The Multiple Signaling Systems Regulating Virulence in
Pseudomonas aeruginosa
Pol Nadal Jimenez,a,b Gudrun Koch,b,c Jessica A. Thompson,a Karina B. Xavier,a,d Robbert H. Cool,b and Wim J. Quaxb
Instituto Gulbenkian de Ciência, Oeiras, Portugala; Department of Pharmaceutical Biology, University of Groningen, Groningen, The Netherlandsb; Institute of Molecular
Infection Biology, Würzburg Universität, Würzburg, Germanyc; and Instituto de Tecnologia Química e Biológica, Oeiras, Portugald
INTRODUCTION
C
ell-to-cell communication by means of diffusible signaling
molecules allows bacteria to trigger coordinated responses to
achieve outcomes that would otherwise remain impossible for individual cells. During the past 2 decades, much attention has been
given to bacterial communication systems due to their involvement in acute and chronic infections. Analyses of the molecular
mechanisms of cell-to-cell communication may help scientists to
develop specific antimicrobial agents that will decrease both the
defensive and offensive traits of pathogens. The signaling network
of Pseudomonas aeruginosa is perhaps one of the most complex
systems known and, to date, is the best studied among all microorganism systems. It consists of multiple interconnected signaling
layers that coordinately regulate virulence and persistence, driving
the emergence of P. aeruginosa from the enormous number of
species that comprise the biodiverse bacterial domain to join an
46 mmbr.asm.org
elite group of a few dozen that pose a major threat to humans. This
review summarizes the major signaling systems regulating virulence and persistence in P. aeruginosa, with special attention to
those involving the production and detection of diffusible signaling molecules. Due to the complexity and diversity of these signaling networks, we define the relevance of each system with regard
to signal integration, adaption responses, and virulence, emphasizing the importance of less-well-studied signals as potential key
elements in the global virulence network of P. aeruginosa. (Gene
and protein numbers in this article refer to the corresponding
numbers from the P. aeruginosa PAO1 genome.)
Address correspondence to Wim J. Quax, w.j.quax@rug.nl.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/MMBR.05007-11
1092-2172/12/$12.00
Microbiology and Molecular Biology Reviews
p. 46 – 65
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INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
THE EMERGING NEED FOR NOVEL AGENTS AGAINST P. AERUGINOSA INFECTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47
AHLS: THE CLASSICAL GRAM-NEGATIVE SIGNALS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47
Further Regulation of the AHL Systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47
Controlling the Activation Threshold . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47
Additional AHLs in P. aeruginosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48
DKPS AND LUXR ACTIVATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48
DKPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48
Additional Properties of DKPs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49
4-QUINOLONE SIGNALING: THE PSEUDOMONAS-BURKHOLDERIA LANGUAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49
PQS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49
Function of HHQ as a Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49
The Additional Role of PQS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49
THE GAC SYSTEM: CONNECTING VIRULENCE, BIOFILM FORMATION, AND SWARMING . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50
The Transition from Acute to Chronic Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50
Multiple Kinases Interact with the GAC System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
PIGMENTED SIGNALS: THE COLORFUL LANGUAGE OF PSEUDOMONAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
Pyoverdine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
Signaling under low-iron conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
Phenazines. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
Pyocyanin as a terminal signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52
Sox box and colony morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52
DIFFUSIBLE SIGNAL FACTORS (DSF-LIKE FACTORS): FATTY ACIDS AS INTERKINGDOM MESSENGERS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54
Cross-Kingdom Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54
DSF Signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55
DSF and biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55
NUCLEOTIDE-BASED SIGNALS: THE SECONDARY MESSENGERS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55
cAMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55
ppGpp and pppGpp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56
c-di-GMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56
c-di-GMP and SCV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .57
TARGETING BACTERIAL SIGNALING. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .57
CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .59
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .59
Cell-to-Cell Signaling in P. aeruginosa
THE EMERGING NEED FOR NOVEL AGENTS AGAINST
P. AERUGINOSA INFECTIONS
AHLS: THE CLASSICAL GRAM-NEGATIVE SIGNALS
N-Acyl homoserine lactones (AHLs) were the first broadly accepted bacterial cell-to-cell signals to be discovered and, to date,
remain the most-studied communication molecules in bacteria
(2, 3, 59, 60, 72, 143). A large number of Gram-negative bacteria
produce and use these signals to control and regulate gene expression in a cell density-dependent manner known as quorum sensing (QS) (68, 205, 208). In general, AHLs consist of fatty acids,
varying in length and substitution, linked via a peptide bond to a
homoserine lactone moiety. AHLs are commonly synthesized by
members of the LuxI family of proteins and are sensed by members of the LuxR family of transcriptional regulators (69, 70). After
a certain concentration of AHLs (the threshold) has been produced (correlating to a certain bacterial cell density), a complex
with the cognate LuxR transcriptional regulator will be formed,
enabling binding to DNA, thereby altering the expression of multiple virulence genes. Two different AHL systems coexist in P.
aeruginosa: the Las and Rhl systems. The Las system produces and
responds to N-3-oxo-dodecanoyl homoserine lactone (3-oxoC12-HSL), which is produced by the LasI synthase (PA1432) and
recognized by the transcriptional regulator LasR (PA1430) (154,
156). The Las system controls the production of multiple virulence factors involved in acute infection and host cell damage,
including the LasA (PA1871) and LasB (PA3724) elastases, exotoxin A (PA1148), and alkaline protease (PA1246) (73, 99, 154,
191). The second AHL system, the Rhl system, produces and responds to N-butanoyl homoserine lactone (C4-HSL) (157). This
molecule is generated by the RhlI synthase (PA3476) and sensed
by the transcriptional regulator RhlR (PA3477), inducing the expression of several genes, including those responsible for the pro-
March 2012 Volume 76 Number 1
Further Regulation of the AHL Systems
In addition to RhlR and LasR, P. aeruginosa possesses several putative LuxR-type homologues lacking a LuxI-type cognate partner; these homologues have been designated orphan LuxR homologues (67). Their function and potential relationship to AHL
signaling remain unknown, with the exception of QscR (PA1898),
which exhibits full conservation with functional LuxR-type proteins and forms complexes with LasR and RhlR. These delay the
expression of quorum sensing-regulated genes, thereby reducing
bacterial virulence both in vitro and in vivo (35, 115). Recently,
Chugani and Greenberg revealed an even higher level of complexity in P. aeruginosa AHL signaling, reporting a set of 37 genes
whose expression was controlled by AHLs in the absence of LasR,
RhlR, and QscR (34). These recent results raise questions about
AHL quorum sensing regulation. What is the identity and mechanism of the protein(s) or recognition factor for these AHL signals? Are these mechanisms present in other species? Does this
system function by recognizing native AHL signals only, or does it
respond to AHLs produced by other bacteria and is thus involved
in interspecies communication?
Given this complexity, it seems obvious that AHL quorum sensing must be tightly regulated to coordinate the correct time and
place of expression of virulence factors. Three regulators have
been found in P. aeruginosa that contribute to the timing and level
of control of AHL-regulated virulence. The first, RsaL (PA1431),
acts as a major transcriptional repressor of the Las system, controlling the maximal levels of AHLs, and also therefore virulence
factors, produced (42). RsaL binds simultaneously with the
3-oxo-C12-HSL–LasR complex to the lasI promoter (163), inhibiting its transcription, while in parallel it controls the repression of
AHL-related virulence by directly binding to the promoters of
pyocyanin and hydrogen cyanide (HCN) genes (165).
Controlling the Activation Threshold
The second regulator, QteE (PA2593), was recently shown to prevent the posttranslational accumulation of LasR by reducing its
stability and also blocking RhlR accumulation by an as yet unknown LasR-independent mechanism (179). This activity inhibits
the induction of virulence phenotypes, as shown by attenuated
infection by P. aeruginosa in plant and Drosophila melanogaster
models upon overexpression of qteE (118). QslA (PA1244) prevents early activation of QS-regulated virulence by forming complexes with LasR that prevent its binding to the target DNA. In
contrast to QteE, QslA does not affect RhlR QS activation or LasR
stability, nor does its absence correlate with an early activation of
QS (176). These newly discovered regulators provide a logical explanation for how a threshold for activation can be created in
bacteria where early activation of QS-dependent virulence is
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Since its initial discovery in the late 19th century (63), the Gramnegative bacterium P. aeruginosa has gained a notorious place in
the list of infamous human pathogens (65, 158, 206). The arrival
of the antibiotic era largely palliated the previously fatal outcome
of acute infections in healthy patients. Only a relative improvement has been achieved in the eradication of chronic infections,
which develop mainly in individuals suffering from cystic fibrosis
or severe burns or who are immunocompromised (30, 74, 98).
Two intrinsically related factors in the fatal outcome of infection
in these patients are the rapid prescription of not always appropriate antibiotic treatments and the development or acquisition of
multidrug-resistant strains. While the use of an appropriate antibiotic(s) has been reported as an essential factor in the eradication
of P. aeruginosa infections (102, 122, 134), conversely, antibiotic
abuse significantly contributes to increasing resistance by exerting
a continuous selective pressure for the acquisition of such capabilities. Antibiotics alone do not account for the high prevalence of
multidrug-resistant variants: P. aeruginosa has multiple, chromosomally encoded intrinsic mechanisms of resistance, including
low permeability of the cell envelope and numerous multidrug
efflux pumps. Another major factor accounting for the successful
invasive behavior and persistence of this bacterium is its high
adaptability, allowing rapid colonization of different environments. To perform these adaptations, P. aeruginosa has evolved a
complex and extensive array of regulatory signaling networks that
detect and react to endogenous and environmental molecules,
triggering massive changes in genetic expression.
duction of rhamnolipids, and repressing those responsible for assembly and function of the type III secretion system (T3SS), a
major virulence determinant in human infections that allows the
release of toxic proteins into the cytoplasm of eukaryotic cells
(12). A hierarchical relationship exists between the Las and Rhl
systems: the Las system controls the Rhl system, as the 3-oxo-C12HSL–LasR complex directly upregulates rhlR transcription (112).
Thus, activation of the LasIR system allows the later activation of
the RhlIR system (Fig. 1). Experiments with mouse models demonstrated that deletion of either AHL synthases or AHL receptors
results in a decrease in infection severity (155, 172, 183).
Nadal Jimenez et al.
HSL is produced, the 3-oxo-C12-HSL–LasR complex binds the promoter regions of multiple genes, activating or repressing their transcription. Among the genes
upregulated by this complex are lasI, which enhances the production of 3-oxo-C12-HSL (autoinduction effect), and rhlR, which increases the production of the
rhl response regulator RhlR, activating the second AHL pathway at an earlier stage. Virulence factors regulated by each respective receptor-ligand complex are
detailed on the left.
avoided during the initial growth phase. It remains possible, although not yet proven, that QteE and/or QslA also actively represses AHL-dependent gene expression during late stationary
phase to conserve energy when AHL-related virulence is no longer
needed.
Additional AHLs in P. aeruginosa
The AHL systems discussed here encompass only the two main
AHLs of P. aeruginosa (C4-HSL and 3-oxo-C12-HSL), although
low concentrations of other distinct AHLs, namely, 3-oxo-C14HSL and 3-oxo-C10-HSL, can be detected in the supernatants of P.
aeruginosa cultures (28). The presence of smaller quantities of
these AHLs may be due to a case of mistaken identity where the
LasI synthase couples the wrong acyl carrier protein (ACP) to
S-adenosylmethionine (SAM). However, these AHLs might also
originate from the action of a different type of AHL synthase. In
addition to the LuxI-type synthase, two other unrelated AHL synthase families have been reported: the LuxM synthase family, exclusive to Vibrio spp. (7, 77), and the more diverse HdtS synthase
(113), originally identified in Pseudomonas fluorescens, with putative homologues in several Pseudomonas spp., including P. aeruginosa.
novel set of molecules capable of binding and activating LuxRtype proteins was confirmed as a reality in 1999, when Holden and
coworkers purified and elucidated several structures of a novel
family of cyclic dipeptides, termed diketopiperazines (DKPs),
from the supernatants of various bacteria, including P. aeruginosa
(Fig. 2) (92). DKPs have been proven to interfere with the
quorum-sensing systems of various bacteria; this interference is
most likely by binding to the LuxR family of receptors, either
activating or antagonizing AHL signals. In the same work, the
authors identified three different DKPs in the supernatants of various bacteria: cyclo(⌬Ala-L-Val) and cyclo(L-Pro-L-Tyr) in P.
aeruginosa, Proteus mirabilis, and Citrobacter freundii; cyclo(⌬AlaL-Val) in Enterobacter agglomerans; and cyclo(L-Phe-L-Pro) in P.
fluorescens and Pseudomonas alcaligenes.
DKPS AND LUXR ACTIVATION
DKPs
A close examination of LuxI and LuxR homologues in all sequenced genomes of Gram-negative bacteria clearly reveals an
increased prevalence of LuxR- over LuxI-type proteins. Whether
this ratio reflects a predominance of eavesdroppers over speakers
or is an indication of the presence of unidentified alternative
LuxR-binding molecules remains a matter of debate. The idea of a
48 mmbr.asm.org
FIG 2 Structures of the two DKPs produced by P. aeruginosa.
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FIG 1 Virulence regulation of and interactions between the two AHL quorum-sensing systems in P. aeruginosa. After a threshold concentration of 3-oxo-C12-
Cell-to-Cell Signaling in P. aeruginosa
Despite the discovery of this novel type of signaling compound
over 10 years ago, little attention has been given to DKPs. Two
possible reasons for this apparent lack of interest are that the high
concentrations of DKPs required to activate LuxR-like quorumsensing systems strengthen the idea of a fortuitous cross talk (91)
and the fact that no bacterium has yet been demonstrated to regulate QS solely by depending on DKPs, undermining the importance of these molecules as essential signaling compounds. However, DKPs may become the focus of more intensive research in
the coming years due to the increasing number of studies on the
additional antimicrobial properties of these compounds.
Additional Properties of DKPs
4-QUINOLONE SIGNALING: THE
PSEUDOMONAS-BURKHOLDERIA LANGUAGE
PQS
Despite 4-quinolones having been discovered in the 1940s (82)
and subsequently studied due to their antibacterial effects (37,
119), their signaling properties were not reported until more than
50 years later, when Pesci and coworkers identified the first signaling role for a 4-quinolone in P. aeruginosa (159). This molecule,
2-heptyl-3-hydroxy-4-quinolone, termed the Pseudomonas quinolone signal (PQS), is synthesized from anthranilate and an
␣-keto-fatty acid by the products of the pqs biosynthesis genes
pqsABCD (PA0996 to PA0999) (18, 61). These synthesize the precursor molecule 2-heptyl-4(1H)-quinolone (HHQ), which is finally converted into PQS by PqsH (PA2587). After a certain
threshold concentration of PQS in the extracellular medium is
reached, this molecule binds to its cognate receptor, PqsR (also
called MvfR [PA1003]). The resulting complex activates the expression of the pqsABCDE and phnAB (PA1001-PA1002) operons, increasing PQS and pyocyanin production (25, 46, 52). The
increase in production of PQS resulting from the PQS-PqsR complex binding to the pqsA promoter region constitutes an autoinduction mechanism similar to that observed in AHL quorumsensing systems. Two additional regulators, MvaT (PA4315) and
March 2012 Volume 76 Number 1
Function of HHQ as a Signal
Although PQS is the major 4-quinolone signaling molecule produced by P. aeruginosa, approximately 50 structurally related
4-quinolones are also produced by the PqsABCD proteins. Most
of these molecules are produced in amounts too small to play a
significant role in cell-to-cell signaling. However, one of them, the
PQS precursor HHQ, has clearly been demonstrated to also act as
a cell-to-cell signal (47). HHQ can be released into the extracellular medium and subsequently taken up by neighboring cells, in
which it either is converted into PQS by PqsH or binds directly to
PqsR, in both cases activating PQS-regulated gene expression to
levels similar to those observed in response to PQS itself (with the
only exception being the phzA1-phzG1 [PA4210 to PA4216]
operon, responsible for pyocyanin biosynthesis, which is activated
by PQS but not by HHQ) (211). Although a study of P. aeruginosa
PAO1 revealed that HHQ conversion to PQS is necessary for driving the expression of the lectin gene lecA (PA2570) (51), studies
using a pqsH mutant in P. aeruginosa UCBPP-PA14 indicated that
the conversion of HHQ into PQS is unnecessary, as HHQ alone
was able to fully activate PqsR-dependent virulence expression
(211). A role of HHQ in cell-to-cell signaling is even more evident
if we take into account that only P. aeruginosa produces PQS,
while other Pseudomonas spp. and Burkholderia spp. rely on HHQ
and other methylated 4-hydroxy-2-alkylquinoline analogues for
4-quinolone signaling (50, 199).
The Additional Role of PQS
Since PQS seems to be dispensable for cell-to-cell signaling, what
is the benefit to P. aeruginosa of having an enzyme (PqsH) to
generate this molecule? Recent studies on PQS may have solved
this question, demonstrating that the presence of the additional
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Lactobacillus plantarum produces two DKPs, namely, cyclo(LPhe-L-Pro), as found in P. fluorescens and P. alcaligenes, and
cyclo(L-Phe-trans-4-OH-L-Pro), and both of these have been reported to display antifungal activity (189). Cyclo(L-Phe-L-Pro)
has also been detected in the supernatants of the human pathogens Vibrio vulnificus, Vibrio cholerae, Vibrio parahemolyticus, and
other related Vibrio spp. (151). An interesting aspect highlighted
by the authors is that while this molecule activates the Lux reporter system of Vibrio fischeri, in V. vulnificus and other related
species this DKP enhances the expression of the outer membrane
protein ompU gene and the cholera toxin ctxAB genes, known to
be under the control of ToxR. The presence of DKPs in V. vulnificus is especially intriguing because this bacterium does not produce AHLs and therefore could use DKPs to activate orphan LuxR
regulators. On the other hand, in a recent study on the QS properties and mode of action of DKPs, Campbell and coworkers
found no evidence of LuxR-like activation or interaction by any of
the DKPs tested (24), including those previously reported by
Holden and colleagues. These controversial results cast serious
doubts on the role of DKPs as bacterial signaling molecules, and
their potential involvement in P. aeruginosa QS-regulated virulence requires further research.
its homologue MvaU (PA2667), are found in several Pseudomonas
spp. and may also be involved in PQS production in P. aeruginosa
(53, 197). An mvaT mutant exhibits more production of PA-IL
lectin and pyocyanin, reduced biofilm formation and swarming
motility, and increased drug resistance (53, 204). The observation
that mvaT and mvaU single mutants increase pyocyanin synthesis
while an mvaT mvaU double mutant abolishes pyocyanin and
PQS production suggests that these regulators work in different
ways to control pyocyanin production, with one involving PQS
production and the other directly controlling pyocyanin synthesis
(117). In addition to the four genes involved in PQS biosynthesis,
the pqsABCDE operon contains a fifth, pqsE (PA1000), encoding a
protein with a metallo--lactamase fold (62) that is not required
for PQS synthesis (71). Despite the recent elucidation of the crystal structure of PqsE (215), little is known about its function and
natural substrate. PqsE is the major virulence effector in the
4-alkyl-quinolone (4-AQ) system, controlling the production of
several virulence factors, such as pyocyanin, lectin, rhamnolipids,
and HCN (52, 71), which are all implicated in toxicity and acute
infection (Fig. 3). The 4-quinolone signaling system is linked in a
hierarchical manner to the AHL signaling systems of P. aeruginosa, as LasR (positively) and RhlR (negatively) control the levels
of PQS by binding to the promoter region of the PqsR regulator
(202). Additionally, PqsE alone is sufficient to regulate its virulence target genes via the rhl QS system intrinsically linked to RhlR
(62). Mutations in either pqsA or pqsE significantly reduce P.
aeruginosa virulence in plant and animal infection models (46,
164).
Nadal Jimenez et al.
hydroxyl group in PQS is essential for the binding of iron (17).
The formation of PQS-iron complexes confers iron-chelating
properties upon this molecule; however, iron-bound PQS is not
transported back into the cell and therefore does not function as a
siderophore (51). Instead, PQS is found at high concentrations in
the outer cell membrane of P. aeruginosa, within membrane vesicles (128, 129). The presence of iron-bound PQS at the membrane may contribute to the accumulation of iron in close proximity to the cell, facilitating the work of the actual siderophores
pyoverdine (PVD) and pyochelin. This mechanism would allow
the bacterium to rapidly and efficiently obtain iron without losing
siderophores in the surrounding environment (51). Additionally,
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PvdS, the major regulator of pyoverdine biosynthesis, has been
proven to play a role in PQS synthesis by controlling the expression of PqsR (147), demonstrating an intrinsic relationship of
PQS production with iron levels.
THE GAC SYSTEM: CONNECTING VIRULENCE, BIOFILM
FORMATION, AND SWARMING
The Transition from Acute to Chronic Infection
In addition to the AHL and PQS systems, P. aeruginosa controls its
lifestyle (free-living and biofilm) and the production of multiple
virulence factors via two-component signal transduction systems.
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FIG 3 Biosynthesis, autoinduction, and virulence regulation by 4-alkyl-quinolones in P. aeruginosa. Biosynthesis of PQS starts with the conversion (by the
PqsABCD proteins) of anthranilate (which originates from either the kynurenine pathway or the PhnAB anthranilate synthase) into HHQ, which is finally
converted into PQS by the PqsH monooxygenase. Both HHQ and PQS bind the PqsR regulator, and the complex activates the transcription of the pqsABCDE and
phnAB operons, increasing the levels of PQS (autoinduction) and pyocyanin production. Additionally, transcription of the PQS operon results in an increase in
the levels of PqsE, an enzyme of uncharacterized function that increases the levels of pyocyanin, lectin, HCN, and rhamnolipids.
Cell-to-Cell Signaling in P. aeruginosa
Multiple Kinases Interact with the GAC System
Two further sensor kinases, LadS (PA3974) and RetS (PA4856),
have been found to modulate gene expression via GacA. LadS (lost
adherence sensor) acts in parallel to GacS, positively controlling
the expression of the pel operon (PA3058 to PA3064), which increases biofilm production, and repressing the expression of genes
involved in the T3SS (198). The third sensor kinase involved in
this pathway, RetS (regulator of exopolysaccharide and type III
secretion), controls GacA in an opposite manner to GacS and
LadS, promoting acute infection and repressing the expression of
genes associated with biofilm production. This was evidenced by a
comparison of gene expression in retS and ladS mutant strains,
which clearly demonstrated reciprocal control of the same set of
genes (198). Furthermore, tests of the effects of these additional
kinases in a mouse model of acute pneumonia revealed that unlike
its parental strain, a P. aeruginosa retS mutant was unable to establish infection (78).
Interestingly, it was found that the effects of RetS on GacAdependent virulence expression are not due to a phosphorylation
cascade, as expected from the protein function, but to a direct
interaction between RetS and GacS. In an elegant study, Goodman
and coworkers demonstrated that the formation of heterodimers
between RetS and GacS blocks the autophosphorylation ability of
the latter, interfering with the consequent phosphotransfer to
GacA and leading to a reduction in RsmZ expression. While RsmA
bound to RsmZ or -Y promotes the expression of genes involved
in biofilm formation, at low concentrations of RsmZ, RsmA promotes the expression of genes involved in acute virulence and
represses the expression of genes involved in chronic infections
(Fig. 4) (79). The GAC system also has a control on the AHL
system via RsmA, by negatively controlling the synthesis of C4HSL and 3-oxo-C12-HSL and of extracellular virulence factors
March 2012 Volume 76 Number 1
controlled by AHLs (105, 160, 168). Furthermore, a recent study
published by Filloux and coworkers (137) revealed that the RetSdependent switch between T3SS and T6SS activities, associated
with the transition to chronic infections, is regulated via cyclic
di-GMP (c-di-GMP) signaling. This finding reveals how these two
pathways regulate a common set of phenotypes, predicting an
exciting series of studies that will ultimately aim to unravel the
exact molecular mechanism of this interaction.
Despite extensive study of the GAC system, the identities of the
signals triggering the phosphorylation response remain unknown.
Finding the activators of these sensor kinases has been a long quest
for many scientists aiming to ultimately control the behavior of
such a relevant bacterial genus. Identifying these signals could
provide physicians and biotechnologists with a molecule capable
of switching bacterial lifestyles to ease antibiotic treatment during
human infections or to control antibiotic production in beneficial
species enhancing crop protection.
PIGMENTED SIGNALS: THE COLORFUL LANGUAGE
OF PSEUDOMONAS
Pyoverdine
Signaling under low-iron conditions. PVDs are the major ironchelating molecules (siderophores) of P. aeruginosa (Fig. 5). The
production of pyoverdine involves the production of multiple
proteins and is therefore likely to be of considerable metabolic
burden to the bacterium, perhaps providing a positive selection
pressure for further exploitation of this molecule in other systems.
Pyoverdine has been found to initiate a signaling cascade responsible for the production of several virulence factors, including exotoxin A (ToxA), PrpL endoprotease (PA4175), and pyoverdine
itself (111).
This signaling cascade involves iron-bound pyoverdine (FePVD), the cell surface receptor protein FpvA (PA2398), and the
anti-sigma factor FpvR (PA2388). Upon binding to iron, the FePVD–FpvA complex interacts with the periplasmic domain of
FpvR, allowing the expression of the regulators PvdS (PA2426)
and FpvI (PA2387). PvdS upregulates the production of ToxA,
PrpL, and pyoverdine, whereas expression of FpvI generates a
positive-feedback loop through increased production of the PVD
receptor FpvA (9). Recently, Shirley and Lamont characterized an
additional protein necessary for the transport and signaling cascade of Fe-PVD (178). TonB1 (PA5531) is a member of a family of
proteins responsible for the energy transduction required for the
import of molecules via outer membrane proteins. Binding of
TonB1 to the TonB box present in FpvA is essential for both the
transport of Fe-PVD and the consequent signaling cascade controlled by this molecule (Fig. 6). While the role of the PVD signaling pathway per se has not been tested in vivo in a P. aeruginosa
infection model, iron availability and iron chelation via PVD play
important roles in infection establishment (5, 180) and the development of chronic infections (141).
Phenazines
Phenazines are pigmented, redox-active, heterocyclic, nitrogencontaining molecules secreted by a considerable number of
bacteria, including multiple fluorescent Pseudomonas spp.
Phenazines display a broad spectrum of (toxic) activity toward
prokaryotic and eukaryotic organisms, varying according to the
nature and position of the substituents on the heterocyclic ring
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These systems act through phosphorylation cascades that induce
conformational changes in regulatory proteins, resulting in global
changes in gene expression. Over 60 two-component systems have
been found in the genome of P. aeruginosa; among them, the GAC
system (global activator of antibiotic and cyanide synthesis) is
perhaps the most interesting and best studied. Initially identified
in Pseudomonas syringae in 1992 (93), the GAC system in the genus Pseudomonas has gained special notoriety due to the rich metabolic pool produced by the species of this genus and its involvement in microbe-host interactions. Particularly in P. aeruginosa,
one of the major features attracting researchers is the main role of
the GAC system in the transition from acute to chronic infection.
In patients suffering from cystic fibrosis, it is not the acute infection mode of P. aeruginosa that poses the major threat but the
highly resistant biofilm lifestyle that leads to recurrent infections
ending in fatal lung failure. The GAC system consists of a transmembrane sensor kinase, GacS (LemA [PA0928]), which upon
autophosphorylation transfers a phosphate group to its cognate
regulator, GacA (PA2586), which in turn upregulates the expression of the small regulatory RNAs RsmZ (PA3621.1) and RsmY
(PA0527.1). Binding of RsmZ and RsmY to the small RNAbinding protein RsmA (PA0905) activates the production of genes
involved in biofilm formation and represses multiple genes involved in acute virulence and motility. As a consequence, in a
mouse model of acute pneumonia, a mutation in RsmA reduced
colonization during the initial infection stages but ultimately favored chronic infection (140).
Nadal Jimenez et al.
to the promoters of multiple genes, enhancing bacterial motility and activating the production of several acute virulence factors while repressing the production
of virulence factors associated with chronic infections. GacA phosphorylation via GacS stimulates the production of the small RNAs RsmZ and RsmY, which bind
to the RsmA protein, releasing the repression of virulence factors associated with chronic infections and repressing the production of acute infection-associated
factors. The sensor kinase LadS works in parallel to GacS, activating RsmZ and RsmY production, while the sensor kinase RetS acts in an opposite manner to LadS
and GacS, forming a protein-protein complex with GacS that blocks RsmY and RsmZ production.
(21, 114, 130). This toxicity confers a clear advantage to phenazine
producers by eliminating competitors and enhancing survival in
highly populated environments such as the rhizosphere (131).
Pyocyanin (5-N-methyl-1-hydroxyphenazine), the first and
most-studied member of the phenazine family, is produced only
by P. aeruginosa (Fig. 7), a specificity that has been useful in the
rapid diagnosis of this opportunistic pathogen (63, 76, 100). This
blue phenazine is one of the major virulence factors in this pathogen, contributing to both acute and chronic infections (123, 207),
as it suppresses lymphocyte proliferation (146), damages epithelial cells as a consequence of hydroxyl radical formation (20, 207),
inactivates protease inhibitors (consequently causing tissue damage by endogenous proteases) (19), and targets multiple cellular
functions (44, 101, 139, 166, 185, 196).
Pyocyanin as a terminal signal. Besides its major function as a
virulence factor and electron transfer facilitator (86), pyocyanin
also serves as a signaling molecule in P. aeruginosa, controlling a
limited set of genes, termed the PYO stimulon (48), during the
stationary growth phase. This includes genes involved in efflux
and redox processes, as well as iron acquisition genes. The efflux
pump genes mexGHI-opmD (PA4205 to PA4208) and the putative
monooxygenase gene PA2274 are among the genes most strongly
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regulated by the PYO stimulon. These genes were initially identified as part of the PQS regulatory cascade and were assumed to be
controlled directly by PQS and PqsE (PA1000) (46). This assumption, however, can now be attributed to the facts that PQS and
PqsE directly control phenazine biosynthesis and that pyocyanin
itself is responsible for the upregulation of these genes, pointing
once again to the complexity of the signaling networks present in
this bacterium. The mechanism behind pyocyanin-controlled upregulation of many genes, including mexGHI-opmD, PA2274, and
PA3718 (encoding a putative MFS transporter), has been demonstrated to occur via the transcriptional regulator SoxR (PA2273).
The absence of sox boxes in the remaining genes of the PYO stimulon indicates that additional regulatory factors in this signaling
cascade remain to be elucidated (48). Similar to the case for PVD,
the main role of PYO in an alternative pathway has limited studies
on the secondary role of these molecules in signaling.
Sox box and colony morphology. Two years after the initial
discovery of the signaling role of pyocyanin, Dietrich and coworkers linked pyocyanin levels to the development of wrinkled colonies, a clear morphotype taking place in the late growth phase
(49). While wild-type P. aeruginosa colonies developed a severely
wrinkled phenotype 4 days after inoculation, a phenazine-null
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FIG 4 The GAC system network in P. aeruginosa controls the reversible transition from acute to chronic infections. The small regulatory protein RsmA binds
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FIG 5 Pyoverdines produced by P. aeruginosa. Each P. aeruginosa strain produces one type of pyoverdine exclusively. The amino acids D-Tyr and L-Glu (bottom
right of each PVD) are further modified during biosynthesis to yield the final pyoverdine. D-Tyr is converted into catechol, and L-Glu into either succinyl,
succinamide, ketoglutaryl, or a free acid, and is thus represented by “-R”.
March 2012 Volume 76 Number 1
mmbr.asm.org 53
Nadal Jimenez et al.
that the Gram-positive bacterium Streptomyces coelicolor, which
contains a soxR homologue as well as genes controlled by sox
boxes, develops a similar colony morphotype and controls gene
expression in response to the presence or absence of the two pigmented antibiotics actinorhodin and undecylprodigiosin. These
data clearly imply that a conserved Sox-dependent transcriptional
regulatory role exists for redox-active pigments in later developmental stages.
DIFFUSIBLE SIGNAL FACTORS (DSF-LIKE FACTORS): FATTY
ACIDS AS INTERKINGDOM MESSENGERS
Cross-Kingdom Signaling
mutant developed the same type of colonies after only 2 days. In
contrast, a wild-type strain overproducing phenazines remained
smooth throughout the 6 days of the experiment. The same results
were obtained with a soxR mutant and a mexGHI-opmD mutant,
indicating a possible role of high intracellular phenazine levels in
colony morphology regulation. The authors also demonstrated
FIG 7 Biosynthesis and signaling system of pyocyanin. Chorismic acid is transformed via the PhzA to -G proteins into phenazine-1-carboxylic acid, which is
subsequently converted into different phenazines by the enzymes PhzH, PhzS, and PhzM. The product of the latter is transformed by PhzS into pyocyanin (PYO).
Next to its role as a virulence factor, PYO acts as a signaling molecule activating a limited set of genes termed the PYO stimulon. A large fraction of the PYO
stimulon genes are controlled by the regulator SoxR, although the mechanism by which PYO activates SoxR, as well as the activation mechanism of SoxRindependent genes, remains unknown.
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FIG 6 PVD signaling pathway in P. aeruginosa. In the absence of Fe-PVD
(left), the signaling system is inactive. Binding of Fe-PVD to the PVD receptor
FpvA (right) initiates a signaling cascade that requires TonB1 and FpvR and
stimulates the production of FpvA, PVD, ToxA, and the PrpL protease.
Considering the enormous biological diversity present in an ecological niche, it would be naïve to assume that bacterial communication is limited to intraspecies or interspecies signaling. Given
the large number of possible interactions in a microcosmos and
the high level of competition among organisms, it seems logical
that bacteria would produce or receive signals enabling communication with fungi, plants, and animals. In recent years, various
signals capable of accomplishing this function have been found.
One interesting example of an interkingdom signal interaction
was discovered between P. aeruginosa and the opportunistic fungal pathogen Candida albicans. These two organisms share ecological niches, and both produce signals capable of interfering with
the production of virulence factors by the other. Production of
3-oxo-C12-HSL by P. aeruginosa inhibits C. albicans filamentation, a crucial virulence adaptation for the development of opportunistic infections (90), while production of the fungal metabolite
farnesol reduces PQS and pyocyanin levels and swarming motility
in P. aeruginosa (39, 132). The slight structural resemblance (C12
Cell-to-Cell Signaling in P. aeruginosa
negative bacteria.
backbone) between the two molecules suggests that the deleterious effects could be due to a competitive inhibition of the organism’s native receptor by the foreign signal molecule. The importance of the fatty acid backbone structure of 3-oxo-C12-HSL and
farnesol gained even more relevance with the discovery of certain
long-chain fatty acids as intra-/inter- and cross-kingdom signaling molecules.
DSF Signals
The first member of the DSF signal family, cis-11-methyl-2dodecenoic acid, was initially discovered in the plant pathogen
Xanthomonas campestris and termed diffusible signal factor (DSF)
because of its ability to cross cell membranes by passive diffusion
(6). In X. campestris, DSF controls the production of multiple
virulence factors and cyclic glucan, and additionally, it induces
biofilm dispersal (6, 57, 200); signaling occurs via a set of genes
called rpf (regulation of pathogenicity factors) genes. DSF synthesis is carried out by the long-chain fatty acyl coenzyme A (CoA)
ligase RpfB and the enoyl-CoA hydratase-like enzyme RpfF (6).
The DSF network involves multiple proteins, including RpfG, a
two-component regulator containing a novel c-di-GMP hydrolytic domain (HD-GYP) (174), indicating that the DSF signaling
circuit is a complex regulatory system with overlapping regulatory
layers. A complete overview of DSF and c-di-GMP interactions in
X. campestris signaling is provided by excellent reviews by Dow et
al. and He and Zhang (58, 83).
Although the DSF signaling cascade has been studied extensively in X. campestris, these signals are not limited to members of
the genus Xanthomonas. Various DSF-related molecules have
been found in other bacteria: DSF signaling controls motility, lipopolysaccharide (LPS) production, and cell aggregation in
Stenotrophomonas maltophila (64) and virulence and insect transmission in Xylella fastidiosa (29), and the DSF-like signal cis-2dodecenoic acid (BDSF) regulates multiple virulence factors and
positively controls biofilm formation in members of the Burkholderia cepacia complex (Bcc) (14, 43). Similar to 3-oxo-C12-HSL,
DSF-related signals are able to disrupt C. albicans filamentation,
thus emerging as a novel class of potential antifungal agents (14).
DSF and biofilms. The DSF-like molecule cis-2-decenoic acid
was isolated from cell-free supernatants of P. aeruginosa (Fig. 8).
Similar to BDSF in B. cepacia, this signal affects biofilm formation
March 2012 Volume 76 Number 1
NUCLEOTIDE-BASED SIGNALS: THE
SECONDARY MESSENGERS
cAMP
In recent years, it has become evident that in addition to diffusible
communication signals, a large group of nucleotide-based molecules plays a crucial role in controlling bacterial physiology. The
first of these signals to be identified in prokaryotes was cyclic AMP
(cAMP), reported for the bacterium Brevibacterium liquefaciens
(later reclassified as Arthrobacter nicotianae) in the early 1960s
(148). Now recognized as an extensively distributed molecule in
bacteria, cAMP is produced by adenylate cyclase enzymes and
binds and activates transcription factors from the CRP family
(cAMP regulator proteins) (80). In P. aeruginosa, synthesis of
cAMP is driven primarily by the adenylate cyclases CyaB
(PA3217) and, to a lesser extent, CyaA (PA5272) (209). cAMP
binding to the CRP-homologous regulator VfR (virulence factor
regulator [PA0652]) directly and indirectly controls the production of multiple virulence factors, upregulating exotoxin A, type
four pili (TFP), the T3SS, and the Las QS system (1, 10) and downregulating flagellar gene expression (40). Modulation of cAMP
levels occurs via the Chp (chemotaxis-like chemosensory system)
gene cluster in P. aeruginosa, where PilG (PA0408), PilI (PA0410),
PilJ (PA0411), ChpA (PA0413), ChpC (PA0415), FimL (PA1822),
and FimV (PA3115) upregulate and PilH (PA0409), PilK
(PA0412), and ChpB (PA0414) downregulate cAMP levels, establishing a link between Chp and TFP (66, 96). Additionally, mutations in mucA (PA0763) and consequent activation of the AlgU
(PA0762) regulon have been reported to inhibit cAMP-VfR signaling (97), demonstrating that cAMP-VfR signaling constitutes a
complex signaling cascade with multiple regulatory inputs. Infection studies using a mouse model of acute pneumonia with vfr,
cyaA, and cyaB mutants revealed a dominant role of CyaB and VfR
during infection (184). In parallel to CyaA and CyaB, a third adenylate cyclase with an intriguing mode of action is produced by P.
aeruginosa. Exoenzyme Y (ExoY [PA2191]) is produced by P.
aeruginosa and delivered directly via the T3SS to host cells, where
it modulates cAMP activity contributing to bacterial virulence
(38, 94, 175, 212).
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FIG 8 DSF-like fatty acids controlling cell-to-cell signaling in various Gram-
and, more interestingly, induces the dispersion of established P.
aeruginosa biofilms as well as biofilms from a wide range of species
(41). As yet, little is known about the synthesis and signaling network of this DSF-like molecule. Given the similarity of the phenotypes observed in bacteria producing DSF signals and the crossreactivity of these signals, it is tempting to speculate that these
compounds may interact with a common substrate-binding domain present in RpfC homologues. In accordance with this hypothesis, mixed-species biofilm experiments using S. maltophila
and P. aeruginosa revealed substantial differences in the architecture of P. aeruginosa biofilms and an increase in resistance of this
bacterium toward polymyxin antibiotics in the presence of DSF
(173). Additionally, it was demonstrated that the sensor kinase
PA1396 is essential for the response to these signals. Given the
importance of this opportunistic pathogen in human infections
and the relevance of antibiotic resistance and biofilm development, it is clear that this research into DSF-like molecules may be
only at an initial stage, with future work potentially analyzing the
efficacy of these molecules as therapeutic agents.
Nadal Jimenez et al.
internal levels of c-di-GMP. Binding of c-di-GMP to its receptor targets stimulates biofilm formation, suppressing motility. In parallel, binding of GTP to its
receptors (i.e., the allosteric site of the PDE FimX) increases the c-di-GMP-degrading activity of PDE, decreasing c-di-GMP levels, suppressing biofilm formation,
and increasing motility.
ppGpp and pppGpp
The second group of nucleotide-based molecules was discovered
in 1970 (27) and comprises the cellular alarmones ppGpp and
pppGpp. Under amino acid starvation conditions, these molecules rapidly accumulate intracellularly, triggering a switch from
cell growth to survival adaptation. In P. aeruginosa, AlgQ (AlgR2
[PA5255]) positively regulates the production of the nucleoside
diphosphate kinase Ndk (PA3807), responsible for the production of these molecules. Deletion of algQ leads to cell death at late
exponential phase, a clear consequence of the inability of this mutant to adapt from exponential-phase cell growth to survival
mode. This phenotype was rescued by overexpressing AlgQ or
Ndk, confirming the role of these proteins in survival adaptation
(107).
c-di-GMP
Another nucleotide-based molecule, cyclic di-GMP (c-di-GMP),
has attracted even more attention due to its major role as a secondary signaling molecule in many species from all kingdoms. In
bacteria, c-di-GMP levels are fine-tuned by the actions of two
types of enzymes: diguanylate cyclases (DGCs) containing
GGDEF domains, responsible for c-di-GMP synthesis, and phosphodiesterases (PDEs) that contain EAL domains, involved in
c-di-GMP degradation (Fig. 9). Together, these enzymes control a
multitude of phenotypes in multiple organisms, including biosynthesis of adhesins and exopolysaccharides, motility, long-term
survival and environmental stress adaptation, synthesis of second-
56 mmbr.asm.org
ary metabolites, regulated proteolysis and cell cycle progression,
and virulence in plant and animal pathogens.
In addition to enzymes containing single c-di-GMP domains,
several proteins contain both GGDEF and EAL domains. These
proteins are thus capable of both synthesis and degradation of
c-di-GMP, leading to the hypothesis that they act by balancing the
internal cellular levels of this molecule. Furthermore, a series of
proteins containing degenerate GGDEF domains have been found
in multiple organisms. These domains no longer generate c-diGMP but instead function as allosteric sites (33) or as c-di-GMP
receptors (144).
In P. aeruginosa, 39 genes have been identified as containing
either a DGC, a PDE, or both GGDEF and EAL domains (110).
One of the best-studied proteins involved in c-di-GMP formation
in this bacterium is the DGC WspR (PA3702). The product of
wspR is a response regulator linked to the wsp operon, which encodes a chemosensory system related to the chemotaxis pathway
of Escherichia coli (171). Mutations in wspF (PA3703), encoding a
putative methylesterase related to the chemotactic response protein CheB (177), result in high levels of c-di-GMP and biofilm
formation, while a wspF wspR double mutant restores the wildtype phenotype. This observation led to the hypothesis that WspF
acts by phosphorylating WspR, which stimulates c-di-GMP synthesis (88).
TFP are essential for twitching motility, adherence, and biofilm
formation in P. aeruginosa. One of the proteins required for TFP
formation is FimX (PA4959), a protein essential in twitching mo-
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FIG 9 c-di-GMP signaling mechanism. Diguanylate cyclases and phosphodiesterases regulate the bacterial lifestyle (free-living versus biofilm) by balancing the
Cell-to-Cell Signaling in P. aeruginosa
c-di-GMP and SCV
A clinically relevant phenotype associated with c-di-GMP levels in
P. aeruginosa is the occurrence of small-colony variants (SCV), an
adaptation morphotype associated with late-stage infections in
the lungs, antibiotic resistance, hyperadherence, and high levels of
EPS production (45, 81, 161, 186, 201). SCV development has
been linked to elevated levels of c-di-GMP, indicating that some of
these morphotypes could arise from mutations enhancing the activity of DGCs such as WspR. In a recent work, Jenal and coworkers identified a novel c-di-GMP-related operon, yfiBNR (PA1119
to PA1121), involved in the regulation of c-di-GMP levels (124).
The operon encodes YfiB (an OmpA-like outer membrane lipoprotein), YfiN (also known as TpbP, a membrane-integral DGC),
and YfiR (a small periplasmic protein). The yfiBNR genes control
the production of EPS by upregulating pel and psl (PA2231PA2245) expression, a common phenotype observed in SCV variants. Jenal et al. proposed a model in which YfiR represses the
activity of the DGC YfiN, reducing the levels of c-di-GMP. Consistent with this model, a cystic fibrosis SCV isolate reverted to the
wild type after overexpression of yfiR, indicating that this SCV
variant arose as a consequence of a mutation in the yfi operon.
Despite its lower fitness in vitro, this mutant persisted for many
weeks in a mouse infection model, demonstrating once again the
role that SCV morphotypes play in chronic infections and their
linkage to mutations that result in elevated c-di-GMP levels.
TARGETING BACTERIAL SIGNALING
A major goal in the study of bacterial cell-to-cell communication
systems is the development of novel and efficient antimicrobial
agents capable of disrupting virulence. As the first-discovered and
most-characterized pathway, AHLs have been the main target of
this research. Multiple approaches have been applied in attempts
March 2012 Volume 76 Number 1
to develop effective AHL-quenching drugs, including research
and development of synthetic and natural AHL mimics, use of
enzymes to degrade AHLs, and, more recently, the development of
AHL antibodies capable of either sequestering or degrading AHLs.
The role of P. aeruginosa in human infections has driven most
of the research on quorum quenching toward this pathogen. One
of the first groups of natural compounds with AHL-quenching
activity to be identified was the furanones, produced by the marine alga Delisea pulchra (126), which block quorum sensing in P.
aeruginosa, among other bacteria (84). This research led to a
plethora of studies on the production of chemically synthesized
furanones with improved quorum-quenching activity (89, 127).
Reports show that some furanones are able to inhibit quorum
sensing, facilitating clearance of P. aeruginosa in a mouse model of
lung infection (210). This observation corroborates those obtained from microarray experiments on the effects of furanones
on the AHL quorum-sensing regulon (85). Although their exact
mode of action remains uncharacterized, it has been suggested
that the effects of furanones are related to a reduction in LuxR
concentration (125). In addition, protein modeling studies on
LuxR receptors (109) and the LasR receptor protein (15) suggest
that furanones bind to LasR in the same position as the lactone
ring of 3-oxo-C12-HSL. Several further natural and synthetic molecules structurally similar and dissimilar to AHLs have been
reported to interfere with AHL quorum sensing by binding to
LuxR-type receptors (11, 75, 138, 167, 181, 182), indicating that
small-molecule interference with AHL QS systems is an interesting field with several potential applications.
A second approach to inhibit QS-regulated virulence is the
identification and improvement of quorum-quenching enzymes
capable of disrupting bacterial communication by degrading bacterial signaling molecules. Three types of AHL-quenching enzymes have been reported to date: lactonases, acylases, and oxidoreductases. The first group of quorum-quenching enzymes was
initially identified in Bacillus spp. (56) and was subsequently
found in the genomes of countless bacteria and eukaryotes (26, 54,
133, 145, 153, 170, 203, 213, 216). AHL lactonases belong to either
the metallo--lactamase family of proteins (108, 121, 190) or the
phosphotriesterase-like family (195). Lactonolysis of the AHL
ring leads to an inactive product that no longer activates the AHL
QS system, with consequent attenuation of virulence, as observed
in various infection models (31, 55, 149, 188).
The second class of quorum-quenching enzymes, AHL acylases, was discovered in Ralstonia spp. (120) and belongs to the
Ntn hydrolase superfamily (16). AHL acylases degrade the amide
bond of AHLs, releasing homoserine lactone and an acyl moiety. A
marked difference between AHL acylases and AHL lactonases lies
in their specificities toward different AHL substrates, in that acylases act upon a more restricted set of AHLs. A comparison between the AHL-binding mechanisms of the Bacillus thuringiensis
AHL lactonase (108) and the AHL acylase PvdQ from P. aeruginosa (13) provides an explanation for this specificity: the long
hydrophobic cavity of PvdQ acts as a major selective determinant
of substrate stability and catalysis. The in vivo virulence reduction
upon expression or addition of AHL acylases has also been proven
(120, 136, 150), demonstrating that AHL lactonases and AHL acylases are interesting candidates in the development of novel antimicrobial drugs.
Finally, oxidoreductases constitute the third and last family of
AHL-quenching enzymes found to date. The oxidoreductase of
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tility (95). FimX contains both an EAL domain and a degenerate
GGDEF domain (GDSIF). Biochemical and structural analyses
revealed that FimX is capable of degrading but not synthesizing
c-di-GMP. The degenerate GGDEF domain exerts an allosteric
role, binding c-di-GMP with a high affinity and thereby controlling FimX function (142). FimX is localized at a single cell pole
(95); mutagenesis of the EAL and GGDEF domains has demonstrated that both are essential for directing FimX to its correct
subcellular location. Slowly but steadily, the mechanism by which
FimX controls motility in this bacterium is being elucidated (106).
Surprisingly, the major flagellar regulator in P. aeruginosa, FleQ,
has also been found to be c-di-GMP responsive, despite the absence of GGDEF, EAL, or any other known degenerate domains
(87). This recent finding demonstrates that the relatively young
c-di-GMP field is examining an extremely complex regulatory system that has still to yield many exciting discoveries.
Furthermore, the commonly occurring linkage between the different signaling systems in P. aeruginosa has once again been demonstrated, this time for c-di-GMP and AHLs. In recently published work, Ueda and Wood demonstrated that the Las system
indirectly controls the levels of c-di-GMP through the tyrosine
phosphatase TpbA (PA3885). Substrate-bound LasR activates expression of tpbA, whose product in turn dephosphorylates and
inactivates the GGDEF protein TpbB (PA1120), resulting in reduced levels of c-di-GMP and, consequently, increases in exopolysaccharide (EPS) production and biofilm and pellicle formation
and a decrease in swarming motility (192, 193).
Nadal Jimenez et al.
remains to be proven. The PVD and GAC systems use membrane-associated proteins to activate signaling in response to their respective signals.
Rhodococcus erythropolis W2 has been reported to reduce 3-oxosubstituted AHLs to 3-OH-substituted AHLs (194). In addition,
CYP102A1, a cytochrome P450 of Bacillus megaterium, was also
reported to act as a quorum-quenching enzyme by efficiently oxidizing bacterial AHLs (32). A major drawback that may have
limited attention to the study of oxidoreductases as quorumquenching enzymes resides in the fact that the products still exhibited (markedly reduced) quorum-sensing activity.
A more recent approach to the development of novel antibacterial drugs targeting cell-cell communication is the production of
antibodies capable of eliciting an immune response upon detection of the bacterial signal. Binding of antibodies to signaling molecules would interfere with cell-cell communication, resulting in a
decrease of virulence. This approach was initially tested in Grampositive bacteria, with very promising results partially favored by
the peptidic nature of their QS signals, which aided in the development of antibodies (4, 152, 214). In order to overcome the small
size of AHLs and to potentiate the maximal response for antibody
generation, Janda and coworkers (104) pioneered a study where
AHLs and AHL analogues were chemically conjugated to keyhole
limpet hemocyanin (KLH) or bovine serum albumin (BSA), leading to efficient production of AHL-sequestering antibodies. These
antibodies have shown very promising efficacy in protecting mice
against P. aeruginosa infections (103, 104, 135).
The potential for novel antimicrobial drugs targeting
4-quinolone-mediated signaling in P. aeruginosa is also being explored. Farnesol, a common sesquiterpene produced by many organisms, was found to inhibit PQS production in P. aeruginosa,
although the high concentrations required may hamper in vivo
applications (39). Synthetic anthranilate derivatives (23, 36, 116)
have been developed and have shown promising results in a P.
aeruginosa mouse infection model (116). In parallel, Fetzner and
coworkers reported PQS-degrading activity by conversion of PQS
to N-octanoylanthranilic acid by the dioxygenase Hod (1H-3-
58 mmbr.asm.org
hydroxy-4-oxoquinaldine 2,4-dioxygenase) of Arthrobacter nitroguajacolicus (162). Hod and the Pseudomonas putida homologue QDO (1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase) (8)
belong to an unusual family of cofactor-independent dioxygenases involved in the breakdown of N-heteroaromatic compounds (187). A major drawback in the use of Hod against P.
aeruginosa infections is its sensitivity to degradation by P. aeruginosa exoproteases (162), a limiting factor that may be overcome in
the following years by protease-resistant variants selected via rational or random mutagenesis.
Besides AHL and 4-AQ systems, little progress has been made
in the development of novel drugs targeting the other cell-cell
communication systems. The two main explanations for this are
that the identities of many signals remain unknown and that little
attention has been given to the less-well-studied pathways. To
provide some examples, pyoverdine-mediated signaling in P.
aeruginosa is likely to be disrupted (directly or indirectly) by the
addition of pyoverdines from different Pseudomonas spp. (22), as
well as by the action of molecules that would interfere with the
FpvA receptor or proteins that could degrade pyoverdine. In the
case of pyocyanin, finding an enzyme capable of efficient degradation of this molecule not only would have consequences on the
cell-cell signaling of P. aeruginosa but also would have potential
biomedical interest due to the cytotoxic effects of pyocyanin
(169); however, such approaches have yet to be investigated fully.
CONCLUDING REMARKS
The initial discovery of AHL quorum sensing, followed by the
discovery of multiple and different bacterial signaling systems,
revolutionized our concept of the control that bacteria have on
their behavior in relation to the environment. For decades, bacteria had been considered simple organisms; this early misconception can be attributed to two main misleading observations: first,
an underestimation based on the size of these organisms, and
Microbiology and Molecular Biology Reviews
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FIG 10 Summary of cell-to-cell signaling systems in P. aeruginosa. Dashed lines indicate that the presence of a signal activating the system has been proposed but
Cell-to-Cell Signaling in P. aeruginosa
TABLE 1 Interactions between signaling systems in P. aeruginosa
Protein
Target system
Effect
Mechanism
Reference(s)
LasR
PqsE
PvdS
RetS
RhlR
RsaL
c-di-GMP
PQS
PYO
PQS
c-di-GMP
PQS
PYO
Activation
Activation
Activation
Activation
Repression
Repression
Repression
192
202
52, 71
147
137
202
42, 163, 165
RsmA
LasI, RhlI
Repression
LasR binding to tbpA promoter
Binding to pqsR promoter
Unknown
Unknown
Unknown
Binding to pqsR promoter
Direct mechanism (binding to the phzA1 and phzM promoters) and indirect
mechanism (binding to the promoter of LasI and inhibiting its
transcription)
Binding to lasI and rhlI promoters
March 2012 Volume 76 Number 1
vival, an elegant strategy to eliminate competition when survival
depends on iron availability.
In parallel to virulence factor production, the second major
threat posed by bacterial pathogens is persistence leading to
chronic infections, which is linked intrinsically to biofilm formation. In P. aeruginosa, the two major systems controlling this transition are the GAC and c-di-GMP systems. Although extracellular
signals have not been found for these two systems, their major role
in virulence in P. aeruginosa, among other bacteria, prompted us
to add them to this review. Together, these two signaling cascades
play a major role in the switch from highly virulent (acute) to
highly persistent (chronic) phenotypes. It is likely that in a versatile bacterium, parallel functions of these systems have been maintained to provide a complex and efficient survival mechanism
which can be fine-tuned according to environmental or endogenous signals. Regardless of the importance of the abovementioned signaling systems due to their implications in infections, it remains crucial to maintain efforts in the search and
understanding of the complete signaling network of this pathogen
(Fig. 10). Some of the signaling systems described here may be
considered less relevant due to their limited influence on virulence. However, until we unveil the full network of signals and
their corresponding pathways, regulation, and interactions with
other systems (Table 1), it is impossible to know the full extent of
their involvement in such phenotypes. Thus, each signaling system discovered contributes through its potential as a novel target
for antimicrobial drugs. Additionally, understanding the complete signaling integration network of a bacterium may prove essential for determining optimal targets for drug research and discovery.
ACKNOWLEDGMENTS
We are very grateful to the European Union for funding the research on
Pseudomonas aeruginosa carried out in our lab, under Antibiotarget contract MEST-CT-2005-020278.
We acknowledge all the participants of the Antibiotarget consortium
and the additional members of these labs for magnificently providing us
with knowledge and excellent discussions that contributed significantly to
the purpose of this research.
REFERENCES
1. Albus AM, Pesci EC, Runyen-Janecky LJ, West SE, Iglewski BH. 1997.
Vfr controls quorum sensing in Pseudomonas aeruginosa. J. Bacteriol.
179:3928 –3935.
2. Bainton NJ, et al. 1992. A general role for the lux autoinducer in bacterial cell signalling: control of antibiotic biosynthesis in Erwinia. Gene
116:87–91.
mmbr.asm.org 59
Downloaded from http://mmbr.asm.org/ on January 24, 2016 by guest
second, the apparent absence of communication systems. The
term “communication” has largely been associated exclusively
with the animal kingdom, whose members are able to transmit
messages using verbal or body language. Nonetheless, it now
seems obvious that ancient organisms have developed the ability
to transfer messages without having to employ sound or movement. Chemistry is unequivocally the most universal cellular language operating in all living organisms; using chemical molecules
as signals, all organisms, including bacteria, can produce and detect a large variety of messages that allow individual members to
sense and react to the constantly changing environment. By doing
so, bacteria have been able to display a dual lifestyle: living as single
organisms in the absence of communal obligations and displaying
complex social interactions when part of a community. Using
chemical signals, bacteria are able to determine population density and diversity, two ecological factors crucial for their survival.
One of the best examples of bacterial adaptation is given by the
Gram-negative bacterium P. aeruginosa. Initially studied for its
implications in human infections, this bacterium soon became a
model organism with which to study bacterial signaling due to the
high complexity, large degree of adaptability, and rich metabolic
diversity that allow its survival in the most hazardous environments, as well as colonization of a large number of hosts. A key
factor in the adaptation of P. aeruginosa is the large number of
signaling proteins encoded in its genome that allow this bacterium
to react rapidly to a wide range of signals. In this review, we have
selected some of the most clinically relevant examples related to
virulence factor production and resistance to conventional antibiotic treatments. The master virulence signaling systems in P.
aeruginosa are the AHL systems Las and Rhl, which together control the expression of multiple virulence factors in response to cell
density. Las and Rhl belong to the Lux-type family of signaling
systems, responsible for AHL production, the most extended signals in Gram-negative bacteria. The presence of two AHL signals
(3-oxo-C12-HSL and C4-HSL) in this bacterium is particularly
intriguing. Short-chain AHLs are more common among Gramnegative bacteria; therefore, having the Las system (3-oxo-C12HSL) at the top of the hierarchy may allow P. aeruginosa to make
its own message prevail over exogenous ones. Another group of
signaling molecules related to virulence factor production but encountered lower in the hierarchy are the 4-quinolones. These molecules are highly specific to members of the Pseudomonas and
Burkholderia families. An intriguing fact is that 4-quinolones are
strongly upregulated under low-iron conditions, connecting virulence factor production (pyocyanin) with adaptation and sur-
105, 160, 168
Nadal Jimenez et al.
60 mmbr.asm.org
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
with a unique self-regulatory mechanism. Proc. Natl. Acad. Sci. U. S. A.
98:14613–14618.
Carlier A, et al. 2003. The Ti plasmid of Agrobacterium tumefaciens
harbors an attM-paralogous gene, aiiB, also encoding N-acyl homoserine lactonase activity. Appl. Environ. Microbiol. 69:4989 – 4993.
Cashel M, Kalbacher B. 1970. The control of ribonucleic acid synthesis
in Escherichia coli. V. Characterization of a nucleotide associated with the
stringent response. J. Biol. Chem. 245:2309 –2318.
Charlton TS, et al. 2000. A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatographymass spectrometry: application to a model bacterial biofilm. Environ.
Microbiol. 2:530 –541.
Chatterjee S, Wistrom C, Lindow SE. 2008. A cell-cell signaling sensor
is required for virulence and insect transmission of Xylella fastidiosa.
Proc. Natl. Acad. Sci. U. S. A. 105:2670 –2675.
Chatzinikolaou I, et al. 2000. Recent experience with Pseudomonas
aeruginosa bacteremia in patients with cancer: retrospective analysis of
245 episodes. Arch. Intern. Med. 160:501–509.
Chen R, Zhou Z, Cao Y, Bai Y, Yao B. 2010. High yield expression of
an AHL-lactonase from Bacillus sp. B546 in Pichia pastoris and its application to reduce Aeromonas hydrophila mortality in aquaculture. Microb. Cell Fact. 9:39.
Chowdhary PK, et al. 2007. Bacillus megaterium CYP102A1 oxidation of
acyl homoserine lactones and acyl homoserines. Biochemistry 46:
14429 –14437.
Christen M, Christen B, Folcher M, Schauerte A, Jenal U. 2005.
Identification and characterization of a cyclic di-GMP-specific phosphodiesterase and its allosteric control by GTP. J. Biol. Chem. 280:
30829 –30837.
Chugani S, Greenberg EP. 2010. LuxR homolog-independent gene regulation by acyl-homoserine lactones in Pseudomonas aeruginosa. Proc.
Natl. Acad. Sci. U. S. A. 107:10673–10678.
Chugani SA, et al. 2001. QscR, a modulator of quorum-sensing signal
synthesis and virulence in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci.
U. S. A. 98:2752–2757.
Coleman JP, et al. 2008. Pseudomonas aeruginosa PqsA is an
anthranilate-coenzyme A ligase. J. Bacteriol. 190:1247–1255.
Cornforth JW, James AT. 1954. Some chemical properties of a naturally
occurring antagonist of dihydrostreptomycin. Biochem. J. 58:xlviii–xlix.
Cowell BA, Evans DJ, Fleiszig SM. 2005. Actin cytoskeleton disruption
by ExoY and its effects on Pseudomonas aeruginosa invasion. FEMS Microbiol. Lett. 250:71–76.
Cugini C, et al. 2007. Farnesol, a common sesquiterpene, inhibits PQS
production in Pseudomonas aeruginosa. Mol. Microbiol. 65:896 –906.
Dasgupta N, Ferrell EP, Kanack KJ, West SE, Ramphal R. 2002. fleQ,
the gene encoding the major flagellar regulator of Pseudomonas aeruginosa, is sigma70 dependent and is downregulated by Vfr, a homolog of
Escherichia coli cyclic AMP receptor protein. J. Bacteriol. 184:5240 –5250.
Davies DG, Marques CN. 2009. A fatty acid messenger is responsible for
inducing dispersion in microbial biofilms. J. Bacteriol. 191:1393–1403.
de Kievit T, Seed PC, Nezezon J, Passador L, Iglewski BH. 1999. RsaL,
a novel repressor of virulence gene expression in Pseudomonas aeruginosa. J. Bacteriol. 181:2175–2184.
Deng Y, Wu J, Eberl L, Zhang LH. 2010. Structural and functional
characterization of diffusible signal factor family quorum-sensing signals
produced by members of the Burkholderia cepacia complex. Appl. Environ. Microbiol. 76:4675– 4683.
Denning GM, Railsback MA, Rasmussen GT, Cox CD, Britigan BE.
1998. Pseudomonas pyocyanine alters calcium signaling in human airway
epithelial cells. Am. J. Physiol. 274:L893–L900.
Deziel E, Comeau Y, Villemur R. 2001. Initiation of biofilm formation
by Pseudomonas aeruginosa 57RP correlates with emergence of hyperpiliated and highly adherent phenotypic variants deficient in swimming,
swarming, and twitching motilities. J. Bacteriol. 183:1195–1204.
Deziel E, et al. 2005. The contribution of MvfR to Pseudomonas aeruginosa pathogenesis and quorum sensing circuitry regulation: multiple
quorum sensing-regulated genes are modulated without affecting lasRI,
rhlRI or the production of N-acyl-L-homoserine lactones. Mol. Microbiol. 55:998 –1014.
Deziel E, et al. 2004. Analysis of Pseudomonas aeruginosa 4-hydroxy-2alkylquinolines (HAQs) reveals a role for 4-hydroxy-2-heptylquinoline
in cell-to-cell communication. Proc. Natl. Acad. Sci. U. S. A. 101:1339 –
1344.
Microbiology and Molecular Biology Reviews
Downloaded from http://mmbr.asm.org/ on January 24, 2016 by guest
3. Bainton NJ, et al. 1992. N-(3-Oxohexanoyl)-L-homoserine lactone regulates carbapenem antibiotic production in Erwinia carotovora.
Biochem. J. 288:997–1004.
4. Balaban N, et al. 1998. Autoinducer of virulence as a target for vaccine
and therapy against Staphylococcus aureus. Science 280:438 – 440.
5. Banin E, Vasil ML, Greenberg EP. 2005. Iron and Pseudomonas aeruginosa biofilm formation. Proc. Natl. Acad. Sci. U. S. A. 102:11076 –11081.
6. Barber CE, et al. 1997. A novel regulatory system required for pathogenicity of Xanthomonas campestris is mediated by a small diffusible signal
molecule. Mol. Microbiol. 24:555–566.
7. Bassler BL, Wright M, Showalter RE, Silverman MR. 1993. Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence. Mol. Microbiol. 9:773–786.
8. Bauer I, Max N, Fetzner S, Lingens F. 1996. 2,4-Dioxygenases catalyzing N-heterocyclic-ring cleavage and formation of carbon monoxide.
Purification and some properties of 1H-3-hydroxy-4-oxoquinaldine 2,4dioxygenase from Arthrobacter sp. Ru61a and comparison with 1H-3hydroxy-4-oxoquinoline 2,4-dioxygenase from Pseudomonas putida
33/1. Eur. J. Biochem. 240:576 –583.
9. Beare PA, For RJ, Martin LW, Lamont IL. 2003. Siderophore-mediated
cell signalling in Pseudomonas aeruginosa: divergent pathways regulate
virulence factor production and siderophore receptor synthesis. Mol.
Microbiol. 47:195–207.
10. Beatson SA, Whitchurch CB, Sargent JL, Levesque RC, Mattick JS.
2002. Differential regulation of twitching motility and elastase production by Vfr in Pseudomonas aeruginosa. J. Bacteriol. 184:3605–3613.
11. Bjarnsholt T, et al. 2005. Garlic blocks quorum sensing and promotes
rapid clearing of pulmonary Pseudomonas aeruginosa infections. Microbiology 151:3873–3880.
12. Bleves S, Soscia C, Nogueira-Orlandi P, Lazdunski A, Filloux A. 2005.
Quorum sensing negatively controls type III secretion regulon expression in Pseudomonas aeruginosa PAO1. J. Bacteriol. 187:3898 –3902.
13. Bokhove M, Nadal Jimenez P, Quax WJ, Dijkstra BW. 2010. The
quorum-quenching N-acyl homoserine lactone acylase PvdQ is an Ntnhydrolase with an unusual substrate-binding pocket. Proc. Natl. Acad.
Sci. U. S. A. 107:686 – 691.
14. Boon C, et al. 2008. A novel DSF-like signal from Burkholderia cenocepacia interferes with Candida albicans morphological transition. ISME J.
2:27–36.
15. Bottomley MJ, Muraglia E, Bazzo R, Carfi A. 2007. Molecular insights
into quorum sensing in the human pathogen Pseudomonas aeruginosa
from the structure of the virulence regulator LasR bound to its autoinducer. J. Biol. Chem. 282:13592–13600.
16. Brannigan JA, et al. 1995. A protein catalytic framework with an
N-terminal nucleophile is capable of self-activation. Nature 378:416 –
419.
17. Bredenbruch F, Geffers R, Nimtz M, Buer J, Haussler S. 2006. The
Pseudomonas aeruginosa quinolone signal (PQS) has an iron-chelating
activity. Environ. Microbiol. 8:1318 –1329.
18. Bredenbruch F, et al. 2005. Biosynthetic pathway of Pseudomonas
aeruginosa 4-hydroxy-2-alkylquinolines. J. Bacteriol. 187:3630 –3635.
19. Britigan BE, Railsback MA, Cox CD. 1999. The Pseudomonas aeruginosa secretory product pyocyanin inactivates alpha1 protease inhibitor:
implications for the pathogenesis of cystic fibrosis lung disease. Infect.
Immun. 67:1207–1212.
20. Britigan BE, et al. 1992. Interaction of the Pseudomonas aeruginosa
secretory products pyocyanin and pyochelin generates hydroxyl radical
and causes synergistic damage to endothelial cells. Implications for
Pseudomonas-associated tissue injury. J. Clin. Invest. 90:2187–2196.
21. Britton G. 1983. Biochemistry of natural pigments. Cambridge University Press, Cambridge, United Kingdom.
22. Buyer JS, Leong J. 1986. Iron transport-mediated antagonism between
plant growth-promoting and plant-deleterious Pseudomonas strains. J.
Biol. Chem. 261:791–794.
23. Calfee MW, Coleman JP, Pesci EC. 2001. Interference with Pseudomonas quinolone signal synthesis inhibits virulence factor expression by
Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 98:11633–11637.
24. Campbell J, Lin Q, Geske GD, Blackwell HE. 2009. New and unexpected insights into the modulation of LuxR-type quorum sensing by
cyclic dipeptides. ACS Chem. Biol. 4:1051–1059.
25. Cao H, et al. 2001. A quorum sensing-associated virulence gene of
Pseudomonas aeruginosa encodes a LysR-like transcription regulator
Cell-to-Cell Signaling in P. aeruginosa
March 2012 Volume 76 Number 1
73.
74.
75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase
expression. J. Bacteriol. 173:3000 –3009.
Gambello MJ, Kaye S, Iglewski BH. 1993. LasR of Pseudomonas aeruginosa is a transcriptional activator of the alkaline protease gene (apr) and
an enhancer of exotoxin A expression. Infect. Immun. 61:1180 –1184.
Gang RK, Bang RL, Sanyal SC, Mokaddas E, Lari AR. 1999. Pseudomonas aeruginosa septicaemia in burns. Burns 25:611– 616.
Geske GD, Wezeman RJ, Siegel AP, Blackwell HE. 2005. Small molecule inhibitors of bacterial quorum sensing and biofilm formation. J.
Am. Chem. Soc. 127:12762–12763.
Gessard C. 1882. Sur les colorations bleues et vertes des linges a pansements. C. R. Acad. Sci. Hebd. Seances Acad. Sci. 94:536 –538.
Gilson L, Kuo A, Dunlap PV. 1995. AinS and a new family of autoinducer synthesis proteins. J. Bacteriol. 177:6946 – 6951.
Goodman AL, et al. 2004. A signaling network reciprocally regulates
genes associated with acute infection and chronic persistence in Pseudomonas aeruginosa. Dev. Cell 7:745–754.
Goodman AL, et al. 2009. Direct interaction between sensor kinase
proteins mediates acute and chronic disease phenotypes in a bacterial
pathogen. Genes Dev. 23:249 –259.
Harman JG. 2001. Allosteric regulation of the cAMP receptor protein.
Biochim. Biophys. Acta 1547:1–17.
Haussler S, Tummler B, Weissbrodt H, Rohde M, Steinmetz I. 1999.
Small-colony variants of Pseudomonas aeruginosa in cystic fibrosis. Clin.
Infect. Dis. 29:621– 625.
Hays EE, et al. 1945. Antibiotic substances produced by Pseudomonas
aeruginosa. J. Biol. Chem. 159:725–750.
He YW, Zhang LH. 2008. Quorum sensing and virulence regulation in
Xanthomonas campestris. FEMS Microbiol. Rev. 32:842– 857.
Hentzer M, et al. 2002. Inhibition of quorum sensing in Pseudomonas
aeruginosa biofilm bacteria by a halogenated furanone compound. Microbiology 148:87–102.
Hentzer M, et al. 2003. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors. EMBO J. 22:3803–3815.
Hernandez ME, Newman DK. 2001. Extracellular electron transfer.
Cell. Mol. Life Sci. 58:1562–1571.
Hickman JW, Harwood CS. 2008. Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Mol.
Microbiol. 69:376 –389.
Hickman JW, Tifrea DF, Harwood CS. 2005. A chemosensory system
that regulates biofilm formation through modulation of cyclic diguanylate levels. Proc. Natl. Acad. Sci. U. S. A. 102:14422–14427.
Hjelmgaard T, Persson T, Rasmussen TB, Givskov M, Nielsen J. 2003.
Synthesis of furanone-based natural product analogues with quorum
sensing antagonist activity. Bioorg. Med. Chem. 11:3261–3271.
Hogan DA, Vik A, Kolter R. 2004. A Pseudomonas aeruginosa quorumsensing molecule influences Candida albicans morphology. Mol. Microbiol. 54:1212–1223.
Holden I, Swift I, Williams I. 2000. New signal molecules on the
quorum-sensing block. Trends Microbiol. 8:101–104.
Holden MT, et al. 1999. Quorum-sensing cross talk: isolation and
chemical characterization of cyclic dipeptides from Pseudomonas aeruginosa and other gram-negative bacteria. Mol. Microbiol. 33:1254 –1266.
Hrabak EM, Willis DK. 1992. The lemA gene required for pathogenicity
of Pseudomonas syringae pv. syringae on bean is a member of a family of
two-component regulators. J. Bacteriol. 174:3011–3020.
Hritonenko V, et al. 2011. Adenylate cyclase activity of Pseudomonas
aeruginosa ExoY can mediate bleb-niche formation in epithelial cells and
contributes to virulence. Microb. Pathog. 51:305–312.
Huang B, Whitchurch CB, Mattick JS. 2003. FimX, a multidomain
protein connecting environmental signals to twitching motility in Pseudomonas aeruginosa. J. Bacteriol. 185:7068 –7076.
Inclan YF, Huseby MJ, Engel JN. 2011. FimL regulates cAMP synthesis
in Pseudomonas aeruginosa. PLoS One 6:e15867.
Jones AK, et al. 2010. Activation of the Pseudomonas aeruginosa AlgU
regulon through mucA mutation inhibits cyclic AMP/Vfr signaling. J.
Bacteriol. 192:5709 –5717.
Jones AM, et al. 2010. Clinical outcome for cystic fibrosis patients
infected with transmissible Pseudomonas aeruginosa: an 8-year prospective study. Chest 137:1405–1409.
Jones S, et al. 1993. The lux autoinducer regulates the production of
exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa. EMBO J. 12:2477–2482.
mmbr.asm.org 61
Downloaded from http://mmbr.asm.org/ on January 24, 2016 by guest
48. Dietrich LE, Price-Whelan A, Petersen A, Whiteley M, Newman DK.
2006. The phenazine pyocyanin is a terminal signalling factor in the
quorum sensing network of Pseudomonas aeruginosa. Mol. Microbiol.
61:1308 –1321.
49. Dietrich LE, Teal TK, Price-Whelan A, Newman DK. 2008. Redoxactive antibiotics control gene expression and community behavior in
divergent bacteria. Science 321:1203–1206.
50. Diggle SP, et al. 2006. Functional genetic analysis reveals a 2-alkyl-4quinolone signaling system in the human pathogen Burkholderia pseudomallei and related bacteria. Chem. Biol. 13:701–710.
51. Diggle SP, et al. 2007. The Pseudomonas aeruginosa 4-quinolone signal
molecules HHQ and PQS play multifunctional roles in quorum sensing
and iron entrapment. Chem. Biol. 14:87–96.
52. Diggle SP, et al. 2003. The Pseudomonas aeruginosa quinolone signal
molecule overcomes the cell density-dependency of the quorum sensing
hierarchy, regulates rhl-dependent genes at the onset of stationary phase
and can be produced in the absence of LasR. Mol. Microbiol. 50:29 – 43.
53. Diggle SP, Winzer K, Lazdunski A, Williams P, Camara M. 2002.
Advancing the quorum in Pseudomonas aeruginosa: MvaT and the regulation of N-acylhomoserine lactone production and virulence gene expression. J. Bacteriol. 184:2576 –2586.
54. Dong YH, Gusti AR, Zhang Q, Xu JL, Zhang LH. 2002. Identification
of quorum-quenching N-acyl homoserine lactonases from Bacillus species. Appl. Environ. Microbiol. 68:1754 –1759.
55. Dong YH, et al. 2001. Quenching quorum-sensing-dependent bacterial
infection by an N-acyl homoserine lactonase. Nature 411:813– 817.
56. Dong YH, Xu JL, Li XZ, Zhang LH. 2000. AiiA, an enzyme that
inactivates the acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia carotovora. Proc. Natl. Acad. Sci.
U. S. A. 97:3526 –3531.
57. Dow JM, et al. 2003. Biofilm dispersal in Xanthomonas campestris is
controlled by cell-cell signaling and is required for full virulence to
plants. Proc. Natl. Acad. Sci. U. S. A. 100:10995–11000.
58. Dow JM, Fouhy Y, Lucey JF, Ryan RP. 2006. The HD-GYP domain,
cyclic di-GMP signaling, and bacterial virulence to plants. Mol. Plant
Microbe Interact. 19:1378 –1384.
59. Eberhard A. 1972. Inhibition and activation of bacterial luciferase synthesis. J. Bacteriol. 109:1101–1105.
60. Eberhard A, et al. 1981. Structural identification of autoinducer of
Photobacterium fischeri luciferase. Biochemistry 20:2444 –2449.
61. Farrow JM, III, Pesci EC. 2007. Two distinct pathways supply anthranilate as a precursor of the Pseudomonas quinolone signal. J. Bacteriol.
189:3425–3433.
62. Farrow JM, III, et al. 2008. PqsE functions independently of PqsRPseudomonas quinolone signal and enhances the rhl quorum-sensing
system. J. Bacteriol. 190:7043–7051.
63. Fordos J. 1859. Receuil des travaux de la Societé d’Emulation pour les
Sciences Pharmaceutiques, vol 3. Societé d’Emulation pour les Sciences
Pharmaceutiques, Paris, France.
64. Fouhy Y, et al. 2007. Diffusible signal factor-dependent cell-cell signaling and virulence in the nosocomial pathogen Stenotrophomonas maltophilia. J. Bacteriol. 189:4964 – 4968.
65. Freeman L. 1916. Chronic general infection with the Bacillus pyocyaneus.
Ann. Surg. 64:195–202.
66. Fulcher NB, Holliday PM, Klem E, Cann MJ, Wolfgang MC. 2010. The
Pseudomonas aeruginosa Chp chemosensory system regulates intracellular cAMP levels by modulating adenylate cyclase activity. Mol. Microbiol. 76:889 –904.
67. Fuqua C. 2006. The QscR quorum-sensing regulon of Pseudomonas
aeruginosa: an orphan claims its identity. J. Bacteriol. 188:3169 –3171.
68. Fuqua C, Greenberg EP. 1998. Self perception in bacteria: quorum
sensing with acylated homoserine lactones. Curr. Opin. Microbiol.
1:183–189.
69. Fuqua C, Winans SC, Greenberg EP. 1996. Census and consensus in
bacterial ecosystems: the LuxR-LuxI family of quorum-sensing transcriptional regulators. Annu. Rev. Microbiol. 50:727–751.
70. 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.
71. Gallagher LA, McKnight SL, Kuznetsova MS, Pesci EC, Manoil C.
2002. Functions required for extracellular quinolone signaling by Pseudomonas aeruginosa. J. Bacteriol. 184:6472– 6480.
72. Gambello MJ, Iglewski BH. 1991. Cloning and characterization of the
Nadal Jimenez et al.
62
mmbr.asm.org
123. Mahajan-Miklos S, Tan MW, Rahme LG, Ausubel FM. 1999. Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas
aeruginosa-Caenorhabditis elegans pathogenesis model. Cell 96:47–56.
124. Malone JG, et al. 2010. YfiBNR mediates cyclic di-GMP dependent small
colony variant formation and persistence in Pseudomonas aeruginosa.
PLoS Pathog. 6:e1000804.
125. Manefield M, et al. 2002. Halogenated furanones inhibit quorum sensing through accelerated LuxR turnover. Microbiology 148:1119 –1127.
126. Manefield M, Welch M, Givskov M, Salmond GP, Kjelleberg S. 2001.
Halogenated furanones from the red alga, Delisea pulchra, inhibit carbapenem antibiotic synthesis and exoenzyme virulence factor production in the phytopathogen Erwinia carotovora. FEMS Microbiol. Lett.
205:131–138.
127. Manny AJ, et al. 1997. Reinvestigation of the sulfuric acid-catalysed
cyclisation of brominated 2-alkyllevulinic acids to 3-alkyl-5-methylene2(5H)-furanones. Tetrahedron 53:15813–15826.
128. Mashburn LM, Whiteley M. 2005. Membrane vesicles traffic signals and
facilitate group activities in a prokaryote. Nature 437:422– 425.
129. Mashburn-Warren L, et al. 2008. Interaction of quorum signals with
outer membrane lipids: insights into prokaryotic membrane vesicle formation. Mol. Microbiol. 69:491–502.
130. Mavrodi DV, Blankenfeldt W, Thomashow LS. 2006. Phenazine compounds in fluorescent Pseudomonas spp. biosynthesis and regulation.
Annu. Rev. Phytopathol. 44:417– 445.
131. Mazzola M, Cook RJ, Thomashow LS, Weller DM, Pierson LS, III.
1992. Contribution of phenazine antibiotic biosynthesis to the ecological
competence of fluorescent pseudomonads in soil habitats. Appl. Environ. Microbiol. 58:2616 –2624.
132. McAlester G, O’Gara F, Morrissey JP. 2008. Signal-mediated interactions between Pseudomonas aeruginosa and Candida albicans. J. Med.
Microbiol. 57:563–569.
133. Mei GY, Yan XX, Turak A, Luo ZQ, Zhang LQ. 2010. AidH, an
alpha/beta-hydrolase fold family member from an Ochrobactrum sp.
strain, is a novel N-acylhomoserine lactonase. Appl. Environ. Microbiol.
76:4933– 4942.
134. Micek ST, et al. 2005. Pseudomonas aeruginosa bloodstream infection:
importance of appropriate initial antimicrobial treatment. Antimicrob.
Agents Chemother. 49:1306 –1311.
135. Miyairi S, et al. 2006. Immunization with 3-oxododecanoyl-L-homoserine
lactone-protein conjugate protects mice from lethal Pseudomonas aeruginosa
lung infection. J. Med. Microbiol. 55:1381–1387.
136. Morohoshi T, Nakazawa S, Ebata A, Kato N, Ikeda T. 2008. Identification and characterization of N-acylhomoserine lactone-acylase from
the fish intestinal Shewanella sp. strain MIB015. Biosci. Biotechnol.
Biochem. 72:1887–1893.
137. Moscoso JA, Mikkelsen H, Heeb S, Williams P, Filloux A. 2011. The
Pseudomonas aeruginosa sensor RetS switches type III and type VI secretion via c-di-GMP signalling. Environ. Microbiol. 13:3128 –3138.
138. Muh U, et al. 2006. A structurally unrelated mimic of a Pseudomonas
aeruginosa acyl-homoserine lactone quorum-sensing signal. Proc. Natl.
Acad. Sci. U. S. A. 103:16948 –16952.
139. Muhlradt PF, Tsai H, Conradt P. 1986. Effects of pyocyanine, a blue
pigment from Pseudomonas aeruginosa, on separate steps of T cell activation: interleukin 2 (IL 2) production, IL 2 receptor formation, proliferation and induction of cytolytic activity. Eur. J. Immunol. 16:434 – 440.
140. Mulcahy H, et al. 2008. Pseudomonas aeruginosa RsmA plays an important role during murine infection by influencing colonization, virulence,
persistence, and pulmonary inflammation. Infect. Immun. 76:632– 638.
141. Nadal Jimenez P, et al. 2010. Role of PvdQ in Pseudomonas aeruginosa
virulence under iron-limiting conditions. Microbiology 156:49 –59.
142. Navarro MV, De N, Bae N, Wang Q, Sondermann H. 2009. Structural
analysis of the GGDEF-EAL domain-containing c-di-GMP receptor
FimX. Structure 17:1104 –1116.
143. Nealson KH, Platt T, Hastings JW. 1970. Cellular control of the synthesis and activity of the bacterial luminescent system. J. Bacteriol. 104:
313–322.
144. Newell PD, Monds RD, O’Toole GA. 2009. LapD is a bis-(3=,5=)-cyclic
dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens Pf0-1. Proc. Natl. Acad. Sci. U. S. A. 106:3461–3466.
145. Ng FS, Wright DM, Seah SY. 2011. Characterization of a phosphotriesteraselike lactonase from Sulfolobus solfataricus and its immobilization for disruption
of quorum sensing. Appl. Environ. Microbiol. 77:1181–1186.
146. Nutman J, Chase PA, Dearborn DG, Berger M, Sorensen RU. 1988.
Microbiology and Molecular Biology Reviews
Downloaded from http://mmbr.asm.org/ on January 24, 2016 by guest
100. Jordan EO. 1899. Bacillus pyocyaneus and its pigments. J. Exp. Med.
4:627– 647.
101. Kamath JM, Britigan BE, Cox CD, Shasby DM. 1995. Pyocyanin from
Pseudomonas aeruginosa inhibits prostacyclin release from endothelial
cells. Infect. Immun. 63:4921– 4923.
102. Kang CI, et al. 2005. Bloodstream infections caused by antibioticresistant gram-negative bacilli: risk factors for mortality and impact of
inappropriate initial antimicrobial therapy on outcome. Antimicrob.
Agents Chemother. 49:760 –766.
103. Kaufmann GF, Park J, Mee JM, Ulevitch RJ, Janda KD. 2008. The
quorum quenching antibody RS2-1G9 protects macrophages from the
cytotoxic effects of the Pseudomonas aeruginosa quorum sensing signalling molecule N-3-oxo-dodecanoyl-homoserine lactone. Mol. Immunol.
45:2710 –2714.
104. Kaufmann GF, et al. 2006. Antibody interference with N-acyl homoserine lactone-mediated bacterial quorum sensing. J. Am. Chem. Soc. 128:
2802–2803.
105. Kay E, et al. 2006. Two GacA-dependent small RNAs modulate the
quorum-sensing response in Pseudomonas aeruginosa. J. Bacteriol. 188:
6026 – 6033.
106. Kazmierczak BI, Lebron MB, Murray TS. 2006. Analysis of FimX, a
phosphodiesterase that governs twitching motility in Pseudomonas
aeruginosa. Mol. Microbiol. 60:1026 –1043.
107. Kim HY, et al. 1998. Alginate, inorganic polyphosphate, GTP and
ppGpp synthesis co-regulated in Pseudomonas aeruginosa: implications
for stationary phase survival and synthesis of RNA/DNA precursors.
Mol. Microbiol. 27:717–725.
108. Kim MH, et al. 2005. The molecular structure and catalytic mechanism
of a quorum-quenching N-acyl-L-homoserine lactone hydrolase. Proc.
Natl. Acad. Sci. U. S. A. 102:17606 –17611.
109. Koch B, et al. 2005. The LuxR receptor: the sites of interaction with
quorum-sensing signals and inhibitors. Microbiology 151:3589 –3602.
110. Kulasakara H, et al. 2006. Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3=-5=)-cyclicGMP in virulence. Proc. Natl. Acad. Sci. U. S. A. 103:2839 –2844.
111. Lamont IL, Beare PA, Ochsner U, Vasil AI, Vasil ML. 2002.
Siderophore-mediated signaling regulates virulence factor production in
Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 99:7072–7077.
112. Latifi A, Foglino M, Tanaka K, Williams P, Lazdunski A. 1996. A
hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links
the transcriptional activators LasR and RhIR (VsmR) to expression of the
stationary-phase sigma factor RpoS. Mol. Microbiol. 21:1137–1146.
113. Laue BE, et al. 2000. The biocontrol strain Pseudomonas fluorescens F113
produces the Rhizobium small bacteriocin, N-(3-hydroxy-7-cistetradecenoyl)homoserine lactone, via HdtS, a putative novel
N-acylhomoserine lactone synthase. Microbiology 146:2469 –2480.
114. Laursen JB, Nielsen J. 2004. Phenazine natural products: biosynthesis,
synthetic analogues, and biological activity. Chem. Rev. 104:1663–1686.
115. Ledgham F, et al. 2003. Interactions of the quorum sensing regulator
QscR: interaction with itself and the other regulators of Pseudomonas
aeruginosa LasR and RhlR. Mol. Microbiol. 48:199 –210.
116. Lesic B, et al. 2007. Inhibitors of pathogen intercellular signals as selective anti-infective compounds. PLoS Pathog. 3:1229 –1239.
117. Li C, Wally H, Miller SJ, Lu CD. 2009. The multifaceted proteins MvaT
and MvaU, members of the H-NS family, control arginine metabolism,
pyocyanin synthesis, and prophage activation in Pseudomonas aeruginosa
PAO1. J. Bacteriol. 191:6211– 6218.
118. Liang H, Duan J, Sibley CD, Surette MG, Duan K. 2011. Identification
of mutants with altered phenazine production in Pseudomonas aeruginosa. J. Med. Microbiol. 60:22–34.
119. Lightbown JW. 1954. An antagonist of streptomycin and dihydrostreptomycin produced by Pseudomonas aeruginosa. J. Gen. Microbiol. 11:
477– 492.
120. Lin YH, et al. 2003. Acyl-homoserine lactone acylase from Ralstonia
strain XJ12B represents a novel and potent class of quorum-quenching
enzymes. Mol. Microbiol. 47:849 – 860.
121. Liu D, et al. 2005. Three-dimensional structure of the quorumquenching N-acyl homoserine lactone hydrolase from Bacillus thuringiensis. Proc. Natl. Acad. Sci. U. S. A. 102:11882–11887.
122. Lodise TP, Jr, et al. 2007. Predictors of 30-day mortality among patients
with Pseudomonas aeruginosa bloodstream infections: impact of delayed
appropriate antibiotic selection. Antimicrob. Agents Chemother. 51:
3510 –3515.
Cell-to-Cell Signaling in P. aeruginosa
147.
148.
149.
150.
151.
153.
154.
155.
156.
157.
158.
159.
160.
161.
162.
163.
164.
165.
166.
167.
168.
169.
170.
March 2012 Volume 76 Number 1
171.
172.
173.
174.
175.
176.
177.
178.
179.
180.
181.
182.
183.
184.
185.
186.
187.
188.
189.
190.
191.
192.
193.
nase QlcA from yet unculturable soil bacteria. Commun. Agric. Appl.
Biol. Sci. 73:3– 6.
Ridgway HG, Silverman M, Simon MI. 1977. Localization of proteins
controlling motility and chemotaxis in Escherichia coli. J. Bacteriol. 132:
657– 665.
Rumbaugh KP, Griswold JA, Iglewski BH, Hamood AN. 1999. Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa
in burn wound infections. Infect. Immun. 67:5854 –5862.
Ryan RP, et al. 2008. Interspecies signalling via the Stenotrophomonas
maltophilia diffusible signal factor influences biofilm formation and
polymyxin tolerance in Pseudomonas aeruginosa. Mol. Microbiol. 68:
75– 86.
Ryan RP, et al. 2006. Cell-cell signaling in Xanthomonas campestris
involves an HD-GYP domain protein that functions in cyclic di-GMP
turnover. Proc. Natl. Acad. Sci. U. S. A. 103:6712– 6717.
Sayner SL, et al. 2004. Paradoxical cAMP-induced lung endothelial
hyperpermeability revealed by Pseudomonas aeruginosa ExoY. Circ. Res.
95:196 –203.
Seet Q, Zhang LH. 2011. Anti-activator QslA defines the quorum sensing threshold and response in Pseudomonas aeruginosa. Mol. Microbiol.
80:951–965.
Sherris D, Parkinson JS. 1981. Posttranslational processing of methylaccepting chemotaxis proteins in Escherichia coli. Proc. Natl. Acad. Sci.
U. S. A. 78:6051– 6055.
Shirley M, Lamont IL. 2009. Role of TonB1 in pyoverdine-mediated
signaling in Pseudomonas aeruginosa. J. Bacteriol. 191:5634 –5640.
Siehnel R, et al. 2010. A unique regulator controls the activation threshold of quorum-regulated genes in Pseudomonas aeruginosa. Proc. Natl.
Acad. Sci. U. S. A. 107:7916 –7921.
Singh PK, Parsek MR, Greenberg EP, Welsh MJ. 2002. A component of
innate immunity prevents bacterial biofilm development. Nature 417:
552–555.
Smith KM, Bu Y, Suga H. 2003. Induction and inhibition of Pseudomonas aeruginosa quorum sensing by synthetic autoinducer analogs. Chem.
Biol. 10:81– 89.
Smith KM, Bu Y, Suga H. 2003. Library screening for synthetic agonists
and antagonists of a Pseudomonas aeruginosa autoinducer. Chem. Biol.
10:563–571.
Smith RS, Harris SG, Phipps R, Iglewski B. 2002. The Pseudomonas
aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)homoserine
lactone contributes to virulence and induces inflammation in vivo. J.
Bacteriol. 184:1132–1139.
Smith RS, Wolfgang MC, Lory S. 2004. An adenylate cyclase-controlled
signaling network regulates Pseudomonas aeruginosa virulence in a
mouse model of acute pneumonia. Infect. Immun. 72:1677–1684.
Sorensen R. 1987. Biological effects of Pseudomonas aeruginosa phenazine pigments. Antibiot. Chemother. 39:113–124.
Starkey M, et al. 2009. Pseudomonas aeruginosa rugose small-colony
variants have adaptations that likely promote persistence in the cystic
fibrosis lung. J. Bacteriol. 191:3492–3503.
Steiner RA, Janssen HJ, Roversi P, Oakley AJ, Fetzner S. 2010. Structural basis for cofactor-independent dioxygenation of N-heteroaromatic
compounds at the alpha/beta-hydrolase fold. Proc. Natl. Acad. Sci.
U. S. A. 107:657– 662.
Stoltz DA, et al. 2008. Drosophila are protected from Pseudomonas
aeruginosa lethality by transgenic expression of paraoxonase-1. J. Clin.
Invest. 118:3123–3131.
Strom K, Sjogren J, Broberg A, Schnurer J. 2002. Lactobacillus plantarum MiLAB 393 produces the antifungal cyclic dipeptides cyclo(L-PheL-Pro) and cyclo(L-Phe-trans-4-OH-L-Pro) and 3-phenyllactic acid.
Appl. Environ. Microbiol. 68:4322– 4327.
Thomas PW, Stone EM, Costello AL, Tierney DL, Fast W. 2005. The
quorum-quenching lactonase from Bacillus thuringiensis is a metalloprotein. Biochemistry 44:7559 –7569.
Toder DS, Gambello MJ, Iglewski BH. 1991. Pseudomonas aeruginosa
LasA: a second elastase under the transcriptional control of lasR. Mol.
Microbiol. 5:2003–2010.
Ueda A, Wood TK. 2009. Connecting quorum sensing, c-di-GMP, pel
polysaccharide, and biofilm formation in Pseudomonas aeruginosa
through tyrosine phosphatase TpbA (PA3885). PLoS Pathog.
5:e1000483.
Ueda A, Wood TK. 2010. Tyrosine phosphatase TpbA of Pseudomonas
mmbr.asm.org 63
Downloaded from http://mmbr.asm.org/ on January 24, 2016 by guest
152.
Suppression of lymphocyte proliferation by Pseudomonas aeruginosa
phenazine pigments. Isr. J. Med. Sci. 24:228 –232.
Ochsner UA, Wilderman PJ, Vasil AI, Vasil ML. 2002. GeneChip
expression analysis of the iron starvation response in Pseudomonas
aeruginosa: identification of novel pyoverdine biosynthesis genes. Mol.
Microbiol. 45:1277–1287.
Okabayashi T, Ide M, Yoshimoto A. 1963. Excretion of adenosine3=,5=-phosphate in the culture broth of Brevibacterium liquefaciens. Arch.
Biochem. Biophys. 100:158 –159.
Ozer EA, et al. 2005. Human and murine paraoxonase 1 are host modulators of Pseudomonas aeruginosa quorum-sensing. FEMS Microbiol.
Lett. 253:29 –37.
Papaioannou E, et al. 2009. Quorum-quenching acylase reduces the
virulence of Pseudomonas aeruginosa in a Caenorhabditis elegans infection model. Antimicrob. Agents Chemother. 53:4891– 4897.
Park DK, et al. 2006. Cyclo(Phe-Pro) modulates the expression of ompU
in Vibrio spp. J. Bacteriol. 188:2214 –2221.
Park J, et al. 2007. Infection control by antibody disruption of bacterial
quorum sensing signaling. Chem. Biol. 14:1119 –1127.
Park SY, et al. 2003. AhlD, an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other bacteria. Microbiology
149:1541–1550.
Passador L, Cook JM, Gambello MJ, Rust L, Iglewski BH. 1993.
Expression of Pseudomonas aeruginosa virulence genes requires cell-tocell communication. Science 260:1127–1130.
Pearson JP, Feldman M, Iglewski BH, Prince A. 2000. Pseudomonas
aeruginosa cell-to-cell signaling is required for virulence in a model of
acute pulmonary infection. Infect. Immun. 68:4331– 4334.
Pearson JP, et al. 1994. Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc. Natl. Acad.
Sci. U. S. A. 91:197–201.
Pearson JP, Passador L, Iglewski BH, Greenberg EP. 1995. A second
N-acylhomoserine lactone signal produced by Pseudomonas aeruginosa.
Proc. Natl. Acad. Sci. U. S. A. 92:1490 –1494.
Perkins RG. 1901. Report of nine cases of infection with Bacillus pyocyaneus. J. Med. Res. 6:281–297.
Pesci EC, et al. 1999. Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A.
96:11229 –11234.
Pessi G, et al. 2001. The global posttranscriptional regulator RsmA
modulates production of virulence determinants and N-acylhomoserine
lactones in Pseudomonas aeruginosa. J. Bacteriol. 183:6676 – 6683.
Proctor RA, et al. 2006. Small colony variants: a pathogenic form of
bacteria that facilitates persistent and recurrent infections. Nat. Rev. Microbiol. 4:295–305.
Pustelny C, et al. 2009. Dioxygenase-mediated quenching of quinolonedependent quorum sensing in Pseudomonas aeruginosa. Chem. Biol. 16:
1259 –1267.
Rampioni G, et al. 2006. The quorum-sensing negative regulator RsaL of
Pseudomonas aeruginosa binds to the lasI promoter. J. Bacteriol. 188:
815– 819.
Rampioni G, et al. 2010. Transcriptomic analysis reveals a global alkylquinolone-independent regulatory role for PqsE in facilitating the environmental adaptation of Pseudomonas aeruginosa to plant and animal
hosts. Environ. Microbiol. 12:1659 –1673.
Rampioni G, et al. 2007. RsaL provides quorum sensing homeostasis
and functions as a global regulator of gene expression in Pseudomonas
aeruginosa. Mol. Microbiol. 66:1557–1565.
Ran H, Hassett DJ, Lau GW. 2003. Human targets of Pseudomonas
aeruginosa pyocyanin. Proc. Natl. Acad. Sci. U. S. A. 100:14315–14320.
Rasmussen TB, et al. 2005. Identity and effects of quorum-sensing
inhibitors produced by Penicillium species. Microbiology 151:1325–
1340.
Reimmann C, et al. 1997. The global activator GacA of Pseudomonas
aeruginosa PAO positively controls the production of the autoinducer
N-butyryl-homoserine lactone and the formation of the virulence factors
pyocyanin, cyanide, and lipase. Mol. Microbiol. 24:309 –319.
Reszka KJ, O’Malley Y, McCormick ML, Denning GM, Britigan BE.
2004. Oxidation of pyocyanin, a cytotoxic product from Pseudomonas
aeruginosa, by microperoxidase 11 and hydrogen peroxide. Free Radic.
Biol. Med. 36:1448 –1459.
Riaz K, et al. 2008. Metagenomics revealed a quorum quenching lacto-
Nadal Jimenez et al.
194.
195.
196.
197.
198.
200.
201.
202.
203.
204.
Pol Nadal Jimenez received his Ph.D. in 2011
from the University of Groningen, The Netherlands. During his doctoral studies in the department of W. J. Quax, he worked on the role of the
quorum-quenching acylase PvdQ in fluorescent pseudomonads. Previous to his Ph.D.
work, he also worked on the use of quorumquenching lactonases in food products and on
the detection and inhibition of quorum sensing
in bacterial biofilms during his master studies in
Ghent, Belgium. In 2011, Pol Nadal Jimenez
joined the group of K. B. Xavier at the Instituto Gulbenkian de Ciência,
Portugal, to study the role of quorum sensing in microbial communities and
microbe-host interactions.
205.
206.
207.
208.
209.
210.
211.
212.
213.
214.
215.
216.
Pseudomonas aeruginosa multidrug efflux operon mexEF-oprN. FEMS
Microbiol. Lett. 255:247–254.
Whitehead NA, Barnard AM, Slater H, Simpson NJ, Salmond GP.
2001. Quorum-sensing in Gram-negative bacteria. FEMS Microbiol.
Rev. 25:365– 404.
Williams EP, Cameron K. 1894. Infection by the Bacillus pyocyaneus a
cause of infantile mortality. Public Health Pap. Rep. 20:355–360.
Wilson R, et al. 1988. Measurement of Pseudomonas aeruginosa phenazine pigments in sputum and assessment of their contribution to sputum
sol toxicity for respiratory epithelium. Infect. Immun. 56:2515–2517.
Withers H, Swift S, Williams P. 2001. Quorum sensing as an integral
component of gene regulatory networks in Gram-negative bacteria.
Curr. Opin. Microbiol. 4:186 –193.
Wolfgang MC, Lee VT, Gilmore ME, Lory S. 2003. Coordinate regulation of bacterial virulence genes by a novel adenylate cyclase-dependent
signaling pathway. Dev. Cell 4:253–263.
Wu H, et al. 2004. Synthetic furanones inhibit quorum-sensing and
enhance bacterial clearance in Pseudomonas aeruginosa lung infection in
mice. J. Antimicrob. Chemother. 53:1054 –1061.
Xiao G, et al. 2006. MvfR, a key Pseudomonas aeruginosa pathogenicity
LTTR-class regulatory protein, has dual ligands. Mol. Microbiol. 62:
1689 –1699.
Yahr TL, Vallis AJ, Hancock MK, Barbieri JT, Frank DW. 1998. ExoY,
an adenylate cyclase secreted by the Pseudomonas aeruginosa type III
system. Proc. Natl. Acad. Sci. U. S. A. 95:13899 –13904.
Yang F, et al. 2005. Quorum quenching enzyme activity is widely conserved in the sera of mammalian species. FEBS Lett. 579:3713–3717.
Yang G, et al. 2006. A novel peptide isolated from phage library to
substitute a complex system for a vaccine against staphylococci infection.
Vaccine 24:1117–1123.
Yu S, et al. 2009. Structure elucidation and preliminary assessment of
hydrolase activity of PqsE, the Pseudomonas quinolone signal (PQS) response protein. Biochemistry 48:10298 –10307.
Zhang HB, Wang LH, Zhang LH. 2002. Genetic control of quorumsensing signal turnover in Agrobacterium tumefaciens. Proc. Natl. Acad.
Sci. U. S. A. 99:4638 – 4643.
Jessica A. Thompson started her scientific career at the University of Cambridge, where she
completed her B.A., before joining the research
group of David Holden at Imperial College
London, United Kingdom, in 2005. While
there, she obtained a Ph.D. in cellular microbiology, researching the interactions between Salmonella virulence factors and macrophage responses to infection in a mouse model of acute
typhoid fever. In 2011, she joined the laboratory
of K. B. Xavier at the Instituto Gulbenkian de
Ciência, Portugal, where she is currently investigating the regulation of bacterial communication in Escherichia coli and Salmonella and its role in colonization, development, and homeostasis of the gastrointestinal flora.
Gudrun Koch earned her M.Sc. in biotechnology from the FH Campus in Vienna, Austria, in
2006. For her doctoral studies, she joined the
group of W. J. Quax at the University of Groningen, The Netherlands, where her work focused on PvdQ, a quorum-quenching acylase
conserved among fluorescent Pseudomonas
spp. After completing her doctoral studies, she
joined the lab of Daniel Lopez at the Institute
for Molecular Infection Biology IMIB at Würzburg University, Germany, where she is currently working on the underlying molecular mechanisms of cell differentiation in Gram-positive bacteria.
64 mmbr.asm.org
Microbiology and Molecular Biology Reviews
Downloaded from http://mmbr.asm.org/ on January 24, 2016 by guest
199.
aeruginosa controls extracellular DNA via cyclic diguanylic acid concentrations. Environ. Microbiol. 2:449 – 455.
Uroz S, et al. 2005. N-Acylhomoserine lactone quorum-sensing molecules are modified and degraded by Rhodococcus erythropolis W2 by both
amidolytic and novel oxidoreductase activities. Microbiology 151:3313–
3322.
Uroz S, et al. 2008. A Rhodococcus qsdA-encoded enzyme defines a novel
class of large-spectrum quorum-quenching lactonases. Appl. Environ.
Microbiol. 74:1357–1366.
Usher LR, et al. 2002. Induction of neutrophil apoptosis by the Pseudomonas aeruginosa exotoxin pyocyanin: a potential mechanism of persistent infection. J. Immunol. 168:1861–1868.
Vallet I, et al. 2004. Biofilm formation in Pseudomonas aeruginosa:
fimbrial cup gene clusters are controlled by the transcriptional regulator
MvaT. J. Bacteriol. 186:2880 –2890.
Ventre I, et al. 2006. Multiple sensors control reciprocal expression of
Pseudomonas aeruginosa regulatory RNA and virulence genes. Proc. Natl.
Acad. Sci. U. S. A. 103:171–176.
Vial L, et al. 2008. Burkholderia pseudomallei, B. thailandensis, and B.
ambifaria produce 4-hydroxy-2-alkylquinoline analogues with a methyl
group at the 3 position that is required for quorum-sensing regulation. J.
Bacteriol. 190:5339 –5352.
Vojnov AA, Slater H, Newman MA, Daniels MJ, Dow JM. 2001.
Regulation of the synthesis of cyclic glucan in Xanthomonas campestris by
a diffusible signal molecule. Arch. Microbiol. 176:415– 420.
von Gotz F, et al. 2004. Expression analysis of a highly adherent and
cytotoxic small colony variant of Pseudomonas aeruginosa isolated from a
lung of a patient with cystic fibrosis. J. Bacteriol. 186:3837–3847.
Wade DS, et al. 2005. Regulation of Pseudomonas quinolone signal
synthesis in Pseudomonas aeruginosa. J. Bacteriol. 187:4372– 4380.
Wang WZ, Morohoshi T, Ikenoya M, Someya N, Ikeda T. 2010. AiiM,
a novel class of N-acylhomoserine lactonase from the leaf-associated bacterium Microbacterium testaceum. Appl. Environ. Microbiol. 76:2524 –
2530.
Westfall LW, et al. 2006. mvaT mutation modifies the expression of the
Cell-to-Cell Signaling in P. aeruginosa
Karina B. Xavier is an Assistant Professor at Instituto Gulbekian de Ciência and Instituto de
Tecnologia Química e Biológica in Oeiras, Portugal. She received her Ph.D. in biochemistry
from the University of Lisbon in 1999 and her
B.S. in biochemistry in 1994, also from the University of Lisbon. Her Ph.D. work was on carbohydrate pathways in hyperthermophilic Archaea. She has been working on bacterial signal
transduction and quorum sensing since 2000,
when she started her postdoctoral training at
Princeton University, NJ, with Bonnie Bassler. In 2006, she became the Principal Investigator of the Bacterial Signaling Laboratory at the Instituto Gulbenkian de Ciência and Instituto de Tecnologia Química e Biológica.
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Robbert H. Cool is currently a lecturer in the
Department of Pharmaceutical Biology at the
University of Groningen, The Netherlands. He
started to work on bacterial elongation factor
Tu at École Polytechnique in Palaiseau, France,
and received his Ph.D. on this topic at the University of Leiden in 1991. After having worked
on the isolation of genes involved in kirromycin
biosynthesis and resistance in Streptomycetes,
he shifted in 1993 to work on Ras signal transduction pathways at the Max Planck Institute
for Molecular Physiology in Dortmund, Germany. In 1999, he moved to the
University of Groningen to work on bacterial and mammalian multidrug
transporter proteins, and since 2002, he has focused on two main research
topics: apoptosis-inducing cytokines in mammalian cells and acylases and
quorum-sensing systems in pseudomonads.
Wim J. Quax is currently Professor of Pharmaceutical Biology at the University of Groningen.
He obtained his Ph.D. in molecular biology at
the University of Nijmegen and worked for several years on the production and engineering of
bacterial enzymes. In 1995, he joined Genencor
International (Palo Alto, CA) to start working
on the regulation of gene expression in both Bacillus subtilis and Pseudomonas spp. During this
period, he studied the role of quorum sensing
both in production of enzymes and in the expression of virulence factors. After his move to Groningen in 1998, he became interested in research on acylases for their use in the biosynthesis of
antibiotics and, more recently, in their role as quorum-quenching enzymes.
Research carried out in his laboratory has focused on the identification of
novel acylases and the development of potent variants with industrial
applications.