiont) tissue, and then were stored at
−20˚C until further molecular analysis. Samples from colony #2
were immediately stored at −20˚C until further processing in the
home laboratory. As the tubeworms from colony #2 exhibited
several morphotypes, these were stored in separate vials.
GULF OF MEXICO TUBEWORM SAMPLE COLLECTION AND
PREPARATION
Gulf of Mexico vestimentiferan tubeworms were sampled from
hydrocarbon seep sites during several research cruises between
1997 and 2011 (Table 2; Figure 2). Lamellibrachia luymesi and
Seepiophila jonesi were collected using the Johnson Sea Link submersible from two sites on the Upper Louisiana Slope from about
540 m depth. Lamellibrachia sp. 1 and Lamellibrachia sp. 2 as
well as Escarpia laminata were collected from three sites on the
Lower Louisiana Slope ranging in depth from 1,975 to 2,604 m.
While on board the research vessel, tubeworms were dissected
and vestimentum (host tissue free of symbionts) and trophosome
(endosymbiont) tissue was preserved at −80˚C or in 95% ethanol
solution. All tissue samples were transported to the Pennsylvania
State University where whole genomic DNA was obtained using a
modified version of the high salt extraction protocol and ethanol
precipitation as in Liao et al. (2007). Isolated DNA is currently
stored at −80˚C at the Pennsylvania State University.
DNA EXTRACTION, PCR AMPLIFICATION, CLONING, AND SEQUENCING
Genomic DNA was extracted from the trophosome and vestimentum tissues of the vestimentiferan tubeworms. The tubeworms
were dissected and washed three times in 0.2 µm filtered seawater prior to DNA extraction. DNA of Mediterranean tubeworm
samples was isolated using the MoBio Power Biofilm Kit (Mo
Bio Laboratories, Carlsbad, CA, USA) according to the protocol
Frontiers in Microbiology | Extreme Microbiology
provided. DNA of GoM tubeworm samples was extracted following the protocol of Liao et al. (2007). Cytochrome c oxidase I
(COI) genes, mitochondrial and bacterial ribosomal (16S rRNA)
genes, as well as cbbM and ACL type II genes were analyzed from
all individuals. Further functional genes and eukaryotic ribosomal
(18S rRNA) genes were analyzed from one individual of colony #1
from the Palinuro volcanic complex.
For all gene amplifications of Mediterranean samples PCR reactions were conducted using Ready-To-Go PCR Beads (GE Healthcare, Munich, Germany) in a total volume of 25 µL. PCR from
GoM samples were conducted using 1 U BioBasic TaqPolymerase
(BioBasic Inc., Markham, ON, Canada) and 1× Thermopol Buffer
(NEB Inc., USA) in a total volume of 50 µL. If not stated otherwise 10 pmol of each primer and 100 ng template DNA was used.
For all amplifications, initial denaturation was 2 min at 94˚C, final
annealing was 1 min at annealing temperature, and final elongation 5 min at 72˚C. For the cycles denaturation was 40 s at 94˚C,
annealing duration 40 s at the respective annealing temperature
and elongation was 1 min at 72˚C. If not stated otherwise, 35
PCR cycles were applied. Fragments of the tubeworms’ 18S rRNA
and mitochondrial 16S rRNA as well as COI genes were amplified using the (i) primer pairs 5′ -start (5′ -GGT TGA TCC TGC
CAG-3′ ) and 1753rev (5′ -GCA GGT TCA CCT ACG G-3′ ) targeting the 18S rRNA gene (30 cycles, 50˚C annealing temperature),
(ii) primer pair 16Sar/16Sbr (Palumbi et al., 2002) targeting the
mitochondrial 16S rRNA gene (30 cycles, 50˚C annealing temperature), and (iii) primers LCO 1490 (5′ -GGT CAA CAA ATC ATA
AAG ATA TTG G-3′ ) and HCO 2198 [5′ -TAA ACT TCA GGG
TGA CCA AAA AAT CA-3′ ; 40 cycles, 47˚C annealing temperature
(Folmer et al., 1994)] using DNA extracted from the symbiontfree vestimentum tissue. Gene fragments of the endosymbiont
were amplified using DNA extracted from trophosome tissue as
the template. Bacterial 16S rRNA gene fragments were amplified
in a 30 cycle PCR at an annealing temperature of 50˚C with the
general bacterial primer set 27F and 1390R (Palinuro samples;
5′ -GAC GGG CRG TGT GTA CAA-3′ ) or 1492R (GoM samples; Lane, 1991). Amplification for fragments of dsrA and aprA
genes was performed using the primer sets rdsrA240F/rdsrA403R
and aps1F/aps4R, respectively (Meyer and Kuever, 2007b; Lavik
et al., 2009). Fragments of soxB were amplified using the primers
soxB432F/soxB1446B [10 cycles with 55˚C annealing temperature
and 25 cycles with 47˚C annealing temperature (Petri et al., 2001)].
For the amplification of fragments of the genes coding for the large
subunit of RubisCO form I and II, the primer sets cbbLF/cbbLR
and cbbM F/cbbM R were used [both include two initial cycles of
2 min annealing at 37˚C and 3 min elongation at 72˚C, as well
as additional 35 cycles of 53 and 58˚C annealing temperature
for cbbL and cbbM respectively (Campbell and Cary, 2004)]. A
fragment of the large subunit of the putative type II ATP citrate
lyase gene was amplified using the newly designed primer acl2F1
(5′ -CGT CGC CAA GGA AGA GTG GTT C-3′ ) and acl2R1 (5′ GGC GAT GGC CTC AAA GCC GTT-3′ ) in a 30 cycle PCR with
annealing temperatures of 45–56˚C (gradient). Fragments of the
hydrogen uptake hydrogenase gene hupL were amplified with the
primer set HUPLX1/HUPLW2 (Csaki et al., 2001). A fragment
of the norCB gene for nitric oxide reductase subunits C and B
was amplified using the primer set norC21mF and norB6R (Tank,
December 2012 | Volume 3 | Article 423 | 4
Sample
Species
Region
Site
name
Depth
Date
(m)
collected
Latitude
Longitude
Endosymbiont
Endosymbiont
Endosymbiont
Host
16S rRNA
cbbM
ACL
mt16S
Reference
Thiel et al.
www.frontiersin.org
Table 2 | Sample identity, geographic origin, and gene sequences accession numbers of tubeworm specimens and their endosymbionts included in this study.
rRNA
634
4/27/2011
39˚32.450′ N
015˚42.410′ E
HE983342-8
HE978225
HE978244
HE974472
This study
630
4/29/2011
39˚32.427′ N
015˚42.384′ E
HE983349-52
HE978226-8
HE978245-6
HE974473
This study
Pos412-
Lamellibrachia
Mediterranean
Pos412-
B1_L1-7
anaximandri
Sea, Palinuro
231
Pos412-
Lamellibrachia
Mediterranean
Pos412-
B2_L1-4
anaximandri
Sea, Palinuro
242
DC673_1211
Lamellibrachia
Gulf of Mexico,
sp. 1
DeSoto Canyon
Lamellibrachia
Gulf of Mexico,
sp. 2
DeSoto Canyon
Escarpia
Gulf of Mexico,
laminata
DeSoto Canyon
Escarpia
Gulf of Mexico,
laminata
Alaminos
et al. (2010),
Canyon
This study
Seamount
Seamount
DC673_1209
DC673_1170
AC601_E6
AC601_L1
Escarpia
Gulf of Mexico,
laminata
Alaminos
DC673
2604
10/30/2006
28˚18.603′ N
087˚18.643′W
HE983327
HE978212
HE978229
HE974464
This study
DC673
2604
10/30/2006
28˚18.603′ N
087˚18.643′W
HE983328
HE978213
HE978230
HE974465
This study
DC673
2604
10/29/2006
28˚18.603′ N
087˚18.643′W
HE983329
HE978214
HE978231
HE974466
This study
AC601
2339
5/27/2002
26˚23.365′ N
094˚30.880′W
HE983330
HE978215
HE978232
GU068203
Miglietta
AC601
2339
5/27/2002
26˚23.365′ N
094˚30.880′W
HE983331-2
HE978216
HE978233-4
HE974467
This study
WR269
1975
5/25/2002
26˚40.672′ N
091˚39.691′W
HE983333
HE978217
HE978235
HE974468
This study
GC852
1437
5/22/2002
27˚05.768′ N
091˚09.897′W
HE983334
HE978218
HE978236
GU068242
Miglietta
Canyon
WR269_E10
GC852_L4
Escarpia
Gulf of Mexico,
laminata
Walker Ridge
Lamellibrachia
Gulf of Mexico,
sp. 1
Green Canyon
et al. (2010),
This study
GC852_L1
GC852_L5
Gulf of Mexico,
sp. 2
Green Canyon
Lamellibrachia
Gulf of Mexico,
sp. 1
Green Canyon
Lamellibrachia
Gulf of Mexico,
sp. 2
Alaminos
GC852
1437
5/22/2002
27˚05.768′ N
091˚09.897′W
HE983335
HE978219
HE978237
HE974469
This study
GC852
1437
5/22/2002
27˚05.768′ N
091˚09.897′W
HE983336
HE978220
HE978238
HE983353
This study
AC601
2335
5/30/2002
26˚23.548′ N
094˚30.849′W
HE983337-8
HE978221
HE978239-40
HE974470
This study
GC234
527
2003
27˚26.839′ N
091˚08.061′W
HE983339
HE978222
HE978241
HE974471
This study
GC184
540
1995
27˚28.171′ N
091˚18.265′W
HE983340
HE978223
HE978242
GU068216
Miglietta
Canyon
GC234_4587 Seepiophila
GC184_L9
Gulf of Mexico,
jonesi
Green Canyon
Lamellibrachia
Gulf of Mexico,
luymesi
Green Canyon
et al. (2010),
This study
GC234_L7
Lamellibrachia
Gulf of Mexico,
luymesi
Green Canyon
GC234
546
1995
27˚26.847′ N
091˚07.986′W
HE983341
HE978224
HE978243
GU068238
Miglietta
et al. (2010),
This study
Carbon fixation in tubeworm endosymbionts
December 2012 | Volume 3 | Article 423 | 5
AC601_L20
Lamellibrachia
Thiel et al.
2005) in a 35 cycle PCR using annealing temperatures of 60–50˚C
(10 touchdown cycles 60˚C/−1˚C, 25 cycles of 50˚C).
Additional primer pairs used in this study include: F2/R5 and
892F/1204R for the two subunits of ATP citrate lyase (Campbell et al., 2003; Hügler et al., 2005); MxaF1003, MxaR1555,
MxaR1561 for methanol dehydrogenase gene mxaF (Neufeld
et al., 2007; Kalyuzhnaya et al., 2008); mmoXA/mmoXB for
genes encoding the conserved alpha-subunit of the hydroxylase
component of the cytoplasmatic soluble methane monooxygenase (sMMO; Auman et al., 2000); and A189F/MB661R for the
particulate methane monooxygenase (pMMO) genes present in
methanotrophs (Costello and Lidstrom, 1999).
All PCR products were purified via gel extraction using
QIAquick gel extraction kit (QIAgen, Hilden, Germany) for
Mediterranean samples, and BioBasic EZ-10 spin columns (BioBasic Inc., Markham, ON, Canada) for GoM samples respectively,
and either directly sequenced by Sanger sequencing (18S rRNA
gene fragments, COI, and functional gene fragments) or cloned
into pCR4-TOPO vectors with the TOPO-TA cloning kit (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer
before sequencing (16S rRNA gene fragments). Sequencing was
conducted using amplification primers and additional internal
primers in the case of 16S rRNA genes (342F, 534R; Muyzer
et al., 1996); 790F (Thiel et al., 2007). Amplification and sequencing of clones was conducted using vector specific primers M13
forward and M13 reverse (PCR) and T3 and T7 (sequencing),
respectively. Sanger sequencing was performed using the BigDye
Terminator v1.1 sequencing kit in a 3730xl DNA Analyzer (Applied
Biosystems, Carlsbad, CA, USA) as specified by the manufacturer.
Sequencing was conducted by the Institut für Klinische Molekularbiologie (IKMB), Universitäts-Klinikum Schleswig-Holstein (UKSH), Kiel, Germany and the sequencing core facility at The
Pennsylvania State University, University Park, PA, USA.
PHYLOGENETIC ANALYSIS
All sequences were edited with ChromasPro c.c1.33 and compared
to the NCBI database using BLAST (Altschul et al., 1997). Functional gene nucleotide sequences were also compared with the
non-redundant protein sequence database using the blastx algorithm. The endosymbiont 16S rRNA gene sequences were aligned
with the ARB software (www.arb-home.de) using the ARB FastAligner utility (Ludwig et al., 2004). The sequence alignment was
manually refined based on known secondary structures. Sequences
of functional genes as well as mitochondrial rRNA genes were
aligned using Clustal X (Thompson et al., 1997) and manually
adjusted using BioEdit (Hall, 1999). Maximum Likelihood based
trees and 100 bootstrap replicates were constructed using PhyML
(Guindon and Gascuel, 2003). In order to verify the tree topology, further phylogenetic analyses using Neighbor-Joining and
Maximum-Parsimony algorithms were conducted using MEGA5
(Tamura et al., 2011).
MICROSCOPY
The morphology of endosymbiotic bacteria in trophosome tissue
of the tubeworms was examined using light microscopy and scanning electron microscopy (SEM). Samples for light microscopy
were prepared by removing small pieces of tissue from different
Frontiers in Microbiology | Extreme Microbiology
Carbon fixation in tubeworm endosymbionts
parts of the trophosome and subsequent squeezing preparation
in a drop of particle-free seawater and examined under 100-fold
magnification using a Zeiss Axiophot Epifluorescence Microscope.
Samples for SEM were prepared by disrupting small trophosome samples in 0.2 µm filtered seawater, and then concentrated by filtration on to 0.2 µm polycarbonate membrane filters
followed by dehydration through ascending concentrations of
ethanol. Subsequently, the samples were critical-point-dried using
a Balzers CPD 030 and CO2 as a transition medium. The filters
were sputter-coated with gold-palladium using a Balzers SCD 004
and observed with a Zeiss DSM 940 electron microscope.
FLUID CHEMISTRY
After recovery of the ROV Mohawk all KIPS fluid samples were
immediately transferred to the onboard ship lab and sub-sampled
for subsequent analyses. Both pH and Eh of the fluids were
determined immediately after sub-sampling using electrochemical techniques after calibration with certified standards. Dissolved
oxygen was determined using standard Winkler titration protocols modified for small volumes. The concentration of dissolved
sulfide was determined in 1 mL aliquots using a zinc acetate
gelatin solution, which precipitates the dissolved sulfide as colloidal zinc sulfide. Subsequently, the color agent, N,N -dimethyl1,4-phenylenediamine-dihydrochloride, and a catalyst, iron chloride solution, were added to form methylene blue (Cline, 1969).
After 1 h, the solutions were measured photometrically at a wavelength of 660 nm using a Genesys Spectra 10 spectrophotometer.
The detection limit was 1 µmol/L. Potential oxidation of dissolved hydrogen sulfide during sampling and sample recovery
cannot be ruled out, but is likely minimal. Nonetheless, hydrogen
sulfide concentration data given in this paper should be considered minimum values. Aliquots for the analysis of nutrients were
stored in polypropylene bottles, sealed, and stored in the dark at
4˚C until analysis. Aliquots for cation and trace element analysis
were pressure-filtrated through 0.2 µm Nucleopore polycarbonate (PC) membrane filters using Sartorius PC filtration units and
high purity nitrogen. Samples were acidified with subboiled nitric
acid to pH <2 and stored in perfluoralkoxy (PFA) bottles until
analysis. Multielement analysis for major ion composition (Cl, B,
Si, Na, K, Ca, Mg, Fe, Mn) of the water samples was performed
with a SPECTRO Ciros SOP ICP-OES spectrometer after 10-fold
dilution and using Y for internal standardization. Trace elements
(As, Li, W) were determined by ICP-MS (Agilent 7500 cs at University of Kiel) after 12.5-fold dilution using both In and Re for
internal standardization. Certified reference materials NIST1643e,
NASS-5, and IAPSO were used for validation and accuracy checks.
CARBON ISOTOPE SIGNATURE
The organic carbon isotopic composition of tubeworm tissue
(δ13 CORG ) was determined via continuous flow EA-IRMS using
an elemental analyzer interfaced to a ThermoFinnigan Delta Plus
isotope ratio mass spectrometer. Briefly, about 40–60 µg of freezedried worm tissue was weighed in tin capsules, combusted to CO2 ,
and chromatographically purified carbon dioxide was transferred
to the mass spectrometer in a He gas stream. Results are reported in
the standard delta notation as per mil difference to the Vienna Pee
Dee Belemnite. Sample measurements were done in duplicate, and
December 2012 | Volume 3 | Article 423 | 6
Thiel et al.
analytical performance was monitored with international reference materials (USGS 24; USGS 40) and lab standards (anthracite;
brown coal) and the reproducibility was generally better than
±0.15‰.
The carbon isotopic composition of dissolved inorganic carbon (δ13 CDIC ) from vent fluids was measured using a Thermo
Finnigan Gas Bench coupled to a Thermo Finnigan Delta Plus
XL. Briefly, 0.5–1.0 mL of hydrothermal fluid was injected into an
exetainer that contained phosphoric acid, liberating DIC as carbon dioxide. Prior to sample injection, the exetainer was flushed
with helium. CO2 was flushed from the exetainer with a stream
of helium and injected into the mass spectrometer. Results are
reported in the standard delta notation as per mil difference to
the Vienna Pee Dee Belemnite. Sample measurements were done
in duplicate, and analytical performance was monitored with a
sodium carbonate lab standard.
Carbon fixation in tubeworm endosymbionts
Table 3 | Composition of seawater-hydrothermal fluid mixtures inside
tubeworm colonies #1 and #2.
T
Fluid
Colony #1,
Colony #2,
Seawater
endmember
232 ROV-2
237 ROV-1
221 CTD
˚C
pH
–
14.7
15.9
14.4
6.9
6.7
8.2
Diss. O2
µM
H2 S
µM
230
139
72
32
Cl
mM
984
–
640
634
626
B
mM
11
0.52
0.52
0.47
Si
mM
1.7
0.02
0.02
0.01
Na
mM
681
528
526
520
K
mM
65
12
11.9
11.5
Ca
mM
78
12.4
12.3
11.8
7.7
Li
µM
0.06
0.06
0.03
Mn
µM
3
<1
<1
NUCLEOTIDE SEQUENCE ACCESSION NUMBERS
Fe
µM
<2
<2
<2
The sequence data have been submitted to EMBL/GenBank/DDBJ
databases under accession numbers HE9744464-85, HE97821246, and HE983327-53.
As
µM
222
9.6
10
<5
W
nM
224
1.6
–
<0.3
RESULTS
BIOGEOCHEMICAL CHARACTERIZATION OF THE TUBEWORM HABITAT
AT PALINURO
For the present study, two colonies of vestimentiferan tubeworms
as well as their biogeochemical environment were sampled by
means of a ROV. The tubeworms occurred within a sediment-filled
depression at the western summit of the Palinuro volcanic complex in water depths of around 630 m and formed small bushes,
up to 1 m2 in diameter, mainly on sedimented surfaces but some
patches also occurred in areas where volcanic rocks were present
at the seafloor. Frequently, shimmering water was observed rising
above the tubeworm colonies suggesting active fluid emanation.
The first colony (colony #1) appeared to be comprised mainly of
equally sized animals. The second colony (colony #2), however,
consisted of animals of different sizes, indicating different ages,
or variation in exposure to hydrothermal fluid at different places
within the colony.
Remotely operated vehicle assisted measurements of water temperature across and within the tubeworm colonies revealed a
maximum recorded temperature of 19.4˚C, about 5˚C above ambient seawater temperatures of 14˚C (Table 1). Diffusely venting
warm hydrothermal fluids reached a maximum temperature of
58.4˚C within small depressions in close proximity to the tubeworms. Chloride concentrations in these fluids were significantly
higher than in ambient seawater indicating influx of hydrothermal brine eventually leading to the formation of small stratified brine pools in these depressions. Chemical analyses of vent
and pore fluids sampled from sediment cores collected in the
same area revealed that local hydrothermal fluids were anoxic,
H2 S-rich, acidic and displayed an elevated salinity. Concentrations of dissolved alkali and alkali earth elements (potassium,
calcium, lithium, cesium), silica, arsenic, and tungsten (“Fluid
end-member” in Table 3) were significantly higher when compared to normal bottom seawater. Fluid samples confirmed that
this hydrothermal fluid was highly diluted with ambient seawater
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as it passed through the two tubeworm colonies leading to partly
oxygenated waters (139 and 227 µmol/L dissolved O2 , Table 3)
and reduced levels of sulfide. Still, dissolved sulfide concentrations of 32 and 72 µmol/L were measured in water samples from
among the tubes in the tubeworm colonies. In contrast, a maximum concentration of dissolved sulfide of 5,172 µmol/L was
measured for the “hottest” hydrothermal fluids (58˚C) sampled
at Palinuro.
CHARACTERIZATION OF THE TUBEWORMS
Characterization of Palinuro tubeworms
A wide range of sizes of vestimentiferan tubeworms were collected from the Palinuro volcanic complex (Figure 3). The tubes
of the collected animals ranged up to 15 cm in length with a maximum exterior diameter of 3 mm at the anterior end, decreasing
slightly to the posterior end. The anterior region was banded reddish brown and white, whereas the posterior region was a more
uniform brownish color. The tube walls were thick and rigid in
the anterior region, becoming thinner and more flexible in the
posterior regions. The vestimentiferan tubeworm hosts were identified by molecular analyses of the 18S rRNA gene as well as the
mitochondrial genes for ribosomal 16S rRNA and the cytochrome
c oxidase I (COI). All three genes were amplified from DNA
extracted from tubeworm vestimentum, which is free of endosymbionts. Based on COI and mitochondrial 16S rRNA nucleotide
analyses, all individuals analyzed from tubeworm colony #1 and
the four individuals exhibiting different morphologies from tubeworm colony #2 obtained from the Palinuro volcanic complex
belonged to the same species. The maximum difference between
the COI gene fragments sequences was two bases (total investigated length 650 bp) and one base for the mitochondrial 16S
rRNA gene (total investigated length 529 bp). In accordance with
18S rRNA gene, mitochondrial 16S rRNA gene and COI sequence
the tubeworm could be identified as the newly described species
Lamellibrachia anaximandri (Southward et al., 2011).
December 2012 | Volume 3 | Article 423 | 7
Thiel et al.
FIGURE 3 | Lamellibrachia sp. tubeworms recovered from the Palinuro
volcanic complex (Mediterranean Sea). (A) in their natural habitat (photo
obtained at Palinuro during cruise Pos340), (B) directly after ROW Mohawk
Characterization of Gulf of Mexico tubeworms
Gulf of Mexico tubeworm samples were identified using mitochondrial 16S rRNA genes amplified from DNA extracted from
the endosymbiont free vestimentum tissue. Phylogenetic results
confirmed the initial morphological characterizations of Lamellibrachia luymesi/Lamellibrachia sp. 1 (van der Land and Nørrevang,
1975), Lamellibrachia sp. 2, Escarpia laminata (Jones, 1985), and
Seepiophila jonesi (Gardiner et al., 2001; Table 2; Figure 4).
CHARACTERIZATION OF THE TUBEWORM ENDOSYMBIONTS
Endosymbionts of L. anaximandri from Palinuro
Microscopic studies on the Palinuro L. anaximandri revealed high
numbers of coccoid bacterial cells in broken trophosome tissue.
These endosymbiotic bacterial cells varied considerably in size (2–
10 µm diameter) and shape (spherical to irregularly coccoid). The
color in the light microscope ranged from light to dark brown.
Different modes of cell division were observed: equal division,
unequal division, and budding (Figure 5). The cell surface of
many endosymbionts showed a characteristic pattern of small
invaginations (0.2–0.5 µm diameter), while others had a completely smooth surface. Frequently it was observed that cells in
the process of budding or unequal cell division had a structured
surface in the larger (older) part of the cell, while the bud was
smooth (Figure 5).
The bacterial endosymbionts of the tubeworms were identified by constructing 16S rRNA gene clone libraries from DNA
extracted from the trophosome tissue of 11 tubeworm individuals. For each specimen at least 20 clones were sequenced and
analyzed. The bacterial 16S rRNA gene sequences of each specimen
had >99% sequence identity, thus representing a single OTU. The
Frontiers in Microbiology | Extreme Microbiology
Carbon fixation in tubeworm endosymbionts
recovery (Pos412) onboard, (C) individual from colony #1 dissected from its
tube (not used for further analysis), (D) stereo-micrograph of plume region,
(E) stereo-micrograph of trophosome region.
consensus sequences (OTUs) from the different individuals were
identical (100% sequence identity over a total length of 1,387 bp),
indicating that only one bacterial endosymbiont phylotype was
present in the Palinuro tubeworms. BLAST analysis revealed the
gammaproteobacterial sulfide-oxidizing “phylotype 2” bacterial
endosymbiont of L. anaximandri from the Eastern Mediterranean
mud volcanoes as the closest relative (FM165438, 99.7% sequence
identity, five nucleotides differences over a total length of 1,387 bp
(Duperron et al., 2009). Other closely related sequences originate from other Lamellibrachia spp. and seep vestimentiferan
endosymbionts from outside the Mediterranean (Figure 6). The
endosymbionts of hydrothermal vent tubeworms Riftia pachyptila
and Tevnia jerichonana were more distantly related and clustered
on a separate branch within the 16S rRNA gene tree (Figure 6).
Phylogeny of seep vestimentiferan endosymbionts from the Gulf of
Mexico
Bacterial endosymbionts from GoM tubeworm specimen were
identified by amplification and direct sequencing of 16S rRNA
genes from DNA extracted from the trophosome tissue.
The GoM vestimentiferan tubeworm’s endosymbionts were
affiliated with the three monophyletic groups of seep vestimentiferan tubeworm endosymbiont sequences described by
McMullin et al. (2003). Three specimens from GoM site DC673
(Lamellibrachia sp. 1, Lamellibrachia sp. 2, and E. laminata
(DC673_1211, DC673_1209, DC673_1170) shared the identical (100% 16S rRNA gene sequence) “group 2” endosymbiont,
very closely related to the sequences from Lamellibrachia sp. 1
and sp. 2 endosymbionts at site GC852 (GC852_L4, GC852_L1,
GC852_L5) and E. laminata endosymbiont sequence from WR269
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Thiel et al.
Carbon fixation in tubeworm endosymbionts
DC673
WR269
GC852
AC601
GC234
GC184
Pos412
Lamellibrachia luymesi GC234_V4L_99, GU068251
Lamellibrachia sp. 1 WR269_102, GU068253
Lamellibrachia luymesi GC234_V6_97, GU068249
Lamellibrachia luymesi BH_LV1P_93, GU068245
Lamellibrachia luymesi GC234_LV6_86, GU068239
100 Lamellibrachia luymesi BH_LA_V9_62 = GC184_L9_L25, GU068216
Lamellibrachia sp. 1 WR269_L9_104, GU068255
Lamellibrachia sp. 1 isolate AC601_L13_107, GU068257
Lamellibrachia sp. 1 DC673_1211
Lamellibrachia sp.1 GC852_L5
50
Lamellibrachia sp. 1 GC852_L4_90, GU068242
Lamellibrachia luymesi GC234_lv7_85 = GC234_L7, GU068238
71 Lamellibrachia sp. 2 GC852_L1
Lamellibrachia sp. 2 AC601_L20
Lamellibrachia sp. 2 GB829_L7_116, GU068263
89 Lamellibrachia sp. 2 GC852_L19_120, GU068267
Lamellibrachia sp. 2 AT340_L39_122, GU068269
Lamellibrachia sp. 2 DC673_1209
Lamellibrachia sp. Pos412 B2_L4
Lamellibrachia sp. Pos412 B1_L1
Lamellibrachia sp. SMH-2007a
isolate 240 Anaximander site, HM746775
Lamellibrachia sp. SMH-2007a
isolate 081 Napoli site, HM746777
Lamellibrachia sp. SMH-2007a
isolate 0061 Central Pockmark, HM746782
Seepiophila jonesi GB647_S6_144, GU068287.1
Seepiophila jonesi GC234_4587
97
Seepiophila jonesi GB647_S2_142, GU068285.1
Seepiophila jonesi GC234_V7_140, GU068283.1
Seepiophila jonesi BH_V7_138, GU068281.1
Escarpia laminata AT340_E8_46, GU068201
100
Escarpia laminata AC601_L7_50, GU068205
Escarpia laminata AC601_E6_48, GU068203
100
Escarpia laminata AT340_E2_52, GU068207
Escarpia laminata DC673_1170
68 Escarpia laminata AC601_L1
Escarpia laminata WR269_E10
0.01
Escarpia laminata AC601_E6, GU068203
FIGURE 4 | Phylogenetic relationship of vestimentiferan
tubeworms based on mitochondrial 16S rRNA gene sequences.
The Maximum Likelihood tree was calculated using the GTR model.
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Numbers at the nodes indicate the proportion of occurrences in 100
bootstrap replicates. The scale represents 0.01 substitutions per
nucleotide site.
December 2012 | Volume 3 | Article 423 | 9
Thiel et al.
FIGURE 5 | Microscopic images of Palinuro Lamellibrachia
anaximandri specimen endosymbionts. (A,B) Light micrographs
showing trophosome content with large spherical prokaryotic cells
(dark) of different size and shape. Various stages of equal and unequal
(WR269_E10). However, the endosymbiont sequence derived
from an E. laminata specimen at site AC601 (AC601_E6) differed and clustered with “group 1” sequences. In two other tubeworm specimens (E. laminata and Lamellibrachia sp. 2) from
site AC601 (AC601_L1, AC601_L20) we detected two different
endosymbionts, one clustering with “group 1” (AC601_L1-PT1,
AC601_L20-PT1) and the other with “group 2” (AC601_L1PT2, AC601_L20-PT2). Endosymbiont sequences derived from S.
jonesi and L. luymesi tubeworms from the shallower sites GC234
(GC234_4587, GC234_L7), and GC184 (GC184_L9) clustered
with “group 3” sequences (Figure 6).
Genes involved in endosymbiont energy metabolism
In order to determine the potential energy-generating pathways for
chemoautotrophic growth of the endosymbiont from the Palinuro L. anaximandri, we tried to amplify fragments of genes coding
for key enzymes involved in the oxidation of sulfur compounds,
hydrogen and methane.
Frontiers in Microbiology | Extreme Microbiology
Carbon fixation in tubeworm endosymbionts
cell division as well as budding can be recognized. (C–F) Scanning
electron micrographs showing endosymbionts with characteristically
structured cell surface. Probable budding stages (C,D) and unequal
cell division (E,F).
The genetic potential for sulfur oxidation of the endosymbiont
was analyzed by amplifying gene fragments coding for dissimilatory sulfite reductase (dsrAB), APS reductase (aprA) – both
enzymes of the APS pathway – and sulfate thiohydrolase (soxB), an
essential component of the Sox multienzyme complex (Friedrich
et al., 2001). Fragments of all three genes (dsrAB, aprA, soxB) were
recovered supporting a sulfide-oxidizing chemotrophic energy
metabolism of the endosymbiont. Sequence similarities as well
as phylogenetic analysis showed them to be very similar to the
endosymbionts of other L. anaximandri (apr within symbiont
cluster) and the vestimentiferans Riftia pachyptila and Tevnia
jerichonana from hydrothermal vents on the East Pacific Rise
(dsrAB, soxB; Figure 7). The 397 bp aprA sequence showed highest
similarity (97% nucleotide similarity, 100% amino acid similarity) to L. anaximandri endosymbiont “phylotype 1” described
from seep specimens at the Amon mud volcano in the Eastern Mediterranean (Duperron et al., 2009). Phylogenetic analysis
places the Lamellibrachia aprA sequences in a cluster of oxidizing
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Thiel et al.
Carbon fixation in tubeworm endosymbionts
Escarpia laminata endosymbiont DC673_1170
Lamellibrachia sp. 2 endosymbiont GC852_L1
Lamellibrachia sp. 2 endosymbtion DC673_1209
Lamellibrachia sp. 1 endosymbiont DC673_1211
Escarpia spicata endosymbiont, U77482
Lamellibrachia barhami endosymbiont, AY129103
Escarpia laminata endosymbiont AC601_L1 PT2
Lamellibrachia sp. 2 endosymbiont AC601_L20 PT1
Escarpia laminata endosymbiont, AY129108
Lamellibrachia sp. 1 endosymbiont, AY129112
Escarpia laminata endosymbiont WR269_E10
Lamellibrachia sp. 1 endosymbiont GC852_L4
88
Lamellibrachia sp. 1 endosymbiont GC852_L5
100
Lamellibrachia barhami endosymbiont, AY129093
Lamellibrachia anaximandri endosymbiont Pos412_B1_L1
89
Lamellibrachia anaximandri endosymbiont Post 412_B2_L4
Lamellibrachia anaximandri endosymbiont PT 2 clone V11.1, FM165438
79
Seepiophila jonesi endosymbiont, AY129087
Seepiophila jonesi endosymbiont GC234_4587
Seepiophila jonesi endosymbiont, AY129104
95
Lamellibrachia cf. luymesi endosymbiont, AY129100
Lamellibrachia luymesi endosymbiont GC234_L7
Lamellibrachia sp. 2 endosymbiont, AY129110
Lamellibrachia sp. 2 endosymbiont, AY129111
Lamellibrachia luymesi endosymbiont GC184_L9
Lamellibrachia anaximandri endosymbiont PT1 clone V1.3, FM165437
Escarpia laminata endosymbiont AC601_E6
Lamellibrachia sp. 2 endosymbiont AC601_L20 PT2
Lamellibrachia barhami endosymbiont, AY129090
Escarpia laminata endosymbiont, AY129106
Lamellibrachia barhami endosymbiont, AY129113
Lamellibrachia columna endosymbiont, U77481
Escarpia laminata endosymbiont AC601_L1 PT1
Lucina nassula gill symbiont, X95229
98
100
Codakia orbicularis gill symbiont, X84979
endosymbiont of Ridgeia piscesae, AY129120
100
Oasisia alvinae endosymbiont, AY129114
Riftia pachyptila endosymbiont, AY129115
0.01
Tevnia jerichonana endosymbiont, AY129117
DC673
WR269
GC852
AC601
GC234
GC184
Pos412
76
Group 1
Group 3
Group 2
Vent group
FIGURE 6 | Phylogenetic relationship of tubeworms endosymbionts based on 16S rRNA gene sequences. The Maximum Likelihood tree was calculated
using the GTR model. Numbers at the nodes indicate the proportion of occurrences in 100 bootstrap replicates. The scale represents 0.01 substitutions per
nucleotide site.
lineage II APS reductase gene sequences of endosymbiotic and
free-living beta- and gammaproteobacteria including endosymbionts of Riftia and Tevnia (Meyer and Kuever, 2007a; Markert
et al., 2011; Gardebrecht et al., 2012). The dsrAB gene fragment
(987 bp) was most closely related to dissimilatory sulfite reductase
genes from the Riftia/Tevnia endosymbiont (NZ_AFZB01000023,
EGW53672, and EGV52261, 80% nucleotide and 84% amino acid
sequence similarity, Figure 7).
Likewise, the 986 bp soxB fragment from L. anaximandri from
Palinuro showed highest similarities to soxB from Candidatus
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Endoriftia persephone (EF618617, EGV50931, and EGW54296,
84% nucleotide similarity, 90% amino acid similarity).
A fragment of the hupL gene, encoding the large subunit of
a [NiFe] uptake hydrogenase was amplified using the primer
set W1 and Wxy. BLAST search as well as phylogenetic analysis
demonstrated highest similarity with reference sequences from
the Riftia/Tevnia endosymbiont (EGV51840, EGW53439, 82%
nucleotide identity, 93% amino acid identity). This enzyme has
been shown to be involved in the oxidation of molecular hydrogen
for energy generation (Petersen et al., 2011; Kleiner et al., 2012).
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Thiel et al.
Carbon fixation in tubeworm endosymbionts
A
B
100
Chlorobium tepidum
100
Chlorobaculum parvum
100
Magnetococcus marinus MC1
Magnetospirillum gryphiswaldense
Tevnia jerichonana endosymbiont
Riftia pachyptila endosymbiont
Lamellibrachia sp. endosymbiont Pos412_B1_L1_drsA
Thiobacillus denitrificans
100
Thiobacillus thioparus
73
Thiothrix nivea
61
Oligobrachia haakonmosbiensis endosymbiont
100
100
Marichromatium purpuratum
100
Thiocapsa roseopersicina
61
Allochromatium vinosum
55
Thiocapsa marina
Candidatus Ruthia magnifica
Candidatus Vesicomyosocius oktuanii
91
0.1
Alkalilimnicola ehrlichii
Halorhodospira halophila
FIGURE 7 | Phylogenetic tree based on dsrA (A) and aprA (B) protein
sequences. The Maximum Likelihood tree was calculated using the JTT
model. Numbers at the nodes indicate the proportion of occurrences in 100
Key genes for enzymes of methane oxidation (mxaF, mmoX,
pmoA) were not successfully amplified with the different primer
sets (MxaF1003, MxaR1555, MxaR1561, mmoXA, mmoXB,
A189F, MB661R; Costello and Lidstrom, 1999; Auman et al., 2000;
Neufeld et al., 2007; Kalyuzhnaya et al., 2008) used in this study.
Genes involved in nitrate reduction
A nitric oxide reductase (norCB) gene sequence was successfully amplified and sequenced from the Palinuro L. anaximandri
endosymbiont indicating the potential to reduce nitrate. The closest relative was again the endosymbiont of Riftia/Tevnia (EMBL
entry ZP_08818090) with 95% amino acid sequence similarity. In
the metagenomes of the Riftia and Tevnia endosymbionts, all genes
needed for a complete respiration of nitrate to dinitrogen gas have
been detected, and it has been suggested that the endosymbionts
of these species could possibly use nitrate as alternative electron
acceptor (Gardebrecht et al., 2012).
Genes involved in carbon fixation
To investigate the autotrophic potential of the endosymbionts, we
tried to amplify key genes of two carbon fixation pathways, the
CBB cycle and the reductive tricarboxylic acid (rTCA) cycle.
The CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is the key enzyme of the CBB cycle.
In proteobacteria, two different types are known, form I, encoded
Frontiers in Microbiology | Extreme Microbiology
0.1
APR lineage I
100
APR lineage I
96
Archaeoglobus infectus
Archaeoglobus profundus
Thermodesulfobacterium commune
100
Thermodesulfatator indicus
Desulfobacterium autotrophicum
60
Desulfonema magnum
70
Desulfotalea psychrophila
72
Desulfocapsa sulfexigens
81
65
Desulfobulbus marinus
Desulfofustis glycolicus
98
95
Desulfonauticus submarinus
Desulfovibrio vulgaris
Lucinoma aff. kazani endosymbiont
Thyasira sp. endosymbiont
99
Oasisa sp. endosymbiont
Riftia pachyptila endosymbiont
Tevnia jerichonana endosymbiont
Lamellibrachia anaximandri endosymbiont
Lamellibrachia sp. endosymbiont Pos412_B1_L1_aprA
Sclerolinum contortum endosymbiont
Ifremeria nautilei symbiont
Thiothrix nivea
98 Oligobrachia haakonmosbiensis G1 endosymbiont
54
Oligobrachia haakonmosbiensis endosymbiont
Thiobacillus denitrificans
50 Lamprocystis purpurea
Thiocapsa roseopersicina
Olavius algarvensis Gamma1 endosymbiont
Beggiatoa sp. PS
Inanidrilus exumae endosymbiont
Pelagibacter ubique
89 Bathymodiolus brevior endosymbiont
53 Bathymodiolus thermophilus endosymbiont
Idas sp. endosymbiont
69 63 Candidatus Ruthia magnifica
Halochromatium salexigens
Thiococcus pfennigii
66
Chromatium okenii
bootstrap replicates. Sequences of tubeworm endosymbionts are depicted
bold. Sequences obtained in this study are depicted colored. The scale
represents 0.1 substitutions per amino acid position.
by the cbbL gene, and form II encoded by cbbM. In accordance
with other studies on Lamellibrachia spp. endosymbionts (Elsaied
and Naganuma, 2001; Elsaied et al., 2002), we failed to amplify
cbbL from the Palinuro tubeworm endosymbiont. In contrast, a
fragment of the cbbM gene was detected supporting the usage
of the CBB cycle for carbon fixation by the endosymbiont as
was also previously demonstrated for other Lamellibrachia spp.
endosymbionts (Elsaied and Naganuma, 2001; Elsaied et al., 2002;
Vrijenhoek et al., 2007). The cbbM sequence displayed high similarity (96% nucleotide similarity, 100% amino acid similarity)
to the bacterial endosymbiont of L. anaximandri from the Eastern Mediterranean (FM165442 and CAQ63473, Duperron et al.,
2009), but was quite different from cbbM sequences of Riftia/Tevnia endosymbionts (AF047688, 78% nucleotide similarity,
75% amino acid similarity).
Based on genomic, proteomic, enzymatic as well as isotopic
data, the Riftia pachyptila endosymbiont uses the rTCA cycle in
addition to the CBB cycle for autotrophic carbon fixation (Markert et al., 2007, 2011; Gardebrecht et al., 2012). Yet a novel type of
ATP citrate lyase (type II ACL) might be active in this case (Hügler
and Sievert, 2011).
Newly designed primers were used to amplify a putative type
II ACL from the endosymbionts of the Palinuro tubeworms.
Unexpectedly, amplified fragments of this type II ACL gene
indicate additional use of the rTCA cycle for carbon fixation
December 2012 | Volume 3 | Article 423 | 12
Thiel et al.
in the Palinuro L. anaximandri endosymbiont as well. BLAST
results with sequence similarities of 78% amino acid identities, as well as the phylogenetic analysis indicate the gene to
be most closely related to the Riftia and Tevnia endosymbiont (NCBI entry ZP_08829917 and ZP_08817421). In contrast genes coding for a conventional ACL could not be
detected using previously published primers for either subunits (aclA or aclB, Campbell et al., 2003; Hügler et al.,
2005).
Carbon fixation genes in seep vestimentiferan endosymbionts from
the Gulf of Mexico
The discovery of ACL genes in the L. anaximandri endosymbiont
from the Palinuro volcanic complex raised the question about
the further distribution of these genes in tubeworm endosymbionts, especially seep species. Thus we analyzed endosymbionts
from 13 tubeworms from six different sites in the GoM. We discovered the type II ACL genes in all tubeworm endosymbionts
investigated, regardless of their host species identity or site in
the GoM (Table 2). Sequence analysis revealed the type II ACL
gene to be highly conserved between the different GoM tubeworm endosymbionts. Three different phylotypes were found
to be present in the 13 tubeworm samples in the GoM, and
all three differed from the sequences found in the Palinuro L.
anaximandri endosymbionts (Figure 8A). All GoM endosymbionts of host specimens from the sites DC673, WR269, and
GC852 (DC673_1211, DC673_1209, DC673_1170, WR269_E10,
GC852_L4, GC852_L1, GC852_L5) shared identical (100% AA
similarity) type II ACL gene sequences (cluster 2) regardless of host
species identity. Endosymbionts of GoM host specimens from the
shallower Green Canyon sites GC234 and GC184 (GC234_4587,
GC184_L9, GC234_L7) also bear one single type II ACL gene
sequence (cluster 3; 100% AA similarity), which differed from the
deep water site sequence in 14 AA. The third sequence type (cluster 1) was retrieved from endosymbionts of Alaminos Canyon site
AC601 (AC601_E6, AC601_L1, AC601_L20). Within AC601_L1
and AC601_L20 a second ACL sequence type identical to the
sequences of cluster 2 was also retrieved. In phylogenetic analysis, the GoM tubeworm endosymbiont ACL type II sequences
formed a cluster together with the Mediterranean L. anaximandri endosymbiont sequences, and were clearly separated from the
Riftia/Tevnia sequences (Figure 8A).
In addition to the acl genes, we also amplified the cbbM gene
of the GoM tubeworm endosymbionts. As expected, all endosymbionts harbored a cbbM gene in addition to the acl gene. Similar
to the 16S rRNA and acl genes, the cbbM genes from the GoM
endosymbionts formed three different clusters (Figure 8B). Cluster 1 comprises the sequences of Alaminos Canyon site AC601
specimens, AC601_E6, AC601_L1, and AC601_L20. The cbbM
sequences of DeSoto Canyon, Walker Ridge, and Green Canyon
specimens DC673_1211, DC673_1209, WR269_E10, GC852_L4,
GC852_L1, GC852_L5 form a second cluster (cluster 2), while
the cbbM sequence of sample DC673_1170 falls in between these
two clusters. Cluster 3 (Green Canyon samples GC234_4587,
GC184_L9, GC234_L7) is clearly separated from the others. The
cbbM sequences from the Mediterranean tubeworm endosymbionts form a separate cluster. Quite interestingly, the cbbM
www.frontiersin.org
Carbon fixation in tubeworm endosymbionts
sequences of the Riftia/Tevnia endosymbionts are only distantly
related (Figure 8B).
ISOTOPIC SIGNATURE
Bulk organic carbon isotopes analyses of gill tissue from two
Palinuro tubeworms resulted in δ13 C values of −22.5 and
−23.4‰, which are in accordance to previous measurements of
Mediterranean Lamellibrachia spp. (Olu-Le Roy et al., 2004; Carlier et al., 2010) but more positive than most Lamellibrachia spp.
from non-Mediterranean hydrocarbon seeps (Becker et al., 2011).
The carbon isotopic composition of dissolved inorganic carbon in
emanating diffuse fluids sampled at the tubeworm colonies display δ13 C values of −0.39 and −0.68‰. The δ13 CDIC values of
additional samples of shimmering water in the area range from
−1.62 to +1.76‰.
DISCUSSION
PHYLOGENY AND BIOGEOGRAPHY OF THE MEDITERRANEAN
TUBEWORMS
The discovery of living vestimentiferan tubeworm colonies associated with active hydrothermal venting during a seafloor survey of
the Palinuro volcanic complex (Mediterranean Sea) in July 2006
(Petersen et al., 2008; Monecke et al., 2009) came as a surprise,
as until then, vent-associated tubeworms were only known from
the Pacific Ocean. Living individuals of the tubeworms were sampled during a research cruise in 2011 and this communication
is the first description of the worms and their endosymbionts.
Phylogenetic analyses of 18S rRNA, COI and mitochondrial 16S
rRNA genes showed that the tubeworms from Palinuro are Lamellibrachia anaximandri recently described from mud volcanoes of
the Eastern Mediterranean (Southward et al., 2011). The highest in vivo temperatures measured among the tubes in tubeworm aggregations at the Palinuro hydrothermal vent field were
15.6–19.4˚C, elevated by as much as 5.4˚C from the surrounding Mediterranean Seawater (14˚C) and the previously published
tubeworm-bearing locations in the Eastern Mediterranean (13–
14˚C), extending the previously described temperature range of
the species (Southward et al., 2011). L. anaximandri has also been
detected in a mud volcano field in the Western Mediterranean
(Hilário et al., 2011), as well as on two ship wrecks in the Eastern
Mediterranean (110 km southeast of Crete, Hughes and Crawford, 2008) and the Southern Tyrrhenian Sea (Gambi et al., 2011;
Figure 1). Even though this species has not been detected in
the Northeastern Atlantic it has been hypothesized to occur at
the West African and Lusitanian margins as well (Hilário et al.,
2011).
The high diversity of habitats for the Mediterranean Lamellibrachia species is in accordance with Escarpia spp. and other
Lamellibrachia spp. Originally regarded as seep species, they were
subsequently found in several non-seep habitats, i.e., at sediment
covered hydrothermal sites in the Pacific (Juniper et al., 1992;
Fujikura et al., 2006; Miake et al., 2006; Miura and Kojima, 2006),
as well as ship wrecks (Dando et al., 1992), and whale falls (Feldman et al., 1998). Considering the high diversity of so-called
“seep” tubeworm habitats a high site-flexibility of these organisms
becomes apparent and supports the importance of different nonseep habitats in their geographic distribution and the stepping
December 2012 | Volume 3 | Article 423 | 13
Thiel et al.
Carbon fixation in tubeworm endosymbionts
A
B
Seepiophila jonesi endosymbiont GC234_4587_acl
Lamellibrachia luymesi endosymbiont GC184_L9_acl
Lamellibrachia luymesi endosymbiont GC234_L7_acl
Lamellibrachia sp. 2 endosymbiont GC852_L1_acl
Lamellibrachia sp. 1 endosymbiont GC852_L5_acl
Escarpia laminata endosymbiont AC601_L1_aclV2
Lamellibrachia sp. 2 endosymbiont AC601_L20_aclV2
99
Escarpia laminata endosymbiont AC601_E6_acl
Escarpia laminata endosymbiont AC601_L1_aclV1
Lamellibrachia sp.2 endosymbiont AC601_L20_aclV1
Azoarcus sp. KH32C
94
98
100
Burkholderia phymatum STM815
Burkholderia sp. SJ98
Burkholderia terrae BS001
91
100
Burkholderia sp. CCGE1002
Methylobacterium nodulans ORS 2060
Microvirga sp. WSM3557
Sinorhizobium fredii HH103
Geobacter metallireducens GS-15
0.1
Desulfobacter postgatei 2ac9
Cluster 3
Magnetococcus marinus MC1
shallow
Tevnia jerichonana endosymbiont
Riftia pachyptila endosymbiont
Cluster MS
Lamellibrachia sp. endosymbiont Pos412_B1_L1_acl
Lamellibrachia sp. endosymbiont Pos412_B2_L4_acl
94
Cluster 2
Lamellibrachia sp. 1 endosymbiont GC852_L4_acl
100
deep
Escarpia laminata endosymbiont DC673_1170_acl
Escarpia laminata endosymbiont WR269_E10_acl
tubeworm endosymbionts
Lamellibrachia sp.1 endosymbiont DC673_1211_acl
Lamellibrachia sp.2 endosymbiont DC673_1209_acl
Escarpia laminata endosymbiont DC673_1170_cbbM
Vestimentiferan tubeworm endosymbiont (ABB70165)
Vestimentiferan tubeworm endosymbiont (ABB70167)
Escarpia laminata endosymbiont AC601_E6_cbbM
Escarpia laminata endosymbiont AC601_L1_cbbM
Lamellibrachia sp.2 endosymbiont AC601_L20_cbbM
94
Lamellibrachia sp. endosymbiont (BAA92188)
Lamellibrachia sp.1 endosymbiont DC673_1211_cbbM
Lamellibrachia sp.2 endosymbiont DC673_1209_cbbM
Escarpia laminata endosymbiont WR269_E10_cbbM
Lamellibrachia sp. 1 endosymbiont GC852_L4_cbbM
Lamellibrachia sp. 2 endosymbiont GC852_L1_cbbM
Lamellibrachia sp. 1 endosymbiont GC852_L5_cbbM
uncultured bacterium (CAL47121)
Sclerolinum contortum endosymbiont (CAP03142)
Thiomicrospira halophila
Thiomicrospira crunogena
Thiomicrospira pelophila
Leptothrix cholodnii
80
magnetite-containing magnetic vibrio (AF442518)
Thiobacillus denitrificans
Halothiobacillus neapolitanus
Acidithiobacillus ferrooxidans
79
Dechloromonas aromatica
Lamellibrachia sp. endosymbiont Pos412_B1_L1_cbbM
Lamellibrachia sp. endosymbiont Pos412_B2_L4_cbbM
Lamellibrachia anaximandri endosymbiont (FM165442)
Rimicaris exoculata epibiont (FN659777)
Lamellibrachia sp. endosymbiont (BAA94433)
100 Seepiophila jonesi endosymbiont GC234_4587_cbbM
Lamellibrachia luymesi endosymbiont GC184_L9_cbbM
74
Lamellibrachia luymesi endosymbiont GC234_L7_cbbM
73 Oligobrachia haakonmosbiensis endosymbiont (CAP03140)
Oligobrachia haakonmosbiensis endosymbiont (CAP03141)
77
Candidatus Vesicomyosocius oktuanii
Candidatus Ruthia magnifica
Rhodobacter sphaeroides
Rhodopseudomonas palustris
100
Tevnia jerichonana endosymbiont
Riftia pachyptila endosymbiont
Rhodospirillum rubrum
Magnetospirillum gryphiswaldense
66 Thiomicrospira kuenenii
76
Mariprofundus ferrooxydans
Thiomicrospira thermophila
Methanocaldococcus jannaschii
Cluster 1
DC673
WR269
GC852
AC601
GC234
GC184
Pos412
0.1
FIGURE 8 | Phylogenetic tree based on aclA (A) and cbbM (B) protein
sequences. The Maximum Likelihood tree was calculated using the JTT
model. Numbers at the nodes indicate the proportion of occurrences in 100
stone hypothesis (Kimura and Weiss, 1964; Smith and Kukert,
1989; Black et al., 1994; Olu et al., 2010). Larval survival of at
least three weeks and about five weeks has been demonstrated for
the vestimentiferans Riftia pachyptila and Lamellibrachia luymesi
respectively, suggesting potential dispersal distances on the order
of 100 km for seep and vent vestimentiferans (Young et al., 1996;
Marsh et al., 2001; Tyler and Young, 2003). A variety of reducing habitats, functioning as dispersal stepping-stones separated
by days or weeks and connected by currents or shared water
masses could facilitate the large species ranges described for many
vestimentiferans, including the seep species L. barhami, which
has been found in seep and low activity vent sites spanning at
least 4,000–6,000 km of geographical distance (McMullin et al.,
2003).
PHYLOGENY OF ENDOSYMBIONTS
The trophosome of the L. anaximandri specimens from Palinuro
analyzed in this study harbored a single gammaproteobacterial phylotype regardless of collection site or morphotype. The
endosymbiont was closely related (99.7%) to the phylotype 2
found in L. anaximandri from the Amon mud volcano east of
the Nile deep-sea fan (Duperron et al., 2009). The dominating
endosymbiont (phylotype 1) of the seep specimen from the Amon
mud volcano was not found in the tubeworms at the Palinuro
hydrothermal vents.
Frontiers in Microbiology | Extreme Microbiology
bootstrap replicates. Sequences of tubeworm endosymbionts are depicted
bold. Sequences obtained in the present study are depicted colored. The
scale represents 0.1 substitutions per amino acid position.
This study is the first to characterize vestimentiferan tubeworm endosymbionts of shallow hydrothermal vents in the
Mediterranean Sea. The gammaproteobacterial endosymbiont
clusters with endosymbionts of other seep-associated tubeworms
and are clearly distinct from the endosymbionts of vent tubeworms like Riftia and Tevnia (Fisher et al., 1997; Nelson and
Fisher, 2000; McMullin et al., 2003; Vrijenhoek et al., 2007). The
phylogenetic affiliation of the Palinuro L. anaximandri endosymbiont with “group 3” endosymbiont 16S rRNA gene sequences,
as well as the affiliation of the dominating phylotype of L. anaximandri specimen obtained from 1,157 m depth at Amon mud
volcano with “group 1,” may indicate separation by depth, as suggested for other seep vestimentiferan endosymbionts (McMullin
et al., 2003). However, both Mediterranean phylotype sequences
show considerable differences to the “group 1” and “group 3”
cluster sequences in signature nucleotide positions (Table A1
in Appendix) and Mediterranean and GoM tubeworms do not
share identical endosymbiont phylotypes. Further, in the Amon
mud volcano specimen, phylotypes of “group 1” and “group 3”
are present, yet in assumed different abundances (deduced from
the numbers of sequences in the clone libraries; Duperron et al.,
2009). Endosymbionts of different groups are also present in
individuals of Lamellibrachia sp. 2 (AC601_L20) and E. laminata (AC601_L1) tubeworms from the GoM site AC601 (this
study). Thus separation by depth alone cannot explain these
December 2012 | Volume 3 | Article 423 | 14
Thiel et al.
observations and further studies are needed in order to reveal the
question how endosymbionts are selected by their vestimentiferan
hosts.
METABOLIC CHARACTERISTICS OF THE ENDOSYMBIONT
Based on the functional gene analyses of this study, the endosymbiont of Lamellibrachia anaximandri from Palinuro is a sulfideoxidizing chemoautotroph. δ13 C values measured in this study
are in consistence with previous studies of L. anaximandri from
Eastern Mediterranean mud volcano fields (Olu-Le Roy et al.,
2004; Carlier et al., 2010), and together with delta δ15 N and δ34 S
from previous studies support a chemoautotrophic endosymbiont
based nutrition for the host tubeworm (Carlier et al., 2010). Due to
the presence of dsrAB and aprA genes, sulfide oxidation most likely
is carried out via the APS pathway with sulfite and adenosine phosphosulfate as intermediates. As in the Riftia pachyptila endosymbiont, soxB is also present in the endosymbiont of L. anaximandri
from Palinuro. In thiosulfate-utilizing bacteria, SoxB functions
as sulfate thiohydrolase. However, since tubeworm endosymbiont
carbon fixation is not stimulated by thiosulfate, its function in the
tubeworm endosymbionts remains uncertain (Fisher et al., 1989;
Markert et al., 2011).
Although high methane fluxes were noted in the habitat of
L. anaximandri in mud volcano habitats (Olu-Le Roy et al.,
2004), genes of methane oxidation were not successfully amplified with the primer sets used in this study. In contrast, the
potential to use hydrogen as an energy source was suggested by
the detection of the key gene for hydrogen oxidation, hupL in
the endosymbiont from the Palinuro L. anaximandri. The hupL
gene was most similar to the respective genes of the Riftia and
Tevnia endosymbionts, where it is even expressed in situ (Markert et al., 2011; Petersen et al., 2011). Hydrogen concentrations
have not been measured in the hydrothermal fluids from Palinuro volcano complex. However, hydrogen is present in the fluids
at many hydrothermal vents, and elevated hydrogen contents
are present at vent systems associated with ultramafic (mantle)
rocks, or, e.g., following a volcanic eruption (Wetzel and Shock,
2000; Allen and Seyfried, 2003; Kumagai et al., 2008; Petersen
et al., 2011). Such H2 -rich fluids found at vent systems associated with ultramafic rocks have recently been shown to be used
as an energy source by the endosymbionts of a mussel, Bathymodiolus puteoserpentis (Petersen et al., 2011). The endosymbionts
in Bathymodiolus spp. mussels are located on the external edge
of the cells of gill filaments that are themselves only two cells
thick. As a result, passive diffusion of the energy source (hydrogen, sulfide, and/or methane in different species) is sufficient
to fuel the chemoautotrophic life style of these animals (Childress and Fisher, 1992). In contrast, the endosymbionts in tubeworms are deep in an interior tissue and must rely on the host
blood to supply the electron donor for chemoautotrophy. Sulfide
is transported in millimolar concentrations to the trophosome,
bound to hemoglobin molecules in vestimentiferan tubeworms.
Transport molecules for hydrogen or methane have not been
found in these animals and thus both hydrogen and methane are
unlikely to contribute significantly to the metabolism of the intact
symbiosis in most environments (Childress and Fisher, 1992).
However, the gammaproteobacterial endosymbiont might use
hydrogen as potential energy source in its free-living stage. Thus,
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Carbon fixation in tubeworm endosymbionts
the potential for use of hydrogen by tubeworm endosymbionts
deserves additional study.
Detection of cbbM sequences coding for a form II RubisCO
in all vestimentiferan tubeworms reviewed in this study indicates
that the potential to fix CO2 via the CBB cycle is widespread in
vestimentiferan tubeworms (Elsaied and Naganuma, 2001; Elsaied
et al., 2002; Naganuma et al., 2005; Vrijenhoek et al., 2007; Duperron et al., 2009) and the detection of all genes of this cycle in the
metagenome of the Riftia endosymbiont suggest this pathway is
fully functional in vestimentiferans (see Markert et al., 2011 for
further details). Enzyme activity measurements of RubisCO and
phosphoribulokinase add further evidence to the usage of the CBB
cycle in Riftia and Lamellibrachia endosymbionts (Felbeck, 1981;
Felbeck et al., 1981). Up to now, there is no evidence for the presence of RubisCO form I (cbbL) in either the Riftia/Tevnia or any
Lamellibrachia endosymbiont (this study; Elsaied and Naganuma,
2001; Elsaied et al., 2002; Naganuma et al., 2005; Duperron et al.,
2009).
In addition, acl genes, coding for ATP citrate lyase, the key
enzyme of the rTCA cycle were recovered from the Palinuro L.
anaximandri endosymbiont using newly designed primers, suggesting the presence of the rTCA cycle as alternate carbon fixation
pathway. The operation of the rTCA cycle in addition to the CBB
cycle was first shown for the Riftia and Tevnia endosymbiont
using a combination of metagenomic, proteomic and enzymatic
approaches (Markert et al., 2007; Robidart et al., 2008; Gardebrecht
et al., 2012). In the case of the Riftia/Tevnia endosymbiont, citrate
cleavage is accomplished by an unusual type of ATP citrate lyase,
tentatively named ACL type II (Hügler and Sievert, 2011). The
recovered acl sequence from the Mediterranean Palinuro L. anaximandri endosymbiont showed high similarities to the sequence of
the Riftia/Tevnia endosymbiont (Figure 8A). Subsequent analyses
of seep vestimentiferan (Escarpia, Seepiophila, and Lamellibrachia)
endosymbionts from different sites at the Gulf of Mexico showed
the presence of type II ACL genes there as well. This implicates
a wider distribution of these genes than previously thought. The
presence of two different carbon fixation pathways – the CBB cycle
and the rTCA cycle – in a single bacterium seems not restricted to
the Riftia/Tevnia endosymbiont, but rather seems to be a common
feature of vestimentiferan tubeworm endosymbionts, regardless of
genus or habitat.
Despite the still rather scarce dataset of type II ACL gene
sequences, these sequences appear to be monophyletic in tubeworm endosymbionts (Figure 8A). Similarly, ribosomal genes
as well as aprA genes support a monophyletic origin for the
tubeworm endosymbionts (Figures 6 and 7). In contrast, a monophyletic origin of the cbbM gene of tubeworm endosymbionts
is not clearly supported by the phylogenetic analyses performed
here (Figure 8B). This could mean that the rTCA cycle is the evolutionary older CO2 fixation pathway in the endosymbionts, and
the cbbM gene is acquired afterward, e.g., via lateral gene transfer.
This evolutionary aspect clearly requires further studies.
The presence of two different carbon fixation pathways
increases the metabolic versatility of the tubeworm endosymbionts. In case of the Riftia/Tevnia endosymbiont proteomic data
suggest the usage of both pathways simultaneously (Markert et al.,
2007, 2011; Gardebrecht et al., 2012). This is also supported by
the isotopic signature of the Riftia tubeworms (Markert et al.,
December 2012 | Volume 3 | Article 423 | 15
Thiel et al.
Carbon fixation in tubeworm endosymbionts
2007). The carbon isotopic fractionation associated with the rTCA
cycle is generally smaller than the one observed for the CBB cycle
(House et al., 2003). Considering the carbon isotopic composition
of ca. −23‰ measured for plume tissue of two L. anaximandri
tubeworms from Palinuro low temperature diffuse vent sites and
a respective carbon isotopic composition of dissolved inorganic
carbon (δ13 CDIC between −0.7 and −0.4‰), the isotopic difference would be consistent with the operation of the CBB cycle for
autotrophic carbon fixation. Neither a greatly attenuated isotopic
fractionation characteristic for the rTCA cycle nor an isotopic signature reflecting a substantial contribution from methane-derived
carbon is discernible at the site studied here. Yet, one has to keep in
mind, that the isotopic signature provides only indirect evidence
and neither the actual fractionation by the enzymes involved in the
rTCA cycle present in tubeworm endosymbionts, nor fractionation during uptake and transport of DIC to the endosymbionts
are known. Thus future studies are needed in order to determine
the conditions for the usage, as well as the regulation of the two
different carbon fixation pathways in vestimentiferan tubeworm
endosymbionts (Hügler and Sievert, 2011).
CONCLUSION
In this study we characterize vestimentiferan tubeworms and their
endosymbionts from the Mediterranean Sea and the GoM. The
tubeworms retrieved from a shallow water hydrothermal vent field
in the Western Mediterranean – Palinuro volcanic complex – represent the first vestimentiferan tubeworms found associated with
hydrothermal venting outside the Pacific Ocean. Our molecular
studies of marker genes (18S rRNA, mitochondrial 16S rRNA, and
COI) identify the tubeworms as the recently described species L.
anaximandri, the only vestimentiferan species described from the
Mediterranean Sea to date.
Based on 16S rRNA gene surveys we conclude that the Palinuro L. anaximandri harbor a single gammaproteobacterial
endosymbiont, closely related to endosymbionts of other Lamellibrachia spp. (Figure 6). Carbon isotopic data and the analysis
of functional genes suggest a sulfide-oxidizing chemoautotrophic
lifestyle. Energy can be generated by oxidizing reduced sulfur
compounds via the APS pathway involving dissimilatory sulfite
reductase and APS reductase. Due to the presence of a hupL gene
one can speculate that the endosymbiont has the potential to use
hydrogen as a supplemental energy source. Nitrate could potentially serve as alternative electron acceptor for the endosymbiont,
as we detected a nitric oxide reductase gene sequence (norCB) and
it was shown, that the metagenome of the Riftia/Tevnia endosymbiont includes all genes needed for the complete reduction of
nitrate to dinitrogen gas (Gardebrecht et al., 2012).
Surprisingly, we were able to detect the key genes of two
alternative carbon fixation pathways, namely cbbM, encoding
RubisCO form II, the key enzyme of the CBB cycle, and a gene
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vent fluids from ultramafic-hosted
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Frontiers in Microbiology | Extreme Microbiology
coding for ATP citrate lyase type II, the key enzyme of the
rTCA cycle. Newly designed primers were used to amplify a gene
sequence of the type II ACL. The presence of the rTCA cycle in
addition to the CBB cycle for carbon fixation was previously shown
for the endosymbiont of the vent-associated tubeworms Riftia
pachyptila and Tevnia jerichonana (Markert et al., 2007; Gardebrecht et al., 2012). However, before this study, only the CBB cycle
was documented as a carbon fixation pathway for Lamellibrachia
spp. (Felbeck, 1981; Felbeck et al., 1981; Elsaied and Naganuma,
2001; Elsaied et al., 2002; Vrijenhoek et al., 2007). We also demonstrate the presence of the key genes of both carbon cycles in the
endosymbionts from Lamellibrachia luymesi, Lamellibrachia sp.
1, Lamellibrachia sp. 2, Escarpia laminata, and Seepiophila jonesi
from the Gulf of Mexico. These results suggest that the occurrence of two carbon fixation pathways in one bacterium may be
a common feature of vestimentiferan tubeworm endosymbionts,
which in turn indicates that this feature is more widely distributed than previously considered. It has already been shown, that
carbon fixation through the rTCA cycle is important at deep-sea
hydrothermal vent sites (cf. Hügler and Sievert, 2011 and references therein). Our study indicates that the rTCA cycle could play
an important role at seep sites as well.
ACKNOWLEDGMENTS
The authors would like to thank the captain and crew of R/V
Poseidon for their support during cruise Pos412. We also gratefully acknowledge the support of ROV pilots Jamie Norman and
Paul Hastings during sampling as well as Peter Buchanan (Oceaneering Inc., Aberdeen, UK) for good cooperation during ROV
Mohawk negotiations and adaptation for research purposes. We
further thank Katrin Kleinschmidt (GEOMAR, Kiel, Germany)
for onboard support. We also thank the captains and crews of
the R/V Seward Johnson and NOAA ship Ronald Brown as well as
the pilots and support personnel for the Johnson Sea Link submersible and ROV Jason II for assistance with the collection of
GoM Tubeworms. For Sanger sequencing, we would like to thank
the teams from the IKMB at the UK-SH, Kiel, Germany and from
the sequencing facility at the Pennsylvania State University, PA,
USA. Artur Fugmann and Ben Hindersmann (WWU Münster,
Germany) are thanked for their assistance during stable isotope
measurements, and Ulrike Westernströer and Karen Bremer (CAU
Kiel, Germany) for help with elemental analysis. Further, we thank
Chunya Huang (Pennsylvania State University, PA, USA) for assistance during COI gene analysis and Costantino Vetriani (Rutgers
University, NJ, USA) for fruitful discussion. JFI, SP, DGS, and HS
acknowledge support through the German Science Foundation
(IM12/18). CRF acknowledges the support of the US NSF, the
US Bureau of Ocean Energy Management and US NOAA Office
of Ocean Exploration for many years of support for the study of
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Conflict of Interest Statement: The
authors declare that the research was
conducted in the absence of any
commercial or financial relationships
that could be construed as a potential
conflict of interest.
Received: 04 October 2012; paper pending
published: 05 November 2012; accepted:
26 November 2012; published online: 14
December 2012.
Citation: Thiel V, Hügler M, Blümel M,
Baumann HI, Gärtner A, Schmaljohann
R, Strauss H, Garbe-Schönberg D,
Petersen S, Cowart DA, Fisher CR and
Imhoff JF (2012) Widespread occurrence of two carbon fixation pathways
in tubeworm endosymbionts: lessons
from hydrothermal vent associated
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Sea. Front. Microbio. 3:423. doi:
10.3389/fmicb.2012.00423
This article was submitted to Frontiers
in Extreme Microbiology, a specialty of
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Copyright © 2012 Thiel, Hügler, Blümel,
Baumann, Gärtner, Schmaljohann,
Strauss, Garbe-Schönberg , Petersen,
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December 2012 | Volume 3 | Article 423 | 19
Thiel et al.
Carbon fixation in tubeworm endosymbionts
APPENDIX
Table A1 | Signature nucleotide of tubeworm endosymbiont 16S rRNA gene sequences from vent- and seep vestimentiferan tubeworms.
E. coli pos.
75
77
79
80
90
92
137
231
250
258
268
286
301
381
Vent
c
g
a
g
t
t
Seep-1
a
t
g
a
c
a
c
t
a
g
c
a
g/a
a/c
t
c
t
a
t
g
g
Seep-2
a
t
g
a
c
c
a
t
c
t
a
t
g
g
Seep-3
a
t
g
a
c
c
a
t
c
t
a
t
g
g
PT-1
a
t
g
c
a
c
a
t
c
a
a
c
g
g
PT-2
a
t
c
g
a
c
a
t
c
t
a
t
g
g
Pos412
a
a
t
g
a
c
a
t
c
t
a
t
g
g
c
E. coli pos.
441
446
457
459
460
461
463
469
488
646
648
650
658
831
Vent
a
g
c
g
a
g
a
c
c
g
a
g
a
c
Seep-1
a
t
c
a
g
a
t
g
a
c
t
a
g
t
Seep-2
a
t
t
a
g
a
t
g
a
c
t
a
g
t
Seep-3
g
t
c
a
g
g
t
a
a
c
t
a
g
t
PT-1
a
t
c
a
g
g
t
a
a
c
t
a
g
t
PT-2
a
t
c
a
g
a
t
a
a
c
t
a
g
t
Pos412
a
t
c
a
g
a
t
a
a
c
t
a
g
t
E. coli pos.
849
859
986
987
998
999
1000
1002
1006
1007
1008
1009
1010
1011
Vent
c
c
a
g
c
c
t
g
c
t
t
t
c
t
Seep-1
t
a
t
a
c
c
t
a
c
t
t
g
t
t
Seep-2
t
a
t
a
t
g
a
a
t
c
c
t
g
t
Seep-3
t
a
t
a
t
g
a
a
t
c
c
a
g
t
PT-1
t
a
t
a
c
c
t
a
c
t
t
g
t
t
PT-2
t
a
t
a
t
g
a
a
t
c
c
a
g
c
Pos412
t
a
t
a
t
g
a
a
t
c
c
a
g
c
E. coli pos.
1019
1020
1021
1022
1023
1036
1037
1040
1041
1042
1043
1121
1135
1152
Vent
g
a
t
t
g
g
c
a
g
t
g
t
g
a
Seep-1
a
c
t
t
g
a
t
a
g
t
g
a
g
t
Seep-2
c
g
g
g
a
–
c
g
a
a
a
t
a
a
Seep-3
c
a
g
g
a
–
c
g
a
a
a
a
g
t
PT-1
a
c
t
t
g
a
t
a
g
t
g
a
g
t
PT-2
c
a
g
g
a
–
c
g
a
a
a
a
g
t
Pos412
c
a
g
g
a
–
c
g
a
a
a
a
g
t
E. coli pos.
1155
1219
1243
1257
1278
Vent
g
t
c
t
c
Seep-1
a
a
c
c
t
Seep-2
a
a
a/c
t
t
Seep-3
a
a
c
c
t
PT-1
a
a
c
c
t
PT-2
a
a
c
c
t
Pos412
a
a
c
c
t
“Vent” vestimentiferan group includes endosymbiont rRNA gene sequences of Riftia pachyptila, Tevnia jerichonana, and Osasisia alvinae. “Seep” vestimentiferan
tubeworm endosymbiont cluster “seep-(1–3)” refer to grouping according to McMullin et al. (2003). PT1 and PT2 refer to Lamellibrachia anaximandri endosymbionts,
phylotype 1 and phylotype 2 according to Duperron et al. (2009). Pos412 refers to the Lamellibrachia anaximandri endosymbiont rRNA gene sequence phylotype
obtained from the Palinuro volcanic complex during cruise Pos412 of R/V Poseidon in this study. The position numbers refer to E. coli gene sequence positions.
Frontiers in Microbiology | Extreme Microbiology
December 2012 | Volume 3 | Article 423 | 20