[Advances in Microbial Physiology] Volume 58 || Quorum Sensing

Quorum Sensing: Regulating the Regulators

Marijke Frederix1 and J. Allan Downie

Molecular Microbiology, John Innes Centre, Norwich, United Kingdom1Present address: Joint BioEnergy Institute, Lawrence Berkeley National Laboratory,

Berkeley, CA, USA

ABSTRACT

Many bacteria use ‘quorum sensing’ (QS) as a mechanism to regulate geneinduction in a population-dependent manner. In its simplest sense thisinvolves the accumulation of a signaling metabolite during growth; thebinding of this metabolite to a regulator or multiple regulators activatesinduction or repression of gene expression. However QS regulation isseldom this simple, because other inputs are usually involved. In thisreview we have focussed on how those other inputs influence QSregulation and as implied by the title, this often occurs by environmental orphysiological effects regulating the expression or activity of the QSregulators. The rationale of this review is to briefly introduce the main QSsignals used in Gram-negative bacteria and then introduce one of theearliest understood mechanisms of regulation of the regulator, namely theplant-mediated control of expression of the TraR QS regulator inAgrobacterium tumefaciens. We then describe how in several species,multiple QS regulatory systems can act as integrated hierarchicalregulatory networks and usually this involves the regulation of QSregulators. Such networks can be influenced by many differentphysiological and environmental inputs and we describe diverse examplesof these. In the final section, we describe different examples of howeukaryotes can influence QS regulation in Gram-negative bacteria.

ADVANCES IN MICROBIAL PHYSIOLOGY, VOL. 58 Copyright # 2011 by Elsevier Ltd.ISSN: 0065-2911 All rights reservedDOI: 10.1016/B978-0-12-381043-4.00002-7

24 MARIJKE FREDERIX AND J. ALLAN DOWNIE

1. In
troduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 2. Q S Signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.1.
N -Acylhomoserine Lactones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 2.2. A utoinducer 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 2.3. P seudomonas Quinolone Signal in Pseudomonas aeruginosa . . . . . 28 2.4. Q S Based on Two-Component Regulators . . . . . . . . . . . . . . . . . . . . . 29
3. P
lasmid Transfer in Agrobacterium tumefaciens:ATale of Two Signals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 3.1. In duction of traR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 3.2. R egulation of TraR Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 3.3. R egulation of AHL Turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
4. H
ierarchical QS and the Rhizobium-Legume Symbiosis . . . . . . . . . . . . . . 33 4.1. R hizobium leguminosarum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 4.2. S inorhizobium meliloti . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
5. O
ther Hierarchically Organized QS Systems . . . . . . . . . . . . . . . . . . . . . . . 37 5.1. Y ersinia Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 5.2. B urkholderia cenocepacia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 5.3. R alstonia solanacearum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
6. In
tegration of QS Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 6.1. P seudomonas aeruginosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 6.2. V ibrio Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
7. E
nvironmental Signals Affecting QS Gene Regulation . . . . . . . . . . . . . . . . 45 7.1. N utrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 7.2. O ther Environmental Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
8. S
ignals from Other Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 8.1. M icrobial Cross-Communication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 8.2. C ommunication with Eukaryotes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 A cknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 R eferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
1. INTRODUCTION

Quorum sensing (QS) regulation allows bacteria to control their geneexpression in response to their population density. The bacteria producesignaling molecules that accumulate during specific stages of growth,although their production level can also be influenced by the environment.At a threshold concentration, the signals activate a regulator that caninduce or repress target genes. Usually processes that are regulated byQS are beneficial when a group of bacteria acts together (for a reviewsee Waters and Bassler, 2005). For example in the marine bacteriumVibrio fischeri, QS regulates luminescence in the squid light organ(Nealson et al., 1970; Eberhard et al., 1981). Similarly, QS regulation canbe used as a strategy to invade hosts successfully: a single bacteriumexpressing virulence genes could be detected and dealt with by the host’s

25QUORUM SENSING

immune response, but a coordinated attack by a population of bacteriamay overwhelm a host before it has a chance to defend itself. Many speciesof bacteria regulate several aspects of life using QS, including biofilmformation, bioluminescence, virulence, DNA exchange, etc. (Parsek andGreenberg, 2000; Whitehead et al., 2001; Winzer and Williams, 2001;Coulthurst et al., 2008; Downie and Gonzalez, 2008; Stabb et al., 2008;Stirling et al., 2008). QS in Gram-positive bacteria is beyond the scope ofthis review (for reviews see Sturme et al., 2002; Podbielski andKreikemeyer, 2004; Novick and Geisinger, 2008).

QS signals may also be a means of detecting diffusion-limited situations(Redfield, 2002). For example, it would be better to secrete extracellularenzymes in an environment that does not allow the enzymes to diffuse away.The concepts of QS as population density sensing and diffusion sensing havebeen unified in the concept of efficiency sensing (Hense et al., 2007).

In this review we describe how inputs from environmental stimuli,nutritional status, and interactions with both eukaryotes and prokaryotescan influence QS regulation in Gram negative bacteria.

2. QS SIGNALS

2.1. N-Acylhomoserine Lactones

N-acylhomoserine lactone (AHL)-based QS requires an AHL-synthase anda LuxR-type regulator whose activity is modified by the AHLs. AHLs canvary (Fig. 1A), although their basic structures are similar, consisting of ahomoserine lactone (HSL) ring and an acyl chain which can vary in lengthand degree of saturation. The third carbon can contain a hydrogen-, oxo-,or hydroxyl-substitution (Churchill and Herman, 2008). This variationprovides a mechanism for some specificity and can enable bacteria todistinguish between their own AHLs and those produced by other species.

There are three known protein families that can synthesize AHLs. TheLuxI-type synthases, found in the a-, b-, and g-proteobacteria (Gray andGarey, 2001), catalyze the ligation of S-adenosylmethionine (SAM) withan acylated acyl-carrier protein (Schaefer et al., 1996b; Val and Cronan,1998; Parsek et al., 1999). The second family, found only inVibrio and relatedspecies, includes LuxM fromVibrio harveyi, AinS fromV. fischeri andVanMfrom Vibrio anguillarum (Gilson et al., 1995; Hanzelka et al., 1999; Miltonet al., 2001). These proteins are very different from the LuxI-type synthases,although both types seem to use the same reaction mechanism (Hanzelkaet al., 1999). The third family, including HdtS in Pseudomonas fluorescens

Figure 1 Different kinds of QS molecules in Gram-negative bacteria.(A) AHLs. The R-group is variable among different species, with changes in lengthand degree of saturation of the carbon chain. In addition, the third carbon atom can


27QUORUM SENSING

(Laue et al., 2000) and Act in Acidithiobacillus ferrooxidans (Rivas et al.,2007), is related to the lysophosphatidic acid acyltransferase protein family,but it is not known how these enzymes synthesize AHLs.

Most AHL regulators belong to the LuxR family and contain an N-termi-nal domain to which the AHLs bind, leading to dimerisation and activation(Choi and Greenberg, 1992; Hanzelka and Greenberg, 1995). The C-termi-nal domain contains a helix-turn-helix (HTH)DNA-bindingmotif that bindsto conserved sequences (the so-called ‘lux boxes’). The structures of LasR(P. aeruginosa) and TraR (A. tumefaciens) complexed with their cognateAHLs have been determined (Vannini et al., 2002; Bottomley et al., 2007;Zou and Nair, 2009). Although they normally induce gene expression (seereview by Nasser and Reverchon, 2007), LuxR-type regulators can also bindto target sequences in the absence of AHLs, thus blocking transcription.Binding of AHLs causes reduction of the DNA-binding activity, therebyinducing gene transcription (Horng et al., 2002;Minogue et al., 2002). In addi-tion to LuxR-type response regulators, AHL-responsive sensor kinases (e.g.,LuxN) have been found inVibrio species as part of a typical two-componentsignaling system (Bassler et al., 1994).

AHLs can diffuse across the membrane (Kaplan and Greenberg, 1985),although transporters for long chain AHLs have also been reported (Pearsonet al., 1999; Chan et al., 2007).AHL concentrations are also influenced by theirdegradation rates. Nonenzymatic degradation is increased by a high tempera-ture and analkaline pH (Byers et al., 2002;Yates et al., 2002). In addition, threeclasses of AHL-degrading enzymes have been identified (Dong and Zhang,2005; Czajkowski and Jafra, 2009): AHL lactonases inactivate AHLs byhydrolysis of the ester bond of the HSL ring (Dong et al., 2000, 2001), AHLacylases hydrolyse the AHL amide bond between the fatty acid and HSLmoieties (Sio et al., 2006) and AHL oxidoreductases inactivate AHLs by ahydrolysis reaction of the 3-oxo group (Uroz et al., 2005).

2.2. Autoinducer 2

Autoinducer 2 (AI-2) is a potential QS signal produced by a wide range ofGram-positive and Gram-negative bacteria (for a review see Federle, 2009).The structure of V. harveyi AI-2 has been determined as the boron ester of

contain a hydrogen-, oxo-, or hydroxyl-substitution (Churchill and Herman, 2008).(B) AI-2 produced by Vibrio species (Chen et al., 2002). (C) AI-2 produced byS. typhimurium (Miller et al., 2004). (D) PQS (Deziel et al., 2004), (E) bradyoxetin(Loh et al., 2002a), (F) 3OH-PAME (Clough et al., 1997b), (G) DSF (Wang et al.,2004), and (H) CAI-1 (Higgins et al., 2007).


(2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydro-furan (Chen et al., 2002),while AI-2 from Salmonella enterica serovar Typhimurium was found to lackthe borate (Miller et al., 2004; Fig. 1B,C). LuxS produces AI-2 by cleavingS-ribosyl-L-homocysteine to generate homocysteine and the AI-2 precursor4,5-dihydroxy-2,3-pentanedione (DHP). DHP then spontaneously cyclises,thus forming AI-2 (Schauder et al., 2001). LuxS in S. enterica serovarTyphimurium can be posttranslationally modified and is transported acrossthe cytoplasmic membrane, despite the lack of an obvious signaling motif.This indicates that the function of LuxS is potentially not limited tosynthesizing AI-2 (Kint et al., 2009).

AI-2 is produced by many bacterial species, but there is discussion aboutthe precise role of AI-2 as a signaling molecule. In Vibrio species a receptorcomplex LuxPQ for AI-2 has been identified (Miller et al., 2002; Henke andBassler, 2004; Sun et al., 2004). In S. enterica serovar Typhimurium andEscherichia coli AI-2 is perceived by an ABC transporter (Lsr) that phos-phorylates AI-2 upon uptake (Xavier et al., 2007). The phosphorylatedAI-2 molecule is thought to bind to the transcriptional regulator LsrR thatactivates further transcription of the lsrACDBFGE operon (Taga et al.,2001, 2003; Xavier et al., 2007). Reports on other species have suggested thatAI-2 has no signaling function but is a secreted metabolite formed by LuxSin the recycling of methionine from S-adenosyl-L-homocysteine (Vendevilleet al., 2005; Rezzonico and Duffy, 2008). In several studies, it was observedthat adding chemically synthesized AI-2 did not restore the phenotype ofluxSmutants and it was concluded that in some bacteria, the changes in geneexpression that occur upon mutation of luxS are a consequence of metabolicchanges (Winzer et al., 2003; Vendeville et al., 2005; Holmes et al., 2009).

2.3. Pseudomonas Quinolone Signalin Pseudomonas aeruginosa

Besides AHLs, Pseudomonas species also produce PQS (Pesci et al., 1999)(Fig. 1). The PQS molecules (3,4-hydroxy-2-heptylquinolines; Deziel et al.,2004) belong to the family of 4-hydroxy-2-alkylquinolines (HAQ) and aresynthesized by the enzymes encoded by pqsABCD and pqsH, via thecondensation of anthranilic acid with b-keto fatty acids. The PQS precur-sor, 4-hydroxy-2-heptylquinoline (HHQ), is converted to PQS by an oxida-tion step catalyzed by PqsH (Bredenbruch et al., 2005). Both PQS andHHQ function as autoinducers as they can bind to the transcriptionalregulator PqsR (also known as MvfR) and activate expression from thepqsA promoter (Cao et al., 2001; Xiao et al., 2006a,b). It has been

29QUORUM SENSING

suggested that PQS might be dispensable as pqsH mutants display normalPqsR-dependent gene regulation (except for pyocyanin production),although HHQ is 100-fold less potent than PQS (Xiao et al., 2006a).In addition to PqsR, two other regulatory mechanisms for PQS signalinghave been proposed. PqsE functions as a PqsR-independent responseeffector and requires the LuxR-type regulator RhlR for function. A pqsEmutant is not capable of producing PQS-controlled virulence factors andthis phenotype can be suppressed by addition of RhlI-made AHLs (Farrowet al., 2008). PQS molecules also have iron-chelating properties and thiscan contribute to the regulation of genes involved in iron scavenging andsiderophore biosynthesis by trapping iron at the cell surface (Bredenbruchet al., 2006; Diggle et al., 2007). The PQS-iron complex is toxic for the host(Zaborin et al., 2009). Although PQS-dependent gene regulation has beenmainly studied in Pseudomonas, other bacteria like Burkholderia pseu-domallei have also been shown to produce HAQ molecules (Diggle et al.,2006).

2.4. QS Based on Two-Component Regulators

2.4.1. Hydroxy-Fatty-Acyl Derivatives

The plant pathogen Ralstonia solanacearum uses 3-hydroxypalmitic acidmethyl ester (3-OH PAME, Fig. 1F) to regulate its virulence factors in apopulation-dependent manner. 3-OH PAME is synthesized by PhcB,which catalyses the conversion of a fatty acid to its methyl ester (Cloughet al., 1997b). 3-OH PAME is sensed by the sensor kinase PhcS and theresponse regulator PhcR relays the information to the regulator PhcA.PhcA is the actual regulator that induces the expression of the virulencegenes at high population densities (Clough et al., 1997b; Flavier et al.,1997). The chemolithoautotroph Ralstonia eutropha regulates expressionof motility and siderophore synthesis by a similar mechanism (Garget al., 2000).

The plant pathogen Xanthomonas campestris produces the autoinducercis-11-methyl-2-dodecenoic acid or DSF (Fig. 1G), which is involved inthe regulation of virulence factors (Wang et al., 2004; Torres et al., 2007).Production of cis-2-dodecenoic acid (BDSF) was also shown inBurkholderia cenocepacia (Ryan et al., 2009). DSF is produced by RpfFand is sensed by the two-component sensor kinase RpfC, which transmitsthe signal to the HD-GYP protein RpfG (Torres et al., 2007). RpfG isnot a DNA-binding protein as is usually the case for a two-component


response regulator, but relies on its HD-GYP domain for its regulatoryactivity (Fouhy et al., 2006; Ryan et al., 2006, 2010). This HD-GYP domainhas phosphodiesterase activity and hydrolyses cyclic-di-guanosine mono-phosphate (cyclic-di-GMP) to cyclic guanosine monophosphate (cGMP;He et al., 2007; Chin et al., 2010). The levels of cyclic-di-GMP and cGMPin the cell are monitored by the DNA-binding regulator Clp, which has acyclic nucleoside monophosphate (cNMP) binding domain (Tao et al.,2010). It is this protein that is responsible for mediating the transcriptionalresponse when DSF is sensed by RpfC.

Vibrio cholerae and Legionella pneumophila produce a-hydroxy ketones(AHKs), respectively cholera autoinducer 1 or CAI-1 (Fig. 1H) andLegionella autoinducer 1 or LAI-1, which is similar to CAI-1 but with a lon-ger acyl chain (Spirig et al., 2008). These AHKs are synthesized by CqsA andLqsA, which are homologous; CqsA uses acyl-CoA and aminobutyrate assubstrates to form an amino-acyl intermediate that is converted into CAI-1independently of CqsA (Kelly et al., 2009). The signals are presumablyrecognized by the transmembrane sensor kinases CqsS and LqsS.

2.4.2. Bradyoxetin in Bradyrhizobium japonicum

In B. japonicum bradyoxetin or CDF (Fig. 1E) accumulates at high popu-lation density (Loh et al., 2002a). Bradyoxetin affects the expression ofnolA and nodD2 and by doing so represses the expression of the nodula-tion genes at high population densities (Loh et al., 2001). Bradyoxetin isdetected by the two-component response regulator NwsB (Loh et al.,2002b). Bradyoxetin activity has been detected in extracts of all testeda-proteobacteria (Loh et al., 2002a).

2.4.3. Autoinducer 3 (AI-3) in Enterohemorrhagic Escherichia coli(EHEC)

In the human pathogen EHEC a new kind of autoinducer, AI-3, was discov-ered. AI-3 is thought to resemble the mammal hormones epinephrine andnorepinephrine, thus providing a means of communication with the eukary-otic host in addition to its role as a QSmolecule (Sperandio et al., 2003). Pro-duction of AI-3 was reported to depend on a luxS gene (Sperandio et al.,2003), but this was later shown to be due to an indirect effect (Walterset al., 2006). AI-3 is perceived by the sensor kinase QseC and its cognateresponse regulator QseB (Clarke and Sperandio, 2005; Clarke et al., 2006).

31QUORUM SENSING

3. PLASMID TRANSFER IN AGROBACTERIUM TUMEFACIENS:A TALE OF TWO SIGNALS

A. tumefaciens is a plant pathogen that carries a tumor-inducing (Ti) plas-mid, which contains oncogenic genes that are transferred to plant cells andintegrated into the nuclear DNA. It is a very unusual pathogen, because, inaddition to reprogramming plant cell growth to induce tumors, theresulting transformed plant cells are programmed to make and exportunusual metabolites (generically referred to as opines) that extracellularagrobacteria can use for growth. So a growth advantage to this pathogenis conferred to those agrobacteria that grow on and around the tumor.Specialized genes are required to degrade opines and the genes requiredfor this are also present on the Ti plasmid, but on the part that is not trans-ferred to the plant. Clearly transfer of such a catabolic capability to otherbacteria has a great selective advantage and in a lovely twist of fate, theregulation of transfer of these plasmids requires both opines and QS regu-lation. In essence this spreads the pathogenicity plasmid into anyagrobacteria that are nearby and simultaneously promotes their growth,hence increasing the pathogenic potential of the population. TraR inducesplasmid transfer, but in addition to activation by 3-oxo-C8-HSL, the activ-ity of TraR is affected by protein degradation, protein folding, multimerformation and interaction with modulatory proteins (Winans, 2008).

3.1. Induction of traR

Conjugation of the Ti plasmid to other Agrobacterium strains is stronglystimulated by compounds such as opines which are nutritional sourcesfor agrobacteria and fall into different chemical groupings such asoctopine, mannopine agrocinopine, etc. (Genetello et al., 1977; Kerret al., 1977). In ‘octopine’-type Ti plasmids, octopine binds to and activatesOccR, the LysR-type regulator of an octopine catabolism operon, toinduce expression of the operon. traR is the last gene in this operon andso is induced when the bacteria catabolise octopine (Habeeb et al., 1991;Fig. 2). In nopaline-type Ti plasmids, accR (agrocinopine catabolism regu-lator) encodes a repressor of the agrocinopine catabolism operon and thisrepression is relieved by agrocinopines. The first gene in this catabolismoperon is traR which is induced when agrocinipines are available (Beckvon Bodman et al., 1992; Piper et al., 1993; Fuqua and Winans, 1994). Thusby two independently evolved mechanisms, opine catabolism is coupled

traRtraM

traI

pTi

TraR

TraI

occR

TraM OccR

Opines

trlR

TrlR

Figure 2 Induction of the tra QS system of A. tumefaciens by plant-madeopines. The Ti plasmid carries the plasmid conjugation genes. Expression of thesegenes is induced by TraR in response to TraI-made AHLs (Fuqua and Winans,1994). Expression of traR is induced by the transcriptional regulator OccR inresponse to plant-made opines (Habeeb et al., 1991). TraM (Chen et al., 2004)and TrlR (Oger et al., 1998) function as antiactivators of TraR.


with traR induction. The expression of TraR leads to the induction of traI,and then as TraI-made AHLs start to accumulate, they activate TraR,resulting in a positive feedback loop that induces high levels of expressionof the plasmid transfer genes (Fuqua and Winans, 1994; Fig. 2). Conjugalplasmid transfer therefore requires both a high population density andthe appropriate plant-made signal.

3.2. Regulation of TraR Activity

TraR is very rapidly proteolytically degraded, but its ligand, 3-oxo-C8-HSLgreatly reduces the level of turnover of the protein (Chai et al., 2001).Determination of the structure of TraR showed that binding of the AHLtriggers a protein conformational change that promotes the formation ofdimers, in which the AHL is an intimate component of the complex(Vannini et al., 2002; Zhang et al., 2002b). These structuralcharacterisations of the complex also included the consensus DNA-bindingsequence known as a tra box.

33QUORUM SENSING

There are two proteins TraM and TrlR that can inhibit TraR activity.TraM forms dimers (Chen et al., 2004; Vannini et al., 2004) that can bindto the TraR–AHL complex (Swiderska et al., 2001) thereby preventing itfrom binding to the tra box. It has been proposed that TraM causes disso-ciation of the TraR–AHL complex, resulting in formation of an inactiveTraM–TraR complex (Chen et al., 2004). The mannopine-degradationgene cluster on the Ti plasmid contains a LuxR-type regulator TrlR (alsoknown as TraS) that is induced by mannopines, which is a kind of opine(Oger et al., 1998; Zhu and Winans, 1998; Chai et al., 2001). TrlR is verysimilar to TraR, but has a frameshift mutation which results in a proteinlacking the DNA-binding domain. TrlR interacts with TraR forming inac-tive heterodimers. Therefore, when mannopines are present, TrlR isinduced and this inhibits conjugation of the Ti plasmid.

3.3. Regulation of AHL Turnover

Plant signals alter the expression levels of AHLs by inducing lactonasesthat degrade the AHLs. Two lactonases are expressed by A. tumefaciens,AttM (Zhang et al., 2002a) and AiiB (Carlier et al., 2003). The attM geneis induced by g-aminobutyrate (GABA), which induces the attKLMoperon involved in the catabolism of GABA produced in wounded tissues.In contrast, aiiB is induced by agrocinopines and an aiiB mutantaccumulated high levels of TraI-made 3-oxo-C8-HSL. Thus there are atleast two plant signals that can affect TraR activity by modulating the turn-over rate of the TraI-made AHLs (Chevrot et al., 2006; Haudecoeur et al.,2009b).

4. HIERARCHICAL QS AND THE RHIZOBIUM-LEGUMESYMBIOSIS

Some species of rhizobia are closely related to agrobacteria and manyrhizobia contain one or more AHL-based QS systems. Different aspectsof the Rhizbobium-legume symbiosis have been shown to be regulatedby QS (Downie and Gonzalez, 2008), such as nodulation efficiency (Cuboet al., 1992; Gao et al., 2006; Zheng et al., 2006; Cao et al., 2009), nodule for-mation (Zheng et al., 2006; Cao et al., 2009), symbiosome development(Daniels et al., 2002), exopolysaccharide production (Marketon andGonzalez, 2002), symbiotic plasmid transfer (Danino et al., 2003) and


nitrogen fixation (Daniels et al., 2002). Nevertheless, many rhizobia seemto be able to establish effective symbioses with their legume hosts aftermutation of their QS genes, indicating that their role is mainly to optimizethe interactions between the bacteria and their host. The role of QS in theRhizobium legume symbiosis has been studied extensively in many species.Many reviews regarding this subject have been written (Wisniewski-Dyeand Downie, 2002; Gonzalez and Marketon, 2003; Sanchez-Contreraset al., 2007; Downie and Gonzalez, 2008).

4.1. Rhizobium leguminosarum

Four different QS regulatory systems have been identified in R.leguminosarum although not all strains have all systems. They operate ina hierarchical network with cinI and cinR at the top regulating the produc-tion of RaiI-, RhiI-, and TraI-made AHLs by the rai, rhi, and tra systems(Lithgow et al., 2000; Wisniewski-Dye et al., 2002). QS genes similar to cinIand cinR were identified in Rhizobium etli and Mesorhizobiumtianshanense (respectively the cinI/R and mrtI/R genes), but the roles ofthese genes appear to be different: whereas mutation of cinI in R.leguminosarum did not affect symbiotic nitrogen fixation, mutation in theequivalent genes in R. etli and M. tianshanense reduced or blocked symbi-otic nitrogen fixation (Daniels et al., 2002; Zheng et al., 2006).

The traI and traR genes on the symbiotic plasmid pRL1JI are homolo-gous to these found in A. tumefaciens, but the induction of traR is different.Expression of traR is induced by CinI-made AHLs, which results in recipi-ent-induced plasmid transfer (Fig. 3). The key to this is the presence ofanother LuxR-type regulator on pRL1JI, BisR, which can act both as aninducer and as a repressor (Danino et al., 2003). In strains carrying pRL1JI(donor strains), BisR represses expression of cinI, thus preventing the syn-thesis of CinI-made 3-hydroxy-C14:1-HSLs (Wilkinson et al., 2002). Instrains that do not carry pRL1JI (potential recipient strains), BisR isabsent and this repression does not occur and therefore CinI produces 3-hydroxy-C14:1-HSLs. When a recipient strain and donor strain come intoclose proximity, BisR in the donor strain is activated by 3-hydroxy-C14:1-HSLs produced by the recipient strain and the activated BisR inducesthe expression of traR (Wilkinson et al., 2002). TraR is then activated byTraI-made AHLs as seen with A. tumefaciens including a role for TraMas an antiactivator of TraR (Danino et al., 2003; McAnulla et al., 2007).The bivalent mode of action of BisR (as an inducer of traR and arepressor of cinI) is therefore responsible for a regulatory mechanism

rhiIrhiABCrhiR

traRtraM traIbisR trb

pRL1JI

RhiR

TraR

BisR

RhiI

TraI

cinR cinI S

CinI-made AHLs(Recipient strain)

Donor strain

TraM

BisR

Figure 3 Recipient induced plasmid transfer in R. leguminosarum. When BisRis present (in donor strains), it represses the expression of cinI, thus preventing thesynthesis of CinI-made AHLs. Recipient strains do not express BisR and can there-fore produce CinI-made AHLs. These are recognized by a donor strain in closeproximity and activate BisR. BisR induces the expression of traR and TraR inducesthe expression of the plasmid transfer genes in response to TraI-made AHLs. TraMrepresses expression of the plasmid transfer genes at low levels of TraI-made AHLsprobably by forming an antiactivator complex with TraR (Danino et al., 2003). Thesymbiotic plasmid pRL1JI also contains the rhiI and rhiR genes. RhiR activates theexpression of the rhiABC genes in response to RhiI-made AHLs (Cuboet al., 1992).

35QUORUM SENSING

that allows the recipient strains to induce plasmid transfer in the presenceof a possible donor strain.

The production of AHLs made by RhiI and RaiI is under the control ofthe cin QS system (Lithgow et al., 2000; Wisniewski-Dye et al., 2002). Forthe induction of raiI, this has been shown to be intimately linked to a smallgene cinS, downstream of, and translationally coupled to cinI (Edwards


et al., 2009). Even in a cinI-cinR double mutant, cinS expressed from a vec-tor promotor induced the expression of raiR and hence raiI, suggestingthat the link between the cin and rai QS systems is mediated by the popu-lation density-dependent accumulation of CinS (Edwards et al., 2009).It has become apparent that CinS also controls rhiR expression in a similarmanner and that CinS acts as an antirepressor, relieving the activity of arepressor that reduces raiR and rhiR expression (Frederix, Edwards, andDownie unpublished data). RhiR induces the rhiABC genes in responseto RhiI-made C6, C7, and C8-HSLs and these genes play a role in rhizo-sphere competence and nodulation (Dibb et al., 1984; Economou et al.,1989; Cubo et al., 1992). It is currently unknown which genes are regulatedby RaiR in R. leguminosarum, but in R. etli RaiR was involved in therestriction of nodule number. In vitro mutation of raiI led to an increasein nodulation numbers and nitrogenase activity, although in planta, no sig-nificant increase in nitrogen fixation could be demonstrated (Rosemeyeret al., 1998). Interestingly, mutation of raiR had no effect on nodulation.

4.2. Sinorhizobium meliloti

In S. meliloti two LuxR-type regulators SinR and ExpR can respond toSinI-made AHLs (ranging in size from C12-HSL to C18-HSL; Marketonet al., 2002; Pellock et al., 2002; Hoang et al., 2004). Mutation of sinI or sinRdelayed nodule formation and reduced the total number of nodules(Marketon et al., 2002; Gao et al., 2005) and it appears that most gene reg-ulation in response to SinI-made AHLs is mediated via ExpR and not viaSinR. ExpR regulates the biosynthesis of the symbiotically importantEPSII and succinoglycan, as well as motility and other processes (Pellocket al., 2002; Marketon et al., 2003; Hoang et al., 2004, 2008; Gao et al.,2005). Gene regulation by ExpR is particularly unusual, because it is capa-ble of influencing gene expression in a versatile way: it can be both depen-dent and independent of SinI-made AHLs and it can have both positiveand negative effects on gene expression (Hoang et al., 2004).

Expression of sinI is absolutely dependent on SinR and SinR inducesmoderate transcription of sinI even in the absence of SinI-made AHLs(McIntosh et al., 2008). ExpR also regulates expression of the sinI and sinRgenes and it does so at two levels, resulting in both a positive and a nega-tive feedback loop. ExpR induces sinI in response to SinI-made AHLs bybinding to a sequence upstream of sinI (Bartels et al., 2007; McIntosh et al.,2008), while it represses the expression of sinR (McIntosh et al., 2009). AnExpR binding site was identified in front of sinR, but this binding site was

37QUORUM SENSING

not required for the repression of sinR by ExpR, showing that theobserved reduction in expression might be due to an indirect effect. Theamount of AHLs in the environment probably determines whether thepositive or the negative feedback mechanism has the upper hand, eventu-ally resulting in an equilibrium state between both at higher populationdensities (McIntosh et al., 2009).

5. OTHER HIERARCHICALLY ORGANIZED QS SYSTEMS

5.1. Yersinia Species

The mammalian enteropathogen Yersinia pseudotuberculosis contains theypsI/R and ytbI/R genes, both of which are involved in the regulation ofcell aggregation and motility (Atkinson et al., 1999). In Y. pseudotuberculo-sis, YtbR induces the expression of ytbI in response to YtbI-made AHLs(C6-HSL, 3-O-C6-HSL, 3-O-C7-HSL, 3-OH-C8-HSL, 3-O-C8-HSL, C8-HSL, and 3-O-C10-HSL). YpsR represses the expression of ypsI and ypsRin response to YpsI-made AHLs (C6-HSL, 3-O-C6-HSL, 3-O-C7-HSL). Inaddition it activates the expression of ytbI and ytbR (Atkinson et al., 2008).YtbR has got a positive effect on motility, while YpsR has got a negativeeffect (Atkinson et al., 2008). The related strain Yersinia pestis has ypsI/Rand ypeI/R genes (Kirwan et al., 2006).

5.2. Burkholderia cenocepacia

Burkholderia species are opportunistic pathogens in people with cysticfibrosis and they use AHLs for the regulation of virulence factors. InB. cenocepacia, the cepI/R (Lewenza et al., 1999) and cciI/R (Malottet al., 2005) genes are organized in a hierarchical fashion. CepI synthesizesprimarily C8-HSL, and minor amounts of C6-HSL (Lewenza et al., 1999;Lewenza and Sokol, 2001). CciI synthesizes primarily C6HSL and minoramounts of C8-HSL (Malott et al., 2005). CepR is an inducer of geneexpression, while CciR is primarily repressing gene expression (includingautorepression of the cciIR operon). Several genes have been found tobe regulated by both regulators reciprocally (O’Grady et al., 2009). CepRinduces the transcription of the cciIR operon in response to C8-HSL. Anegative feedback loop is formed by two mechanisms: repression of cepIexpression by CciR (Malott et al., 2005) and inactivation of CepR in the


presence of high levels of C6-HSL (Weingart et al., 2005). The closelyrelated species Burkholderia vietnamiensis contains the cepI/R and bviI/Rgenes. Similar to the situation in B. cenocepacia, CepR is required forthe expression of bviI (Malott and Sokol, 2007). Different species ofBurkholderia have multiple QS regulatory systems but there is not a com-mon theme of cross regulation (Suarez-Moreno et al., 2010).

5.3. Ralstonia solanacearum

The plant pathogen R. solanacearum, which was previously categorized tothe Pseudomonas genus, produces two autoinducers: 3-OH PAME andSolI-made C8 and C9-HSL. The 3-OH PAME system is hierarchically ontop of the sol QS system. The 3-OH PAME signal is detected by the PhcShistidine sensor kinase, which relays the information via the response reg-ulator PhcR to the transcriptional regulator PhcA. PhcA induces solRexpression and SolR induces gene expression in response to SolI-madeAHLs (Clough et al., 1997a,b; Flavier et al., 1997).

6. INTEGRATION OF QS SYSTEMS

6.1. Pseudomonas aeruginosa

P. aeruginosa is an opportunistic, bronchial human pathogen, associatedwith infection of immuno-compromised patients. Its pathogenicity iscaused by secretion of extracellular virulence factors, such as proteases,heamolysins, exotoxinA, exoenzyme S and pyocyanin that cause extensivetissue damage. The regulation of the expression of these virulence factorsis tightly regulated by QS (Passador et al., 1993; Willcox et al., 2008;Winstanley and Fothergill, 2009). Because of its importance in pathogenic-ity, QS has been studied extensively in P. aeruginosa PAO1. In this species,at least three QS systems are present and their expression is organized in ahierarchical fashion (Fig. 4).

P. aeruginosa PAO1 contains the rhlI/R (Ochsner et al., 1994; Ochsnerand Reiser, 1995; Pearson et al., 1995) and the lasI/R QS genes (Gambelloand Iglewski, 1991; Pearson et al., 1994). RhlI synthesizes C4-HSL and LasIsynthesizes 3-oxo-C12-HSL. Together, the rhl and las genes regulate, eitherdirectly or indirectly, the expression of about 6% of the P. aeruginosagenome (Schuster et al., 2003). LasR induces the expression of lasI in

rhlR rhlI

RhlR RhlI

lasR lasI

LasR LasI

rsaL

RsaL

pqsA pqsB pqsC pqsD pqsE phnA phnB pqsR

pqsHPQSPqsR

PqsE

Figure 4 Hierarchical organization of QS systems in P. aeruginosa. The lasI andlasR genes regulate gene expression of target genes and are on top of a hierarchicalQS network. LasR activates lasI expression in response to LasI-made AHLs. RsaL,which is encoded between lasI and lasR, represses the expression of lasI. ActivatedLasR also induces the expression of rhlR and pqsR. RhlR induces expression of tar-get genes in response to RhlI-made AHLs, but represses the expression of thepqsABCDE operon and pqsR (for a review see Schuster and Greenberg, 2008).PqsR induces gene expression in response to PQS molecules, which are synthesizedby the proteins encoded by pqsABCD (Diggle et al., 2003). Activated PqsR inducesthe expression of the pqsABCDE operon and rhlI. pqsE is cotranscribed withpqsABCD but its product is not involved in PQS biosynthesis. PqsE functions asa response effector: PqsE requires RhlR for function, but the mechanism by whichthis happens has not yet been determined (Farrow et al., 2008).

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response to LasI-made AHLs. A second transcriptional regulator RsaL isencoded between lasI and lasR and represses transcription of lasI(de Kievit et al., 1999; Rampioni et al., 2006, 2007). LasR and RsaL bindto adjacent sites in the lasI promoter and the repressor activity of RsaLis dominant over the inducer activity of activated LasR. In addition, RsaLaffects the QS response by binding some of the promoters of genes that arecontrolled by QS (Rampioni et al., 2007).


In most studies, the lasI/R genes have been found to be hierarchically ontop of the rhlI/R genes, but other studies indicated that this hierarchy isdependent on the environmental conditions used (Duan and Surette,2007). Expression of rhlR is induced by LasR when it is activated byLasI-made AHLs (Latifi et al., 1996; Pesci et al., 1997). Further regulationof the rhl genes by the las genes is exerted posttranslationally: at low po-pulation densities, the activation of RhlR by RhlI-made AHLs is inhibitedby competitive binding of LasI-made AHLs to RhlR. Only at higher celldensities are the RhlI-produced AHLs able to outcompete the LasI-madeAHLs (Pesci et al., 1997). Both mechanisms of control probably serve toensure that the las and rhl genes are switched on in the right order: firstthe las genes and then the rhl genes. Recently, the repressor QteE wasidentified that inhibits both the lasI and rhlI genes from being activatedbefore the right population density is reached, by reducing the stabilityof LasR and RhlR (Siehnel et al., 2010).

P. aeruginosa also possesses two other LuxR-type regulators, both ofwhich affect the expression of the rhlI and lasI genes. QscR (quorum sens-ing control repressor) negatively affects the production of RhlI- and LasI-made AHLs in an AHL-independent manner, despite being able to bindAHLs (Chugani et al., 2001). QscR is thought to function through the for-mation of inactive dimers with LasR and RhlR, by titering out AHLs(Chugani et al., 2001; Ledgham et al., 2003b). QscR has also been shownto have DNA-binding activity (Lee et al., 2006). VqsR (virulence and quo-rum sensing regulator) is another LuxR-type regulator, which has a keyrole in the Pseudomonas QS regulatory cascade. Microarray analysisshowed that in a vqsR mutant the expression of lasI is greatly reduced(Juhas et al., 2004, 2005). In addition, it was shown that the expression ofvqsR itself is under the control of LasR (Li et al., 2007).

P. aeruginosa contains a PQS system, which is AHL independent andseems to be in an intermediate position between las and rhl (Diggleet al., 2003). The pqs genes are induced by LasR at two levels. First, LasRcontrols the amount of PQS signal that is produced, by inducing theexpression of pqsH (which catalyses the final step in PQS synthesis; Pesciet al., 1999; Gallagher et al., 2002; Deziel et al., 2004) although under somecircumstances, PQS synthesis can occur independently of LasR (Diggleet al., 2003). Second, the expression level of pqsR (also known as mvfR),which encodes a transcriptional regulator that is activated by PQS, is underdirect control of activated LasR (Wade et al., 2005; Xiao et al., 2006a).Cross-regulation between the PQS and rhl QS systems has also beenobserved, as activated PqsR induces rhlI expression (McKnight et al.,2000), while activated RhlR represses the expression of pqsR and the

41QUORUM SENSING

pqsABCDE operon (Wade et al., 2005; Xiao et al., 2006a). Induction of thepqs operon also affects the production of RhlI-made AHLs by means ofthe response effector PqsE, which is believed to act as a response effectorprotein that requires RhlR for function (Farrow et al., 2008; Yu et al.,2009). PQS molecules only accumulate at late stationary phase; thereforeit is not likely that the PQS system induces gene expression in the samepopulation density-dependent way as AHLs (McKnight et al., 2000).It probably functions to link the induction of the rhl and las genes, providingan extra means of control in the hierarchical cascade to ensure that therhl genes are only switched on after the las genes have been activated.

The three QS systems in P. auruginosa are part of a complex regulatorynetwork, and many other regulators that affect their expression and activityhave been identified: MvaT (Diggle et al., 2002), GidA (Gupta et al., 2009),the YebC-like protein PmpR (Liang et al., 2008a), AlgQ (Ledgham et al.,2003a), AlgR (Morici et al., 2007), VqsM (Dong et al., 2005), PA1196(Liang et al., 2009), PpyR (Attila et al., 2008), PtxR (Carty et al., 2006),PPK1 (Fraley et al., 2007), and Lon protease (Bertani et al., 2007; Takayaet al., 2008).

In several other Pseudomonas species multiple QS systems have beenfound, but their organization appears to be different from that seen inP. aeruginosa PAO1. In the plant-growth-promoting strain P. aeruginosaPUPa3, the lasI/R and rhlI/R genes are present, but their induction doesnot occur in a hierarchical fashion (Steindler et al., 2009). Likewise, inthe plant pathogen Pseudomonas aureofaciens, the phzI/R and csaI/Rgenes are also not induced hierarchically. However, in this species theAHLs produced by PhzI cross-react with CsaR, and the AHLs producedby CsaI can interact with PhzR (Zhang and Pierson, 2001).

6.2. Vibrio Species

6.2.1. Vibrio harveyi

In the marine bacterium V. harveyi, three autoinducers have beenidentified (Fig. 5A). These control the expression of bioluminescence, typeIII secretion and metalloprotease production (Henke and Bassler, 2004).The use of three different autoinducers, HAI-1, CAI-1, and AI-2, providesa way for the bacteria to decipher which species of bacteria are present intheir occupied niche (Henke and Bassler, 2004; Waters and Bassler, 2006).HAI-1 (harveyi autoinducer-1, 3-OH-C4-HSL) is produced by the LuxMAHL synthase (Cao and Meighen, 1989; Bassler et al., 1993). HAI-1 is only

V. harveyi V. cholerae V. fisheri

LuxPQ

luxS luxPQ

LuxS

AI-2

AinR

ainS ainR

AinS

C8-HSL

LuxU

LuxO

Qrr’s

LitR

Hfq

luxI luxR

LuxRLuxI

cAMP–CRP

ArcA/B

3–O–C6–HSL

LuxPQ

luxS luxPQ

LuxS

AI-2

CqsS

cqsA cqsS

CqsA

CAI-1

cAMP–CRP

CqsS

LuxU

LuxO

Qrr’s

HapR

Hfq

s 54

Fis

FliA

CsrA

VarA/S

CsrBCD

LuxN

luxM

LuxM

HAI-1

s 54

cAMP–CRP

MetR

LuxU

LuxO

Qrr’s

LuxR

Hfq

LuxPQ

luxS luxPQ

LuxS

AI-2

cqsA cqsS

CqsA

CAI-1

CqsS

luxN

A B C

42

MARIJKEFREDERIX

AND

J.ALLAN

DOWNIE

43QUORUM SENSING

produced by V. harveyi and its close relative Vibrio parahaemolyticus, andit is therefore proposed to be an intraspecies signal. The secondautoinducer produced by V. harveyi, CAI-1 (cholerae autoinducer-1), isproduced by CqsA (Miller et al., 2002; Higgins et al., 2007; Kelly et al.,2009). CAI-1 has been shown to be produced by many differentVibrio species, and therefore could act as an intragenus signal. The thirdautoinducer is AI-2, which is produced by LuxS in many bacterialspecies and could act as an interspecies signaling molecule (Bassler et al.,1997; Schauder et al., 2001; Chen et al., 2002). Each of these autoinducersis detected by its own two-component system sensor histidine kinase:HAI-1 by LuxN (Bassler et al., 1993; Freeman et al., 2000), CAI-1 byCqsS (Miller et al., 2002; Higgins et al., 2007) and AI-2 by the sensor histi-dine kinase complex LuxPQ (Bassler et al., 1994; Neiditch et al., 2005).The three sensor histidine kinases transmit information through aphosporylation step into the same protein, LuxU, which subsequentlyrelays the signal to LuxO (Freeman and Bassler, 1999a,b). This mechanismallows three autoinducer signals, each of which is sensing a differentaspect of the microbial community, to be integrated into one response.

At low population densities (when no autoinducers are present toactivate the cascade) LuxN, CqsS, and LuxQ function as kinases,phophorylating LuxU. LuxU-P relays the phosphate to LuxO, whichcauses this protein to be activated (Freeman and Bassler, 1999a; Lilleyand Bassler, 2000). Activated LuxO-P then induces the expression of quo-rum regulatory RNAs (Qrrs; Tu and Bassler, 2007). The Qrrs interact with

Figure 5 QS inVibrio species.V. harveyi (A),V. cholerae (B), andV. fischeri (C)all produce more than one kind of autoinducer, and when these are perceived, thesignals are integrated in one central signaling cascade which is similar in all threeorganisms; for a review see (Milton, 2006). V. harveyi produces HAI-1 (LuxM),CAI-1 (CqsA), and AI-2 (LuxS), which are perceived by the sensor histidine kinasesLuxN, CqsS, and LuxPQ, respectively.V. cholerae produces CAI-1 (CqsA) and AI-2(LuxS), which are perceived by the sensor histidine kinases CqsS and LuxPQ, respec-tively. V. fischeri produces C8-HSL (AinS) and AI-2 (LuxS) and 3-oxo-C6-HSL(LuxI), which are perceived by AinR and LuxPQ, respectively. At low populationdensities, LuxO-P is phosphorylated and induces the expression of the Qrr sRNA’s,which repress translation of theQS regulator LitR and interact withHfq.At high pop-ulation densities, the sensor kinases are activated, which leads to dephosphorylationof LuxU. LuxU subsequently dephosphorylates LuxO, thus reducing the expressionof the Qrrs and inducing the expression of the QS master regulators LuxR(vh) (A),HapR (B), and LitR (C). In V. fischeri (C) LitR induces the expression of ainS andthe luxR-type regulator luxR(vf). LuxR(vf) is activated by LuxI-made 3-oxo-C6-HSL. Other components that affect gene regulation have been identified, includings54, Fis, cAMP–CRP, VarA/S, FliA, MetR, and ArcA/B.


the sRNA chaperone Hfq, and together they bind to the luxR(vh) mRNA,thus blocking translation of the QS ‘master’ regulator LuxR(vh)(Showalter et al., 1990; Lenz et al., 2004; Tu and Bassler, 2007; Fig. 5A).To avoid confusion in nomenclature with V. fischeri LuxR (which will bediscussed later), V. harveyi LuxR is represented as LuxR(vh) while V.fischeri LuxR is represented as LuxR(vf) in this text. V. harveyi is capableof responding gradually to the presence of Qrrs (Tu and Bassler, 2007),which allows for the integration of the QS response with other environ-mental queues at the level of Qrr transcription. At high population den-sities, the presence of the autoinducer molecules switches the function ofLuxN, CqsS, and LuxQ to phosphatases, ultimately leading to a dephos-phorylation of LuxO-P, and thus repressing the expression of the Qrrs.As a consequence, LuxR(vh) protein is produced and this regulatory pro-tein is responsible for the activation or repression of QS responsive genes(Showalter et al., 1990; Swartzman et al., 1992; Pompeani et al., 2008).

6.2.2. Vibrio cholerae

QS in the human pathogen V. cholerae is very similar to V. harveyi, but itonly produces CAI-1 and AI-2 and not HAI-1 (Miller et al., 2002; Fig. 5B).At high population densities, V. cholerae QS represses biofilm formationand the expression of the virulence genes (Zhu et al., 2002; Hammer andBassler, 2003; Higgins et al., 2007). As in V. harveyi, the CAI-1 and AI-2signals are transmitted to LuxU and LuxO to affect the expression levelof the Qrr sRNA’s. While the Qrrs of V. harveyi function in an additiveway, the Qrrs of V. cholerae function redundantly (Lenz et al., 2004). Thismeans that V. cholerae is extremely sensitive to the presence ofautoinducers, and only one Qrr needs to be present for full repression ofthe QS regulator hapR.

Even in the absence of LuxU, the response regulator LuxO can controlgene expression in a population-dependent way (Miller et al., 2002). Thesmall nucleoid protein Fis, which is highly expressed at low populationdensities (Ishihama, 1999) is required for the expression of the V. choleraeQrr sRNA’s and this occurs probably due to direct binding of Fis to thepromoter region of the Qrr sRNAs (Lenz and Bassler, 2007). The Qrrsbind to and inactivate the stability of hapR mRNA, which encodes theQS master regulator HapR (Kovacikova and Skorupski, 2002; Zhu et al.,2002). HapR represses its own expression in two ways: at high populationdensities HapR binds directly to its own promoter (Lin et al., 2005), whileat low population densities HapR activates the transcription of the QrrsRNA’s, thus indirectly destabilizing hapR mRNA (Svenningsen et al.,2008). The latter is thought to speed up the inactivation of the QS response

45QUORUM SENSING

of V. cholerae cells when the population density reduces, for example afterinvasion of a host. This provides a mechanism to evade the host’s immuneresponse, as it is important that upon invasion the QS controlled virulencegenes are inactivated as quickly as possible.

6.2.3. Vibrio fischeri

In the squid symbiont V. fischeri the situation is slightly different from thatin V. harveyi or V. cholerae. V. fischeri contains three QS systems, encodedby ainS/R (Gilson et al., 1995), luxI/R (Eberhard et al., 1981; Engebrechtand Silverman, 1984) and luxS/PQ (Lupp and Ruby, 2004; Fig. 5C). Theseare responsible for regulating the expression of the luminescence genesand colonization factors in the light organ of the squid and are organizedin a hierarchical fashion (Lupp et al., 2003; Lupp and Ruby, 2005).

LuxI synthesizes 3-oxo-C6-HSL, which activates the LuxR(vf) regulator(Engebrecht and Silverman, 1984). Note that this LuxR(vf) regulator is nothomologous to that described previously for V. harveyi LuxR(vh). The V.fisheri ainS/R and luxS/PQ genes are similar to the V. harveyi luxM/N andluxS/PQ genes and they function similarly: AinS makes C8-HSL, which issensed by the sensor histidine kinase AinR (Kuo et al., 1994; Gilson et al.,1995), while LuxS-madeAI-2 is sensed by the LuxPQ sensor histidine kinasecomplex (Lupp and Ruby, 2004). As inV. harveyi andV. cholerae, high pop-ulation density is sensed by AinR and LuxPQ to induce a phosphorelay viaLuxO to relieve repression of the transcriptional regulator LitR (Miyashiroet al., 2010), which is homologous to LuxR(vh) in V. harveyi and HapR inV. cholerae (Fidopiastis et al., 2002; Miyamoto et al., 2003).

Expression of luxS/PQ and ainS/R leads to the induction of the productionof LuxI-made AHLs and control is exerted at two levels. First, AinS-madeAHLs are able to weakly activate LuxR(vf) (Lupp et al., 2003). The AinS-made AHLs may compete with the LuxI-made AHLs, and this could ensurethat higher population densities are reached before full activation of LuxR.Second, LitR induces luxR(vf) (Fidopiastis et al., 2002) and also induces ainS,thus establishing a positive feedback loop (Lupp and Ruby, 2004).

7. ENVIRONMENTAL SIGNALS AFFECTING QS GENEREGULATION

Transcriptome analysis of QS regulatory genes in different species hasshown that factors like medium composition, temperature, oxygen avail-ability, pH, glucose availability, osmolarity, and redox state have a drastic


impact on the expression of QS regulatory and QS-regulated genes(Surette and Bassler, 1999; Bollinger et al., 2001; DeLisa et al., 2001;Wagner et al., 2003; Bazire et al., 2005; Kim et al., 2005; McGowan et al.,2005; Duan and Surette, 2007; Sonck et al., 2009). Understanding howthe expression of QS genes is modified by environmental factors mightgive clues for new antivirulence approaches that combat the activation ofQS. In many cases the regulatory mechanisms behind these changes inexpression are not clear and can probably be attributed to a generalchange in metabolic activity in the cell or lactonolysis of AHLs by pH ortemperature. For example, in most Erwinia carotovora species, highertemperatures caused a reduction in production of AHLs (Hasegawaet al., 2005; McGowan et al., 2005). In Yersinia pseudotuberculosis,increased temperatures caused degradation of AHLs, thus reducing theQS-dependent expression of the flagella genes (Yates et al., 2002). This isthought to provide a mechanism for the bacteria to swim until they areinside the host, but stop swimming thereafter.

7.1. Nutrients

Although many effects of nutrient limitation on QS can probably partiallybe attributed to a change in metabolic state, there are specific regulatorymechanisms in place that couple nutrient sensing and QS gene regulation.Low nutrient availability can prevent bacteria from growing to high popu-lation densities and so, in some circumstances, it is beneficial to elicit a QSresponse at low population densities. In addition, many pathogenic bacte-ria seem to trigger QS in response to the low levels of some nutrients likeMg2þ, phosphate, etc. Bacteria have different regulatory systems in placeto sense and respond to nutrient conditions.

7.1.1. The Stringent Response

When confronted with low nutrient availability, bacteria switch to a spe-cific metabolic state and this is known as the ‘stringent response’ (for areview see Jain et al., 2006). This state is characterized by the inhibitionof stable RNA (ribosomal and transfer RNA) synthesis as a result fromthe building up of high levels of guanosine 30,50-bidiphosphate (ppGpp)in the cell. When high concentrations of ppGpp are reached, it binds tothe b-subunit of RNA polymerase, thereby altering the promoter selectiv-ity of the RNA polymerase. In E. coli the ribosome-associated protein

47QUORUM SENSING

RelA functions as a ppGpp synthase, while SpoT functions both as both appGpp synthase and a ppGpp hydrolase. RelA mainly responds to aminoacid starvation, while SpoT responds to other starvation conditions. The‘stringent response’ encompasses an inhibition of several other cellularprocesses and in several bacteria it is involved in modifying the expressionof QS genes in a low nutrient environment.

In P. aeruginosa the stringent response causes the premature inductionof QS and virulence genes (van Delden et al., 2001), some of which encodetissue-degrading enzymes. Therefore early activation of QS can enablethe bacteria to access different nutrients during infection (Winstanleyand Fothergill, 2009). Increased production of ppGpp by RelA leads toincreased production of RhlI- and LasI-made AHLs via a transcriptionaleffect on the expression of rhlR and lasR (van Delden et al., 2001; Ericksonet al., 2004). Changes in the fluidity of the cell membrane underextreme environmental conditions can also trigger ppGpp synthesis andsubsequent activation of the QS genes (Baysse et al., 2005). DksA, origi-nally identified as a repressor of rhlI (Branny et al., 2001) can stabilizethe interaction between ppGpp and RNA polymerase (Jude et al., 2003;Paul et al., 2004; Perron et al., 2005).

A role for the stringent response in modulating QS was also found inR. etli and A. tumefaciens. In R. etli mutation of relA reduced the levelsof both CinI- and RaiI-made AHL molecules (Moris et al., 2005). In con-trast, in A. tumefaciens the stringent response is responsible for the activa-tion of the lactonase AttM upon starvation, leading to a decrease in AHLproduction and conjugation of the Ti plasmid (Zhang et al., 2004).

7.1.2. Catabolite Repression

Catabolite repression was originally identified in E. coli and allows bacte-ria to adapt quickly to the presence of different carbon sources. In thepresence of multiple carbon sources, bacteria can selectively use the onethey prefer by inhibiting the expression of enzymes that catabolise non-preferred carbon sources (Stulke and Hillen, 1999; Bruckner andTitgemeyer, 2002). In E. coli the preferred carbon source is glucose, whichis taken up by the phosphoenolpyruvate phosphotransferase system andglucose inhibits adenylate cyclase (which converts ATP to cyclic adeno-sine-monophosphate or cAMP). Conversely, when glucose is absent, levelsof cAMP increase (Deutscher et al., 2006) and bind to the cAMP receptorprotein (Crp). The activated cAMP–Crp complex induces the promoters ofenzymes that catabolise less preferred carbon sources (Fic et al., 2009).


cAMP–Crp mediated carbon catabolite repression is a modulator of QSgene expression, causing an increase in AHL production when less of thepreferred substrates are present. In V. harveyi cAMP–Crp induces QS bybinding to the luxR(vh) promoter (Chatterjee et al., 2002; Fig. 5A).In V. cholerae cAMP–Crp induces biosynthesis of CAI-1 autoinducersindirectly by influencing the stability of the cqsA mRNA (Liang et al.,2007, 2008b; Fig. 5B). In V. fisheri the cAMP–Crp complex is requiredfor expression of luxR(vf), but it has not yet been shown at which level thisregulation occurs (Dunlap, 1999; Fig. 5C).

In E. coli, cAMP–Crp influences QS gene regulation in two ways (Wanget al., 2005). cAMP–Crp stimulates the production of the Hfq-bindingsRNA CyaR, which can bind to and destabilize luxS mRNA, reducingLuxS and AI-2 levels (De Lay and Gottesman, 2009). In addition,cAMP–Crp induces the lsr AI-2 uptake system gene by binding to its pro-moter (Xavier and Bassler, 2005). AI-2 is thus synthesized during earlyexponential growth (when glucose is present), but upon stationary phaseit production ceases. Instead, AI-2 is being transported into the cells,possibly to be used as an alternative carbon source.

A role for cAMP–Crp in QS gene regulation was also identified in thephytopathogen Erwinia chrysanthemi, which contains the expI/R genes.ExpR activates the virulence genes in response to ExpI-made AHLs(Nasser et al., 1998; Reverchon et al., 1998). cAMP–Crp decreases expIexpression, but increases expR expression. This could explain the observa-tion that production of AHLs decreases after a quorum has reached andwhen the bacteria enter stationary phase (Nasser et al., 1998).

Vfr, the homologue of Crp in P. aeruginosa was originally identified as avirulence factor regulator (West et al., 1994), but it appears to act differentlyfromCrp inE. coli (Suh et al., 2002) and themain regulator of carbonmetab-olism and catabolite repression in P. aeruginosa is Crc (Wolff et al., 1991).The effect of Vfr on the induction of the virulence genes was due to inductionof lasR; Vfr bound to the lasR promoter in the presence of cAMP (Albuset al., 1997). In a vfr mutant, transcription of rhlR is also reduced, but it hasnot yet been established whether this is due to a direct or indirect effect(Medina et al., 2003a). A recent study showed that Crcmodulates the expres-sion of several QS-regulated virulence genes (Linares et al., 2010).

7.1.3. Nitrogen Limitation

Sigma factors are subunits of RNA polymerase required for gene transcrip-tion to occur. The expression ofmost genes in a bacterial cell is dependent on

49QUORUM SENSING

the expression of the ‘housekeeping’ sigma factor s70, but bacteria canexpress different sigma factors in response to different environmentalconditions. These alternative sigma factors are involved in adaptation to spe-cified niches, such as interactions with eukaryotic hosts. In many bacteria alink between one of these alternative sigma factors and QS gene regulationhas been found. Under nitrogen starvation conditions the alternative sigmafactor RpoN (s54) is activated and induces the expression of genes that areinvolved in nitrogen assimilation (Hendrickson et al., 2001).

In V. cholerae and V. harveyi, the activity of the response regulatorLuxO-P requires RpoN to induce the transcription of the Qrr sRNA’s(Klose et al., 1998; Lilley and Bassler, 2000; Lenz et al., 2004). Increasedtranscription of the qrr genes causes a destabilization of the QS master reg-ulator, and thus RpoN has a negative effect on the expression ofQS-regulated genes (Fig. 5A and B).

RpoN was reported to reduce production of RhlI and LasI-made AHLsin P. aeruginosa probably due to indirect effects, as RpoN induced expres-sion of vfr and repressed expression of gacA (Heurlier et al., 2003). In con-trast RpoN was observed to increase production of RhlI-made AHLs byinduction of rhlI expression (Thompson et al., 2003) and RpoN activatedexpression of rhlR (Medina et al., 2003b).

7.1.4. Iron Limitation

There is a link between QS and iron deprivation in P. aeruginosa, asexpression of lasI and lasR increased under iron-limited conditions(Bollinger et al., 2001; Kim et al., 2005; Jensen et al., 2006). Since invasionof the host is usually characterized by a shift to low iron conditions, thiscould serve as a signal for activation of the QS genes followed by the viru-lence genes. When host tissues become damaged as a consequence of thevirulence factors, the resulting increase in iron concentrations shoulddownregulate the production of virulence factor, which could favor hostsurvival. Uptake of iron is controlled by a large set of genes, includingsiderophores, ferric uptake regulators, and sigma factors (Cornelis et al.,2009). The QS regulators PqsR (Deziel et al., 2005), VqsR (Cornelis andAendekerk, 2004; Juhas et al., 2004, 2005), LasR and RhlR (Schusteret al., 2003) induce the expression of many iron responsive genes.

An effect of low iron concentration on the induction of the QS genes isexerted at different levels. Under iron-limiting conditions the expression oflasR is increased (Kim et al., 2005) and expression of pqsR increased inresponse to the iron starvation sigma factor PvdS (Ochsner et al., 2002).


In addition, under low iron conditions, the ferric uptake regulator (Fur)increases expression of two small regulatory RNAs encoded by prrF1and prrF2. The PrrF sRNAs destabilize the mRNA of the antABC genesthat are responsible for the degradation of the PQS-precursor anthranilate,thus sparing anthranilate for PQS production and activating QS (Oglesbyet al., 2008). Finally, the ability of PQS to trap iron is likely to reduce theamount of available iron in the cell (Bredenbruch et al., 2006).

Iron deficiency also causes an activation of QS gene regulation asdescribed in B. japonicum, as production of bradyoxetin was found to bemaximal under low iron conditions (Loh et al., 2002a).

7.1.5. Phosphate

PhoR is a histidine sensor kinase that senses the amount of available inor-ganic phosphate in the environment by interacting with the ABC-typephosphate-specific transport system (Pst): at low phosphate concentrationsPhoR is activated by autophosphorylation, and then phosphorylates theresponse regulator PhoB. When there is sufficient phosphate, the Pst sys-tem is thought to form a repressing complex with PhoR, thus preventingactivation of PhoB (Lamarche et al., 2008). PhoB is not only activated byits partner histidine kinase PhoR, but also by other histidine kinases. Forexample, in E. coli the EnvZ sensor protein can activate PhoB in responseto acetylphosphate in the absence of PhoR (Kim et al., 1996). Such crosstalk allows the integration of other environmental queues through PhoB.

PhoR homologues modulate QS gene regulation in several differentbacterial species. In S. meliloti low phosphate can trigger early inductionof QS by induction of the transcriptional regulator sinR (Krol and Becker,2004) and this was due to a regulatory effect exerted by PhoB. The mech-anism by which this occurs has not yet been identified. A pho box wasfound upstream region of sinR, but deletion of this sequence did not abol-ish regulation by phoR, indicating that potentially the regulatory effect ismediated via an unidentified intermediate regulator (McIntosh et al.,2009). The premature induction of QS at low phosphate concentrationscan be beneficial: in the soil, phosphate levels are usually low due to phos-phate uptake by plants which creates a zone of phosphate depletion in therhizosphere. Therefore, without induction by PhoB, the rhizobia might notbe able to grow to a sufficiently high population density to activate QS(Schachtman et al., 1998).

Other species in which low phosphate conditions induced QS have beenidentified. In Serratia sp. ATCC39006 phosphorylated PhoB induced the

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AHL synthase encoded by smaI (Gristwood et al., 2009), while inP. aeruginosa low phosphate conditions induced expression of rhlR andpqsR (Jensen et al., 2006; Zaborin et al., 2009).

7.1.6. Magnesium

The PhoP/Q two-component regulatory system mediates the adaptation ofan organism to Mg2þ concentrations, with PhoP serving as the responseregulator and PhoQ as the histidine kinase sensor protein. It was firstdescribed in Salmonella enterica serovar Typhimurium where it controlsthe expression of the virulence factors (Kier et al., 1979; Miller et al.,1989; Groisman, 2001). PhoP induced the expression of the AHL synthaseencoded by pcoI in P. fluorescens 2P24 in response to low Mg2þ con-centrations (Yang et al., 2009). Under the same conditions, increasedexpression of PQS biosynthesis genes and lasI was observed inP.aeruginosa, but it has not yet been investigated whether the PhoP isresponsible for this (Guina et al., 2003).

7.1.7. Amino Acids

V. harveyi contains the LysR-type regulator MetR that monitors the aminoacids in the environment. In response to homocysteine MetR causes adecrease in luminescence, and this was shown to be a direct effect as MetRbinds to the luxR promoter (Chatterjee et al., 2002; Fig. 5A).

7.2. Other Environmental Conditions

7.2.1. Oxygen

As explained previously, in X. campestris QS gene regulation relies on therecognition of DSF by the sensor kinase RpfC (Fig. 6). The signal is trans-mitted to the HD-GYP protein RpfG, causing hydrolysis of cyclic-di-GMPto cGMP. The altered levels of cGMP are sensed by the transcriptionalregulator Clp, which induces target gene expression (Fouhy et al., 2006;Ryan et al., 2006; He et al., 2007; Tao et al., 2010). Therefore the presenceof other enzymes that modulate the levels of cGMP in the cell could alterthe QS response.

RpfC

RpfF

RpfG

cyclic di-GMP cGMP

Clp

RavS

Low oxygen

RavR

rpfG

clp

rpfCrpfF

Figure 6 QS in X. campestris. DSF is produced by RpfF and is sensed by thetwo-component sensor kinase RpfC. RpfC transmits the signal to the HD-GYPprotein RpfG. RpfG has a HD-GYP domain hydrolyses cyclic-di-GMP to cGMP.The regulator Clp senses cGMP and induces gene expression in response (Wanget al., 2004). cGMP levels are also modulated by the presence of the RavS sensorkinase, which senses low oxygen conditions (Kaplan et al., 2009).


Intracellular cyclic-di-GMP levels are typically modulated in two ways:proteins containing a GGDEF domain are responsible for the synthesisof cyclic-di-GMP, while proteins that contain an EAL or HD-GYP domaindegrade cyclic-di-GMP (Fouhy et al., 2006; Schirmer and Jenal, 2009).In addition, some proteins contain both a GGDEF and an EAL domain.In X. campestris the proteins containing GGDEF, EAL, or HD-GYPdomains were all analyzed by deletion mutagenesis studies. Only the dele-tion of ravR altered the DSF induced virulence response (He et al., 2009).RavR contains both a GGDEF and an EAL domain and is activated bythe sensor kinase RavS (Fig. 6). The RavR EAL domain degradedcyclic-di-GMP to cGMP, while the GGDEF domain (normally responsiblefor synthesizing cyclic-di-GMP) was found to be not functional. RavS issimilar to the oxygen-sensing protein FixL from rhizobia and containstwo domains with a conserved fold and key residues involved in haembinding (Gong et al., 1998; Key and Moffat, 2005; He et al., 2009). ThusRavR increases the amount of intracellular cGMP in response to lowoxygen tension (Fig. 6), and this is subsequently detected by the

53QUORUM SENSING

transcriptional regulator Clp, which can modulate the QS-induced viru-lence response (He et al., 2009; Chin et al., 2010).

Oxygen levels also modulate the QS response in P. aeruginosa via thetranscriptional regulator ANR, which belongs to the FNR (fumarate andnitrate reductase regulator) family and is activated under low oxygenconditions (Spiro, 1994). ANR is thought to function synergistically withLasR and RhlR (Pessi and Haas, 2000). In addition expression of LasRwas increased under oxygen stress (Kim et al., 2005).

Bioluminescence in V. fischeri is under QS control and light is generatedby induction of the luxICDABEG genes. The reaction requires oxygen andreducing power so it was predicted that the expression of the lux geneswould be under redox control (Visick et al., 2000; Timmins et al., 2001).Good candidates to mediate redox-dependent gene regulation werehomologues of ArcA and ArcB, which were originally identified in E. colias part of a redox-sensitive two-component system (Georgellis et al., 1997).In V. fischeri growing in planktonic conditions (no oxidative stress), ArcAis phosphorylated by ArcB and binds to the luxICDABEG promoter, thusblocking access of LuxR. Upon colonization of the light organ, oxidativeconditions are established, possibly due to host-generated reactive oxygenspecies. When this happens, ArcB dephosphorylates ArcA, which no lon-ger binds to the luxICDABEG promoter allowing LuxR to bind, thusinducing AHL-based QS (Bose et al., 2007; Fig. 5C).

7.2.2. FliA: Sensing Arrival at Colonization Site

V. cholerae uses the alternative sigma factor FliA to sense that it hasreached its site of colonization in the small intestine, to control QS induc-tion of the virulence genes. This control depends on the breakage offlagella during passage of the bacteria through the mucosal layer thatcovers the small intestine epithelial cells. Loss of the flagella leads to therelease of the antisigma factor FlgM, which causes the release of FliA. FliArepresses transcription of the QS regulator hapR and this causes a loss ofHapR-mediated repression (hence activation) of the virulence genes thatare under QS control (Liu et al., 2008; Tsou et al., 2008; Fig. 5B).

7.2.3. varA/S (gacA/S)

The best studied two-component system with regards to its effect on QSgene regulation is the gacA/S system in P. aeruginosa (Reimmann et al.,


1997), which is called varA/S inV. cholerae (Lenz et al., 2005). Pseudomonasand Vibrio species that grow to high population densities secreteGacA-activating signals, which are chemically unrelated to AHLs orAI-2. Because the signal accumulates at high population densities, theGacA/S two component system has been proposed to function as a QS sys-tem itself (Dubuis and Haas, 2007). In V. cholerae, VarA and VarS controlthe transcription of three sRNAs (CsrB, CsrC, and CsrD) that are homolo-gous to the E. coli carbon storage regulator sRNAs CsrB and CsrC. ThesesRNAs bind to and inactivate CsrA (Lenz et al., 2005), which post-transcriptionally regulates the levels of LuxO and thus the expression ofthe Qrr sRNAs. At low population densities VarS is not activated, andhence there is no transcription of the csr sRNA’s. This means CsrA isactive and increases the amount of the response regulator LuxO-P byincreasing the levels of luxO mRNA, which leads to the induction of theQrr sRNA’s. The effect of CsrA on the amount of luxO mRNA is proba-bly not direct, but appears to be mediated by an as yet unidentified protein(Lenz et al., 2005; Fig. 5B). The influence of the VarA on QS is not con-served in all Vibrio species, as in V. fischeri no effect of VarA and VarSon AHL production could be observed (Whistler and Ruby, 2003).

In several Pseudomonas species GacA and GacS induce the productionof AHLs (Reimmann et al., 1997; Chancey et al., 1999; Quinones et al.,2004; Kay et al., 2006). In P. aeruginosa GacA induced the expression ofthe secondary metabolite sRNAs (RsmY and RsmZ; Heurlier et al.,2004; Kay et al., 2006) that are capable of binding and inactivating RsmA,a sRNA binding protein homologous to CsrA. When active, RsmAreduces the expression of rhlI and lasI and the amount of RhlI- andLasI-made AHLs (Pessi and Haas, 2001; Kay et al., 2006) probably dueto reduced expression of rhlR and lasR (Reimmann et al., 1997). Theglobal RNA chaperone Hfq binds to and stabilizes RsmY (Sonnleitneret al., 2006). GacA and GacS also affect QS by inducing the expressionof the luxR-type regulator qscR (Ledgham et al., 2003a). Two other sensorkinases-response regulator hybrids, LadS and RetS, control the expressionof the sRNA RsmZ, affecting the activity of RsmA (Ventre et al., 2006).Thus LadS, RetS, and GacS represent three different sensor kinases, whichintegrate different signals into one central signaling cascade. GacA andGacS regulation of QS genes was also observed in other pseudomonads,like P. aureofaciens (Chancey et al., 1999; Zhang and Pierson, 2001),Pseudomonas syringae (Kitten et al., 1998; Quinones et al., 2004), Pseudo-monas sp. M18 (Wang et al., 2008), and P. fluorescens (Yan et al., 2009).

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7.2.4. RpoS: Different Stresses

The sigma factor RpoS (s38) is activated in stationary phase (Lange andHengge-Aronis, 1991) and in response to stresses like UV radiation, acid,temperature, osmotic shock, oxidative stress and nutrient deprivation(Klauck et al., 2007; Durfee et al., 2008). The role of RpoS in P. aeruginosaQS seems to be dependent on the experimental conditions used. Initialstudies backed up by microarrays showed that RhlR activated transcriptionof rpoS (Latifi et al., 1996; Schuster et al., 2003, 2004; Wagner et al., 2003).Other studies found that rpoS expression was not regulated by QS(Whiteley et al., 2000; Bertani et al., 2003), but instead RpoS repressed rhlIexpression (Whiteley et al., 2000). Repression of QS by RpoS was alsoobserved in P. syringae (Chatterjee et al., 2007) and P. fluorescens (Yanet al., 2009) and in stationary phase, more than 40% of the P. aeruginosagenes that were controlled by QS were also controlled by RpoS (Schusteret al., 2004). RpoS repressed the expression of some QS-induced genes likehcnABC and phzABC (Whiteley et al., 2000), while another reportsuggested that RpoS induced the expression of QS-induced genes likerhlAB (Medina et al., 2003b). To explain these seemingly contradictoryobservations a model was proposed (Schuster et al., 2004) in which RpoSand QS-regulated genes were divided into different categories, dependingon whether the regulatory effects of both factors are direct or indirect.Other factors can modulate QS gene regulation in Pseudomonas spp.through their regulatory effects on the sigma factor RpoS. The long chainfatty acid sensor PsrA increased AHL production, probably via an indirecteffect, by induction of rpoS expression (Kojic and Venturi, 2001; Girardet al., 2006; Chatterjee et al., 2007; Kang et al., 2009).

In several other species, a role for RpoS modulating QS has beenshown. In Edwardsiella tarda RpoS repressed the expression of the AI-2synthase luxS (Xiao et al., 2009). In R. solanacearum RpoS induced expres-sion of solR and solI (Flavier et al., 1998). In E. coli RpoS had a dual effect,repressing lsr expression, resulting in reduced uptake of AI-2 and inducingthe luxS homologue ygaG (Lelong et al., 2007). In V. anguillarum RpoSinduced the expression of the QS master regulator VanT (the homologueof the regulator LuxR(vh) in V. harveyi and HapR in V. cholerae). Thiseffect was indirect and mediated through the repression of the expressionof hfq, destabilizing the Qrrs and thus stabilizing vanT mRNA (Weberet al., 2008).


8. SIGNALS FROM OTHER SPECIES

8.1. Microbial Cross-Communication

In natural conditions, bacteria usually occur as a mixture of species, whichhave developed means of communicating with each other. Bacteria arethought to use AI-2 for interspecies communication and the variable chem-ical nature of AHLs allows intraspecies communication, as described ear-lier for Vibrio species. However LuxR-type regulators can also interactwith noncognate AHLs, and such interactions could lead to an unwantedactivation or inhibition of QS (Schaefer et al., 1996a; McClean et al.,1997; Zhu et al., 1998; Welch et al., 2000). This could explain why otherQS signals, like PQS in Pseudomonas species, 3-OH PAME inR. solanacearum, bradyoxetin in B. japonicum and DSF in Xanthomonasspecies have been adopted, as a lack of cross talk could provide a selectiveadvantage. In addition, several species have LuxR-type transcriptional reg-ulators, although they do not produce any AHLs, which could enable themto listen into conversations between other bacteria. For example, E. coliand S. enterica serovar Typhimurium contain the LuxR-type regulatorSdiA, which can be activated by AHLs produced by other bacteria, possi-bly to indicate their arrival in the right environment to induce their viru-lence genes (Ahmer et al., 1998; Kanamaru et al., 2000; Michael et al.,2001). Another mechanism to alter the bacterial QS response is by the pro-duction of autoinducer-degrading enzymes as described above.

8.2. Communication with Eukaryotes

Examples of QS gene regulation being used to influence the relationshipbetween bacteria and their eukaryotic hosts include virulence gene expres-sion in the human pathogens P. aeruginosa (Bjarnsholt and Givskov,2007), V. cholerae (Higgins et al., 2007), pathogenic E. coli (Sircili et al.,2004), and S. enterica serovar Typhimurium (Choi et al., 2007). Examplesof bacterial QS influencing interactions in the plant kingdom include thoseseen with interactions between plants and their nitrogen-fixing rhizobialsymbionts (Downie and Gonzalez, 2008) and with the plant pathogensR. solanacearum (Genin et al., 2005), E. carotovora (Barnard and Salmond,2007), A. tumefaciens (White and Winans, 2007), X. campestris (He andZhang, 2008) and S. marcescens (Coulthurst et al., 2004). In the marineenvironment, bioluminescence produced in the squid light organ by

57QUORUM SENSING

V. fischeri (Fidopiastis et al., 2002) and V. harveyi (Bassler et al., 1993), isimportant for squid competitiveness and V. anguillarum QS signalsenhance biofilm formation by the green seaweed Ulva (Joint et al., 2006).It is therefore not surprising that the eukaryotic hosts have developedmechanisms to modulate bacterial QS. Eukaryotic signals have beenidentified that interfere with bacterial QS (‘quorum quenching’), eitherby affecting the expression of the QS genes, the production of AHLmimics or by the production of autoinducer-degrading enzymes, thus alter-ing the level of autoinducers that are perceived by the bacteria rather thendirectly altering their level of production.

Expression of the virulence genes in the plant pathogen Pantoeaagglomerans (also known as Erwinia herbicola) is induced by the pagI/RQSgenes.Upon formation of a plant tumor, the plant produces the plant hor-mones indole acetic acid (IAA) and cytokinin and these modulate theexpression of pagI and pagR (Chalupowicz et al., 2009). In E. chrysanthemithe transcriptional regulator PecS, which can bind to plant-made urate (Per-era and Grove, 2010), repressed the AHL synthase gene expI (Reverchonet al., 1998). Rhodopseudomonas palustris produces a new class of AHLmolecules (coumaroyl-AHLs), that require plant-produced coumaric acidrather than fatty acids as substrate for the biosynthesis (Schaefer et al.,2008). Many other examples of plant metabolites that affect bacterial QShave been found in plant essential oils and extracts, but is often not knownhow they function (Al-Hussaini and Mahasneh, 2009; Bodini et al., 2009;Feldman et al., 2009; Khan et al., 2009; Truchado et al., 2009).

In some cases a direct interaction of the compounds (AHL mimics) withthe autoinducer receptor has been shown. The marine red alga Deliseapulchra inhibitis QS gene regulation by production of halogenated furanonesthat interact with bacterial AHL receptors, leading to degradation of thereceptor (Givskov et al., 1996; Manefield et al., 2002). Chlamydomonas spe-cies produce a variety of AHLmimics, capable of activating some QS genes,while repressing others (Teplitski et al., 2004; Rajamani et al., 2008). Med-icago sativa produces L-canavanine, which can interferewithQS in S.meliloti(Keshavan et al., 2005). Other higher plants, such as Medicago truncatula,pea, vetch, soybean, tomato, and rice have been shown to produce AHLmimics (Teplitski et al., 2000; Gao et al., 2003; Degrassi et al., 2007). InXanthomonas oryzae and Xanthomonas campestris currently unidentifiedcompounds in plant exudates were shown to activate the orphan LuxR-typeregulators OryR and XccR (Ferluga and Venturi, 2009).

Animal metabolites also modulate QS gene expression. For example,upon infection with P. aeruginosa, host stress can cause the release of themorphine-like chemical dynorphin, which can induce the pqs genes to


induce virulence (Zaborina et al., 2007). In enterohemorrhagic E. coli(EHEC), autoinducer AI-3 is thought to resemble the chemical structureof the mammalian hormones epinephrine and norepinephrine (Sperandioet al., 2003). Therefore, when EHEC is present in the human colon, itcan recognize these hormones and use them for the activation of thevirulence genes (Clarke et al., 2006). The bacterivorous nematodeCaenorhabditis elegans produces compounds that inhibit PseudomonasQS (Kaplan et al., 2009).

Eukaryotes can also affect autoinducer degradation. For example, inA. tumefaciens plant signals alter the expression levels of AHL lactonasesthat are encoded in the bacterial genome as a manner of defense againstinvasion (Chevrot et al., 2006; Haudecoeur et al., 2009a,b). Other examplesinclude the inactivation of Pseudomonas AHLs by human airway epithelia(Chun et al., 2004) and the degradation of AHLs by mammal paraoxonases(Draganov et al., 2005; Ozer et al., 2005; Teiber et al., 2008).

ACKNOWLEDGMENTS

We thank Anne Edwards for comments on the text. J.A.D. and M.F. aresupported by the Biotechnology and Biological Sciences Research Counciland M.F. received a Marie-Curie short term EST fellowship (019727) fromthe EU.

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[Advances in Microbial Physiology] Volume 58 || Quorum Sensing