Signalling: Listening in on bacteria: acyl-homoserine lactone signalling

Roughly 35 years ago, two papers were published thatreached the same surprising conclusion: that individualbacteria could use chemical signals to communicate andto co-ordinate group activities. One publication indi-cated that the Gram-positive bacterium Streptococcuspneumoniae controlled factors for GENETIC COMPETENCE by aself-produced chemical cue called the competencefactor1. The other publication described the control ofluminescence in a marine Gram-negative bacterium,now called Vibrio fischeri, by a different self-produced sig-nal that was described as an autoinducer2.

These publications were ahead of their time — biolo-gists were not ready for the idea that bacteria were talkingto each other. Nevertheless, that is exactly what manybacteria do. The genetic and chemical details of the V. fis-cheri and S. pneumoniae signalling systems are now rea-sonably well understood, and many microbes are knownto use cell-to-cell signalling mechanisms. Although ittook nearly 30 years, once microbiologists accepted thefact that communication between individuals was notrestricted to ‘higher organisms’or ‘special bacteria’ (suchas the fruiting myxobacteria), the floodgates opened.

Signalling is generally described as quorum sensing,in reference to the frequent observation that the signalsonly accumulate in environments that support a suffi-ciently dense population — a quorum — of signal-generating bacteria3,4. When a quorum-sensing signalmolecule reaches a critical level, the population at large

responds, usually through the co-ordinated expressionof specific target genes. In V. fischeri, the targets are theluminescence (lux) genes; in S. pneumoniae, the targetgenes code for genetic competence (com). We nowknow that quorum sensing is a common attribute ofmany bacterial species, and that it might in fact be auniversal feature of bacteria. New signals and new sys-tems are being discovered at a rapid pace.

The details of the V. fischeri quorum-sensing systemwere revealed first. In 1981, the ‘autoinducer’ signal wasfound to be a specific acylated derivative of homoser-ine lactone — N-3-oxo-hexanoyl-L-homoserine lac-tone5. Fortuitously, in 1983, the genes for the signal-generating enzyme, the signal receptor and the luxgenes that they regulate were isolated together on a sin-gle DNA fragment6 (FIG. 1). Even more surprisingly, reg-ulation of the lux genes on this fragment in Escherichiacoli was very similar to their regulation in V. fischeri. Inthe early 1990s, homologues of the lux-signal-generat-ing enzyme, the signal receptor and acylated homoser-ine lactones (acyl-HSLs) were discovered3,7. There arenow over 50 known examples of such components inbacterial species, thus far restricted to members of thePROTEOBACTERIA8.

In this review, we concentrate on the current mecha-nistic understanding of V. fischeri LuxR–LuxI-systemhomologues, their relevance to microbial activity andpotential uses of this understanding in the control of

LISTENING IN ON BACTERIA:ACYL-HOMOSERINE LACTONESIGNALLINGClay Fuqua* and E. Peter Greenberg‡

Bacterial cell-to-cell signalling has emerged as a new area in microbiology. Individual bacterialcells communicate with each other and co-ordinate group activities. Although a lot of detail isknown about the mechanisms of a few well-characterized bacterial communication systems,other systems have been discovered only recently. Bacterial intercellular communication hasbecome a target for the development of new anti-virulence drugs.

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 3 | SEPTEMBER 2002 | 685

*Department of Biology,Indiana University, JordanHall 142, 1001 East 3rdStreet, Bloomington,Indiana 47405, USA.‡Department ofMicrobiology, University ofIowa, Iowa City, Iowa52242, USA.Correspondence to E.P.G.e-mail: [email protected]:10.1038/nrm907

GENETIC COMPETENCE

The ability to take up nakedDNA from the externalenvironment through theprocess of transformation.

PROTEOBACTERIA

A large, physiologically diversephylogenetic group of Gram-negative bacteria, also known asthe purple bacteria.

R E V I E W S

S I G N A L L I N G

© 2002 Nature Publishing Group

686 | SEPTEMBER 2002 | VOLUME 3 www.nature.com/reviews/molcellbio

R E V I E W S

homoserine through an amide bond. There is consider-able structural variety between acyl-HSLs from differentbacteria and even between different acyl-HSLs synthesizedby the same bacterium. The acyl chain lengths can varybetween 4 and 16 carbons, usually by increments of twocarbon units (C4, C6, C8 etc.); the C16-HSL is producedby Rhodobacter capsulatus (A. L. Schaefer and E.P.G.,unpublished observations). The third carbon in the acylchain might either be a fully oxidized carbonyl, carry ahydroxyl group or be fully reduced.The variety at the thirdposition reflects the derivation of the acyl chains fromfatty-acid biosynthesis (see below).Two of the longer acyl-HSLs have a single unsaturated bond in the middle of theacyl chain9,10. Recently, an acyl-HSL with a seven-carbonacyl chain was identified, but the source of this odd-num-bered acyl chain has not been elucidated11.

Acyl-HSLs have been abbreviated in several differentways, based on their microbe of origin and limitedaspects of their chemistry. In this review, we indicate theoxidation state of the third acyl chain carbon with a pre-fix (3-oxo, 3-OH or no prefix), followed by the chainlength (C4, C6, C8 etc.) and the abbreviation HSL. Forexample, N-3-oxo-hexanoyl-homoserine lactone isabbreviated as 3-oxo-C6-HSL and N-butyryl-homoser-ine lactone is C4-HSL.

Acyl-HSL signallingIn Vibrio fischeri. The essential components of the V. fis-cheri system are shown in FIG. 1 and FIG. 2. The V. fischerisignal is enzymatically synthesized by the product of agene called luxI and can diffuse freely across biologicalmembranes6,12. The signal receptor encoded by the luxRgene is an acyl-HSL-responsive transcriptional activatorthat is not a transmembrane protein but, rather, residesin the cytoplasm6,13. LuxR binds to a 20-bp element (nowcalled the lux box) centred −42.5 bp from the transcrip-tion start of the first gene in the luminescence operon(FIG. 1a) and interacts with RNA polymerase (RNAP) tostimulate transcription of the operon14,15.

Why does V. fischeri have this specific, elegant sig-nalling system? This symbiotic bacterium colonizes thelight organs of a range of marine fishes and squids, inwhich it grows to very high densities (1010–1011 cells perml) and produces light16. It also exists at low densities inother marine environments and can be found free inseawater at low densities (up to a few hundred cells perml)17,18. The V. fischeri quorum-sensing system allowsindividual cells to distinguish between low- and high-population-density environments. In the high-densitylight-organ environment, lux genes will be activatedand the bacteria will produce light, which is beneficialto the animal. In exchange, the animal provides the bac-teria with nutrients. At low population densities in theseawater environment, the V. fischeri signal cannotaccumulate and luminescence is not induced. There isno known function for light production outside thehost organism (the amount of light produced by a fewhundred cells per ml is too weak to be detected biologi-cally). When free in seawater, a relatively nutrient-poorenvironment, the bacteria do not expend energy onunnecessary light production.

microbial pathogenesis (BOXES 1, 2). Although there areseveral recognized interbacterial signalling mechanisms,the LuxR–LuxI-type systems are among the best under-stood at the molecular level.Also, because they often reg-ulate functions required for host–microbe interactions,LuxR–LuxI systems are of great significance for combat-ing infectious disease and, conversely, for harnessingbeneficial microbial activities. Several other emergingsignalling systems are described in BOXES 3 AND 4.

Structural diversity of acyl-HSLsAcyl-HSLs have been identified in many differentProteobacteria. In general, these signal molecules arecomposed of a fatty acyl chain ligated to a lactonized

Figure 1 | Vibrio fischeri lux-gene organization and symbiotic bioluminescence. a | The luxoperon contains luxI followed by five genes that are required for light production (luxCDABE) and anadditional gene of unknown function (luxG). The luxC, luxD and luxE genes code for components ofan acid reductase that converts the long-chain fatty acid tetradecanoic acid into the fatty-aldehydesubstrate (tetradecanal) for the light-producing enzyme luciferase. The luxA and luxB genes encodethe α and β subunits of luciferase. The luxI gene encodes the enzyme (autoinducer (AI) acyl-homoserine lactone synthase) that produces the quorum-sensing signal 3-oxo-C6-HSL. The singlegene transcribed in the opposite direction, luxR, encodes the signal-responsive transcriptionactivator of the luxICDABEG operon. Modified with permission from REF. 113 © (1999) CambridgeUniversity Press. b | An Australian pinecone fish (~12 cm long). The red organ on the lower jaw is alight organ that contains ~1010 V. fischeri cells per ml fluid. The light organ appears red in thisphotograph because the light-organ tissue is highly vascularized. Australian pinecone fish arenocturnal reef dwellers and they use the light organ to search for prey at night. c | A Hawaiian bobtailsquid. This adult squid is ~2 cm long. There is a V. fischeri light organ close to the ink sac within themantle cavity of the animal. This light organ contains ~1011 V. fischeri cells per ml. These nocturnalsquid emit light downwards through the mantle cavity and, by matching the intensity of the moon-and starlight above, they become invisible to predators below them. Images b and c kindly providedby Edward G. Ruby (University of Hawaii, USA).

R I C D A B E G

Transcriptionactivator

AI synthase Luciferase

Acid reductase

0 1 2 3 4 5 6 7 8Kilobase pairs

?

b

a

c



R E V I E W S

hierarchy23,34–36. In addition, promoters that are rec-ognized by RhlR are also recognized to a lesser extentby LasR, but not vice versa37. This contributes to thedominance of the LasR system over the RhlR system.Mutations in either quorum-sensing system reducethe virulence of P. aeruginosa38,39.

Acyl-HSL synthasesMore than one family? Members of the LuxI family ofproteins are synthases that catalyse the production ofacyl-HSLs. The acyl portion of the acyl-HSL is derivedfrom fatty-acid precursors conjugated to the ACYL CARRIER

PROTEIN (acyl-ACP), and the HSL moiety is derived fromS-adenosylmethionine (SAM)40,41. Many different LuxI-type proteins have been identified from a range ofProteobacteria; these are 190–230 amino acids long andshare 30–35% pairwise identity. Ten residues conservedwithin most LuxI-type proteins cluster in the amino-terminal 110 amino acids (FIG. 3).

There is no simple correlation between the acyl-HSLsynthesized and the level of sequence identity betweenproteins. However, it has been noted that many of theLuxI-type proteins that direct the synthesis of 3-oxo-acyl-HSLs have a conserved threonine residue at posi-tion 143 (numbering relative to LuxI). Consistent withthis, recent structural and mutational analysis of theEsaI acyl-HSL synthase from Pantoea stewartii (for-merly Erwinia stewartii) indicates that this threonineresidue might be involved in stabilizing interactionswith fatty-acyl biosynthetic precursors carrying a car-bonyl group at the third position in the acyl chain (incontrast to derivatives that have a hydroxyl group or arefully reduced at this position)42. The crystal structurealso reveals that EsaI and other acyl-HSL synthasesshare structural similarity with the N-acetyltransferases(enzymes from eukaryotic organisms that use a fatty-acyl precursor similar to that of acyl-HSL precursors).The structural data from EsaI should facilitate the devel-opment of new antimicrobial compounds that targetacyl-HSL synthesis.

The LuxM, AinS and VanM proteins of Vibrio har-veyi, V. fischeri and Vibrio anguillarum, respectively,define a second distinct, albeit small, family of acyl-HSLsynthases19,20,43,44. These proteins are required for thesynthesis of 3-OH-C4-HSL, C8-HSL and 3-OH-C6-HSL, respectively, but show no sequence similarity toLuxI. Purified AinS protein directs the synthesis of C8-HSL from SAM and C8-ACP, as observed with LuxI-type proteins20. In contrast to LuxI-type proteins, how-ever, AinS can also use an octanyl-coenzyme-Aconjugate as efficiently as the ACP conjugate, implyingsome mechanistic divergence between these two fami-lies of synthases.

Recently, it was reported that the HdtS protein ofPseudomonas fluorescens directs the synthesis of smallamounts of an acyl-HSL when produced in E. coli45.HdtS is homologous to neither LuxI- or AinS-type pro-teins but rather to the lysophosphatidic-acid-acyl-trans-ferase family, members of which are generally involvedin the biosynthesis of the phospholipid precursor phos-phatidic acid in diverse prokaryotic and eukaryotic

Much more recently, a gene called ainS was isolatedfrom V. fischeri, the translation product of which cansynthesize C8-HSL19–21. Although there is no LuxR-typeprotein associated with ainS, there is an adjacent gene,tentatively designated ainR, that encodes a homologueof sensor kinases from TWO-COMPONENT SYSTEMS and is pos-tulated to be involved in C8-HSL regulation19. However,no target genes under the direct influence of C8-HSL orainR have been identified, and the function of this sys-tem remains unclear.

In other bacteria. We now know that manyProteobacteria have homologues of LuxR and LuxI,and produce and respond to acyl-HSLs, but differentbacteria regulate different target genes with these acyl-HSL quorum-sensing systems8,22 (TABLE 1). A well-studied example is Pseudomonas aeruginosa. This bac-terium has two LuxR–LuxI quorum-sensing systems,which are responsible for the global control of per-haps 2–5% of its genes23. The P. aeruginosa genes con-trolled by quorum sensing encode a range of func-tions including EXOENZYME synthesis, virulence-factorsynthesis, secondary metabolism and the develop-ment of sessile multicellular structures calledBIOFILMS23–26 (BOX 2).

P. aeruginosa uses two quorum-sensing systems:the 3-oxo-C12-HSL–LasR–LasI system and the C4-HSL–RhlR–RhlI system27–30. The Las system was orig-inally discovered as a regulator of the virulence factorelastase but is now known to regulate many differentgenes24. The Rhl system was identified as a regulatorof rhamnolipid surfactant biosynthesis and is alsonow recognized to have many additional target func-tions30–33; indeed, the main quorum-sensing controlof elastase is through the Rhl system23. The LasR–LasIsystem is required for induction of the RhlR–RhlI sys-tem, thereby creating a quorum-sensing regulatory

TWO-COMPONENT SYSTEMS

A common bacterial signal-transduction system that iscomposed of at least twocomponents — a sensor kinase(which alters its rate ofautophosphorylation inresponse to specificenvironmental conditions) and aresponse regulator (to which thephosphate group is transferred,and which transduces theregulatory signal to cellularprocesses such as geneexpression).

EXOENZYME

An enzyme that is secreted to theexternal environment across thebacterial envelope.

BIOFILMS

Surface-adherent microbialpopulations, usually embeddedwithin a self-produced matrixmaterial.

ACYL CARRIER PROTEIN

Conserved protein required forfatty-acid biosynthesis. Fatty-acid intermediates are covalentlyassociated with acyl carrierprotein through thephosphopantethiene prostheticgroup.

Box 1 | Microbial ‘biowarfare’ and biotechnology

A mechanism to confound bacterial communication pathways might involve thespecific degradation or modification of the signal molecules. Certain members of thegenus Bacillus were shown to degrade acyl-homoserine lactones (acyl-HSLs) throughlactonase enzymes that attack the homoserine-lactone ring structure83. Variovoraxparadoxus strains use acyl-HSLs as a source of energy, carbon and nitrogen84, and(instead of using lactonases) cleave the acyl group from the signals as the first step oftheir catabolism.

Why should bacteria produce acyl-HSL-inactivating enzymes? Although it is plausiblethat they use acyl-HSLs as growth substrates, the signal molecules are not typicallypresent at concentrations that might support the reasonable growth of bacteria. Perhapsthese enzymes allow the metabolism of other substrates, and acyl-HSL degradation iscoincidental cross-recognition? The most intriguing possibility is that these enzymes areused to inactivate the signalling systems of competing bacteria in specific habitats —that is, they function in a form of bacterial species warfare.

Acyl-HSL-degrading enzymes and the genes that encode them might have a brightfuture in biotechnology. Recently, a cloned Bacillus lactonase gene was used to generatetransgenic plants85. The recombinant plants were challenged with Erwinia carotovora(which requires quorum sensing for virulence) and were much more resistant to E.carotovora than control plants that did not produce the lactonase86. This approach is novelbecause it does not involve killing or general growth inhibition of the pathogen,highlighting the potential use of quorum sensing as a molecular target for disease control.



R E V I E W S

on the methionine moiety of SAM and the first car-bon of the acyl chain conjugates these precursors andprecedes the release of apo-ACP. Lactonization of thehomoserine ring allows the release of C4-HSL.5′-methylthioadenosine derived from SAM is the finalproduct released from the enzyme, which can then initi-ate a new round of synthesis. Although many reactionsuse ACP conjugates to supply acyl groups, this is, to ourknowledge, a novel mechanism for SAM use and mighttherefore provide a unique target for the inhibition ofacyl-HSL quorum sensing.

Mechanism of acyl-HSL synthases. Different LuxI-typeproteins recognize the common precursor SAM, butmust specifically recognize acyl-ACPs of different chainlengths and reduction states. The ten amino-acidresidues conserved among the amino termini of all LuxI-type proteins probably function in directing the com-mon aspects of the enzymatic mechanism — recogniz-ing SAM and ACP (FIG. 3). Mutational studies of severalLuxI-type proteins have identified all of these residues asbeing important for catalysis49,50. Seven of the tenresidues carry charged side chains and are absolutelyrequired to direct the reaction. Mutation of the threeremaining conserved residues results in functional pro-teins with reduced efficiency, which indicates that theyhave less stringent roles, perhaps in the architecture of

organisms. It has not been unequivocally shown thatHdtS is a true acyl-HSL synthase, nor that it functions inthis capacity in P. fluorescens. However, if it is then itmight represent a third family of acyl-HSL synthasesand hence reveal an astonishing level of mechanisticdiversification for the generation of these cell-to-cell sig-nalling molecules.

Synthesis of acyl-HSLs. SAM is used as the HSL precur-sor by LuxI- and AinS-type synthases20,40,41,46. Studies ofP. aeruginosa RhlI indicate that acyl-HSL synthases useSAM in an enzymatically distinct way from otherenzymes that catalyse reactions involving SAM. Theprimary roles of SAM in the cell are to serve as a pre-cursor for the membrane phospholipid phosphatidyl-choline and to donate methyl groups for nucleic acidand other methylation reactions47. In addition, SAM isa substrate for other synthetic reactions (biotin andpolyamines) and for several unusual transfer-RNA basemodifications48. Acyl-HSL synthases share nodetectable similarity with other SAM-dependentenzymes, and the acyl-HSL biosynthetic reaction, asunderstood at present, is enzymatically different fromother reactions that use SAM46.

Interaction with SAM initiates the reaction catalysedby RhlI, followed by the binding of butyryl-ACP (FIG. 2).Amide-bond formation between the amino nitrogen

Box 2 | Quorum sensing and biofilms

Many bacteria can develop into sessile antimicrobial-agent-resistant biofilm communities. Microbial biofilmsare loosely defined as multicellular assemblages ofmicroorganisms attached to abiotic or biotic surfaces,and embedded within a self-produced matrix material87.Pseudomonas aeruginosa causes chronic biofilminfections in, for example, the lungs of people with thegenetic disease cystic fibrosis88. P. aeruginosa biofilmdevelopment involves a programmed pathway89,90.Under at least some conditions, the last step in thepathway involves the conversion of microcolonies on asurface into a mature biofilm with cells encased in a self-produced ‘slime’. Quorum sensing has a role in thisstep25. We do not yet know which quorum-sensing-controlled genes are involved in biofilm development. Itis also clear that the requirement for quorum sensingduring P. aeruginosa biofilm maturation can vary withdifferent environmental conditions. When theconditions do not allow the surface assemblages to formstructurally complex biofilms, quorum-sensing mutantsand the wild type are similar in appearance91. Recentstudies have shown that quorum sensing also has a rolein the biofilm development of Burkholderia cepacia92

and Aeromonas hydrophila93, which are bothopportunistic human pathogens. How consistentlyquorum sensing is involved during the maturation ofbiofilms among other bacteria is not yet known.

The figure shows quorum sensing and biofilm development. a | The steps involved in biofilm development.b | Confocal-microscope images of a P. aeruginosa biofilm developing over time on a microscope slide. The cells areproducing the green fluorescent protein. The mushroom- and tower-like structures that appear by 8 days are 100 µmhigh. Images in b kindly provided by M. Welsh, P. Singh and E.P.G (University of Iowa, USA).

Microcolonies

Attachment

Planktonic bacteria(free living)

Mature biofilm community

Quorumsensing

8 days

3 days

1.5 days

8 hours

a

b

Biofilm development



R E V I E W S

implicated membrane efflux pumps in acceleratingthe transport of certain acyl-HSLs across mem-branes51–53. The role of efflux systems seems to bemost relevant for acyl-HSLs with longer, morehydrophobic acyl chains, which would otherwisepartition into the lipid bilayer.

LuxR-type proteinsBasic structure and function. Most members of theLuxR protein family are acyl-HSL-responsive tran-scriptional activators, although members of oneemerging subfamily seem to function as induciblerepressors. LuxR-type proteins can be subdivided intotwo functional domains (FIG. 4) based on clusters ofsequence conservation, biochemical analysis of severalrepresentative members and the recent three-dimen-sional structure of TraR from Agrobacterium tumefa-ciens. A conserved cluster of residues in the amino-terminal portion of LuxR-type proteins comprises anacyl-HSL-binding region, and mutations in this areaabolish the binding of 3-oxo-C6-HSL to LuxR54,55. Allfunctional LuxR homologues also contain a helix–turn–helix (HTH) motif in their carboxyl terminus,

the catalytic pocket42. The EsaI structure reveals thatmany of these residues lie within the presumptive cat-alytic cleft of the enzyme.

Additional residues, which are likely to lie in themore divergent carboxyl terminus, must providespecificity for the appropriate acyl-ACP conjugate.Support for this idea is derived from mutationalanalysis of LuxI, in which several alterations in themore weakly conserved region of the protein abolishactivity49. Also consistent with this idea, threonineresidues in the carboxy-terminal region (Thr143 inLuxI) provide specificity for 3-oxo-ACPs42. Continuedstructural analyses of LuxI-type proteins associatedwith the acyl-ACP and SAM substrates should pro-vide considerably more insight into the function ofthese enigmatic enzymes.

Transmembrane signal transitStudies of the V. fischeri lux system show that 3-oxo-C6-HSL can diffuse passively across the BACTERIAL

ENVELOPE12. In a similar way, other acyl-HSLs seem tobe able to traverse the membrane unassisted.However, several lines of evidence have recently

BACTERIAL ENVELOPE

The cell wall and cytoplasmicmembrane of a bacterial cell.

Box 3 | Gram-positive bacteria often use peptide signals

To date, acyl-homoserine lactone (acyl-HSL) production has not been shownfor any Gram-positive bacterium,although the antibiotic-producingfilamentous Streptomyces use acylatedlactones (called γ-butyrolactones) assignals94. Aside from this specializedgroup, Gram-positive bacteria often usepeptides as signals for cell-to-cellcommunication95. Certain species ofStreptococcus and Enterococcus produceunmodified linear peptides, and severalother linear-peptide signals can be post-translationally modified, such as thoseproduced by members of the genusBacillus96,97. Some species ofStaphylococcus and Enterococcussynthesize and release cyclicpeptides98,99. As a generalization, boththe linear and the cyclic peptide signals are synthesized as an unprocessed translation product and are subsequentlyprocessed and secreted into the external environment.

The regulatory systems that facilitate response to the extracellular peptide signals vary between different bacteria, butthey often involve a two-component signal-transduction cascade100. As an example, we depict the accessory generegulation (Agr) cyclic-peptide signalling system of Staphylococcus aureus, which regulates virulence-factor production.The cyclic peptide called autoinducing peptide (AIP) is processed and then cyclized by linking a cysteine residue to thecarboxy-terminal amino acid101. Inducing concentrations of AIP activate the synthesis of a small regulatory RNA that,either directly or indirectly, activates the production of a battery of virulence factors, including toxins. AIP also signals therepression of factors involved in the initial attachment of S. aureus to host-tissue surfaces99. Interestingly, different isolatesof S. aureus produce different varieties of cyclic peptides, and these peptides are often potent inhibitors of other Agrsystems102.

The figure shows S. aureus Agr quorum sensing. The agr genes are linked to the hla (RNAIII) locus, which they activate.The agrD gene codes for a prepropeptide that is processed by and secreted through the product of the agrB gene. Theprocessed AIP peptide is cyclic and interacts with a histidine sensor kinase receptor — AgrC — of a two-componentregulatory module. The response regulator is the product of the agrA gene, which activates transcription of the regulatoryRNA (RNAIII). RNAIII, in turn, controls the production of Agr virulence factors. SarA is an additional transcriptionfactor that activates both promoter 2 (P2) and promoter 3 (P3) in conjunction with AgrA.

AIP

AIP

AgrD

AgrC

AgrA

AgrA

P

P

P2 P3

agrA agrC agrBagrD hla

AgrB

a AgrD is processed and pumped out of cell

b The cyclic peptide product interacts with the sensor histidine kinase AgrC

c Phosphate is transferred to the response regulator AgrA

d Increased expression of the Agr operon and regulator RNA (RNA III)

Cell wall and membrane

SarA



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A general model for LuxR-type protein function basedon several lines of evidence suggests that these proteinsreside in the cytoplasm or are loosely associated with theinner leaflet of the cytoplasmic membrane (FIG. 2).Interaction with the acyl-HSL through contacts withinthe amino-terminal domain triggers conformational

which is required for DNA binding55,56. Specificresidues in the HTH and flanking sequences are wellconserved in the LuxR family and also in the widerFixJ–NarL superfamily of prokaryotic transcriptionfactors, which all share similar HTH motifs and addi-tional sequences that flank the HTH57.

Box 4 | ‘Bacterial esperanto’ — a universal language?

The initial description of Vibrio fischeri quorum sensing was paralleled by a similar description in the relatedluminescent marine bacterium Vibrio harveyi103. Before we had any mechanistic understanding of acyl-homoserinelactone (acyl-HSL) signalling, it was shown that many other marine bacteria made something that signalled V. harveyi toinduce its luminescence genes104. It seemed that V. harveyi might measure the total bacterial load in its local environmentrather than simply its own population size104.

There are, in fact, two integrated quorum-controlled circuits that govern the V. harveyi lux genes, either of which caninduce luminescence independently43. The signal for one is the acyl-HSL 3-OH-C4-HSL105. The second quorum-sensingsystem is based on a signal originally described as autoinducer-2 (AI-2), and it is this system that responds to interspeciesbacterial signals106,107. There is an increasing amount of evidence that bacteria other than V. harveyi respond to AI-2-typesignals and that, by analogy with V. harveyi, these microbes might also monitor the abundance of other AI-2-synthesizingbacteria in their local environment108.

A gene called luxS, which is conserved in a diverse range of bacteria, is responsible for the production of AI-2 byEscherichia coli109. LuxS is an enzyme that can synthesize a molecule derived from S-ribosylhomocysteine, anintermediate in methionine recycling110,111. Despite this information and tremendous efforts, the true nature of the AI-2signal remained elusive. Only recently have Bonnie Bassler and colleagues identified the enigmatic signal, associated withits receptor protein: receptor-bound AI-2 is a furanosyl borate diester112.Apparently, the sugar from S-ribosylhomocysteine is cyclized and an atom of boron is incorporated to form the diester. Not only does this workprovide at least one view of the interspecies signal, but it also suggests an unexpected role for elemental boron in thesignalling pathway.

Figure 2 | Model of acyl-homoserine-lactone (acyl-HSL) quorum sensing in a single generalized bacterial cell. Tentativemechanisms for acyl-HSL synthesis and acyl-HSL interaction with LuxR-type proteins are shown. Double arrows with filled yellowcircles at the cell envelope indicate the potential two-way diffusion of acyl-HSLs into and out of the cell. The proposed dimerizationof LuxR (red) is based on genetic evidence and biochemical analysis of TraR; other LuxR-type proteins might form higher-ordermultimers. Binding of the acyl-HSL to LuxR and multimerization are represented as distinct events, although they might occursimultaneously. The LuxI label indicates LuxI-type proteins. 5′-MTA, 5′-methylthioadenosine; ACP, acyl carrier protein; SAM, S-adenosylmethionine. Modified with permission from REF. 22 © (2001) Annual Reviews.

Lactonizationand release

Acylation LuxR-typeproteins

Acyl-HSLbound Multimer

ACP-SH

Acyl-SAM

Acyl-ACP SAM 5′-MTA

5′-MTASAM

Acyl-ACP

Target gene(s)

lux-type box

MTA release

Acyl-HSL

Substrate binding

(+)

Acyl-HSL interaction with LuxR-type proteinsLuxI

LuxI

LuxILuxIAcyl-HSLsynthesis

Cell wall and membrane



R E V I E W S

associate with TraR during protein synthesis58. This con-trasts with the typical concept of receptor–ligand inter-action, in which receptors have exposed ligand-bindingsites that recognize their substrate and modify someaspect of their activity in response to the binding event.The TraR studies indicated that it is the nascent, activelyfolding form of TraR that is actually the receptor for 3-oxo-C8-HSL, which is consistent with the observationthat 3-oxo-C8-HSL is fully caged within the acyl-HSL-binding domain of TraR, with little opportunity for sol-vent interaction63. However, pre-folded LuxR from V.fischeri seems to release and bind its acyl-HSL signalafter it has been synthesized (M. Urbanowski andE.P.G., unpublished observations). It is therefore plausi-ble that the unusual, co-translational mechanism ofacyl-HSL interaction is unique to TraR.

The carboxy-terminal domain of TraR is a four-helixbundle encompassing the HTH motif 63. The recogni-tion helix associated with the DNA-binding sequence isprojected into the major groove of DNA, providingbase-specific contacts (FIG. 4). Interestingly, there is aregion of TraR that corresponds to a transcriptional-activation motif that was genetically defined in LuxR ofV. fischeri; this TraR region is in a position on the DNA-bound TraR dimer that would facilitate its interactionswith RNAP64.

Mode of action of LuxR proteins. The TraR structureprovides great insight into the function of LuxR-typeproteins. However, as with any crystal structure, this is asingle snapshot of the protein and any dynamic alter-ations in the molecular structure can only be inferred.Genetic and biochemical analyses have provided amodel that describes the action of LuxR-type proteins,and this must now be integrated with the structuralinformation.

Precise ablation of the amino-terminal domains ofLuxR (LuxR∆N) from V. fischeri and LasR (LasR∆N)from P. aeruginosa results in proteins that activate tran-scription of their target genes constitutively65,66. Thissuggests a mechanism in which association of the acyl-HSL with the amino-terminal binding site relieves inhi-bition of the DNA-binding/transcriptional activationdomain. It is also clear that multimerization is animportant consequence of acyl-HSL binding. In vitro

changes that stimulate multimerization and DNA bind-ing53,58. The multimeric LuxR-type proteins recognizeconserved DNA sequences upstream of target genes.These DNA sequences are often perfect or imperfectinverted repeats, which are located proximal to the regu-lated promoter. The sequences share recognizable pri-mary sequence similarity with the LuxR targetsequence, upstream of the V. fischeri lux genes, and aretherefore called lux-type boxes14,15,59. Transcriptionalactivation of the associated promoter follows binding ofthe lux-type box60–62.

Structural insights. A crystal structure of TraR associ-ated with its cognate ligand (3-oxo-C8-HSL) and anoligonucleotide carrying a canonical binding site hasrecently been published, providing insights about TraRand, by extension, other LuxR homologues63. Many pre-dictions based on genetic and biochemical studies ofdiverse LuxR-type proteins are confirmed by analysis ofthe TraR structure. The exquisite detail provided by theTraR structure with regard to the acyl-HSL interactionand DNA binding will allow rapid progress to be madetowards rational drug design.

The ligand-bound TraR protein is a dimer, with eachmonomer being composed of two folded domainsattached by a flexible linker region. As predicted frommolecular-genetic studies of LuxR family members, theamino-terminal domain contains the acyl-HSL-bindingregion. This domain forms an α–β–α sandwich that co-ordinates ligand interactions through the central β-sheet. Many of the residues that have been identified asimportant for acyl-HSL interaction, and are conservedbetween LuxR-type proteins, are involved in binding tothe acyl-HSL (FIG. 4).

One of the most striking structural features of theamino-terminal domain is that the acyl-HSL (3-oxo-C8-HSL) is completely buried within the protein63. Thisobservation is consistent with biochemical studies sug-gesting that pre-folded TraR (apo-TraR) cannot bind toits acyl-HSL ligand, but rather that the acyl-HSL must

Table 1 | Examples of acyl-homoserine lactone quorum sensors*

Bacteria Regulators‡ Signal Target function

Vibrio fischeri LuxR–LuxI 3-oxo-C6-HSL BioluminescenceAinR–AinS§ C8-HSL Bioluminescence, ?

Pseudomonas LasR–LasI 3-oxo-C12-HSL Virulence and biofilm developmentaeruginosa RhlR–RhlI C4-HSL Virulence and rhamnolipids

Agrobacterium TraR–TraI 3-oxo-C8-HSL Virulence plasmid copy number tumefaciens and conjugal transfer

Erwinia CarR–CarI 3-oxo-C6-HSL Carbapenem antibiotics and caratovora ExpR exoenzymes

Pantoea EsaR–EsaI 3-oxo-C6-HSL Exopolysaccharide stewartii

Rhodobacter CerR–CerI ∆7-C14-HSL|| Aggregationsphaeroides

Vibrio anguillarum VanR–VanI 3-oxo-C10-HSL None yet identified

* See reference 114 and references on individual proteins and acyl-HSLs. ‡ Regulators are denotedas ‘transcriptional regulator–acyl-HSL synthase’. All of these quorum-sensing regulatory proteins aremembers of the LuxR–LuxI family except where indicated. § AinR is a two-component sensor kinase,AinS is an alternate acyl-HSL synthase (see text).|| Has an unsaturated bond between positions 7 and8 on the acyl chain. Acyl-HSL, acyl-homoserine lactone.

Figure 3 | Structure and function of LuxI-type acyl-homoserine-lactone (acyl-HSL) synthases. Residuesconserved in all LuxI-type proteins are labelled with an asterisk.Residues whose mutation in LuxI and RhlI results in significantloss of activity are shown in red; residues for which inactivatingmutations have been isolated in LuxI only are shown in blue;residues for which an inactivating mutation has been isolated inRhlI only are shown in green. The threonine residue that isconserved in LuxI homologues that synthesize 3-oxo-acyl-HSLderivatives is shown in grey. Numbering is relative the LuxIsequence. Blue and red bars define the areas that are proposedto be involved in catalysis and specificity, respectively.

R25*F2

9*W

35*E44

*D46

*D49

*D60G67R70

*F8

4*E10

1*

R104*

A133T1

43E15

0G16

4



R E V I E W S

of most lux-type boxes is likely to reflect the symmetri-cal binding domain of its corresponding regulator, anal-ogous to TraR. The HTH motif is positioned within themajor groove of the TraR DNA-binding site, allowing itto recognize specific base chemistries within thesequence63. The DNA-binding domain of other LuxR-type proteins will probably function similarly but theirDNA sequence specificities will be imparted by keyamino acids within the recognition helix that differbetween LuxR homologues.

In many cases, binding of the multimeric LuxR-type protein to its DNA-recognition sequence posi-tions the regulator directly upstream of the −35 REGION

of the regulated promoter and facilitates interactionswith RNAP. LuxR and TraR are sufficient to activatetranscription of their target promoters in vitrothrough purified RNAP, suggesting that no other fac-tors are necessary for activation of transcription61,62,68.Transcriptional activation in prokaryotes oftendepends on appropriate contacts with the α-subunitcarboxy-terminal domain (αCTD) of RNAP, or its σSUBUNIT69. Extensive analysis of LuxR interactions withthe lux box suggests that it might, in fact, contactboth the αCTD and the σ subunit as a so-calledambidextrous activator15. The efficiency of transcrip-tional activation by LuxR is reduced in the presenceof RNAP carrying specific amino-acid substitutionsin the αCTD or σ, consistent with a requirement forcontact with these regions of RNAP70 (A. M. Stevens,personal communication). In addition, there aremutations within the DNA-binding domain of LuxRthat abolish transcriptional activation but do notaffect DNA binding64.

LuxR-type repressors. LuxR functions as an acyl-HSL-dependent repressor of a lacZ promoter that has beenreconstructed with a lux box between the −10 REGION andthe –35 region. Also, TraR functions as an acyl-HSL-dependent repressor of an artificial promoter with a trabox overlapping the –10 sequence60,71. These engineeredrepression systems have been successfully used to discrimi-nate in vivo DNA-binding activity and transcriptional acti-vation for these regulators. They also suggest that acyl-HSL-dependent repression might occur in naturalsystems,although no such system has been identified.

Perhaps more interesting is the evidence that severalLuxR homologues from plant-pathogenic species ofErwinia and Pantoea function as repressors, and thatacyl-HSL binding relieves repression72,73. The regulatorypattern is the same as that for the transcriptional activa-tors — accumulation of the acyl-HSL results in target-gene expression — but the mechanism is different. Thesimple model for these systems is that the transcriptionfactor binds to its target DNA sequences in the absenceof acyl-HSL and that interaction with the ligand causesdissociation from DNA, resulting in derepression oftarget genes. In fact, recent in vitro analysis of EsaRfrom P. stewartii is consistent with this inductionmodel74. Comparison of these proteins with other LuxRhomologues does not reveal any striking differences inprimary sequence, and elucidation of the mechanism of

studies with TraR of A. tumefaciens and CarR from theplant pathogen Erwinia carotovora have shown thatthese LuxR-type proteins increase their multimerizationstate in the presence of the ligand — from monomers todimers for TraR, and from dimers to higher-order mul-timers for CarR53,58. Within one monomer subunit ofTraR in the active ligand-bound dimer, there are no con-tacts between the amino-terminal domain and the car-boxy-terminal domain. This observation is consistentwith a model in which binding of the acyl-HSL is mutu-ally exclusive with contact between the two domainswithin the same monomer and instead promotes inter-actions between different monomers, mediating forma-tion of the active dimer. However, there is no direct evi-dence for the presumed inhibitory interactions betweendomains within the same monomer in the absence ofacyl-HSL.

The acyl-HSL-associated form of most LuxR-typeactivators is proficient in binding to lux-type-box DNAsequences61,62. All LuxR-type proteins thus far character-ized regulate the expression of genes through interac-tions with the housekeeping (that is, the ‘vegetative’ σ70

type) RNAP. Although lux-type boxes of different quo-rum-sensing microbes are recognizably similar to eachother, each sequence is unique3,67. As with many otherprokaryotic transcription factors, the twofold symmetry

Figure 4 | Structure and function of LuxR-type transcription factors. a | Importantresidues that are shown above the filled yellow bar were identified in studies of LuxR and bycomparing LuxR homologues (numbering relative to LuxR sequence). Residues labelled withan asterisk are conserved in most members of the LuxR protein family. The grey trianglesindicate approximate truncation sites on LuxR that result in the production of a constitutivelyactive protein. The upper shaded grey bar indicates a region that has been genetically definedas being required for LuxR multimerization. Important residues that have been identifiedthrough genetic and structural studies of TraR are shown below the filled yellow bar(numbering relative to TraR). The lower shaded grey bars indicate regions that form contactsbetween TraR dimers. For both LuxR and TraR, residues involved in interactions with therelevant acyl-homoserine lactone (acyl-HSL) are shown in red; residues involved in DNAbinding are shown in blue; and residues involved in transcription activation are shown ingreen. b | A 3-oxo-C8-HSL molecule showing the specific TraR residues that are thought toco-ordinate each position of this acyl-HSL (Y61 interacts along the acyl chain). c | The tra box(the DNA-binding site for TraR) is shown with the two residues (R206 and R210) that makespecific base contacts with this site. Symmetrical contacts are made with the DNA sequencein each half-site.

Y70*D79

*P80

*V87 V10

9G11

7T1

20G12

1*

F122

I126*

T184

E187*

A192*

C195G19

7*

W20

1D20

2I20

6H21

7R23

0W

66*

A10 A38G12

3T1

29M19

1N20

3S20

4R20

6V20

7R20

8R21

0K22

1Y53 Y61D70

W57

LuxR

TraR

Amino-terminal domain Carboxy-terminal domain

ATGTGCAGATCTGCACAT

TACACGTCTAGACGTGTA

R210

R210

R210

R210

R206

R206

a

b c

NH

OO

OO

T129

A38 Y53W57

D70

Y61

tra box3-oxo-C8-HSL

−35 REGION

Consensus sequence region ofstandard prokaryotic promoters~35 base pairs upstream of thetranscriptional start site.

σ SUBUNIT

Subunit of the prokaryotic RNApolymerase holoenzyme thatrecognizes and binds to thepromoter sequence anddissociates after transcriptionbegins.

−10 REGION

Consensus sequence region ofstandard prokaryotic promoters~10 base pairs upstream of thetranscriptional start site.



R E V I E W S

the general idea that eukaryotic hosts might deliberatelyinterfere with the communication pathways of coloniz-ing microbes.

Conclusions and future directionsThe past 10–15 years have witnessed the developmentof an active and exciting new subdiscipline in micro-biology — bacterial communication. We now believethat most, if not all, bacterial species have communi-cation systems that allow signalling and responsebetween individuals. A cornerstone in the develop-ment of this new subdiscipline was an increasedunderstanding of how the marine luminescent bacter-ial species V. fischeri uses a quorum-sensing signal toregulate the transcription of genes that code for lightproduction. This knowledge allowed the discovery ofelements that are related to the V. fischeri quorum-sensing elements in other bacteria. The long-standingdemonstration that streptococci showed signallingwas confirmed by the identification of the signal andthe genetic elements required for signal productionand detection. There is now a rich body of informa-tion on acyl-HSL signalling in Proteobacteria andpeptide signalling in Gram-positive bacteria.

In this review, we have focused on the V. fischeri-typeacyl-HSL signalling systems. Research on these systemshas become quite sophisticated. Crystal structures of anacyl-HSL signal generator42 and a signal receptor63 havebeen published this year. We now understand that manypathogens use acyl-HSL signals to prompt virulence-gene expression and that these quorum-sensing systemsrepresent a unique target for drug discovery. During thenext 10–12 years, we can expect to see the discovery ofhitherto-unknown bacterial signalling systems and tocontinue to develop our understanding of interspeciescommunication.Although our understanding of bacter-ial communication has increased dramatically, this fieldis still young.

Note in proofA second paper115 on the crystal structure of TraR hasrecently been published, which adds additional detailto our understanding of how this LuxR family mem-ber functions.

acyl-HSL-dependent induction is an interesting area ofcurrent study.

Hierarchies and integrationAs with any regulatory pathway that controls physiolog-ical processes, acyl-HSL quorum sensing is integratedwith other bacterial control circuitry. Additional envi-ronmental signals influence quorum sensing, mostoften by affecting the expression of the genes thatencode the LuxI- and LuxR-type proteins. Such regula-tion often modulates or restricts the activity of the quo-rum sensor under specific environmental conditions.Further integration is sometimes achieved throughadditional regulatory proteins that directly impinge onthe activity of LuxR-type proteins, such as defectiveLuxR homologues that form inactive heterodimers andother regulators that form inhibitory complexes withLuxR proteins75–78.

Specific LuxI–LuxR systems can also influence theactivity of other quorum-sensing elements, establish-ing interconnected regulatory pathways. An excellentexample of this overlapping regulation is the hierar-chical Las and Rhl quorum-sensing systems of P.aeruginosa, in which LasR controls the expression ofthe rhlR gene34,36. Therefore, as discussed above, genesthat are indirectly under the control of LasR throughRhlR are influenced by two separate quorum-sensingsignals. Another example of hierarchical LuxI–LuxR-type quorum sensing has been reported for the plantsymbiotic bacterium Rhizobium leguminosarum11,and more will probably be identified. Bacterialgenome sequence analysis suggests that multipleorthologues of LuxR or LuxI, or both, are oftenencoded within the same cell. Ongoing functionalgenomic analysis of quorum sensing should help usto understand how all these quorum-sensing regula-tors are integrated with each other and with addi-tional aspects of cellular physiology.

Certain metazoan organisms seem to be able to inter-fere with acyl-HSL quorum sensing. The eukaryotic redalga Delisea pulchra releases halogenated furanones thatcan inhibit acyl-HSL-based signalling pathways79–81.Quorum-sensing antagonists have also been isolatedfrom several terrestrial plants82. These findings support

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AcknowledgementsWe thank our many colleagues, who make the area of cell-to-cellbacterial communication so exciting. Specifically, we gratefullyacknowledge S. von Bodman, M. Churchill, A. Stevens and S.Winans for sharing their unpublished results, and D. Bartels, AuroraBiosciences, for help in drafting the Box 3 figure. C.F. receives sup-port from the National Science Foundation and the United StatesDepartment of Agriculture. E.P.G. acknowledges the generous sup-port of the National Institutes of Health, the National ScienceFoundation, the Cystic Fibrosis Foundation, Procter & Gamble andthe W. M. Keck Foundation.

Online links

DATABASESThe following terms in this article are linked online to:SwissProt: http://ca.expasy.org/sprot/agrA | agrB | AgrC | agrD | ainR | ainS | CarR | EsaI | HdtS | hla |lacZ | LasI | LasR | luxA | luxB | luxC | luxD | luxE | LuxI | LuxR | luxS |RhlI | RhlR | TraR | VanMAccess to this interactive links box is free online.


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Signalling: Listening in on bacteria: acyl-homoserine lactone signalling