Curr. Issues Mol. Biol. 8: 1–10. Online journal at www.cimb.org

Quorum Sensing and the Lifestyle of

Steven Atkinson1, R. Elizabeth Sockett2, Miguel implies a positive feedback or autoregulatory mechanism Cámara1 and Paul Williams1* of action. However this is frequently not the case and therefore the term can be misleading and will be avoided 1Institute of , Immunity and Infammation, Centre here (Cámara et al., 2002). The accumulation of a for Biomolecular Sciences, University of Nottingham, diffusible signal molecule also indicates the presence of Nottingham NG7 2UH, UK a diffusion barrier, which ensures that more molecules 2Institute of Genetics, University of Nottingham, Queen’s are produced than lost from the micro-habitat (Winzer et Medical Centre, Nottingham NG7 2UH, UK al., 2002b; Redfeld, 2002). This could be regarded as a type of ‘compartment sensing’, where signal molecule Abstract accumulation is both the measure for the degree of Bacterial cell-to-cell communication (‘quorum sensing’) compartmentalization and the means to distribute this is mediated by structurally diverse, small diffusible information among the entire population. Similarly, signal molecules which regulate gene expression as a diffusion of quorum sensing signal molecules between function of cell population density. Many different Gram- spatially separated bacterial sub-populations may convey negative animal, plant and fsh pathogens employ N- information about their physiological state, their numbers, acylhomoserine lactones (AHLs) as quorum sensing and the individual environmental conditions encountered. signal molecules which control diverse physiological At the molecular level, quorum sensing requires processes including bioluminescence, swarming, a synthase together with a signal transduction system antibiotic biosynthesis, plasmid conjugal transfer, bioflm for producing and responding to the signal molecule development and virulence. AHL-dependent quorum respectively. The latter usually involves a response sensing is highly conserved in both pathogenic and non- regulator and/or sensor kinase protein (Swift et al., 2001; pathogenic members of the genus Yersinia. Yersinia Cámara et al., 2002). While quorum sensing systems pseudotuberculosis for example, produces at least eight are ubiquitous in both Gram-negative and Gram-positive different AHLs and possesses two homologues of the , there is considerable chemical diversity in the LuxI family of AHL synthases and two members of the nature of the signal molecules involved which range from LuxR family of AHL-dependent response regulators. In all post-translationally modifed peptides to quinolones, Yersinia species so far examined, the genes coding for lactones and furanones (Cámara et al., 2002). In addition, LuxR and LuxI homologues are characteristically arranged siderophores, which previously were considered only convergently and overlapping. In Y. pseudotuberculosis in the context of iron transport, may also function in AHL-dependent quorum sensing is involved in the control the producer organism as signal molecules capable of of cell aggregation and swimming motility, the latter via controlling genes unrelated to iron acquisition (Lamont et the fagellar regulatory cascade. This is also the case al., 2002). While there is, as yet, no clear cut evidence for swimming and also swarming motility in Yersinia for a molecularly conserved quorum sensing system enterocolitica. However the role of AHL-dependent quorum throughout the bacterial kingdom, the LuxS protein and sensing in remains to be determined. the furanone generated from the ribosyl moiety of S- ribosylhomocysteine (termed AI-2 for autoinducer-2) Introduction have been suggested to fulfl such a role (Bassler, 2002). Until relatively recently, cell-to-cell communication was However, many bacteria (e.g. the pseudomonads) do not rarely considered to constitute a major mechanism for possess luxS and hence do not produce AI-2 (Winzer facilitating bacterial adaptation to an environmental et al., 2002a). Furthermore, as LuxS is a key metabolic challenge. However, it is now clear that diverse bacterial enzyme in the activated methyl cycle responsible for genera communicate using specifc, extracellular signal recycling S-adenosylmethionine (SAM) (Winzer et al., molecules, which facilitate the coordination of gene 2002b; Winzer et al., 2002a) phenotypes associated with expression in a multi-cellular fashion. Signalling can be mutation of luxS are often not a consequence of a defect linked to specifc environmental or physiological conditions in cell-to-cell communication but the result of the failure and is employed by bacteria to monitor their cell population to recycle SAM metabolites (Winzer et al., 2002b; Winzer density. The term quorum sensing is commonly used to et al., 2002a). To date, only in Vibrio harveyi is there any describe the phenomenon whereby the accumulation direct experimental data to support the function of LuxS of a diffusible, low molecular weight signal molecule as a quorum sensing signal molecule synthase. Thus (sometimes called an ‘autoinducer’) enables individual although Yersinia spp. possess a luxS gene and produce bacterial cells to sense when the minimum number or AI-2 (unpublished data) this does not constitute evidence quorum of bacteria has been achieved for a concerted for the presence of a quorum sensing system based on response to be initiated (Williams et al., 2000; Swift et AI-2. Here we examine the nature and contribution of N- al., 2001; Cámara et al., 2002). The term ‘autoinducer’, acylhomoserine lactone (AHL)-mediated quorum sensing to the lifestyle of Yersinia spp. *For correspondence: [email protected]

© Horizon Scientifc Press. Offprints from www.cimb.org 2 Atkinson et al.

N-Acylhomoserine lactone-dependent quorum sensing a short acyl side chain, or are pumped out of the cell if the The most intensively investigated family of quorum AHLs have a long, e.g. C12 acyl side chain (Pearson et sensing signal molecules in Gram-negative bacteria are al., 1999). the AHLs. These are produced by many different bacterial Apart from the AHL synthase, the second regulatory genera including Aeromonas, Agrobacterium, Brucella, component of an AHL-dependent quorum sensing circuit Burkholderia, Chromobacterium, Erwinia, Enterobacter, is usually a member of the LuxR family of transcriptional Pseudomonas, Rhizobium, Serratia, Vibrio and Yersinia regulators (Swift et al., 2001; Cámara et al., 2002; Fuqua although not in all strains and species of the same genus et al., 1996). AHLs bind to and activate LuxR proteins (Swift et al., 2001). AHLs consist of a homoserine lactone such that the LuxR/AHL complex is responsible for either ring covalently linked via an amide bond to an acyl side activating or repressing target structural genes (Swift et chain (Fig. 1). To date, naturally occurring AHLs with chain al., 2001; Cámara et al., 2002; Zhang et al., 2002). The lengths between 4 and 18 carbons have been identifed V. fscheri LuxR protein for example, consists of an N- which may be saturated or unsaturated and with or terminal, cytoplasmic membrane-associated regulatory without a hydroxy-, oxo- or no substituent on the carbon domain that binds AHLs and a cytoplasmic C-terminal at the 3 position of the N-linked acyl chain (Fig. 1). AHLs domain that binds DNA. LuxR is considered to activate are usually synthesized by enzymes belonging to the transcription as a dimer and a number of studies LuxI family, which employ the appropriately charged acyl have identifed regions within the protein required for acyl-carrier protein (acyl-ACP) as the major acyl chain multimerization (Choi and Greenberg, 1992a), AHL binding donor while S-adenosyl methionine (SAM) provides the (Shadel et al., 1990; Slock et al., 1990; Hanzelka and homoserine lactone moiety (Jiang et al., 1998; Watson Greenberg, 1995), DNA binding, transcriptional activation et al., 2002, Moré et al., 1996). Recent data derived (Choi and Greenberg, 1991; Choi and Greenberg, 1992b) from the crystal structure of EsaI from Pantoea stewartii and autorepression (Choi and Greenberg, 1991). The V. indicates that LuxI proteins share structural similarities fscheri LuxR protein appears unable to associate with with eukaryotic N-acetyltransferases which employ similar DNA until a conformational change has been induced via fatty acid precursors as substrates (Watson et al., 2002). an interaction between the N-terminal AHL binding-domain Once synthesized, AHLs either move across the bacterial and the cognate AHL molecule(s) (Choi and Greenberg, cell envelope by simple diffusion in the case of AHLs with 1992b). However, among the growing family of LuxR homologues, it is now clear that there are variations between their respective mechanisms of transcriptional A activation (Luo and Farrand, 1999). In addition, certain O LuxR homologues function as repressors rather than activators (e.g. EsaR) (von Bodman et al., 1998). To date O the structure of only one LuxR homologue has been solved, N that of TraR from Agrobacterium tumefaciens (Zhang et H H al., 2002). These data revealed that TraR forms a dimer O and confrmed that the amino-terminal domain contains the AHL-binding region. However, the cognate AHL, N- B 3-(oxooctanoyl)homoserine lactone (3-oxo-C8-HSL) is O O completely buried within the protein and it appears that the AHL associates with nascent, actively folding TraR O N during protein synthesis. However this ‘co-translational’ mechanism may prove to be unique to TraR and does not H H appear to be representative of all LuxR homologues. O The DNA sequence of the LuxR binding site has been determined and is called the lux box. This is a 20 C O nucleotide inverted repeat, which, in V. fscheri is situated within the intergenic region between luxR and luxI, and is O N required for the primary regulation of lux gene expression. H This DNA element has features that are conserved with H the binding sites of other LuxR-type proteins and share O the consensus RNSTGYAXGATNXTRCASRT where N = A, T, C or G; R = G or A; S = C or G; Y = C or T and X D O O = N or a gap in the sequence. (Stevens and Greenberg, 1997). In for example, lux box- O like elements have been identifed in the promoter regions N of many different target structural genes (Whiteley and H H Greenberg, 2001) and purifed LuxR proteins such as TraR (from A. tumefaciens) and ExpR (from Erwinia O chrysanthemi) have been shown to bind in vitro to lux Fig. 1. Structures of AHLs produced by Y. pseudotuberculosis. (A) N- box-type sequences (Zhu and Winans, 1999; Nasser et hexanoyl homoserine lactone. (B) N-(3-oxohexanoyl)homoserine lactone. al., 1998; Reverchon et al., 1998). (C) N-octanoyl homoserine lactone. (D) N-(3-oxododecanoyl)homoserine lactone. Quorum Sensing and the Lifestyle of Yersinia 3

AHL-controlled multicellular behaviour in Gram- be positive in the AHL bioassay were subjected to high- negative bacteria includes a variety of physiological resolution tandem mass spectrometry (MS-MS) which processes such as bioluminescence, swarming, revealed the presence of two AHLs in an approximately swimming and sliding motility, antibiotic biosynthesis, 50:50 ratio. These AHLs were identifed as 3-oxo-C6- bioflm maturation, plasmid conjugal transfer and the HSL and N-hexanoyl homoserine lactone (C6-HSL) production of virulence determinants in animal, fsh and respectively (Fig. 1) (Throup et al., 1995) and are easily plant pathogens (for reviews see Swift et al., 1999; Swift separated and visualized by thin layer chromatography et al., 2001; Williams., 2002). (TLC) as purple spots on a white background using the Chromobacterium violaceum CV026 biosensor in an agar The search for AHL-mediated quorum sensing in overlay (see Fig. 2 for an example of this TLC overlay Yersinia originally described by McClean et al., 1997). Because of their relative hydrophobicities AHLs can readily Since AHL synthesis depends on the presence of an be concentrated from culture supernatants by partitioning AHL synthase, a plasmid-based in trans complementation into organic solvents such as dichloromethane or ethyl strategy in which a Y. enterocolitica chromosomal gene acetate. However, since they lack good chromophores, library was transformed into E. coli [pSB401] was devised relatively high concentrations are required for detection (Swift et al., 1993; Throup et al., 1995). This approach via their ultra violet spectroscopic properties following yielded a highly bioluminescent clone indicative of an separation by HPLC. As a consequence, a number insert carrying a gene whose product was capable of sensitive bioassays have been developed which of inducing the AHL reporter fusion. This clone was facilitate detection of AHL production by bacterial subsequently shown to be producing both 3-oxo-C6-HSL colonies on plates, in microtitre plate assays, on thin and C6-HSL and to contain two convergently transcribed layer chromatograms (TLC) and in fractions collected and overlapping open reading frames (Fig. 3). The frst after HPLC-based separation (Bainton et al., 1992; of these genes was termed yenI, the product of which Throup et al., 1995; McClean et al., 1997; Shaw et al., exhibited homology to the LuxI protein family while the 1997; Winson et al., 1998). For example, Throup et al. product of the second, termed yenR belongs to the (1995) constructed a recombinant AHL reporter plasmid LuxR protein family. TLC in conjunction with the CV026 which coupled luxR and the luxI promoter region from biosensor showed that inactivation of yenI by deletion Vibrio fscheri to the luxCDABE structural operon of mutagenesis abolished AHL production in Y. enterocolitica Photorhabdus luminescens. When introduced into E. coli, confrming the function of yenI as an AHL synthase. this lux-based AHL reporter (termed pSB401) is dark but In V. fscheri luxI expression is governed by a positive responds to the presence of exogenous AHLs by emitting feedback loop as a consequence of its induction by LuxR light. The advantage of this AHL reporter compared with activated by 3-oxo-C6-HSL (Meighen, 1994). However, in earlier versions employing luxAB alone (Bainton et al., Y. enterocolitica, yenI is not subject to such autoinduction 1992; Swift et al., 1993) is that it does not require the since inactivation of yenI did not abolish production of addition of a long chain fatty aldehyde (which is required yenI mRNA (Throup et al., 1995). This observation was by the luciferase) since the inclusion of the luxCDE genes provides for in situ synthesis of the aldehyde. Using this AHL bioassay, cell free supernatants from several species of Yersinia including isolates of belonging to serotypes O:3, O:8, 3-oxo-C6-HSL O:9, O:10K, O:1(2a, 3), a non-typeable strain along with Yersinia pseudotuberculosis serotype III pIB1, C5-HSL Yersinia frederiksenii, Yersinia kristensenii, and Yersinia intermedia were screened. All of these strains induced bioluminescence in the reporter offering preliminary C6-HSL evidence that Yersinia spp. were AHL producers (Throup et al., 1995). Subsequently, Yersinia pestis (Swift et al., 1999) and Yersinia ruckeri (unpublished data) were also C7-HSL confrmed as AHL-producers using the same strategy. C8-HSL AHL-mediated quorum sensing in Y. enterocolitica Although E. coli [pSB401] incorporates LuxR from V. fscheri, the cognate AHL for which is N-(3- A B C oxohexanoyl)homoserine lactone (3-oxo-C6-HSL), it responds, with differing sensitivities, to a variety of AHLs Fig. 2. TLC chromatogram of the AHLs present in cell-free supernatants (Winson et al., 1998). While a positive bioassay result of Y. pseudotuberculosis grown with shaking in LB at 22ºC and detected confrms the presence of AHLs, it does not indicate either using the C. violaceum CV026 AHL biosensor. Lane A, 3-oxo-C6-HSL; which AHL or indeed how many different AHLs may be 1.4 x 10–8 mol; lane B from top, AHL standards, N-pentanoylhomoserine lactone (C5-HSL) (5.4 x 10–8 mol), C6-HSL (5 x 10–9 mol), C7-HSL present in the sample. To characterize unequivocally the (4.7 x 10–7 mol) and C8-HSL (2.2 x 10–8 mol); lane C, Y. pseudotuberculosis AHLs present in spent Y. enterocolitica culture supernatants, supernatant extract from cultures grown at 22º C showing the presence they were frst concentrated by solvent extraction and of 3-oxo-C6-HSL, C6-HSL, C7-HSL and C8-HSL. It should be noted that subjected to preparative HPLC. The fractions found to CV026 does not respond to long chain AHLs and the size of the spots are not equivalent to the concentration of AHL present. 4 Atkinson et al.

ypsR CTATTTAG the formation of bioflms and in bacterial pathogenesis ypsI GATAAATC (Fraser and Hughes, 1999). In the Y. enterocolitica yenI mutant, swimming and swarming could both be restored by complementation with a plasmid-borne copy of yenI. Although Y. enterocolitica strains 10460 and 90/54 are yenR ACTTATTAAACCTATTTAATAATACCAGGT TGAATAATTTGGATAAATTATTATGGTCCA yenI not isogenic, these data do provide preliminary evidence linking quorum sensing with the virulence plasmid even though the loss of motility and swarming are both likely ypeR CTATTTAG to involve changes in expression of the chromosomally GATAAATC ypeI encoded fagellar genes. In bacteria such as Serratia liquefaciens (Lindum et al., 1998), Burkholderia cepacia (Huber et al., 2001) and Pseudomonas aeruginosa yukR TTATACCAGAACTGGTTTAA (Kohler et al., 2000) the loss of swarming motility in yukI AATATGGTCTTGACCAAATT quorum sensing mutants is associated with the loss of biosurfactant production. For mutants defective in AHL ytbR TTAAGCCGATTCTGGCATAA production, swarming can be restored by the provision ytbI AATTCGGCTAAGACCGTATT of either the appropriate AHL signal molecule(s) or a biosurfactant. In addition, although such mutants do not swarm they remain capable of swimming motility. In the vanI AGTGTCAATGCATAAGCCTTATAAATAGGGATT Y. enterocolitica 90/54 yenI mutant, swarming could not TCACTGTTACGTATTCGGAATATTTATCCCTAA vanR be restored by providing an exogenous surfactant and since the mutant also failed to swim, this suggests that AHL-mediated quorum sensing in this organism is directly esaI CTACCTGGCCGCTGACGCTGCCGGTCTGA linked to fagellin expression/function given that fagellar esaR GATGGACCGGCGACTGCGACGGCCAGACT are required for both swimming and swarming motility. Fig. 3. Organization of genes coding for LuxR and LuxI homologues in When analysed by SDS-PAGE the 90/54 yenI mutant Yersinia spp. Hyphenated inverted repeat sequences identifed within fails to produce fagellin when compared to the parent and convergently transcribed luxR/I homologues. Stop codons are underlined. using RT-PCR the expression of fhCD, fiA and fiC was Bold bases indicate inverted repeats. Key: ypsR/I and ytbR/I, Yersinia examined in the Y.enterocolitica parent and yenI mutant. pseudotuberculosis; yenR/I, Yersinia enterocolitica; ypeR/I, Yersinia pestis; yukR/I, Yersinia ruckeri; vanR/I, Vibrio anguillarum; esaR/I, These data suggested that quorum sensing regulates Pantoea (Erwinia) stewartii motility at the level of fiC expression (unpublished observations). Furthermore, given that motility is required confrmed by examining the expression of yenI’::luxAB to initiate host cell invasion by Y. enterocolitica (Young transcriptional fusions as chromosomal insertions in both et al., 2000), it is possible that AHL-mediated quorum the parent and yenI mutant respectively. In addition, the sensing may also turn out to play a role in this context. promoter region of yenI contains no matches for the lux box consensus sequence. Thus the expression of yenI is AHL-mediated quorum sensing in Y. pseudotuberculosis not part of an autoinducible system analogous to that of V. TLC analysis of Y. pseudotuberculosis spent fscheri but is independent of growth phase. culture supernatants grown at (22°C) extracted with A preliminary analysis of potential Y. enterocolitica dichloromethane using both C. violaceum CV026 (Fig. 2) target genes which may be controlled by AHL-dependent and E. coli [pSB401] revealed the presence of at least three quorum sensing was undertaken by comparing 2D AHLs, the chemical identities of which were subsequently SDS-PAGE profles of the Y. enterocolitica strains 90/54 confrmed using HPLC and MS as 3-oxo-C6-HSL, C6-HSL (serotype O:9) and 10460 (biotype 3, serotype O:1 (2a, and N-octanoyl-L-homoserine lactone (C8-HSL) (Fig. 2). 3); NCTC 10460) and their corresponding yenI mutants. However, when grown to stationary phase at 37°C in While no obvious differences between the 10460 parent LB medium, Y. pseudotuberculosis appears to produce and mutant were observed, the 90/54 yenI mutant profle extremely low levels of these AHLs. Since temperature lacked a number of proteins that were present in the plays an important role in the regulation of virulence parent strain. However, as yet, none of these proteins has genes in Yersinia (Kapperud et al., 1985; Cornelis et al., been identifed. The major differences between 10460 and 1987; Pepe and Miller, 1993; Pierson and Falkow, 1993; 90/54 relate to virulence since the former lacks the pYV Cornelis and Wolf-Watz, 1997; Bleves and Cornelis, virulence plasmid and this implies a possible role for AHL- 2000), AHL production was examined in stationary phase mediated quorum sensing in controlling plasmid-borne cultures grown for 24 h at 28°C and 22°C (Yates et al., functions. Although we were unable to demonstrate a role 2002). By comparing the AHL profles of cultures grown at for AHL-mediated quorum sensing in regulating type III Yop the three different temperatures, AHL levels were found secretion in Y. enterocolitica (Throup et al., 1995) the yenI to be markedly dependent on growth temperature. The mutant of 90/54, in contrast to the 10460 yenI mutant was higher levels appeared to be greater after growth at 22°C, unable to swim or swarm (unpublished data). Swarming substantially reduced at 28°C and almost undetectable motility is a fagellum-dependent behaviour that facilitates after growth at 37°C. Initially this suggested that either bacterial migration over solid surfaces and is distinct from the expression or function of the corresponding AHL swimming motility, which occurs in fuid environments. synthase(s) or substrate availability might be temperature Such multicellular behaviour has been implicated in dependent, implying that quorum sensing was temperature Quorum Sensing and the Lifestyle of Yersinia 5 dependent. However, it subsequently became clear that identifed as, N-heptanoylhomoserine lactone (C7- there was a simple physico-chemical explanation. This HSL) while YpsI generates 3-oxo-C6-HSL and C6-HSL is because the AHL ring is highly susceptible to pH- (Atkinson et al., 1999 and unpublished data). These dependent lactonolysis. Ring opened compounds are data suggest that there is a difference in the AHLs made unable to activate LuxR-type proteins and therefore do via YtbI and YpsI in an E. coli genetic background and not activate AHL biosensors. By simply acidifying spent those made in Y. pseudotuberculosis since in the latter culture supernatants to below pH 2.0, lactonolysis can be homologous background, YtbI can clearly produce all reversed and the AHLs recovered from cultures grown three major AHLs in the absence of YpsI (Atkinson et at 37°C (Yates et al., 2002). In LB, which is unbuffered, al., 1999). This fnding was unexpected since the AHL the growth of Y. pseudotuberculosis rapidly renders profle of recombinant LuxI proteins from other bacteria the pH alkaline as a consequence of the metabolism of expressed in E. coli has previously corresponded closely amino acids and the release of ammonia. The rate of pH- with the AHL profle in the original organism (Atkinson dependent lactonolysis is further enhanced by raising the et al., 1999). Notwithstanding our observations with incubation temperature. Thus, these parameters must respect to pH-dependent lactonolysis, these data be taken into consideration for any study which seeks to indicate that YtbI is able to vary the AHL profle produced quantitate AHL levels in culture supernatants. Furthermore, in Y. pseudotuberculosis in a temperature-dependent the AHL profle is also dependent on the structure of the manner and in the absence of YpsI. AHL production in Y. AHLs present. In the context of lactonolysis, the longer the pseudotuberculosis is abolished when both ypsI and ytbI acyl side chain of an AHL then the more stable it will be are mutated but not after deletion of both ypsR and ytbR towards increases in pH and temperature such as those indicating that, as in Y. enterocolitica, AHL production is encountered in vivo in mammalian hosts. Indeed the not dependent on the presence of a functional YpsR or data obtained by Yates et al. (2002) revealed that to be YtbR protein (Buckley, 2002). functional under physiological conditions in mammalian Much of the analysis of AHL production in Y. tissue fuids, AHLs require an acyl side chain of at least 4 pseudotuberculosis was undertaken using AHL biosensors carbons in length. This explains why homoserine lactone which preferentially respond to short chain (C4-C8) AHLs itself cannot function as a quorum sensing signal molecule (Fig. 2). Recently, we have re-investigated AHL production as it rapidly undergoes lactonolysis at pHs above 2 (Yates in Y. pseudotuberculosis using an AHL biosensor et al., 2002). This in turn suggests that the production of (pSB1075) based on the LuxR homologue, LasR from a range of AHL signal molecules with different acyl side Pseudomonas aeruginosa, the cognate AHL for which is chain lengths provides a pathogen with greater versatility the long chain N-(3-oxododecanoyl)homoserine lactone in adapting to changing environments. (3-oxo-C12-HSL). In pSB1075, we have incorporated lasR and the lasI promoter fused to luxCDABE (Winson Y. pseudotuberculosis produces multiple short and long et al., 1998). When E. coli [pSB1075] was used as an chain AHLs overlay in conjunction with a TLC system optimized for the Using the same trans-complementation strategy chromatography of long chain AHLs employing reverse described by (Throup et al., 1995) i.e. the introduction of phase RP-2 TLC plates and 45% v/v methanol in water a genomic library into the AHL biosensor E. coli [pSB401], as the mobile phase (Yates et al., 2002), dichloromethane Atkinson et al (1999) cloned a luxI homologue from Y. extracts of the Y. pseudotuberculosis wild type were found pseudotuberculosis by screening for bioluminescent to contain three long chain AHLs. These were tentatively clones. This approach facilitated the identifcation of a identifed as N-(3-oxodecanoyl)homoserine lactone (3- LuxI homologue, YpsI and a LuxR homologue, YpsR. oxo-C10-HSL), N-(3-oxododecanoyl)homoserine lactone Deletion insertion mutations were introduced into both (3-oxo-C12-HSL) and N-(3-oxotetradecanoyl) homoserine ypsI and ypsR and an analysis of the AHL profles of both lactone (3-oxo-C14-HSL). None of these AHLs were made mutants revealed that after growth at 37°C, the mutants by the ypsI/ytbI double mutant nor by the ytbI mutant produced the same three AHLs (3-oxo-C6-HSL, C6-HSL suggesting that YtbI was the synthase responsible for and C8-HSL) as the wild type. However, the ypsI mutant their synthesis. This was confrmed by demonstrating that produced no 3-oxo-C6-HSL at 28°C while the ypsR E. coli expressing ytbI but not ypsI also produces the long mutant produced reduced levels of 3-oxo-C6-HSL and C6- chain AHLs (Buckley, 2002). HSL and no C8-HSL could be detected (Atkinson et al., 1999). These data suggested that Y. pseudotuberculosis AHL-mediated quorum sensing controls motility and cell possessed an additional AHL synthase, the expression of aggregation in Y. pseudotuberculosis which was likely therefore to be controlled by YpsR. When grown in LB at 28°C or 37°C, Y. pseudotuberculosis To locate this second putative AHL synthase, ypsR mutant cells aggregate, a phenomenon which the same complementation strategy used above was does not occur at 22°C or in either the parent or ypsI employed but using genomic DNA prepared from the Y. mutant at any temperature (Atkinson et al., 1999) (Fig. pseudotuberculosis ypsI mutant (Atkinson et al., 1999). 4). This phenotype implied the involvement of surface This approach yielded a clone containing two genes components which were likely to be regulated via AHL- termed ytbI and ytbR which when translated, exhibited dependent quorum sensing. SDS-PAGE of cell surface homology to YpsI and YpsR and were therefore additional extracts prepared from parent, ypsI and ypsR mutants members of the LuxR and LuxI protein families. When revealed that the expression of numerous proteins expressed in E. coli, YtbI is responsible for the production was affected. In particular, a prominent protein of of C6-HSL and C8-HSL and a molecule we tentatively approximately 42 kDa was substantially up-regulated 6 Atkinson et al.

A C motile. These data indicate that the ytbR/I locus may be

t 1.0 µm an activator of motility while the ypsR/I locus functions t n n a to repress motility. The two quorum sensing circuits also t a t u

u appear to be linked since YpsR appears to be involved in t M M n the control of ytbI expression since ypsR mutants fail to R e I r s s a

p produce the YtbI-generated AHL, C8-HSL during growth p y P y at 28°C. Since ypsI/ytbI and ypsR/ytbR double mutants are non-motile while the ypsR and ypsI single mutants exhibit early motility in batch culture when compared to the wild type, it is likely that the quorum sensing circuitry controls the expression of key regulatory genes involved in the control of swimming motility. In enteric bacteria, fagellar 500 nm gene transcription is arranged as a hierarchy with genes B in three regulatory levels or classes which are responsible for producing and maintaining functional fagella and the chemotaxis apparatus (MacNab, 1996). FlhD and FlhC activate the class II structural genes encoding the basal body and hook proteins, along with an activator FliA (σF) and a repressor FliM (anti-σF) of class III genes. Class III genes encode the proteins which constitute the mature fagellar flament, chemotactic sensors and motor and are transcribed from FliA-dependent promoters 10 µm in a temperature dependent manner (MacNab, 1996). Although the fhDC master regulatory system was once Fig. 4. Mutation of ypsR causes cell aggregation in Y. pseudotuberculosis. considered to be exclusively involved in regulating the (A) 24 h Static LB cultures of the parent ypsI and ypsR mutants production of fagella for swimming motility (Fraser and following growth for 24 h shaking at 200 rpm. (B) Light and (C) electron micrographs of the ypsR mutant prepared from overnight cultures grown Hughes, 1999) it is now known to play a more global role with shaking at 22°C. in regulating diverse physiological processes (Pearson et al., 2000; Tan et al., 1999). In Y. enterocolitica (Young et al., 1999) as well as S. liquefaciens (Givskov et al., in the ypsR mutant (Atkinson et al., 1999). N-terminal 1997) and P. mirabilis (Fraser and Hughes, 1999), fhDC sequence analysis indicated that this protein exhibited is required for fagellin production, swimming motility and signifcant homology to the major fagellin structural swarming motility. proteins , FleB and FleC of Y. enterocolitica (Kapatral An analysis of the Y. pseudotuberculosis fhDC and and Minnich, 1995). These data implicated ypsR in the fiA promoter/operator region revealed the presence of control of fagellar mediated motility. On swimming motility putative lux-box like elements. By employing reporter gene plate assays, both the ypsR and ypsI mutants, but not fusions in single and double ypsR/I and ytbR/I mutants we the parent strain, were motile after 24h when grown at have established that, for example fhDC expression is 22°C. None of the strains was motile after 24 h or 48 h repressed by YpsR and YtbR at 37°C. In addition, YtbI at 37°C by phase contrast microscopy or on swarming appears to be involved as an activator of fhDC expression motility plate assays. By following the induction of motility at temperatures below 28°C (unpublished data). in liquid medium over the growth curve, it is clear that at In a Y. enterocolitica fhDC mutant (Bleves et al., 22°C, motility is induced after 6 h in both ypsI and ypsR 2002) noted an increase in Yop expression and a loss mutants but delayed until 26 h in the parent (Atkinson et of the temperature dependency usually associated with al., 1999). Thus cell density appears to be one important Yop expression at 37°C. Since the Y. pseudotuberculosis environmental cue for activating swimming motility in Y. quorum sensing circuitry appears to control fhDC, this pseudotuberculosis (Atkinson et al., 1999). Furthermore, implies the existence of a regulatory link between type although the ypsR/I locus is involved in the repression III secretion and motility. However such a link has to be of motility, mutation of this locus does not overcome the demonstrated experimentally. temperature dependence of swimming motility. Although the molecular basis behind the clumping To determine whether the ytbR/I locus is also involved phenotype associated with the ypsI and ypsR mutants is in the control of fagellar-mediated motility, both ytbI and currently unknown this observation lead to an investigation ytbR genes were inactivated. We speculated that if the into the relationship between bioflm formation and the ytbR/I system was not involved in the regulation of motility, quorum sensing system of Y. pseudotuberculosis using a the mutants would display the hypermotile phenotype biotic surface model. In this model Y. pseudotuberculosis characteristic of the ypsR/I mutants. However, in common produces bioflms after 24 h whereas the ypsI ytbI and with the parent strain, both ytbI and ytbR mutants were non ypsR ytbR double mutants are attenuated for bioflm motile even after 48 h at 22°C. While this initially implied formation (unpublished observations). The implications that the ytbR/I system was not involved in controlling for these fndings are far reaching given the fact that motility, the corresponding double I or double R mutants in Y.pestis bioflms have been shown to develop in the i.e. the ypsI ytbI and ypsR ytbR mutants were also non- proventriculus and fore gut of the fea post infection and Quorum Sensing and the Lifestyle of Yersinia 7 bioflm development is associated with the expression Quorum sensing in Y. pestis and Y. ruckeri of components of the hms locus (Jarrett et al., 2004). Given the close genetic relationship between Y. However, under the conditions tested and using a triple pseudotuberculosis and Y. pestis, it is perhaps not Y. pestis mutant defective for ypeI, yepI and luxS, Jarrett surprising to fnd two pairs of LuxRI homologues, the et al (2004) were unable to establish a link between the genes for which have been termed ypeRI and yepRI in quorum sensing system of Y. pestis and the hms locus. The the latter (Isherwood, 2001; Swift et al., 1999). In addition, relationship between the hms locus and quorum sensing preliminary studies on Y. ruckeri, which is responsible for has yet to be characterized in Y. pseudotuberculosis but enteric red mouth disease in salmonid fsh, have also once tested may stimulate further investigations into the revealed the presence of a luxRI pair, termed yukRI differential control of bioflm formation in both yersinia (unpublished data). Each of these Yersinia LuxR and LuxI species. homologues share signifcant homology with each other and each genetic locus is organized similarly in that the The biological signifcance of long chain AHLs produced two genes are convergently transcribed and overlapping by Y. pseudotuberculosis by either 8 or 20 bp in the respective host species (Fig. Although only C6-HSL, 3-oxo-C6-HSL and C8-HSL 3). Neither the genetic nor physiological consequences of were initially identifed and fully chemically characterized such a gene arrangement are clear since luxR and luxI (Atkinson et al., 1999), it is also clear, as indicated homologues in different organisms can be transcribed above that Y. pseudotuberculosis produces three long in tandem, divergently or convergently (Salmond et al., chain AHLs, 3-oxo-C10-HSL, 3-oxo-C12-HSL and 3- 1995; Swift et al., 1999) oxo-C14-HSL. These AHLs which are also produced via Using AHL biosensors such as C. violaceum CV026, recombinant YtbI expressed in E. coli are not produced by initial cross streak analysis of Y. pestis isolates taken recombinant YpsI. They are present in spent supernatants from a range of environmental and geographic sources prepared from ypsI mutants but absent from spent culture revealed that AHL production is conserved in this Yersinia supernatants prepared from ytbI mutants or ypsI ytbI species. Subsequent analysis indicated that Y. pestis, double mutants, the latter of which make no detectable in common with Y. pseudotuberculosis produces C6- AHLs (Buckley, 2002). While the signifcance of this HSL, 3-oxo-C6-HSL and C8-HSL (Swift et al., 1999). fnding in the context of quorum sensing dependent gene Furthermore, YpeI (which is more closely related to YpsI regulation in Y. pseudotuberculosis is not yet clear, it is and YenI than YtbI), directs the synthesis of C6-HSL and worth noting that 3-oxo-C12-HSL (which is also produced 3-oxo-C6-HSL suggesting that the second Y. pestis LuxI via LasI in P. aeruginosa; (Passador et al., 1993; Pearson homologue, YepI is responsible for C8-HSL synthesis. et al., 1994) possesses potent, dose dependent, pro- Sequence analysis indicates that YepI is more closely infammatory, immune modulatory and vasorelaxant related to YtbI than YpsI which is consistent with the properties (Telford et al., 1998; Lawrence et al., 1999; nature of the respective AHLs produced. Whether YpeI Smith et al., 2001; Chhabra et al., 2003). For example in Y. pestis is also responsible for producing additional 3-oxo-C12-HSL modulates both T and B cell functions, AHLs including compounds with long (C10-C14) acyl blocks lipopolysaccharide-stimulated production of tumour side chains is not yet known. For Y. ruckeri, preliminary necrosis factor alpha (TNF-α) by peritoneal macrophages TLC analysis of the recombinant YukI expressed in E. coli and mediates switching of the T-helper-cell response from suggests that this fsh pathogen also produces C6-HSL, the antibacterial Th-1 response (characterized by IL-12 3-oxo-C6-HSL and C8-HSL (unpublished data). Indeed and gamma interferon production) to a Th-2 response. In although analysis of the translated sequence for a given addition, 3-oxo-C12-HSL also exerts a pharmacological LuxI homologue provides little information on the nature effect on the cardio-vascular system suggesting that of the corresponding AHL(s) produced, the presence of a host cardiovascular function may be modulated, or threonine at position 143 in the carboxy terminal region infuenced, by bacterial quorum sensing molecules. relative to the V. fscheri LuxI protein (Watson et al., 2002) In isolated porcine coronary arteries, 3-oxo-C12-HSL is characteristic of synthases which direct the synthesis caused a concentration dependent relaxation effect on of 3-oxo-substituted AHLs. This observation appears to thromboxane mimetic-induced contractions (Lawrence et hold for all six Yersinia LuxI homologues all of which are al., 1999) and also induces a marked bradycardia in live capable of 3-oxo-C6-HSL production. conscious rats (Gardiner et al., 2001). Structure activity As yet, the target structural genes controlled via studies have indicated that the immune modulatory the yukR/I locus in Y. ruckeri have not been identifed properties of AHLs are optimal in compounds with 11 to and no mutants have been constructed. However in Y. 13 acyl chain carbons and a 3-oxo substituent (Chhabra pestis, both ypeI and ypeR have both been mutated. et al., 2003). Short chain (4 to 8 acyl carbons) AHLs have Despite the observation that quorum sensing is a key little activity in this context. Thus, 3-oxo-C12-HSL and to regulator of motility in both Y. pseudotuberculosis and Y. a lesser extent 3-oxo-C10-HSL and 3-oxo-C14-HSL may enterocolitica, Y. pestis does not exhibit any motility under contribute to virulence directly through manipulation of the conditions tested at all temperatures even though the eukaryotic cells and tissues to maximize the provision of genome contains one full set of fagella genes plus a nutrients via the bloodstream while down regulating host second partial set (Parkhill et al., 2001; Isherwood, 2001). defence mechanisms Despite the similarity between ypsI and ypeI, Y. pestis ypeI mutants are also non-motile (Isherwood, 2001). Analysis 8 Atkinson et al. of Y. pestis virulence gene expression in parent, ypeI and Buckley, C. M. F. G. (2002) Quorum Sensing in Yersinia ypeR mutants grown at 28°C and 37°C did not reveal any pseudotuberculosis. Nottingham University. Thesis. differences with respect to V-antigen, pH 6 antigen, the Cámara, M., Williams, P., and Hardman, A. (2002). coagulase/fbrinolytic protein Pla or lipopolysaccharide Controlling infection by tuning in and turning down the profle (Swift et al., 1999). However in a mouse infection volume of bacterial small-talk. Lancet Infect. Dis. 2, model an increase in time to death was observed for mice 667–676. challenged with the mutant when compared to the parent. Chhabra, S.R., Harty, C., Hooi, D.S.W., Daykin, M., For example at a challenge dose of approximately 104 Williams, P., Telford, G., Pritchard, D.I., and Bycroft, cfu the mean time to death increased from 5.4 days for B.W. (2003). Synthetic analogues of the bacterial signal mice challenged with Y. pestis GB to 6.7 days for mice (quorum sensing) molecule N-(3-oxododecanoyl)-L- challenged with the ypeR mutant implying a possible role homoserine lactone as immune modulators. J. 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