Regulation of Quorum Sensing In: Pseudomonas

Regulation of quorum sensing in Pseudomonas
Vittorio Venturi
International Centre for Genetic Engineering and Biotechnology, Trieste, Italy
Correspondence: Vittorio Venturi, Abstract

Bacteriology Group, International Centre for
Genetic Engineering & Biotechnology, Area
Bacteria use small signal molecules in order to monitor their population density
Science Park, Padriciano 99, 34012 Trieste, and coordinate gene regulation in a process called quorum sensing. In Gram-
Downloaded from https://academic.oup.com/femsre/article/30/2/274/2367810 by guest on 30 November 2020

Italy. Tel.: 139040 3757317; fax: 139040 negative bacteria, the most common signal molecules are acylated homoserine
226555; e-mail: venturi@icgeb.org lactones. Several Pseudomonas species produce acylated homoserine lactones that
control important functions including pathogenicity and plant growth promotion.
Received 9 June 2005; revised 5 August 2005; Many reports indicate that the quorum sensing systems of Pseudomonas are
accepted 9 September 2005 significantly regulated and interconnected with regulons of other global regulators.
First published online 18 November 2005.
The integration of quorum sensing into additional regulatory circuits increases the
range of environmental and metabolic signals beyond that of cell density, as well as
doi:10.1111/j.1574-6976.2005.00012.x
further tuning the timing of the response. This review will focus on the regulation
Editor: Simon Cutting
of quorum sensing in Pseudomonas, highlighting a complex response that might
serve a given species to adapt in its particular environment.
Keywords
quorum sensing; acyl homoserine lactone;
global regulation; Pseudomonas.
Introduction system of AHL QS is fairly simple, being most commonly

mediated by two proteins belonging to the LuxI-LuxR
In bacteria, a major level of gene regulation involves families (Fuqua et al., 2001; Whitehead et al., 2001). LuxI-
cell cell communication via the production of small signal- type proteins are the major class of cytoplasmic enzymes
ling molecules. Many signal molecules that have been responsible for AHL synthesis, which then accumulates both
identified are involved in a form of regulation known as intra- and extra-cellularly in proportion to cell number. At
quorum sensing (QS) (Fuqua et al., 1994), which allows the critical cell density, when the quorum concentration of
bacteria to monitor their population density by responding AHL has been reached, AHL interacts directly with the
to the extracellular concentration of the signal molecule they LuxR-type protein; LuxR-AHL complexes can then bind at
produce (Bassler, 2002). The concentration of the signal specific promoter DNA sequences called lux-boxes of QS-
molecule increases concomitantly with bacterial cell density, regulated genes affecting their expression. Many Gram-
and, upon reaching a critical concentration, when a suffi- negative bacterial species have been reported to produce
cient number of cells are present, results in the alteration of AHLs differing only in the length of the acyl-chain moiety
the expression of target genes. This cell-density response has and substitution at position C3, which is either unmodified
evolved as a means to provide advantages to a group of cells, or carries an oxo- or hydroxyl group. Preferential binding of
such as improving access to environmental niches or enhan- an AHL by its cognate LuxR-type protein guarantees a good
cing their defence capabilities against other microorganisms degree of selectivity. In some cases, a single bacterium
or eukaryotic host-defence mechanisms. For instance, QS in possesses multiple AHL signal molecules and LuxI/R-type
bacteria regulates virulence factor gene expression, sporula- proteins. Among the most studied AHL QS systems are the
tion, biofilm formation, swarming, swimming, antibiotic Agrobacterium tumefaciens TraI/R system controlling Ti
biosynthesis and bioluminescence. plasmid conjugation, the Erwinia carotovora CarI/R system
In Gram-negative bacteria, the most common signal regulating exoenzymes and antibiotic production, and the
molecule used is an acylated homoserine lactone (AHL) Pseudomonas aeruginosa LasI/R and RhlI/R systems control-
molecule, which was first described in the marine biolumi- ling biofilm formation and virulence factors (Fuqua et al.,
nescent bacterium Vibrio fischeri, in which QS regulates light 2001; Whitehead et al., 2001; von Bodman et al., 2003; Smith
production (reviewed by Ruby, 1996). The basic molecular & Iglewski, 2003; Lazdunski et al., 2004).

c 2005 Federation of European Microbiological Societies FEMS Microbiol Rev 30 (2006) 274–291
Published by Blackwell Publishing Ltd. All rights reserved
Regulation of quorum sensing in Pseudomonas 275
The scope of this review is to outline the current knowl- remarkable catabolic potential, metabolic and physiological
edge of how the AHL QS systems are regulated in Pseudo- versatility, and, in addition to being found in the rhizosphere,
monas sp. Recently, many studies have shown that they are have been isolated from different environments including soil
integrated with other global regulatory responses present in and fresh water. Most attention from scientists is, however,
bacteria and can thus be influenced by environmental focused on the opportunistic human pathogen P. aeruginosa;
factors, by the metabolic status of the cell, and by other environmental isolates can infect humans, undergo rapid
signal molecules. This review will not cover the molecular adaptation, and cause nosocomial pneumonia, sepsis in burn
mechanisms of AHL QS, for example AHL structure and wounds, urinary-tract infections and chronic pulmonary
synthesis, LuxI-type proteins, LuxR-type proteins, Lux- inflammation in hosts rendered susceptible by cystic fibrosis
R–AHL interaction and AHL QS regulons; these aspects of (www.pseudomonas.com). Scientists investigating P. aerugi-
QS have been recently reviewed elsewhere (Fuqua et al., nosa can benefit from fully sequenced, well-annotated and

2001; Whitehead et al., 2001; Bassler, 2002; von Bodman constantly updated genome data, state of the art microarray
et al., 2003; Lazdunski et al., 2004). facilities, and accessibility to a complete set of mapped
genomic mutants (www.pseudomonas.com; www.genome.
washington.edu/UWGC/pseudomonas/index.cfm).
Quorum sensing systems of Pseudomonas A list of the AHL QS systems identified in Pseudomonas is
Pseudomonads are ubiquitous Gram-negative bacteria that summarized in Table 1. The most extensive studies on AHL-
can live in several environmental niches and have the ability dependent QS have been performed on P. aeruginosa,
to undergo transitions to become important and dangerous making this one of the most studied systems in bacteria.
human pathogens. Pseudomonads are studied for their roles Two AHL QS systems, the las and rhl systems, are present in
as plant pathogens (e.g. Pseudomonas syringae) and as human P. aeruginosa; in the las system, the lasI gene product directs
opportunistic pathogens, as in the case of P. aeruginosa the synthesis of N-(3-oxo-dodecanoyl)-L-homoserine lac-
infections. They are also studied for their ability to colon- tone (3-oxo-C12-AHL), which interacts with LasR and
ize plant-related niches, such as the rhizosphere (e.g. activates target promoters. In the rhl system, rhlI directs the
P. aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, synthesis of N-(butanoyl)-L-homoserine lactone (C4-AHL),
Pseudomonas aureofaciens and Pseudomonas chlororaphis), which interacts with the cognate regulator RhlR and acti-
where they can act as plant-beneficial bacteria by antagoniz- vates target gene promoters. The Las and Rhl systems are
ing plant-deleterious microorganisms and through the pro- intimately connected and have been found to regulate the
duction of traits that directly influence plant disease production of multiple virulence factors including elastase,
resistance and growth. These latter pseudomonads have a alkaline protease, exotoxin A, rhamnolipids, pyocyanin,
Table 1. AHL quorum-sensing systems of Pseudomonas

Quorum-
sensing
Pseudomonas sp. genes Signal molecule Quorum-sensing-regulated function(s) References
Pseudomonas lasI, lasR 3-oxo-C12-AHL C4-AHL Biofilm formation, elastase, Xcp secretion, lipase, Whiteley et al. (1999); Hentzer
aeruginosa rhlI, rhlR exotoxin A, lectins, alkaline protease, hydrogen et al. (2003); Schuster et al.
cyanide, swarming, twitching, pyocyanin, (2003); Smith & Iglewski, (2003);
rhamnolipids, virulence, other cellular functions Wagner et al. (2003); Juhas et al.
(2005)
Pseudomonas ahlI, ahlR 3-oxo-C6-AHL Cell aggregation, epiphytic fitness Dumenyo et al. (1998); Quinones
syringae et al. (2004)
Pseudomonas phzI, phzR C6-AHL currently unknown Phenazine antibiotics cell-surface components, Pierson et al. (1994); Wood et al.
aureofaciens csaI, csaR rhizosphere colonization (1997); Zhang & Pierson, (2001)
Pseudomonas phzI, phzR C6-AHL Phenazine-1-carboxamide biosynthesis Chin et al. (2001, 2005)
chlororaphis
Pseudomonas ppuI, ppuR 3-oxo-C12-AHL Biofilm development Bertani & Venturi (2004); Steidle
putida et al. (2002)
Pseudomonas mpuI, Long acyl-chain-AHL Mupirocin biosynthesis El-Sayed et al. (2001)
fluorescens mpuR
Pseudomonas hdtS 3-OH-C14:1-AHL Currently unknown Laue et al. (2000)
fluorescens
The listed quorum-sensing-regulated functions of the P. aeruginosa quorum-sensing systems are controlled by either the lasI/R or rhlI/R system or by
both.
FEMS Microbiol Rev 30 (2006) 274–291

c2005 Federation of European Microbiological Societies
276 V. Venturi
lectins, superoxide dismuatses and biofilm formation found to be present in P. aureofaciens 30-84 (Zhang &
(Smith & Iglewski, 2003). The effects of the two AHL QS Pierson, 2001). This system is not involved in phenazine
systems have been tested in various models of P. aeruginosa regulation, but was shown to be involved in rhizosphere
infection. Several mouse models were used, including burn colonization and in regulating the biosynthesis of cell-
infection, acute pneumonia and chronic lung infection surface components. Similarly in P. chlororaphis strain
models, all of which showed less virulence when infected PCL1391, which is also a plant-beneficial rhizobacterium,
with P. aeruginosa strains carrying mutation in the AHL QS AHL QS has been shown to regulate the production of the
genes. Similarly, attenuation of virulence was also observed antifungal compound phenzaine-1-carboxamide (PCN).
using alternative infection models of Caenorhabditis elegans, The expression of PCN biosynthesis genes is regulated in a
Arabidopsis thaliana and Dictyostelium discoideum (reviewed population density manner via the PhzI and PhzR system,
recently by Smith & Iglewski, 2003; Juhas et al., 2005). which also produces and responds to C6-AHL (Chin et al.,

Whiteley et al. (1999) reported the first systematic in- 2001). Pseudomonas chlororaphis PCL1391 produces other
vestigation on the QS regulon of P. aeruginosa, highlighting types of AHL molecules (N-butanoyl-L-homoserine lactone,
that it is a more global regulation system, as through a C4-AHL, and N-octanoyl-L-homoserine lactone, C8-AHL),
genetic screen it was observed that 47 genomic loci were but the genetic determinants for the production and
transcriptionally activated by AHLs. Since then, transcrip- response to these AHLs are currently not known (Chin
tional profiling experiments using microarrays have been et al., 2001, 2005). In Pseudomonas taxonomy, P. chloroaphis
performed in three other laboratories, enabling comparisons and P. aureofacies are considered synonyms and are thus
between QS AHL regulons to be made (Hentzer et al., 2003; regarded as the same species.
Schuster et al., 2003; Wagner et al., 2003). In each study, over Two AHL QS systems have been recently isolated from
300 genes were found to affected, of which 97 were identified beneficial rhizosphere P. putida strains WCS358 and IsoF
in all three studies. The differences can probably be attrib- (Steidle et al., 2002; Bertani & Venturi, 2004). These systems,
uted to differences in experimental set-up, including media designated PpuI/R, are orthologues and are responsible for
composition and growth phase at which the RNA was the synthesis and response to 3-oxo-C12-AHL. They share
prepared for transcriptome analysis. The conclusion from the highest identity (approximately 50%) with the LasI/R
P. aeruginosa is, however, that AHL QS is a global regulation system of P. aeruginosa. The PpuI/R system of strain IsoF has
system, controlling expression not only of virulence factors been shown to influence structural biofilm development,
but also of primary metabolic processes. The other Pseudo- whereas no PpuI/R-regulated phenotype has yet been found
monas pathogen for which an AHL QS system has been for strain WCS358. AHL QS has also been characterized in
reported is the plant pathogen P. syringae pv. syringae, which two P. fluorescens strains. In strain NCIMB 10586, a system
is the causal agent of brown spot in bean (Dumenyo et al., designated MupI/R has been isolated for its involvement in
1998; Quinones et al., 2004). Pseudomonas syringae strain the regulation of the polyketide antibiotic mupirocin, which
B728a synthesizes through ahlI, N-(3-oxo-hexanoyl)-L- is used clinically as a topical treatment for staphylococcal
homoserine lactone (3-oxo-C6-AHL), which then interacts infections (El-Sayed et al., 2001). In the rhizosphere-colo-
with AhlR, and this AHL system has been shown to be nizing biocontrol strain P. fluorescens strain F113 it has been
important for cell-aggregation and epiphytic fitness for in reported to produce three AHL molecules [a N-(3-hydroxy-
planta growth and disease (Quinones et al., 2004). 7-cis-tetradecenoyl) homoserine lactone, 3-OH-C14 : 1-AHL,
Several other AHL QS systems have been identified in N-decanoylhomoserine lactone, C10-AHL, and a C6-AHL]
beneficial pseudomonads; none, however, has been thor- through a novel AHL synthase termed HdtS. This synthase
oughly investigated. One of the first reports concerned the potentially represents a new family, as it does not belong to
AHL QS PhzI/R system of the wheat-plant growth-promot- either of the known AHL synthase families (LuxI and
ing rhizosphere-colonizing P. aureofaciens strain 30-84 LuxM); the AHL-interacting response regulator and a
(Pierson et al., 1994; Wood et al., 1997). The AHL synthase phenotype have not yet been linked to the three identified
PhzI directs the synthesis of N-hexanoyl-L-homoserine AHLs of strain F113 (Laue et al., 2000). Importantly, a broad
lactone (C6-AHL), which interacts with cognate regulator survey study has highlighted that there is heterogeneity in
PhzR, which at high cell densities regulates transcription the presence of AHL QS systems in soil pseudomonads
through binding to lux-box-like sequences in the promoter (Elasri et al., 2001). In this study, AHL production was
region of the phenazine operon. The production of phena- reported to be common among plant-associated but was not
zine antibiotics in the wheat rhizosphere by this strain is present in free-living soil isolates, thus implicating an
important for its biocontrol properties because it antago- important role of AHL QS in plant–bacteria interactions, as
nizes the fungus Gaeumannomyces graminins var. tritici, evidenced in some of the studies mentioned above. Many
which is the causal agent of take-all disease of wheat. A more studies are, however, required in order to establish
second AHL QS system, designated CsaI/R, was recently also the role played by these systems in soil fluorescent

pseudomonads and whether there is conservation in the influence QS. Studies with other Pseudomonas spp. are
systems and their mode of action. beginning to indicate that the complexity of QS regulation
is probably a common theme in Pseudomonas.
The Pseudomonas quorum-sensing systems are

regulated Targets for regulation of quorum sensing
Pseudomonads are very versatile bacteria that grow in a The regulation of QS can occur via transcriptional and/or
variety of habitats as well as on plant and animal tissue. In post-transcriptional regulation of the luxI and luxR homo-
fact, the genome of several Pseudomonas spp. has been logues, which will influence the protein levels of the AHL
sequenced, evidencing a large genome size (over six million synthase and AHL-sensor regulator. The stability and activity
base pairs) with over 5500 ORF being markedly larger than of the LuxI and LuxR proteins can also be regulatory targets

most other sequenced bacterial genomes (Stover et al., 2000; as well as AHL mimic compounds produced by other bacteria
Nelson et al., 2002). The genetic complexity is also high- or eukaryotes, which can interfere with the QS cascade. The
lighted by the presence of a large proportion of regulatory latter molecules could act as agonists or antagonists interfer-
genes, which make up almost 9% of the total number of ing with the functioning of the LuxR-type proteins. Another
ORFs, further highlighting metabolic plasticity and capabil- target is the AHL molecule itself through the action of AHL-
ity of adaptation to several environments. Several recent degrading enzymes that can affect the overall available con-
studies have shown that QS in Pseudomonas is integrated centration and hence the QS response.
with certain aspects of cell physiology and that it responds to
other environmental signals. This is also stressed by the
Regulation of quorum sensing in
overlap of the AHL QS regulon with other regulons of other
global regulators, necessitating a high degree of intercon-
Pseudomonas aeruginosa
nectivity among different signalling networks. The integra- The presence of two AHL QS systems, the LasI/R and RhlI/
tion of QS into additional regulatory circuits increases the R, in Pseudomonas aeruginosa led scientists to study whether
range of environmental and metabolic signals that affect QS they were connected via cross-regulation. Importantly, the
gene expression beyond cell density as well as further tuning Las and Rhl systems are not compatible, meaning that the
the timing of the QS response. Most studies on QS regula- RhlI-produced C4-AHL molecule cannot activate LasR, and
tion have been done on P. aeruginosa, and have indicated that LasI-produced 3-oxo-C12-AHL cannot activate RhlR
that a large number of factors (summarized in Fig. 1) (Pearson et al., 1995, 1997). Some of the QS-regulated genes
GacA MvaT
Vfr RpoN
RsaL RsmA
VqsR
PprB
lasR rsaL lasI
QscR LasR LasI

DksA
RpoS
Vfr PQS RpoN
PprB
GacA PprB
rhlR rhlI
RhlR RhlI
Fig. 1. Regulation of the Las and Rhl quorum-sensing systems in Pseudomonas aeruginosa. The Las system is at the top of the hierarchy regulating PQS
production and the Rhl system. Arrows on the promoter regions of the indicated genes mean positive regulation, whereas a short parallel line indicates
negative regulation. QscR is an antiactivator protein of LasR. See text for full details of the regulators and signals involved with this circuitry.

c 2005 Federation of European Microbiological Societies
278 V. Venturi
are under the control of both systems, whereas others are Regulation in response to growth phase
regulated specifically by either the Las or the Rhl system. The
Quorum sensing is defined as a mechanism by which
two systems are auto-regulated and intimately connected
bacteria regulate specific target genes in response to a critical
forming a regulatory cascade (Latifi et al., 1996; Pesci et al.,
concentration of signal molecules, which is a measurement
1997). The lasR gene is expressed throughout the growth
of the cell density of a bacterial population. For many AHL-
phase, increasing its expression upon entry to the stationary
QS-regulated genes, a quorum concentration of AHL signal
phase. LasR/3-oxo-C12-AHL then positively regulates lasI,
molecule is necessary but not sufficient for their expression;
creating a positive induction loop (Seed et al., 1995). LasR/
the growth phase of the bacteria could play an important
3-oxo-C12-AHL also regulates rhlR expression, and RhlR/
role and prevents early expression even at quorum concen-
C4-AHL then regulates rhlI transcription resulting in signal
trations of AHLs. Several regulatory controls are present that
amplification (Seed et al., 1995; Latifi et al., 1996). LasR and
ensure the timely expression of qsc genes in relation to AHL

RhlR transcription regulators bind to specific lux-box-like
concentration and metabolic status of the cell.
sequences, which in P. aeruginosa have been called las-boxes,
being regulated by either RhlR, LasR or both (Whiteley et al.,
1999; Schuster et al., 2004). LasR forms a multimer and
Role of the stationary-phase sigma factor RpoS
binds las-boxes only in the presence of 3-oxo-C12-AHL
and alternative sigma factor RpoN
(Kiratisin et al., 2002), whereas RhlR dimerizes and binds
DNA both in the presence and absence of C4-AHL (Medina RpoS (also known as ss or s38) is a central regulator in
et al., 2003; Ventre et al., 2003). response to several stresses that lead to reduction or cessation
The observation in 1999 by Whiteley et al. (Whiteley et al., of growth thus bringing the cells into stationary phase
1999) that many identified QS-controlled genes (qsc) had to (Hengge-Aronis, 2002; Ishihama, 2000). In P. aeruginosa
be classified based on the temporal pattern of their response RpoS was reported to be involved in the regulation of
in cells grown in the presence of Las AHL signal and/or Rhl extracellular alginate, pyocyanin, exotoxin A and to provide
AHL signal was an important indication that additional stress resistance following exposure to heat, low pH, high
regulation was superimposed on the QS control. Some qsc osmolarity, hydrogen peroxide and ethanol. It is, however,
genes respond immediately to exogenously added signals, not as important in providing resistance to these conditions
whereas others can only respond to the AHL signal when the as the Escherichia coli counterpart (Jorgensen et al., 1999; Suh
bacteria are in the stationary phase of growth; that is, the et al., 2002; Venturi, 2003; Schuster et al., 2004). Since RpoS
AHL-dependent expression cannot be advanced to the loga- levels increase considerably at high cell densities (Kojic &
rithmic growth phase by exogenous addition of AHLs. A Venturi, 2001) as bacteria enter stationary phase, scientists
recent study has shown that in P. aeruginosa the growth were interested in determining if there was a regulatory
media can have a profound effect on the early activation of connection with AHL QS. Initial reports were contradictory
qsc genes by exogenous addition of AHLs (Yarwood et al., and two possible interacting scenarios were reported: that
2005). Apparently, complex medium contains inhibitor(s) AHL QS through RhlIR activates rpoS transcription (Latifi
that affect the early activation of some qsc genes; this effect et al., 1996); and that the RpoS negatively regulates AHL QS
can be relieved if the complex medium is conditioned by through repression of rhlI expression (Whiteley et al., 2000).
growth of a non-AHL-producing P. aeruginosa mutant. Since These two observations report a major link between the two
these important initial observations, several global regulatory globally regulatory systems, and the differences can probably
factors have been shown to influence and regulate AHL QS in be explained through different experimental set-ups. Re-
P. aeruginosa, including the cAMP receptor regulatory pro- cently, however, the role of RpoS in AHL QS regulation was
tein Vfr, the stationary-phase sigma factor RpoS, the alter- re-addressed using transcriptional profiling (Schuster et al.,
native sigma factor RpoN, the stringent response protein 2004). This study found that 800 genes are regulated by RpoS
RelA, two LuxR homologues (QscR and VqsR), the post- in response to stationary phase, including a high percentage
transcriptional regulators RsmA and DksA, the transcrip- (30–40%) of the genes previously determined (also through
tional regulatory proteins RsaL and MvaT, and the global transcriptional profiling) to be regulated by AHL QS, mean-
two-component regulatory system GacA-GacS. In addition, ing that there is considerable regulon overlap between QS and
the quinolone signalling molecule 2-heptyl-3-hydroxy-4-qui- RpoS. Through these transcriptional profiling studies it is
nolone (PQS) adds a further level of control in the QS believed that there is some cross-regulation between the two
network because it provides a link between the Las and Rhl systems, with all data consistent with AHL-QS-regulating
systems. All these elements (depicted in Fig. 1) provide rpoS transcription, albeit by only two-fold (Wagner et al.,
additional controls to the QS circuitry, allowing it to be 2003; Schuster et al., 2004). It is most probable that most
additionally regulated by growth phase, alarmones, environ- genes belonging to the two regulons are independently
mental signals and as yet unidentified stimuli. regulated by both RpoS and AHL QS, while some are

indirectly regulated through either AHL or RpoS. Consider- sequence data banks, making this a unique protein. The
able additional experimentation is required in order to rsaL gene has been found intergenically also between the
formulate more precisely the mechanisms of how the regulon ppuI/R AHL QS systems of P. putida IsoF and WCS358
overlap is controlled by the two systems and whether at the strains (see below; Steidle et al., 2002; Bertani & Venturi,
molecular level there is indeed cross-regulation between RpoS 2004). The ppuI-rsaL-ppuR loci of P. putida show the highest
and AHL QS genes. It is probable that the two systems act degree of homology to the lasI-rsaL-lasR loci of P. aeruginosa
independently, because AHL QS is a cell-density-related (approximately 50% identity), and thus far it is the only
response whereas RpoS is a stationary-phase response, which location where rsaL (or rsaL-like) appears to be found in
in nature in most cases does not involve high cell densities but bacteria. RsaL of P. aeruginosa is believed to act directly on
other stressful growth conditions such as limitation of an lasI to downregulate its transcription, thus acting as a
essential nutrient. repressor of the las AHL system (de Kievit et al., 1999). In

The alternative sigma factor RpoN (also known as s54) fact, the over-expression of rsaL by P. aeruginosa results in a
regulates several metabolic functions and is important in the 20-fold reduction of 3-oxo-C12-AHL production; this RsaL-
regulation of virulence in P. aeruginosa (Hendrickson et al., repressive effect was considerably relieved when P. aerugino-
2001), which is believed to be mainly a result of RpoN sa was grown with exogenously added 3-oxo-C12-AHL. The
regulating pili and flagella, which are important motile and current model for the RsaL mode of action is that it is in
adherence factors (O’Toole & Kolter, 1998). In a nutrient- binding competition to the lasI promoter with LasR/3-oxo-
rich medium, RpoN knock-out mutants produce two-fold C12-AHL; upon increase of AHL concentration, LasR-AHL
more 3-oxo-C12-AHL and five-fold more C4-AHL in early is able to out-compete RsaL, and consequently lasI tran-
and late logarithmic phases of growth, and similarly lasRI scription increases. The putative role of RsaL is therefore to
and rhlRI transcriptional fusion constructs display consider- repress lasI transcription at low AHL concentration, and
ably higher induction levels (Heurlier et al., 2003). The hence at early phases of growth; at present, however,
mode of action of RpoN on the regulation of QS has not verification of this model needs considerable further experi-
been deciphered; however, it has been postulated that RpoN mentation.
could regulate other regulatory components of the AHL QS Another regulatory protein called MvaT has been impli-
cascade, thus indirectly affecting regulating QS (see below; cated to play a role in transcriptional repression of QS at low
Heurlier et al., 2003). A putative RpoN promoter recogni- cell densities. The identification of MvaT was a result of a
tion sequence has, however, been found in the rhlI promoter systematic study designed to identify genetic loci that when
sequence at 144 bp upstream of the translational start codon inactivated result in premature expression of AHL-QS-
and overlapping a previously identified las-box and house- regulated loci (Diggle et al., 2002). The AHL-QS-regulated
keeping s70-binding site (Heurlier et al., 2003; Thompson lecA gene was used for this experiment; it encodes for a
et al., 2003). This putative RpoN binding site, however, does lectin that is an important virulence factor as it can bind the
not play a role in the negative regulation by RpoN (Heurlier most common human antigens and is cytotoxic to respira-
et al., 2003). The rhlI gene, on the other hand, is under tory epithelial cells in primary cultures (Bajolet-Laudinat
positive regulation by RpoN when P. aeruginosa was grown et al., 1994). Expression of the lecA gene, similarly to the case
in minimal medium, rhlI promoter activity and C4-AHL for many other AHL-QS-regulated genes, cannot be ad-
accumulation being considerably reduced in the rpoN vanced to early phases of growth by exogenous addition of
knock-out mutant (Thompson et al., 2003). This nutrient- quorum concentrations of AHLs, and thus they are also
dependent RpoN regulation of rhlI transcription needs growth-phase-regulated (Winzer et al., 2000; Diggle et al.,
further study in order that the molecular mechanisms and 2002; Yarwood et al., 2005). The mvaT mutant was isolated
biological significance can be understood. as it resulted in a greater-than-twofold increase in expres-
sion of a transcriptional lecA-lux fusion; in addition, in
contrast to the parental P. aeruginosa strain, addition of
Negative transcriptional regulation by RsaL and
exogenous AHLs significantly advanced lecA expression
MvaT
(Diggle et al., 2002). Other AHL-QS-regulated phenotypes
In P. aeruginosa, lasR and lasI are separated by a 367-bp were altered in a similar fashion in the mvaT mutant,
intergenic region and are transcribed from independent including pyocyanin and elastase production. The P. aerugi-
promoters in the same direction (Seed et al., 1995; Pesci nosa mvaT knock-out mutant produces higher quantities of
et al., 1997). In the intergenic region, a gene designated rsaL both AHLs: MvaT is therefore acting as a repressor of AHL
is located on the strand opposite lasR and codes for a QS. At present the MvaT target gene(s) in the AHL QS
protein, RsaL, of 10.6 kDa (de Kievit et al., 1999). The system is not known. MvaT has a high identity to the P16
primary structure of RsaL displays no significant similarity subunit of the Pseudomonas mevalonii heteromeric tran-
to any functionally characterized protein contained in scriptional regulator MvaT, which regulates mevalonate

280 V. Venturi
catabolism in Pseudomonas mevalonii (Rosenthal & Rod- induces dissociation of QscR oligomers (Ledgham et al.,
well, 1998). Recently, it has been proposed that MvaT could 2003). The current model is therefore that QscR inhibits LasR
be part of a novel class of H-NS-like proteins (Tendeng et al., and RhlR at low concentrations of AHL through formation of
2003). HN-S-like proteins are widespread in Gram-negative inactive heterodimers with LasR and/or RhlR, thus inhibiting
bacteria and control the expression of many genes involved the expression of QS-regulated genes. The increase of AHL
in metabolism and environmental adaptation (Hommais concentration at late logarithmic/early stationary phases of
et al., 2001). In Pseudomonas sp., surprisingly, no H-NS- growth results in a shift of the equilibrium to LasR and/or
related protein has been reported, and interestingly MvaT RhlR homodimers bound to 3-oxo-C12-AHL and C4-AHL,
shares similar structural and functional organization with respectively, and hence towards regulated expression of target
members of the H-NS family despite rather low amino-acid genes. This type of protein–protein interaction control by
conservation. H-NS-related proteins have the capacity to QscR of LasR and/or RhlR closely resembles what occurs in A.

bind DNA via their C-terminal part and the propensity to tumefaciens, where two different proteins modulate by
form oligomers by their N-terminal domain (Tendeng et al., protein–protein interaction TraR-AHL-dependent transcrip-
2003). Recently, other genes have been identified as being tional activation of QS-regulated genes. A. tumefaciens
regulated by MvaT in P. aeruginosa, making it a more global regulates Ti plasmid conjugal transfer by the TraI/R–3-oxo-
regulator. MvaT regulates a gene cluster called chaperone- C8-AHL QS system (reviewed by von Bodman et al., 2003).
usher pathway (cupA), which is involved in the assembly of The TraM and TrlR proteins regulate TraR activity through
fimbrial structures and is important for biofilm formation formation of inactive complexes with TraR (Chai et al., 2001;
(Vallet et al., 2004). MvaT is also involved in the positive Fuqua et al., 1995; Qin et al., 2004). Interestingly, TrlR, unlike
regulation of ptxS through specific binding at its gene TraM, has high identity with TraR, as it resembles a truncated
promoter; PtxS in turn is a regulator of exotoxin A produc- form of TraR having 88% identity with the first 181 amino
tion in P. aeruginosa (Westfall et al., 2004). acids of TraR (Chai et al., 2001).
This control of AHL QS LuxR-type proteins via protein–
protein interaction relies on the relative protein concentra-
Negative regulation by the LuxR family member
tions, because slight overexpression can have a drastic effect
QscR
on the regulation of the AHL QS system. From this
The genome of P. aeruginosa revealed the presence of a gene perspective, the finding that qscR expression in P. aeruginosa
encoding for a LuxR family member homologue to LasR and is regulated by the global two-component signal transduc-
RhlR, but no cognate or other additional LuxI family homo- tion system GacA-GacS is significant as it could add other
logues are present (Chugani et al., 2001). Since this LuxR environmental parameters to the AHL QS control in P.
family member has the conserved features of AHL-sensor/ aeruginosa (see below; Ledgham et al., 2003).
responsive proteins, scientists were led to study the possible
role that it might play within the AHL QS circuitry of P.
Post-transcriptional regulation by RsmA and by
aeruginosa. This luxR family member, designated qscR, was
DksA
inactivated in the P. aeruginosa genome and the mutant
displayed de-regulated expression of several AHL-QS-regu- A regulatory parameter that is also recognized to be of
lated genes/phenotypes including pyocyanin, hydrogen cya- critical importance in the activation of growth-phase-de-
nide and elastase production; it overproduced these pendent secondary metabolite production in several bacter-
compounds and started to synthesize them at lower culture ia occurs at the post-transcriptional level through the small
density than the wild type (Chugani et al., 2001; Ledgham 7-kDa RNA-binding global regulator protein repressor of
et al., 2003). In fact, P. aeruginosa lacking QscR prematurely secondary metabolism (RsmA); (reviewed by Haas et al.,
transcribes the lasI and rhlI AHL signal generator genes (and 2002). RsmA-like proteins are highly conserved in eubacter-
hence produces both AHLs ahead of time). QscR is therefore ia (e.g. CsrA in Escherichia coli, RsmA in Erwinia carotovora,
involved in the repression of lasI, which retards the produc- RsmA in P. fluorescens) and regulate post-transcriptionally,
tion of 3-oxo-C12-AHL, and since this AHL and LasR are acting as translational repressors most probably by control-
activators of rhlI-rhlR, repression of lasI also retards the ling access to the ribosome binding site and by altering
RhlIR/C4-AHL QS system. The mechanism by which QscR mRNA stability. The translational repression by RsmA is
represses lasI transcription at early phases of growth has modulated by the action of small regulatory RNAs whose
recently been elucidated (Ledgham et al., 2003). By using expression in Pseudomonas is controlled by the global two-
fluorescent anisotropy and in vivo cross-linking it was shown component system GacA-GacS (Heeb et al., 2002; Valverde
that, in the absence of AHLs, heterodimers can be formed et al., 2003). Studies have been performed on P. aeruginosa
between QscR and LasR or QscR and RhlR; the increase of in order to determine if RsmA also regulates QS-regulated
AHL concentration results in QscR–AHL interaction and secondary metabolites and if it plays a role in the regulation

of the AHL QS circuitry (Pessi et al., 2001). In P. aeruginosa, Positive regulation of lasR and rhlR by the global
the rsmA knock-out mutant produces considerably more c-AMP-dependent transcriptional regulator Vfr
pyocyanin and hydrogen cyanide during the logarithmic
phase of growth. Production of both of these compounds is The transcriptional regulator Vfr of P. aeruginosa is 67%
also under AHL QS control (Pessi et al., 2001). In fact, the identical and 91% similar to the cAMP receptor protein
rsmA mutant overproduces, with respect to the parent (CRP) of E. coli. It was identified in P. aeruginosa by West et
strain, both 3-oxo-C12-AHL and C4-AHL in the logarithmic al., (West et al., 1994) as a virulence factor regulator as a
phase, and conversely overexpression of rsmA results in result of its effect on the production of several virulence
significantly lower production of both AHLs. These regula- factors such as protease and exotoxin A. Vfr is mechan-
tory effects by RsmA occur at the level of translation, istically closely related to CRP with respect to cAMP
because a lasI–lacZ translational fusion is induced prema- binding, recognition of binding sites and interaction with

turely by 10-fold in the rsmA mutant when compared with RNA-polymerase (West et al., 1994); however, its role in P.
the wild-type (Pessi et al., 2001). Similarly, in Erwinia aeruginosa physiology differs considerably from that of its
carotovora ssp. carotovora, the AHL QS system, RsmA and CRP counterpart (Suh et al., 2002; Wolfgang et al., 2003).
cognate regulatory RNAs are intimately connected in reg- The regulation by Vfr of several virulence factors occurs in
ulating extracellular protein production and pathogencity part through the AHL QS system, because Vfr regulates the
(Cui et al., 1995; Chatterjee et al., 2002). The precise expression of lasR, which then triggers the expression of
environmental signals to which the RsmA responds to many virulence factors and of the RhlI/R system (Albus
remain unclear; it is likely, however, that the integration of et al., 1997). The lasR promoter region undergoes auto-
this system with AHL QS allows the bacteria to better adapt regulation in a cell-density AHL-dependent manner and
and respond to several environments at high cell densities. contains two transcriptional start sites (T1 and T2) located
Bacteria starved from amino acids initiate the stringent at 201 and 231 from the translational start codon.
response that is triggered by elevated concentrations of Upstream of T1 there is a potential las-box, positioned at
guanosine tetraphosphate (ppGpp), which complexes with 37 bp from the transcriptional start, which could be
RNA polymerase and regulates transcription levels positively involved in the positive auto-regulation resulting in signal
or negatively. Recently it has been established that the DksA amplification, which is very common in AHL QS systems. In
protein increases ppGpp activity through binding to RNA addition, the lasR promoter contains a functional CRP-
polymerase (Paul et al., 2004; Perederina et al., 2004; Perron binding consensus sequence (CCS) centred at nucleotides
et al., 2005). It has been observed that overexpression of 77.5 and 47.5 from the T1 and T2 start sites respec-
dksA in P. aeruginosa results in the inhibition of rhlI tively; Vfr was shown to bind to this region, and a Vfr
transcription, suggesting that overproduction of DksA mutant results is a drastic reduction of lasR transcription
downregulates the signal generator gene rhlI and hence QS- (Albus et al., 1997). The expression of lasR in P. aeruginosa is
dependent virulence factor production (Branny et al., 2001). probably the first step in the QS cascade, and thus Vfr is
In agreement with these observations, it was also later probably playing a crucial role in initiating it. At present it is
described that a P. aeruginosa dksA knock-out mutant not known what precise signal is required for Vfr to act on
displayed increased rhlI expression during the logarithmic the lasR promoter, although high cell density is most
phase of growth (Jude et al., 2003). DksA also has an effect probably a very important determinant.
on the translation efficiency of two QS-regulated genes, Recently, the rhlR promoter has also been studied in some
namely lasB, encoding Las elastase, and rhlAB, responsible detail, demonstrating that the regulation of rhlR is complex
for rhamnolipid biosynthesis (Jude et al., 2003). While and involves the participation of several regulators whose
transcription of both loci was unaffected in the dksA function is influenced by medium composition (Medina
mutant, the translation was significantly reduced. The et al., 2003). Four separate transcriptional start sites are
molecular mechanism of this effect is currently unknown. present in the rhlR promoter (P1-P4): transcription from P1
Future work needs to be performed in order to decipher and P4 involves positive regulation by LasR/3-oxo-C12-
how QS is regulated in P. aeruginosa by DksA during the AHL, transcription from promoter P3 is RpoN-dependent,
stringent response. The levels of the stringent response and transcription from P2 is negatively influenced by the
alarmone ppGpp have an effect on the production of the presence of RhlR. The P4 transcription start site is also
two AHLs of P. aeruginosa (van Delden et al., 2001). Over- positively regulated by Vfr, because, in the vfr mutant,
expression of relA (which is responsible for ppGpp synth- primer extension experiments demonstrated that transcrip-
esis) in P. aeruginosa leads to premature lasR and rhlR gene tion from this promoter is considerably reduced (Medina
expression and hence to high AHL production in earlier et al., 2003). Unlike the case for the lasR promoter, at present
phases of growth, indicating that the stringent response it is not known whether Vfr directly activates the rhlR
plays a role in activating AHL QS (van Delden et al., 2001). promoter through a Vfr binding site or occurs indirectly

282 V. Venturi
via another regulator(s). This complexity of the rhlR gene unknown, and at present it is also not known if the response
promoter highlights the fact that AHL QS can be modulated regulator GacA directly regulates the lasR and rhlR promo-
by several global regulators in response to different environ- ters. A gacA mutant in another P. aeruginosa strain did not
mental responses and growth conditions. result in a significant difference in QS regulation (Parkins
et al., 2001).
Recently, a response regulatory protein, designated PprB,
Positive regulation by the LuxR family member
part of a two-component system of P. aeruginosa, was
VqsR
identified as being involved in the positive regulation of
A fourth LuxR family regulator, which like QscR is an extracellular protease, pyocyanin, elastase, and hameolytic
orphan of a cognate LuxI family member, has recently been activity as well as being necessary for swimming and
reported to be involved in the positive regulation of QS in P. swarming motility (Dong et al., 2005). Microarray tran-

aeruginosa (Juhas et al., 2004, 2005). This new LuxR family scriptional profiling using the response regulator pprB
member has been designated virulence and quorum sensing knock-out mutant (Dong et al., 2005) revealed that 91/113
regulator (VqsR), because the vqsR knock-out mutant does (i.e. 85%) of the PprB-activated genes were also reported to
not produce any of the AHLs, decreases the production of be QS-activated (Schuster et al., 2003; Wagner et al., 2003).
extracellular virulence factors, and reduces pathogenicity in In fact, PprB positively regulates the transcription of QS
a nematode infection model system (Juhas et al., 2004). The genes lasI, rhlI, rhlR and rsaL (PprB does not affect lasR
absence of vqsR decreases lasI mRNA levels by ten-fold and transcription) to the extent that, in the pprB mutant, 3-oxo-
does not affect lasR mRNA levels. Thus the positive control C12-AHL and C4-AHL are hardly detectable (Dong et al.,
of QS exerted by VqsR occurs directly or indirectly via lasI 2005). PprB has been reported to be involved in the
(Juhas et al., 2004, 2005). The vqsR gene contains a putative regulation of membrane permeability and sensitivity to
las-box in its promoter region, which could place the gene various antibiotics (Wang et al., 2003). Interestingly, the
under QS control, thus creating a positive feedback loop pprB mutant displays substantial reduction in 3-oxo-C12-
further indicating an intimate association between AHL QS AHL influx, whereas it was not affected with respect to C4-
and VqsR. A transcriptome analysis revealed that a high AHL uptake; PprB, on the other hand, does not play a role in
proportion of the genes regulated by QS in P. aeruginosa are the efflux of either AHL. It is therefore likely that PprB is
also regulated by VqsR (Juhas et al., 2004). involved in the regulation of transporter genes important
for the active influx of 3-oxo-C12-AHL (Dong et al., 2005).
At present, the cognate environmental signal for PprB is
Environmental regulation via two-component
unknown, as is the cognate sensor protein of this two-
systems
component system involved in the regulation of QS.
In the P. aeruginosa genome there is an astonishing number
of putative two-component regulatory proteins, including
PQS: a signal molecule providing a regulatory link
55 sensors and 89 response regulators (Stover et al., 2000),
between the Las and Rhl systems
and only a very small proportion have been studied. Such a
high number of these systems allows P. aeruginosa to Pseudomonas aeruginosa synthesizes a quinolone signalling
respond to a wide variety of changes in their environment. molecule, namely 2-heptyl-3-hydroxy-4-quinolone (termed
The GacA/GacS of P. aeruginosa is a two-component global PQS for Pseudomonas quinolone signal), which is integrated
regulatory system regulating several secondary metabolites in the AHL QS response governed by the Las and Rhl
and is an essential virulence factor for pathogenesis in plant, systems (Pesci et al., 1999). PQS belongs to the family of 4-
nematode and insect models of infection (Heeb & Haas, hydroxy-2-alkylquinolines (HAQs), which are known for
2001). GacA/GacS also positively regulates lasR and rhlR their antimicrobial activity (Deziel et al., 2004), and requires
gene expression, and gacA knock-out mutants produce a P. aeruginosa-produced biosurfactant to increase its solu-
considerably less C4-AHL. Furthermore, P. aeruginosa with bility and hence its bioactivity as a signal molecule (Calfee
multiple copies of gacA displays higher lasR and rhlR et al., 2005). Biosynthesis of PQS starts from anthranilate,
promoter activities and accumulates more C4-AHL, affect- which is synthesized from chorismate by anthranilate
ing several RhlI/R-regulated phenotypes (Reimmann et al., synthase encoded by the phnAB operon (Cao et al., 2001;
1997). The accumulation of 3-oxo-C12-AHL was not af- Gallagher et al., 2002; Deziel et al., 2004). Anthralinate is
fected in the gacA mutant, regardless of the fact that GacA/ then converted by the pqsABCD gene products of the
GacS positively regulates lasR expression. This is probably pqsABCDE operon to HHQ (4-hydroxy-2-heptyl-quino-
because lasR/LasR undergoes post-transcriptional and/or line), the immediate precursor of PQS (Deziel et al., 2004).
post-translational control. The environmental signal to HHQ is believed to be itself an intercellular signalling
which the histidine sensor kinase GacS responds to is still molecule because it can be released by a cell in a bacterial

population and taken up by another and converted into PQS through the Las and Rhl systems, because MvfR does not
through the gene product of pqsH (Deziel et al., 2004). The mediate the regulatory activity of lasRI nor that of rhlRI
synthesis and response to PQS require the MexGHI-OpmD (Deziel et al., 2005).
multidrug efflux pump, because its inactivation leads to a
reduction in PQS production, believed to be a result of the
PQS regulates the RhlI/R system
intracellular accumulation of anthralinate, which is toxic for
the cell (Aendekerk et al., 2005). PQS signalling plays an The first indication that PQS was also intertwined with the
important role in P. aeruginosa pathogenesis because: (i) it Rhl system was that the exogenous bioactivity of PQS
regulates many virulence factors (Pesci et al., 1999; Calfee (measured through the ability of PQS to activate the elastase
et al., 2001; Diggle et al., 2003; Deziel et al., 2004; Deziel lasB–lacZ fusion) needed a functional RhlR gene, suggesting
et al., 2005); (ii) the PQS molecule has been detected in the that PQS is acting through the Rhl QS system (Pesci et al.,

airways of cystic fibrosis patients (Collier et al., 2002; Guina 1999). It was then established that PQS positively regulated
et al., 2003) and (iii) pqsH and PQS transcriptional regulator rhlI expression, thus influencing C4-AHL accumulation,
mvfR knock-out mutants are less virulent in the nematode and, furthermore, that PQS and C4-AHL cooperatively
and mouse model for infection (Gallagher & Manoil, 2001; induce target gene expression (McKnight et al., 2000).
Deziel et al., 2005). PQS is closely linked to the AHL QS Recently it was observed that the activation of the
hierarchy, because the Las system regulates PQS production, pqsABCDE operon, and hence the production of PQS,
which in turn regulates the Rhl system, forming a bridge depended on the ratio of the two AHL molecules, 3-oxo-
between the two QS systems of P. aeruginosa. The molecular C12-AHL (i.e. the Las system) having a positive effect, and
mechanism by which PQS controls gene expression is C4-AHL (i.e. the Rhl system) having a negative effect
currently unknown. (McGrath et al., 2004). RhlR has a negative effect on pqsR
transcription; however, at present it is not known whether
this control is direct (Wade et al., 2005). Consequently it
Regulation of PQS biosynthesis: involvement of the
appears that the Las and Rhl systems act positively and
LasI/R QS system
negatively respectively on pqsR regulation, making this an
The production of PQS requires the LasR/3-oxo-C12-AHL as intermediate regulator in the signalling chain. PQS itself acts
originally established during the discovery of the PQS as a co-inducer for PqsR, resulting in a positive induction
intercellular signalling molecule of P. aeruginosa (Pesci loop (Wade et al., 2005).
et al., 1999). These observations mean that the LasI/R system
is the dominant P. aeruginosa QS system, because it is
Possible role of AHL-degrading enzymes
responsible for initiating the PQS and the RhlI/R QS
systems. The regulation of PQS biosynthesis by LasR/3- Acylated homoserine lactone molecules are known to be very
oxo-C12-AHL occurs through transcriptional regulation of stable at pH values of 5–6; however, they can be chemically
pqsH, which is responsible for converting HHQ to PQS inactivated via alkaline hydrolysis (Schaefer et al., 2000). Two
(Gallagher et al., 2002). Apparently, however, under certain different bacterial enzymes have been reported to have AHL-
growth conditions there can be considerable LasR-indepen- degrading activity (reviewed by Roche et al., 2004): an AHL-
dent biosynthesis of PQS (Diggle et al., 2003). The regula- lactonase initially found in Bacillus sp. (Dong et al., 2000),
tion of the other PQS biosynthetic genes, i.e. the phnAB and and an AHL-acylase first reported in Variovorax paradoxus
pqsABCDE operons, occurs through the LysR family tran- that can also use AHL as a unique source of carbon and
scriptional regulator called MvfR or PqsR (Gallagher et al., energy (Leadbetter & Greenberg, 2000). Neither of these
2002; Deziel et al., 2004; McGrath et al., 2004), which in turn bacteria produce AHL molecules, and hence do not have
is positively directly regulated by LasR/3-oxo-C12-AHL AHL QS systems; however, similar enzymes have recently
(Wade et al., 2005). PqsR positively regulates the transcrip- been found in AHL-producing bacteria such as Ralstonia,
tion of the phnAB and pqsABCDE operons directly, because Agrobacterium and Pseudomonas sp. (reviewed by Roche
purified PqsR protein can bind to the phnAB and pqsA et al., 2004). Pseudomonas aeruginosa strains were reported
promoters (Cao et al., 2001; Wade et al., 2005). Transcrip- to degrade long-chain AHLs initially via an AHL-acylase
tion profiling and transcriptional fusion studies determined activity, and eventually to utilize the AHL as a carbon source,
that the pqsABCDE operon is regulated by MvfR (Deziel albeit very inefficiently (Huang et al., 2003). This signal decay
et al., 2004; McGrath et al., 2004). MvfR (PqsR) is therefore may have a function in the regulation of the LasI/R-3-oxo-
the master regulator of PQS biosynthesis, and a recent mvfR C12-AHL QS system; however, future work is required in
transcriptome study revealed that as many as 55% of MvfR- order to determine whether the AHL-degrading enzymes are
dependent genes are also regulated by AHL QS in P. playing this role or whether these enzymes have an alternative
aeruginosa (Deziel et al., 2005). This overlap does not go natural function (Huang et al., 2003; Roche et al., 2004). AHL

284 V. Venturi
degradation can potentially modulate AHL QS (Zhang & ppuI/R genes of P. putida have, just like lasI/R in P.
Dong, 2004), because overexpression of a Bacillus sp. lacto- aeruginosa, in between them a repressor gene called rsaL,
nase in P. aeruginosa significantly reduced the accumulation which negatively controls ppuI and lasI gene expression (see
of AHL signals affecting the AHL QS response (Reimmann above; de Kievit et al., 1999; Steidle et al., 2002; Bertani &
et al., 2002). Interestingly, A. tumefaciens contains an endo- Venturi, 2004). In P. putida, ppuI expression is under strong
genous AHL lactonase, designated AttM, of which the negative transcriptional regulation by RsaL; in a rsaL knock-
corresponding gene is regulated in response to the stringent out mutant, ppuI expression (and hence AHL production)
response through a repressor protein called AttJ. Agrobacter- increases dramatically and the QS response is anticipated to
ium tumefaciens can consequently use this signal-decay earlier phases of growth (Bertani & Venturi, 2004). Genetic
cascade as a way to interrupt AHL-QS-dependent Ti plasmid studies have indicated that in P. putida the RsaL protein
conjugal transfer (Zhang et al., 2004). plays a major role in keeping the AHL system at very low

expression levels, probably through competition for ppuI
promoter binding with PpuR/3-oxo-C12-AHL. This model
AHL interference by eukaryotic cells
needs to be substantiated by the molecular analysis of events
One question that scientists are now addressing is that given taking place at the ppuI promoter. RsaL appears to play a
the importance of AHL QS in bacterial virulence, have similar role in lasI gene expression in P. aeruginosa (see
eukaryotic hosts developed defence mechanisms that target above; de Kievit et al., 1999); however, in P. putida there
this communication system? It has recently been reported appears to be tighter control imposed by RsaL, resulting in
that human airway epithelia contain a membrane-associated lower expression levels of AHL QS. The biological role of
activity that inactivates/transforms 3-oxo-C12-AHL of P. this strong repression played by RsaL is currently unknown.
aeruginosa, a process that could play a role in pathogen–host It is tempting to speculate that it could be responding, via
interaction, keeping in mind that P. aeruginosa causes de-repression of ppuI, to an environmental and/or meta-
chronic lung infections in patients with cystic fibrosis (Chun bolic signal. Similarly to what occurs in P. aeruginosa, ppuI
et al., 2004). Natural compounds have also been isolated that expression in P. putida is positively regulated by the GacA/
can block QS in P. aeruginosa (Hentzer et al., 2003). These GacS two-component system and undergoes cross-regula-
are halogenated furanones produced by the macroalgae tion with the stationary-phase RpoS sigma factor (Bertani &
Delisea pulchra, which are similar in structure to AHLs and Venturi, 2004).
have somehow the ability to interfere with AHL QS: the
approach of using a compound that disrupts QS could be a
promising strategy for the treatment of bacterial infections Pseudomonas aureofaciens/Pseudomonas
(Hentzer & Givskov, 2003; Rice et al., 2005). It has been chlororaphis
demonstrated through transcriptome microarray studies The biocontrol strain P. aureofaciens 30-84 employs AHL QS
that these halogenated furanones significantly interfere with via the PhzI/R-C6-AHL system to activate directly the
the expression of AHL-QS-regulated genes (Hentzer et al., expression of the phenazine biosynthetic operon phzXY-
2003). AHL-interfering compounds are also present in a FABCD (Pierson et al., 1994; Wood et al., 1997; Pierson
number of higher plants, most of which have stimulatory et al., 1995). Phenazine biosynthesis also requires a func-
effects on LuxR-type proteins (Teplitski et al., 2000, 2004; tional GacA/GacS two-component signal transduction sys-
Gao et al., 2003; Bauer & Mathesius, 2004). The chemical tem, because mutants in the system are deficient in
identities of these mimic compounds are still unknown, as is phenazine production (Chancey et al., 1999). In addition,
their potential to disrupt/interfere with QS regulation in GacA/GacS is also required for phzI transcription and hence
bacteria. Our understanding of how eukaryotes interfere AHL production in P. aureofaciens 30-84. However GacA/
and/or respond to bacterial QS signals is at a very early stage, GacS does not regulate phenazine gene expression through
and this will be an important research topic to address in the the QS AHL system. This indicates that GacA/GacS regulates
near future. phenazine production at different levels: through AHL QS
and through an as yet unidentified regulatory system(s)
(Chancey et al., 1999). Pseudomonas aureofaciens 30-84
Regulation of quorum sensing in other
contains a second AHL system designated CsaI/R, which
Pseudomonas has been implicated in the regulation of the biosynthesis of
cell-surface components (Zhang & Pierson, 2001). The two
Pseudomonas putida
AHL QS systems, PhzI/R and CsaI/R, are not organized in a
The AHL QS system designated PpuI/R has been reported in hierarchical way like the Las and Rhl systems in P. aerugino-
two P. putida plant-beneficial rhizobacteria, as mentioned sa; however, the AHL produced by PhzI can interact with
earlier (Steidle et al., 2002; Bertani & Venturi, 2004). The CsaR and the AHL produced by CsaI can activate PhzR. The

two systems are cooperatively involved in the regulation ism (i.e. quorum concentrations of 3-oxo-C6-AHL interact
exoprotease production and colonization of the wheat with AhlR and activate the ahlI promoter) typical of other
rhizosphere. The CsaI/R system is also under positive AHL QS systems. This observation that ahlI expression can
regulation by GacA/GacS (Zhang & Pierson, 2001). be rescued in both gacA and aefR mutants by the addition of
The plant-beneficial tomato rhizosphere isolate P. chlor- exogenous 3-oxo-C6-AHL indicates that neither regulator is
oraphis strain PCL1391 controls the production of the acting through a direct interaction at the promoter se-
biocontrol trait phenazine-1-carboxamide (PCN) via the quence. The GacA/GacS system has also been reported to
PhzI/R-C6-AHL system, which is highly similar to the one affect AHL accumulation in P. syringae pv. tomato strain
present in P. aureofaciens (Chin et al., 2001). The phzI/R DC3000, and also regulates a large number of other global
genes are under negative transcriptional regulation by a TetR regulatory systems, placing it at the top of regulatory
family regulator called Pseudomonas sigma regulator (PsrA) cascades (Chatterjee et al., 2003).

(Chin et al., 2005). PsrA also negatively regulates an as yet
unidentified AHL QS system(s) in P. chlororaphis, because
psrA knock-out mutants result in the appearance on AHL
Concluding remarks
molecules which are normally not produced by the wild- In the last few years there has been considerable progress in
type strain. PsrA in P. chlororaphis is also intimately con- the study of the regulation of QS systems of several
nected with the RpoS stationary-phase sigma factor, because Pseudomonas, especially of P. aeruginosa. The AHL QS
PsrA positively regulates rpoS expression (Chin et al., 2005). system of P. aeruginosa is one of the best studied, and a
In fact, PsrA was originally identified in P. aeruginosa and P. unique feature, tailored to its versatility, is the integration of
putida as a direct positive regulator of rpoS expression, QS with many other regulatory circuits (listed in Table 2 and
because it binds and activates transcription at rpoS promo- depicted in Fig. 1). Several systematic studies on P. aerugi-
ters (Venturi, 2003; Kojic & Venturi, 2001; Kojic et al., 2002). nosa have demonstrated that QS is a global regulatory
PsrA does not play a major role in AHL QS regulation in P. network controlling the expression of over 300 genes
aeruginosa and P. putida; psrA knock-out mutants result in (Whiteley et al., 1999; Hentzer et al., 2003; Schuster et al.,
only a slight increase in AHL production (Bertani & Venturi, 2003; Wagner et al., 2003). Recent regulon studies of other
2004). The negative effect exerted on phzI/R transcription by global regulators (e.g. RpoS, VqsR, PprB) in P. aeruginosa
PsrA could therefore occur through RpoS. The psrA gene is have highlighted the fact that they are significantly inter-
itself positively regulated by the two-component GacA/GacS connected with the QS regulon, meaning that several global
system upon induction of an as yet unknown environmental regulators are regulating a similar set of genes, thus increas-
signal that will be important in the signal cascade governing ing the response capability of P. aeruginosa to different
AHL production in P. chloroaraphis (Chin et al., 2005). environmental and growth conditions. These global regula-
tors are cross-regulating each other, as, for example, the
PprB two-component response regulator and LuxR-family
Pseudomonas syringae
family member VqsR are positively regulating QS genes and
Plant pathogen P. syringae pv. syringae strain B728a is a thus they could also be regulating genes through the QS
causal agent of bacterial brown spot of beans and possesses system. The extent of regulation by a global regulator
an AHL QS system called AhlI/R which produces and directly or via another global regulator is a question that
responds to 3-oxo-C6-AHL (Kitten et al., 1998; Kinscherf & will need to be resolved in the future, and is one that in
Willis, 1999; Quinones et al., 2004). It was observed that two addition to genomic-based methodologies will require mo-
regulators are independently involved in the positive regula- lecular studies.
tion of ahlI expression (Quinones et al., 2004). First, the Several regulators of QS are employed to ensure the
GacA/GacS system is involved in the regulation of AHL QS correct timing of the QS response, thus avoiding early
in P. syringae through regulation of ahlI expression (Qui- activation at low cell densities. Transcriptional regulators
nones et al., 2004; Kitten et al., 1998). Secondly, a genetic such as RsaL, MvaT, RpoS, RsmA and RpoN repress the
screen identified a transposon mutant that displayed sig- expression of lasI and rhlI AHL synthase genes at low cell
nificant reduction in AHL production; this mutant was densities and/or during favourable growth conditions, mak-
inactivated in an ORF coding for a TetR family transcrip- ing sure that the QS system is shut off. In addition, there is
tional regulator that was designated AefR (Quinones et al., also the action of the anti-activator protein QscR towards
2004). AefR positively regulates ahlI expression indepen- LasR, which through protein–protein interaction blocks the
dently from GacA/GacS. The expression of ahlI was com- LasR function. At high cell densities, de-repression by the
pletely restored in gacA and aefR mutants by the exogenous negative regulators occurs in concomitance with positive
addition of 3-oxo-C6-AHL, indicating that ahlI expression regulation of the expression of lasR and rhlR genes by
was responsive to AHL through a positive feedback mechan- regulators such as GacA, Vfr and PprB, resulting in rapid

286 V. Venturi
Table 2. Regulators of quorum sensing (QS) in Pseudomonas aeruginosa

Regulators Target(s) Additional comments References
RpoS Negative transcriptional Considerable overlap between RpoS and QS Latifi et al. (1996); Whiteley et al. (2000); Schuster
regulator of rhlI regulons et al. (2004)
RpoN Negative transcriptional In minimal medium RpoN positively regulates rhlI Heurlier et al. (2003); Thompson et al. (2003)
regulator of lasRI and rhlRI expression
RsaL Negative transcriptional In competition with LasR/3-oxo-C12-AHL for lasI de Kievit et al. (1999); Bertani & Venturi (2004)
regulator of lasI transcription; Avoids early activation of QS
MvaT Negative transcriptional Avoids early activation of QS Diggle et al. (2002)
regulator; target currently
unknown
QscR Negative regulator (anti- Avoids early activation of QS; LuxR-family Chugani et al. (2001); Ledgham et al. (2003)

activator) of LasR protein member
RsmA Negative translational Avoids early activation of QS Pessi et al. (2001)
regulator of lasI
DksA Negative transcriptional Avoids early activation of QS Branny et al. (2001); van Delden et al. (2001);
regulator of rhlI Jude et al. (2003)
Vfr Positive transcriptional Binds to lasR promoter, not known if also binds to Albus et al. (1997)
regulator of lasR and rhlR rhlR promoter
VqsR Positive transcriptional Considerable overlap between VqsR and QS Juhas et al. (2004)
regulator of lasI regulons; LuxR-family member
GacA/GacS Positive transcriptional Two-component signal transduction system; Reimmann et al. (1997); Parkins et al. (2001)
regulator of lasR and rhlR Environmental stimulus unknown
PprB Positive transcriptional Response regulator of a two-component signal Dong et al. (2005)
regulator of lasI, rhlI and transduction system; Overlap of PprB and QS
rhlR regulons; Environmental stimulus unknown
PQS Positive transcriptional PQS is a signal molecule, its production is Pesci et al. (1999); McKnight et al. (2000);
regulator of rhlI regulated by the Las system Gallagher et al. (2002); Diggle et al. (2003);
McGrath et al. (2004)
AlgR2 Negative transcriptional Also major regulator of alginate production; Ledgham et al. (2003); Westblade et al. (2004)
regulator of lasR and rhlR Homologous to E. coli Rsd protein which is and
anti-s70 factor
With the exception of QscR and Vfr it is not known if the regulator operates on the target(s) directly or indirectly; see text for details.
activation of the QS response. The general picture that is P. syringae two major positive regulators of QS have been
emerging is therefore that there is negative regulation identified. As all of these pseduomonads are extremely
mainly of lasI and rhlI, which can quickly change through versatile organisms, they will probably undergo complex
their de-repression and activation of lasR and rhlR. The regulation of QS, ensuring a timely control under different
three QS systems of P. aeruginosa (Las, Rhl and PQS) are growth conditions. Each will probably present unique
intimately connected, with LasI/R being at the top of the features because they have evolved to adapt to and colonize
cascade because it positively regulates the PQS and RhlI/R different niches and environments. Understanding the mo-
system. The advantages of this cascade system are still not lecular mechanisms of regulation of QS in Pseudomonas will
entirely clear; however, having three systems organized help us to understand how it serves a given species in its
hierarchically probably means that many genes can be particular environment and to design ways to interrupt it in
regulated at different levels: cell densities, growth phases order to find new antibacterial molecules.
and environmental conditions.
Studies on the regulation of QS in other Pseudomonas lag
behind; however, initial studies have indicated that the
Acknowledgements
complexity present in P. aeruginosa is likely to occur in I thank co-researchers C. Aguilar, I. Bertani, G. Degrassi, G.
other Pseudomonas as well. For example, in plant-growth- Devescovi, M. Kojic, M. Sevo, S. Ferluga for their contribu-
promoting P. aureofaciens/P. chlororaphis more than one tion to the results of this review, and L. Leoni for a critical
AHL QS system is present, and these systems have been reading of the manuscript. This work was supported by
shown to be under regulation. In P. putida, the PpuI/R AHL ICGEB (International Centre for Genetic Engineering and
QS system is highly identical to the LasI/R system, and in Biotechnology, Trieste, Italy).

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Regulation of Quorum Sensing In: Pseudomonas

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Regulation of quorum sensing in Pseudomonas

Correspondence: Vittorio Venturi, Abstract

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Introduction system of AHL QS is fairly simple, being most commonly

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Table 1. AHL quorum-sensing systems of Pseudomonas

FEMS Microbiol Rev 30 (2006) 274–291

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The Pseudomonas quorum-sensing systems are

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QscR LasR LasI

FEMS Microbiol Rev 30 (2006) 274–291

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FEMS Microbiol Rev 30 (2006) 274–291

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FEMS Microbiol Rev 30 (2006) 274–291

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FEMS Microbiol Rev 30 (2006) 274–291

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FEMS Microbiol Rev 30 (2006) 274–291

Table 2. Regulators of quorum sensing (QS) in Pseudomonas aeruginosa

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References subsp. carotovora by affecting its stability. J Bacteriol 184:

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FEMS Microbiol Rev 30 (2006) 274–291

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FEMS Microbiol Rev 30 (2006) 274–291

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FEMS Microbiol Rev 30 (2006) 274–291

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