The LuxM Homologue VanM from Vibrio anguillarum Directs the Synthesis of...

JOURNAL OF BACTERIOLOGY,0021-9193/01/$04.0010 DOI: 10.1128/JB.183.12.3537–3547.2001

June 2001, p. 3537–3547 Vol. 183, No. 12

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

The LuxM Homologue VanM from Vibrio anguillarum Directs theSynthesis of N-(3-Hydroxyhexanoyl)homoserine Lactone

and N-Hexanoylhomoserine LactoneDEBRA L. MILTON,1* VICTORIA J. CHALKER,2† DAVID KIRKE,2 ANDREA HARDMAN,2

MIGUEL CAMARA,2 AND PAUL WILLIAMS2,3

Department of Cell and Molecular Biology, Umeå University, S-901 87 Umeå, Sweden,1 and School of Pharmaceutical Sciences,University of Nottingham, University Park, Nottingham NG7 2RD,2 and Institute of Infections and Immunity,

University of Nottingham, Queen’s Medical Center, Nottingham NG7 2UH,3 United Kingdom

Received 14 February 2001/Accepted 12 March 2001

Vibrio anguillarum, which causes terminal hemorrhagic septicemia in fish, was previously shown to possessa LuxRI-type quorum-sensing system (vanRI) and to produce N-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a vanI null mutant still activated N-acylhomoserine lactone (AHL) biosensors, indicatingthe presence of an additional quorum-sensing circuit in V. anguillarum. In this study, we have characterizedthis second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry andchemical analysis, we identified two additional AHLs as N-hexanoylhomoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase V. anguillarum spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, andC6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore, vanM, the gene respon-sible for the synthesis of these AHLs, was characterized and shown to be homologous to the luxL and luxMgenes, which are required for the production of N-(3-hydroxybutanoyl)homoserine lactone in Vibrio harveyi.However, resequencing of the V. harveyi luxL/luxM junction revealed a sequencing error present in thepublished sequence, which when corrected resulted in a single open reading frame (termed luxM). Downstreamof vanM, we identified a homologue of luxN (vanN) that encodes a hybrid sensor kinase which forms part of aphosphorelay cascade involved in the regulation of bioluminescence in V. harveyi. A mutation in vanMabolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL wasabolished in the vanM mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of3-oxo-C10-HSL via vanRI. However, a vanN mutant displayed a wild-type AHL profile. Neither mutationaffected either the production of proteases or virulence in a fish infection model. These data indicate that V.anguillarum possesses a hierarchical quorum sensing system consisting of regulatory elements homologous tothose found in both V. fischeri (the LuxRI homologues VanRI) and V. harveyi (the LuxMN homologues,VanMN).

Diverse gram-negative and gram-positive bacteria commu-nicate intercellularly to regulate the transcription of multipletarget genes in concert with cell density. This type of commu-nication, termed quorum sensing, is mediated through the pro-duction of diffusible signal molecules, termed autoinducers orpheromones, which effectively enable a bacterium to monitorits own population density (for reviews, see references 13, 17,21, and 44). Quorum sensing is now known to regulate diversephysiological processes such as bioluminescence, swarming,antibiotic biosynthesis, plasmid conjugal transfer, and the pro-duction of exoenzyme virulence determinants in human, ani-mal, and plant pathogens. In gram-negative bacteria, the mostintensively investigated autoinducer molecules are N-acylho-moserine lactones (AHLs) that vary in the length and satura-

tion state of the N-acyl side chain (molecules with from 4 to 14carbons have been characterized) and in the presence or ab-sence of an acyl chain C-3 substituent (oxo- or hydroxy-).

The cell density-dependent regulation of bioluminescence inVibrio (Photobacterium) fischeri (31, 32) is frequently used asthe paradigm for quorum sensing. In this marine symbiont, asthe bacterial cell population density increases, the level of theautoinducer, N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) (14), accumulates until a critical threshold concen-tration is reached. 3-Oxo-C6-HSL then binds to the transcrip-tional activator protein, LuxR, and the resulting LuxR–3-oxo-C6-HSL complex triggers transcription of the luminescence(lux) operon, resulting in the emission of light.

In common with V. fischeri, the free-living marine bacteriumVibrio harveyi also regulates bioluminescence in a cell density-dependent manner at the level of transcription, involving theproduction and sensing of N-(3-hydroxybutanoyl)-L-homo-serine lactone (3-hydroxy-C4-HSL [for a review, see reference32]). However, the regulation of the V. harveyi system is verydifferent and appears to be more complex than in V. fischeri.Based on genetic analyses, Freeman and Bassler (15, 16) have

* Corresponding author. Mailing address: Department of Cell andMolecular Biology, Umeå University, S-901 87 Umeå, Sweden. Phone:46-90-785-3782. Fax: 46-90-771420. E-mail: [email protected].

† Present address: Department of Pathology and Infectious Dis-eases, Royal Veterinary College, North Mymms AL9 7TA, UnitedKingdom.

3537

proposed a model for the regulation of bioluminescence in V.harveyi that involves two signaling systems and two autoinducermolecules. The first quorum-sensing system relies on 3-hy-droxy-C4-HSL (9), the synthesis of which is directed by luxLand luxM. Interestingly, the gene products of luxL and luxMshow no homology to the LuxI family of AHL synthases (4).Regulation of bioluminescence via the second quorum-sensingsystem utilizes another, as yet unidentified signal molecule(AI-2), which is chemically distinct from AHLs and is synthe-sized via LuxS (51). The sensors for 3-hydroxy-C4-HSL andAI-2, named LuxN and LuxQ, respectively, resemble proteinsbelonging to two-component signaling systems (4, 6) and pos-sess both a histidine kinase and a response regulator domain.At low cell densities, in the absence of signal molecules, LuxNand LuxQ are suggested to work in parallel by relaying thephosphates from their response regulator domains to a sharedphosphorelay protein, LuxU (16). The phosphate from LuxU isthen transferred to the response regulator domain of the s54

activator LuxO, which, when phosphorylated, represses biolu-minescence by activating the expression of an unidentifiedrepressor (5, 15, 28). In contrast, at high cell densities, thesignal molecules accumulate and are believed to bind to theirrespective sensors (15, 16). It is thought that 3-hydroxy-C4-HSL may bind directly to LuxN, whereas AI-2 is postulated tobind to LuxQ via interaction with a putative periplasmic pro-tein LuxP. Binding of the signals is suggested to switch thesensor kinase activities of LuxN and LuxQ into phosphatases,leading to the dephosphorylation of LuxO and derepression ofthe lux operon.

It is now known that V. fischeri also possesses multiple quo-rum-sensing circuits and produces at least three AHLs, whichare involved in the regulation of bioluminescence (18, 26).While the production of both 3-oxo-C6-HSL and N-hexanoyl-homoserine lactone (C6-HSL) is mediated via LuxI, an addi-tional protein, AinS, is responsible for the synthesis of N-octanoylhomoserine lactone (C8-HSL) (18, 26). AinS ishomologous to LuxM of V. harveyi but not to LuxI of V.fischeri, suggesting the existence of a new family of AHL syn-thases.

Vibrio anguillarum, a bacterial pathogen that causes hemor-rhagic septicemia in marine fish, was previously shown to con-tain a LuxI and a LuxR homolog, termed VanI and VanR,respectively (36), and to produce N-(3-oxodecanoyl)homo-serine lactone (3-oxo-C10-HSL), the synthesis of which is me-diated by VanI. Although a null mutation in vanI abolished theproduction of 3-oxo-C10-HSL, this mutant was still capable ofweakly activating AHL biosensors, suggesting that V. anguilla-rum possesses multiple quorum-sensing systems. In the presentstudy, we describe the isolation and characterization of twoadditional AHLs, N-(3-hydroxyhexanoyl)homoserine lactone(3-hydroxy-C6-HSL) and C6-HSL. The protein responsible forthe synthesis of these AHLs, VanM, is shown to share homol-ogy with both LuxLM and AinS from V. harveyi and V. fischeri,respectively. In addition, we characterized a second gene,vanN, the product of which is homologous to LuxN from V.harveyi. A null mutation of vanM abolished the production ofboth 3-hydroxy-C6-HSL and C6-HSL. Surprisingly, however,the production of 3-oxo-C10-HSL was also downregulated,suggesting that the vanM quorum-sensing system is involved in

regulating the vanIR locus responsible for the synthesis of3-oxo-C10-HSL.

MATERIALS AND METHODS

Strains, phage, plasmids, and media. Bacterial strains and plasmids are de-scribed in Table 1. V. anguillarum NB10 (serotype O1) is a clinical isolate fromthe Gulf of Bothnia outside Umeå Marine Center, Norrbyn, Sweden (39). Esch-erichia coli JM109 (45) was used as the bacterial background for the purificationof AHLs. The wild-type V. harveyi strain BB120 (7) was used for resequencingthe junction between luxL and luxM. E. coli SY327 (33) was used for transfor-mation after subcloning fragments into either the pNQ705-1 or pDM4 vector. Allplasmids to be conjugated into V. anguillarum were transformed into E. coliS17-1 (49), which was used as the donor strain. Plasmid transfers from E. coli toV. anguillarum were done as previously described (35). E. coli XL1-Blue (Strat-agene) was used for bacteriophage lambda infections and for routine cloning.The medium routinely used for E. coli was Luria broth, which contains BactoTryptone (10 g/liter), Bacto Yeast Extract (5 g/liter), and sodium chloride (10g/liter). For V. anguillarum, Trypticase soy medium (BBL) was used for routinegrowth. For purification and identification of AHLs, V. anguillarum was grown at20°C with shaking in M9 medium (45) supplemented with 2% (wt/vol) NaCl. Forpurification of AHLs produced via recombinant VanM, E. coliJM109(pBSVanMN) and E. coli JM109(pDKVanM) were grown at 30°C withshaking in M9 medium containing ampicillin (100 mg/ml).

Plasmid pVanM-2 is a pSup202P (34) derivative which contains the vanMgene. A fragment containing vanM and its promoter (bp 108 to 1627 [Fig. 1]) wasobtained by PCR. A restriction enzyme site, either NheI or BglII, was added tothe 59 end of each PCR primer to aid cloning of the PCR product. This fragmentwas ligated into the SpeI and BglII sites of pSup202P.

AHL reporter assays. AHLs were detected using a Tn5-generated Chromobac-terium violaceum mutant termed CV026 which responds to a range of AHLs(with from C4 to C8 acyl side chains) by inducing the synthesis of the purplepigment violacein (27, 29, 57). For this assay, AHL-producing strains are cross-streaked vertically against a horizontal streak of CV026 on Trypticase soy agarplates. Alternatively, spent culture supernatants, solvent extracts, or high-pres-sure liquid chromatography (HPLC) fractions (see below) were analyzed for thepresence of short- and long-chain AHLs using the direct and reverse CV026seeded agar plate assays, respectively, as described previously (29, 36). For themore sensitive detection of long-chain AHLs (with C10 to C14 acyl side chains),a bioluminescent E. coli lux-based AHL biosensor termed E. coliJM109(pSB1075), which contains an intact lasR gene and the lasI promoter fromPseudomonas aeruginosa fused to luxCDABE from Photorhabdus luminescens,was used (58). Thin-layer chromatography (TLC) was also used to separateAHLs and overlaid with soft top agar seeded with the appropriate biosensor asdescribed by McClean et al. (29). For analysis of short-chain AHLs, reverse-phase aluminum-backed RP18 F254S TLC plates (20 by 20 cm; Merck) and amobile phase of 60% (vol/vol) methanol in water were used. Long-chain AHLswere analyzed on aluminum-backed Silica Gel 60 F254 normal-phase TLC plates(20 by 20 cm; Merck) using a 45%-55% (vol/vol) hexane-acetone mix as themobile phase.

Isolation, purification, and chemical characterization of AHLs. AHLs werepurified and characterized as described by Bainton et al. (3) and Camara et al.(8). Essentially, spent supernatants (4 liters) from stationary-phase cultures of V.anguillarum NB10, E. coli JM109(pBSVanMN), or E. coli JM109(pDKVanM)were extracted with dichloromethane, and solvent extracts were separated bysemipreparative reverse-phase HPLC as previously described (36). Fractionswere tested for the presence of AHLs using the AHL biosensor C. violaceumCV026 or E. coli(pSB401) in conjunction with TLC as described before (29).Following preparative HPLC, the active subfractions were analyzed by HPLC-mass spectrometry (LC-MS) as described previously (2). The spectra obtainedwere compared with those for synthetic AHL standards subjected to the sameLC-MS conditions.

Synthesis of AHLs. The AHLs 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL were synthesized, purified, and characterized as described previously (8,10). Each compound was subjected to MS, proton nuclear magnetic resonancespectroscopy, and infrared spectroscopy. For 3-hydroxy-C6-HSL and C6-HSL,spectroscopic data are provided in references 8 and 36 for 3-oxo-C10-HSL.

Quantification of AHL production. To determine the relative levels of 3-hy-droxy-C6-HSL, C6-HSL, and 3-oxo-C10-HSL in V. anguillarum wild type andvanM (DM27)- and vanI (DM21)-negative mutants, 100 ml of each stationary-phase culture supernatant was extracted with dichloromethane as describedabove, evaporated to dryness, and resuspended in 100 ml of acetonitrile. Eachsample was subjected to LC-MS, and the concentration was determined by

3538 MILTON ET AL. J. BACTERIOL.

comparison with a calibration curve constructed for molecular ion abundance,using each of the appropriate AHL synthetic standards.

Cloning of the vanMN DNA locus. To identify genes homologous to luxLMNfrom V. harveyi (4) and to ainSR from V. fischeri (18), protein comparisons wereperformed and two degenerate inosine-containing oligonucleotides were de-signed to complement both the ainR and the luxN DNA sequences and to containa restriction endonuclease site, either SacI or SpeI, at each 59 end. These two

primers were used in PCR to generate a 320-bp fragment from the chromosomeof V. anguillarum (Fig. 1). This 320-bp fragment was cloned into the SpeI andSacI sites of pBluescript to give pBSVanN-320. The DNA sequence of thisfragment was similar to that of the partial ainR gene of V. fischeri (18) and theluxN gene of V. harveyi (4). The 320-bp fragment was used as a probe to screena Lambda Zap II (Stratagene)-based gene bank of V. anguillarum (34) as pre-viously described (35). From a positive plaque, pBluescript containing the chro-mosomal fragment was excised from the bacteriophage DNA as described pre-viously (34). This plasmid, pBSVanN10-1, contained a 7-kb chromosomal insertwith the entire vanM gene and all but the last 39 bp of the vanN gene. Thus, asecond screening was done as above, using a 500-bp KpnI fragment from the 39

end of the vanN gene as the probe (Fig. 1). The excised plasmid, pBSVanN10-5,contained a 6.5-kb chromosomal insert with only 368 bp of the 39 end of vanN.The overlap was sufficient to complete the sequencing of both the vanN andvanM genes. From the sequence information, pBSVanMN, a pBluescript deriv-ative that contained the entire putative vanMN operon, was designed. To createpBSVanMN, pBSVanN10-1, which was missing only the last 39 bp of this puta-tive operon, was used. To make the chromosomal fragment insert smaller,pBSVanN10-1 was digested with BamHI and MluI to remove approximately1,800 bp upstream of vanM. The vector fragment containing vanM was gelpurified, and the ends were filled in using Klenow enzyme and then ligatedtogether. To create a complete vanN gene on the now smaller chromosomalfragment insert, approximately 500 bp was removed from the 39 end of thefragment insert using the internal KpnI site, which is about 500 bp from the 39

end of vanN, and the KpnI site in the vector polylinker. The vector ends werethen dephosphorylated using calf intestinal phosphatase, and a PCR fragmentcontaining the vanN sequence from the internal KpnI site to 320 bp downstreamof vanN was ligated to the KpnI-cut pBSVanN10-1. This region was sequenced toensure that it was identical to the wild-type sequence.

FIG. 1. Genetic map of the vanMN locus of V. anguillarum. ThevanN gene overlaps the vanM gene by 8 bp. The horizontal arrowsindicate the direction of transcription. The open arrowhead indicateswhere pNQVanN2 was inserted into vanN to make strain DM34. Thedotted line indicates the region of vanM that was deleted in frame tomake strain DM27. The line labeled P1 indicates the region of vanNthat was amplified using the degenerate primers complementary toainS and luxN. This P1 region of vanN was used as the probe for firstscreening of the gene library from which pBSVanN10-1 was isolated.The line labeled P2 indicates the region of vanN that was used as aprobe in the second screening of the gene library from whichpBSVanN10-5 was isolated.

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Genotype and/or relevant markers References or source

E. coli strainsJM109 recA1 supE44 endA1 hsdR17 gyrA96 relA1 thi D(lac-proAB) 46SY327 D(lac pro) argE(Am) rif malA recA56 34S17-1 thi pro hsdR hsdM1 recA RP4-2-Tc::Mu-Km::Tn7 50XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 D(lac-pro) [F9 proAB lacIq lacZDM15

Tn10(Tetr)]Stratagene

C. violaceum CV026 Kmr; strain ATCC 31532 defective in C6-HSL production due to a mini-Tn5 insertion in thecviI gene

28, 30, 58

V. harveyi BB120 Wild type 7

V. anguillarum strainsNB10 Wild type, serotype O1 40DM21 In-frame deletion of vanI 37DM27 In-frame deletion of vanM This studyDM34 Cmr; polar mutation in vanN This study

PlasmidspBluescript Apr; ColE1 origin StratagenepBSVanN-320 Apr; pBluescript containing a 320-bp PCR fragment from vanN (bp 1706–1990) This studypBSVanN10-1 Apr; pBluescript containing a cloned fragment with the putative vanMN operon to bp 4061 This studypBSVanN10-5 Apr; pBluescript containing a cloned fragment with vanN from bp 3731 to 4099 This studypBSVanMN Apr; pBluescript containing subcloned fragments that give a complete putative vanMN

operonThis study

pSup202P Cmr, Tcr; pSup202 derivative (RP4 Mob1) with a polylinker cloned into PstI within the Apr

gene35

pVanM-2 Cmr, Tcr; pSup202P derivative containing the vanM gene and its promoter (bp 108–1627) This studypDM4 Cmr; suicide vector with an R6K origin and the sacBR genes from Bacillus subtilis 36pDMVanM1 Cmr; pDM4 derivative containing vanM bp 108–324 fused in frame to bp 1309–1518 This studypNQ705-1 Cmr; suicide vector that contains an R6K origin 31pNQVanN2 Cmr; pNQ705-1 derivative containing vanN bp 1548–1787 This studypGEM-T Easy Apr; ColE1 origin PromegapDKVanM Apr; pGEM-T Easy derivative containing vanM This studypSB401 Tcr; contains a fusion of luxRI9::luxCDABE in pACYC184 58pSB1075 Apr; contains a fusion of lasRI9::luxCDABE in pUC18 59

VOL. 183, 2001 MULTIPLE QUORUM-SENSING CIRCUITS IN V. ANGUILLARUM 3539

Construction of pDKVanM. The vanM gene, from 10 bp upstream of the startcodon through the stop codon, was amplified from V. anguillarum NB10 chro-mosomal DNA by PCR and then cloned into the plasmid pGEM-T Easy (Pro-mega) by T/A cloning as described in the manufacturer’s instructions. A clone(pDKVanM) was selected in which the vanM gene was under the control of theplasmid-borne lacZ promoter.

DNA techniques and sequencing. Oligonucleotides for primers were synthe-sized using a model 394 Applied Biosystems DNA/RNA synthesizer. Unlessotherwise stated, all conditions for DNA techniques and enzymatic reactionswere done as described by Sambrook et al. (45) or as suggested by the manu-facturers. Double-strand DNA sequencing was performed either by the dideoxy-chain termination method with T7 DNA polymerase (Pharmacia Biotech) or byautomated sequencing using an Applied Biosystems 373A sequencer. UsingpBSVanN10-1 and pBSVanN10-5 plasmid DNA, both strands of a 4,514-bpfragment containing vanM and vanN were sequenced by primer walking in twodirections.

PCR conditions. PCRs were performed as previously described (30). Forresequencing of the junction between the V. harveyi luxL and luxM genes, twoprimers were designed from the DNA sequence (GenBank accession numberL13940) to amplify bp 587 to 1035. PCR began with template denaturation for 5min at 95°C before the addition of Taq DNA polymerase. After enzyme addition,the PCR was continued for 29 cycles of 2 min at 55°C, 1 min at 72°C, and 1 minat 95°C, followed by one cycle of 55°C for 2 min and 72°C for 10 min.

Construction of the vanM and vanN mutations. The vanN null mutant DM34was made by the integration of a suicide plasmid, pNQVanN2, a derivative ofpNQ705-1 (30), into the vanN gene as described previously (34). Using SacI andSpeI, a 239-bp PCR fragment (residues 1548 to 1787 [Fig. 1]) complementary tovanN was cloned into the SacI and SpeI sites of pNQ705-1, creating pNQVanN2.The entire pNQVanN2 was inserted 28 bp downstream of the vanN start codon.Since the PCR fragment cloned onto pNQVanN2 did not contain the vanNpromoter or start codon, this plasmid insertion created a null mutation of vanN.The small piece of the vanN gene carried on pNQVanN2 used to target theinsertion of the suicide plasmid allows for only the first 89 amino acids of VanNto be translated. The insertion of the plasmid was checked by PCR analysis usinga previously described primer (34) complementary to a region on the plasmid justoutside the polylinker region and a primer complementary to a chromosomalDNA region just outside the PCR fragment cloned into pNQVanN2. The PCRfragments obtained from the mutant were analyzed by restriction mapping toensure that the PCR fragments obtained were from the correct region of thechromosome. Stability of the insertion mutation was tested by growth for 30generations in the absence of chloramphenicol. Of 100 colonies tested, no loss inchloramphenicol resistance was seen.

The vanM mutant DM27 contains an in-frame deletion created by allelicexchange as described previously (35). To create the new deletion allele, overlapPCR was performed joining vanM residues 108 to 324 to residues 1309 to 1518.By using NheI and BglII, this PCR fragment was cloned into the SpeI and BglIIsites of pDM4, creating pDMVanM1. After allelic exchange using pDMVanM1,the start codon was fused to the last 72 amino acid codons of vanM (Fig. 2). Toconfirm that an in-frame deletion was made in DM27, this region from thechromosome was PCR amplified and cloned. Several clones were then se-quenced to ensure that the deletion was made and that no errors were insertedwithin the new allele.

Computer analysis. Database searches were done using the sequence analysissoftware of the Genetics Computer Group, Inc. (University of Wisconsin) (12).

Fish infections. Rainbow trout (Oncorhynchus mykiss) with an approximateweight of 10 to 15 g were infected with V. anguillarum either by intraperitonealinjections or by immersion of the fish in seawater containing V. anguillarum aspreviously described (35). The immersion and intraperitoneal infections weredone at least two times. Five fish were infected for each bacterial dilution used.The 50% lethal doses (LD50s) were calculated as described by Reed and Muench(43). The LD50s recorded are an averaged number of all infections for eachstrain. To aid comparative analysis between strains, the standard deviation of thewild-type LD50 was calculated for both routes of infection. LD50 values werecollected from previous studies in our lab and used in determining a standarddeviation for the wild-type strain. For infection by immersion, 37 LD50 valueswere used to give a standard deviation of 3.9 3 103 bacteria per ml of seawater.For the intraperitoneal route, 35 LD50 values were used, giving a standarddeviation of 29 bacterial cells.

Nucleotide sequence accession number. The vanM and vanN DNA sequencehas been submitted to GenBank with accession number AF288163. The se-quence from V. harveyi with the amended sequence for luxLM sequence in V.harveyi has also been submitted; its accession number is AF286004.

RESULTS

Cloning and sequencing of the vanMN locus. Using AHLbioassays, we have previously detected AHLs in bacterial cul-ture supernatants from both V. anguillarum NB10 and DM21(36), a vanI mutant which does not produce 3-oxo-C10-HSL.This result suggests that V. anguillarum most likely contains asecond quorum-sensing system which may be responsible forthis activity. Since V. anguillarum is related to V. harveyi and V.fischeri and since both V. harveyi and V. fischeri contain lux-LMN quorum-sensing systems (4, 18), we rationalized that V.anguillarum may also have a luxLMN-type circuit. To deter-mine whether V. anguillarum contains a second quorum-sens-ing system, degenerate PCR primers were designed from theluxN and ainR genes, which code for AHL sensor proteins in V.harveyi (4) and V. fischeri (18), respectively. A DNA fragmenthighly homologous to both luxN and ainR was amplified fromthe V. anguillarum chromosome and used as a probe to screena V. anguillarum chromosomal gene bank. Positive clones weresequenced revealing the presence of two open reading frames(ORFs), which we termed vanM and vanN. Further analysisshowed that the last 8 bp of vanM overlapped with the start ofvanN, suggesting that these genes may share the same pro-moter. Figure 1 shows a genetic map of this DNA locus.

The predicted amino acid sequences for VanM and VanNwere compared with the their homologues in both V. harveyiand V. fischeri. The amino terminus of VanM is 58% identicalto LuxL, and the carboxy terminus of VanM is 66% identical toLuxM (4). Over the whole protein, VanM is 33% identical toAinS (Fig. 2A) (18). Figure 2B shows that VanN is 76% iden-tical to the entire LuxN protein (4) and 38% identical to thepartial sequence of AinR that is available (18). Like LuxN (4,16), VanN is also homologous to numerous members of thetwo-component family of adaptive response regulators (for areview, see reference 40), with the highest similarity to hybridsensor kinases that contain both sensor histidine kinase andresponse regulator domains (40). This group includes proteinssuch as BvgS from Bordetella pertussis (1), LemA (GacS) fromPseudomonas syringae pv. syringae (22), and RpfC from Xan-thomonas campestris (54), all of which regulate virulence geneexpression, as well as RcsC, BarA, and ArcB from E. coli,which regulate capsule production, gene expression in re-sponse to changes in osmolarity, and gene expression in re-sponse to oxygen levels, respectively (23, 38, 50).

The V. harveyi luxL and luxM genes are within a single ORF.The sequence homology analyses and the functional data (seebelow) presented in this report show that VanM is the thirdmember of a new family of AHL synthases. However, we wereintrigued as to why a single protein is sufficient for AHL syn-thesis in V. anguillarum and V. fischeri whereas two proteins,LuxL and LuxM, are apparently required in V. harveyi. Giventhe similarity of VanM to LuxL and LuxM and the fact that thecombined molecular mass of LuxL and LuxM (42.5 kDa) isapproximately equal to that of AinS (43.5 kDa) and VanM(44.1 kDa), we wondered whether there is a sequencing errorin the junction between luxL and luxM that leads to a frame-shift which results in two ORFs instead of one. To test thishypothesis, PCR was used to amplify the junction between theluxL and luxM genes from V. harveyi BB120 (7). PCR productsfrom three independent reactions were sequenced, and all


showed an extra base pair at the 39 end of luxL compared tothe previously published sequence (GenBank accession num-ber L13940). The addition of this base pair alters the predictedpolypeptide sequences of LuxL and LuxM, fusing them intoone 44-kDa protein which will be called LuxM (B. Bassler,personal communication). However, for ease of discussion, wewill call the 44-kDa protein LuxM* in this report. VanM is

64% identical to LuxM*, indicating that VanM and LuxMp aremore similar to each other than to AinS (Fig. 2A).

Identification of AHLs synthesized by the vanMN and vanMDNA loci in E. coli JM109. To determine what AHLs, if any,are synthesized via this DNA locus, the vanMN genes weresubcloned into pBluescript and introduced into E. coli JM109.Large-scale solvent extractions were performed on spent su-

FIG. 2. Protein sequence alignments. (A) The corrected V. harveyi LuxM* protein sequence, the V. fischeri AinS protein sequence, and the V.anguillarum VanM protein sequence were aligned and compared for similarities. The LuxL-LuxM fused protein will be called LuxM (Bassler,personal communication), but for the sake of discussion it is called LuxM* in this report. (B) The V. harveyi LuxN protein sequence, the V. fischeriAinR partial protein sequence, and the V. anguillarum VanN protein sequence were aligned and compared for similarities. Asterisks indicateamino acids that are identical in the V. harveyi and V. anguillarum protein sequences; plus signs indicate amino acids that are identical in all alignedprotein sequences. The amino acids within boxes represent the various conserved motifs important for the function of hybrid sensor kinases (10).


FIG. 3. Mass spectra (LC-MS) of two compounds purified from the spent culture supernatant of E. coli JM109(pBSVanMN) (B and D) areindistinguishable from those of synthetic 3-hydroxy-C6-HSL (A) and C6-HSL (C), respectively.


FIG. 3—Continued.


pernatant from stationary-phase E. coli JM109(pBSVanMN)cultures grown in M9 culture medium. The concentrated crudeextract was fractionated by HPLC using a linear acetonitrile-in-water gradient. The resulting fractions were assayed forAHL activity using the CV026 biosensor in conjunction withTLC. Two active fractions (fractions 2 and 3) were then ana-lyzed by LC-MS. The LC-MS spectrum for the moleculepresent in fraction 2 revealed a molecular ion of 215 and afragmentation product of 102 (which corresponds to the ho-moserine lactone moiety). This suggests that the AHL presentis 3-hydroxy-C6-HSL (Fig. 3). Furthermore, we observed apeak at 197 arising from the expected loss of a molecule ofwater which is characteristic of 3-hydroxy AHL derivatives(48). 3-Hydroxy-C6-HSL was synthesized as described previ-ously (10) and shown to possess chromatographic and spectralproperties identical to those of the natural compound. Thepresence of C6-HSL in fraction 3 was deduced from the pres-ence of a molecular ion of 200 (M 1 1) together with frag-mentation ions of 102 and 99, which correspond to the homo-serine lactone moiety and the hexanoyl side chain, respectively(Fig. 3). To confirm that vanM alone was responsible for theproduction of 3-hydroxy-C6-HSL and C6-HSL, the gene wasamplified from the V. anguillarum NB10 chromosome by PCRand cloned into pGEM-T to create pDKVanM. Cell-free cul-ture supernatant extracts were subjected to TLC and overlaidwith either CV026 or E. coli(pSB401). In both cases, two spotswere obtained which migrated with the same Rf values as thesynthetic 3-hydroxy-C6-HSL and C6-HSL standards (data notshown).

To determine the relative levels of the three AHLs producedby V. anguillarum NB10, cell-free culture supernatants wereextracted with solvent and subjected to LC-MS. The concen-tration of each AHL present was determined by reference to acalibration curve constructed for each synthetic standard. Fig-ure 4 reveals that 3-oxo-C10-HSL and 3-hydroxy-C6-HSL arethe major AHLs and are present at approximately 8.5 and 9.5nM, respectively. C6-HSL is a minor component present ataround 0.3 nM.

VanM is required for synthesis of C6-HSL and 3-hydroxy-C6-HSL in V. anguillarum. To determine whether vanM andvanN are required for AHL synthesis, isogenic mutations thatresulted in the inactivation of either of these genes in V. an-guillarum were constructed. The vanM mutant DM27 con-tained an in-frame deletion removing codons 2 through 336 ofVanM (Fig. 1). Analysis of spent cell-free culture supernatants,using the CV026/TLC bioassays and LC-MS (Fig. 4 and 5A),revealed that DM27 did not produce either 3-hydroxy-C6-HSLor C6-HSL. Interestingly, further analysis using the E. co-li(pSB1075) lux-based sensor showed that the production of3-oxo-C10-HSL, which is driven by VanI, had also been down-regulated (Fig. 5B). Using LC-MS, no 3-oxo-C10-HSL couldbe detected in the vanM mutant (Fig. 4). These results dem-onstrate that VanM directs the synthesis of C6-HSL and 3-hy-droxy-C6-HSL in V. anguillarum and that these AHLs arerequired for the production of 3-oxo-C10-HSL. To confirmthat the lack of all three AHLs was due to the loss of vanM,pVanM-2, containing the wild-type vanM gene, was introducedinto the vanM isogenic mutant DM27. TLC analysis showedthat the production of C6-HSL, 3-hydroxy-C6-HSL, and 3-oxo-C10-HSL was restored in the transcomplemented mutant (Fig.

5). Furthermore, growth of DM27 in medium previously con-ditioned by growth with the vanI mutant DM21 (which doesnot produce 3-oxo-C10-HSL but still produces C6-HSL and3-hydroxy-C6-HSL [Fig. 4]) restored production of 3-oxo-C10-HSL in the vanM mutant (data not shown).

To assess the role of vanN in AHL production, a vanNisogenic null mutant was constructed. A suicide plasmid,pNQVanN2, was inserted 28 bp downstream of the vanN startcodon, resulting in mutant DM34 (Fig. 1). Using CV026 and E.coli(pSB1075) bioassays, cell-free supernatants from DM34displayed wild-type levels of all three AHLs, suggesting thatVanN does not play a role in the regulation of AHL produc-tion (data not shown).

Virulence analysis. Since VanN is homologous to severaltwo-component regulatory proteins involved in controlling vir-ulence in other bacteria, the LD50s for the vanM and vanNmutants were compared to that of the wild type in a fishinfection model. Both the immersion and intraperitoneal in-fection routes were used. For the immersion route, the LD50swere 4 3 103, 3 3 104, and 4 3 103 bacteria per ml of seawaterfor the wild type, the vanM mutant (DM27), and the vanNmutant (DM34), respectively. A sevenfold difference in viru-lence was seen for the vanM mutant; however, the standarddeviation of the wild-type LD50 was determined to be 63.9 3103, indicating that this sevenfold difference may not be signif-icant. For the intraperitoneal route, the LD50s were similar: 34(standard deviation of 629), 74, and 70 bacteria for the wildtype, the vanM mutant (DM27), and the vanN mutant (DM34),respectively. These data suggest that the virulence of V. anguil-larum is likely not affected by the vanM and vanN mutations.

Protease production. The metalloprotease, EmpA, of V. an-guillarum is 69% identical to Hap, the V. cholerae hemagglu-tinin protease (33). The hap gene is regulated via HapR, ahomologue of the LuxR protein of V. harveyi (25). Therefore,since LuxR is a component of the same quorum sensing cir-cuitry as LuxM* and LuxN, we reasoned that empA expressionin V. anguillarum may be affected in the vanM and vanN mu-tants. However, on skimmed milk agar plates, the zones ofclearance were similar for both mutants and the wild type.

DISCUSSION

In recent years, diverse gram-negative bacteria have beenshown to produce AHLs that stimulate LuxI/LuxR-type regu-latory circuits. Some of these bacteria possess multiple LuxI/LuxR-type regulatory circuits and produce multiple AHLs,which are involved in the regulation of multiple target genes(for a review, see reference 13). Despite the low similarity(;30%) among members of the LuxI family, these homo-logues appear to have similar enzymatic activities. LuxI homo-logues utilize S-adenosylmethionine and an acyl-acyl carrierprotein (acyl-ACP) in the synthesis of AHLs (19, 37, 41, 47,55), although some have also been shown to use the appropri-ately charged acyl-coenzyme A (acyl-CoA) as an alternativesource of the acyl side chain (24, 41). However, the molecularbasis by which different LuxI homologues are able to select theappropriate acyl chain from ACP or CoA derivatives to pro-duce the desired AHL(s) is not known. Recently, a secondfamily of AHL synthases, which bears no homology to LuxI,has been described (18). To date, members of this new family


have been identified in V. harveyi (LuxM*) (4) and V. fischeri(AinS) (18), where they direct the synthesis of 3-hydroxy-C4-HSL and C8-HSL, respectively. Hanzelka et al. (20) haveshown that AinS utilizes substrates and has enzyme kineticssimilar to those for LuxI. However, in addition to octanoyl-ACP, AinS also utilized octanoyl-CoA as the acyl group donor.Consequently, Hanzelka et al. (20) speculated that possessionof two different types of AHL synthases is beneficial to V.fischeri, as it may permit the production of AHLs and quorumsensing even when the prevailing environmental conditions arenot optimal for AHL synthesis via LuxI.

The results presented in this study add V. anguillarum to thelist of gram-negative bacteria that produce multiple AHLs andpossess multiple quorum-sensing regulatory circuits. Specifi-cally, we have identified two additional AHLs that are pro-duced by V. anguillarum. Moreover, we have characterized thegene responsible for their synthesis, vanM. VanM is homolo-gous to AinS of V. fischeri and LuxM* of V. harveyi. Thus, like

V. fischeri, V. anguillarum contains an AHL synthase from theLuxI (36) and the LuxM*/AinS families.

Protein sequence comparisons revealed that VanM andLuxM* are 64% identical, whereas AinS is only 35% identicalto LuxM*. Thus, AinS appears to be significantly differentfrom VanM and LuxM*. Accordingly, AinS could represent adifferent subset of synthases within this new family. Eventhough LuxM* and VanM are closely related, they direct thesynthesis of different AHLs. However, both make 3-hydroxy-lated AHLs, in contrast to the AHL synthesized via AinS,which lacks a C-3 acyl side chain substituent.

VanN belongs to a family of hybrid sensor kinases thatcontain two or more signaling domains within a single polypep-tide (for a review, see reference 40). Like LuxN, VanN con-tains nine possible membrane-spanning regions at its aminoterminus, suggesting that VanN is a membrane-bound protein.This region (Fig. 2B) may contain the signal input domain thatresponds to the AHLs produced by VanM. Attempts to com-plement a V. harveyi luxN mutant with vanN have not beensuccessful. However, spent culture supernatants from V. an-guillarum NB10 do not activate either the wild-type V. harveyior a luxQ-negative mutant (unpublished data), suggesting thatvanN is unlikely to complement a luxN mutation unless vanMor exogenous 3-hydroxy-C6-HSL is also provided. As shown inFig. 2, VanN appears to contain a centrally located sensorkinase domain required for autophosphorylation, nucleotidebinding, and autokinase activity (reviewed in reference 39). Atypical response regulator domain is found at the carboxy ter-minus that contains the characteristic asparagine, which re-ceives a phosphoryl group from a histidine residue in a sensorkinase domain. A Lys-109 is also present, which may play a rolein triggering a conformational change in the protein uponphosphorylation of the asparagine residue (reviewed in refer-ence 39). In V. harveyi, the phosphoryl group of LuxN is passedto an additional phosphotransfer protein, LuxU, and then tothe s54 activator LuxO (15, 16). Once phosphorylated, LuxO,together with s54, activates the expression of an unidentifiedrepressor of bioluminescence (5, 15, 28). Thus, V. anguillarummay contain homologues of the V. harveyi proteins LuxU andLuxO. Furthermore, V. anguillarum may possess a gene withhomology to luxR from V. harveyi. Jobling and Holmes (25)have shown, by Southern blot analysis, that the hapR gene fromV. cholerae, also a homologue of the V. harveyi luxR gene,cross-hybridizes to chromosomal DNA from V. anguillarum.

In addition to the VanMN system identified in this study, V.anguillarum may also possess homologues of LuxS, LuxP, andLuxQ from V. harveyi (6, 51). These proteins are part of asecond quorum-sensing system that works in parallel to theLuxLMN circuit (15, 16) by transferring a phosphate grouponto the same phosphorelay protein, LuxU. The presence ofthis second circuit may provide an explanation as to why pro-tease production and/or virulence were not affected in thevanM and vanN mutants. In V. harveyi, both sensory channelsneed to be inactivated for loss of bioluminescence; if only onechannel is inactivated, the second channel will compensate,ensuring that the bacterium still produces light (4, 6). In ourstudies, we have made mutations in only one of two possiblesensory channels. If V. anguillarum does indeed contain a sec-ond sensory channel as does V. harveyi, then we would notexpect a loss of function in the mutants that we have made.

FIG. 4. Quantification of AHLs produced by V. anguillarum wildtype (WT) and vanM (DM27) and vanI (DM21) mutants (M- and I-)in stationary-phase supernatant determined by LC-MS. Each samplewas subjected to LC-MS, and the concentration was determined bycomparison with a calibration curve constructed for molecular ionabundance using each of the corresponding AHL synthetic standards.

FIG. 5. TLC analyses of the V. anguillarum vanM mutant (DM27)showing the loss of 3-hydroxy-C6-HSL, C6-HSL (A), and 3-oxo-C10-HSL (B) and the restoration of AHL synthesis in the vanM mutant(DM27) complemented with plasmid-borne copies of vanM[DM27(pVanM-2)]. For this assay, stationary-phase, cell-free super-natants together with synthetic standards were analyzed by TLC inconjunction with the AHL biosensor, C. violaceum CV026 (A) or E.coli JM109(pSB1075) (B), for the detection of short or long-chainAHLs, respectively. (A) C6-HSL (lane 1), wild type (lane 2), DM27(lane 3), DM27(pVanM-2) (lane 4), and 3-hydroxy-C6-HSL (lane 5);(B) 3-oxo-C10-HSL (lane 1), wild type (lane 2), DM27 (lane 3), andDM27(pVanM-2) (lane 4).


Therefore, we cannot rule out that quorum sensing does notregulate virulence genes or protease production until we havedetermined whether a second sensory channel is present.Moreover, Denkin and Nelson (11) have shown that the ex-pression of the metalloprotease gene, empA, is ninefold higherin broth containing fish gastrointestinal mucus than in conven-tional broth. Consequently, metalloprotease production maybe affected differently in vanM and vanN mutants grown in thepresence of fish gastrointestinal mucus.

The AHL profiles for the vanM and vanN mutants, however,may conflict with the presence of a putative dual-sensory-chan-nel model in V. anguillarum. A mutation in vanM not onlyabolished the production of C6-HSL and 3-hydroxy-C6-HSLbut also affected 3-oxo-C10-HSL production, suggesting thatthe VanMN system, in some way, regulates VanIR. In contrast,the vanN mutant showed wild-type AHL production. However,in a dual-pathway model, a mutation in vanM should not affectthe production of 3-oxo-C10-HSL. There are several possibil-ities to explain how a vanM mutation may affect the productionof 3-oxo-C10-HSL. First, the two-sensory-channel model maynot exist in V. anguillarum, and quorum sensing may not reg-ulate virulence genes or protease production. If this is true,then the vanN mutant should also be negative for AHL pro-duction, as there would be no second compensatory system asin V. harveyi. Second, the V. anguillarum VanR protein, ahomolog of LuxR from V. fischeri (36), may bind 3-hydroxy-C6-HSL and/or C6-HSL as well as 3-oxo-C10-HSL to becomean active transcriptional activator of vanI, which directs thesynthesis of 3-oxo-C10-HSL. However, for the LuxR homo-logues studied to date, the cognate AHL seems to be requiredfor maximal activation of gene expression (42, 46). Alterna-tively, multiple AHL signals may be needed for activation ofVanR. Finally, perhaps there is an additional, unidentifiedprotein or signal molecule in this system that allows theVanMN circuit to function independently of a second sensorychannel.

Thus far, the only clear function of either of the two quo-rum-sensing circuits in V. anguillarum is that the VanMN quo-rum-sensing circuit regulates the production of 3-oxo-C10-HSL via the VanRI quorum-sensing circuit, which couldpotentially regulate the expression of target genes. Additionalphenotypic analyses have not yet revealed any further role forthe AHLs produced by V. anguillarum. However, given thepotential complexity of the V. anguillarum quorum-sensing sys-tems, the mutant strains that we are using may not be sufficientto permit the complete elucidation of the role of quorumsensing in this organism. Characterization of any additionalgenes in the VanMN circuit should help to identify AHL-regulated genes in V. anguillarum. One possible role of 3-oxo-C10-HSL may be as an effector of genes found in other bac-teria besides V. anguillarum. Interestingly, 3-oxo-C10-HSL hasbeen shown to antagonize protease activities of Aeromonashydrophila (53) and Aeromonas salmonicida (52). These pro-teases are known to contribute to virulence and to be regulatedby quorum sensing. Since these organisms are all fish patho-gens, Swift et al. (53) have suggested that a possible role for3-oxo-C10-HSL may be to antagonize the quorum-sensing de-pendent virulence of A. hydrophila and A. salmonicida, whichin turn may provide V. anguillarum with a competitive edgeduring fish infection. Another possibility is that the long-acyl-

chain AHL, 3-oxo-C10-HSL, has an effect on eukaryotic cellbehavior. Previously it has been shown that 3-oxo-C12-HSL,produced by the human pathogen P. aeruginosa, has immuno-modulatory and vasodilator activities in various animal modelsprocesses (for a review, see reference 56). Furthermore, activ-ity seems to be dependent on the presence of a long acyl chainsince 3-oxo-C6-HSL was inactive. These studies imply that3-oxo-C12-HSL plays a role not only in regulating P. aerugi-nosa virulence gene expression but also in the orchestration ofeukaryotic cells to maximize the provision of nutrients via thebloodstream while downregulating host defense mechanisms.Since V. anguillarum produces a long-acyl-chain AHL, it ispossible that 3-oxo-C10-HSL may regulate eukaryotic cell be-havior and modulate the disease process within fish.

ACKNOWLEDGMENTS

We thank Karen McGee for making many of the mutants, MavisDaykin for HPLC analysis, Cath Otori for LC-MS analysis, and RamChhabra and Chris Harty for the synthesis of AHLs.

This work was supported by grants from the Swedish Council forForestry and Agricultural Research, the Swedish Research Council forEngineering Sciences, and the Carl Tryggers Foundation, Sweden (toD.L.M.), and by grants and a studentship from the Biotechnologyand Biological Sciences Research Council, United Kingdom (toA.H., M.C., and P.W.), which are gratefully acknowledged.

REFERENCES

1. Arico, B., J. F. Miller, C. Roy, S. Stibitz, D. Monack, S. Falkow, R. Gross,and R. Rappuoli. 1989. Sequences required for expression of Bordetellapertussis virulence factors share homology with prokaryotic signal transduc-tion proteins. Proc. Natl. Acad. Sci. USA 86:6671–6675.

2. Atkinson, S., J. P. Throup, G. S. A. B. Stewart, and P. Williams. 1999. Ahierarchical quorum-sensing system in Yersinia pseudotuberculosis is involvedin the regulation of motility and clumping. Mol. Microbiol 33:1267–1277.

3. Bainton, N. J., P. Stead, S. R. Chhabra, B. W. Bycroft, G. P. C. Salmond,G. S. A. B. Stewart, and P. Williams. 1992. N-(3-Oxohexanoyl)-L-homoserinelactone regulates carbapenem antibiotic production in Erwinia carotovoraBiochem. J. 288:997–1004.

4. Bassler, B. L., M. Wright, R. E. Showalter, and M. R. Silverman. 1993.Intercellular signalling in Vibrio harveyi: sequence and function of genesregulating expression of luminescence. Mol. Microbiol. 9:773–786.

5. Bassler, B. L., M. Wright, and M. R. Silverman. 1994. Sequence and functionof LuxO, a negative regulator of luminescence in Vibrio harveyi. Mol. Mi-crobiol: 12:403–412.

6. Bassler, B. L., M. Wright, and M. R. Silverman. 1994. Multiple signallingsystems controlling expression of luminescence in Vibrio harveyi: sequenceand function of genes encoding a second sensory pathway. Mol. Microbiol.13:273–286.

7. Bassler, B., E. P. Greenberg, and A. M. Stevens. 1997. Cross-species induc-tion of luminescence in the quorum-sensing bacterium Vibrio harveyi. J.Bacteriol. 179:4043–4045.

8. Camara, M., M. Daykin, and S. R. Chhabra. 1998. Detection, purificationand synthesis of N-acyl homoserine lactone quorum sensing molecules.Methods Microb. Bacter. Pathog. 27:319–330.

9. Cao, J.-G., and E. A. Meighen. 1989. Purification and structural identificationof an autoinducer for the luminescence system of V. harveyi. J. Biol. Chem.264:21670–21676.

10. Chhabra, S. R., P. Stead, N. J. Bainton, G. P. C. salmond, G. S. A. B. Stewart,P. Williams, and B. W. Bycroft. 1993. Autoregulation of carbapenem bio-synthesis in Erwinia carotovora by analogues of N-(3-oxohexanoyl)-L-homo-serine lactone. J. Antibiot. 46:441–449.

11. Denkin, S. M., and D. R. Nelson. 1999. Induction of protease activity inVibrio anguillarum by gastrointestinal mucus. Appl. Environ. Microbiol. 65:3555–3560.

12. Devereux, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set ofsequence analysis programs for the VAX. Nucleic Acids Res. 12:387–395.

13. Dunny, G. M., and S. C. Winans. 1999. Cell-cell signaling in bacteria. Amer-ican Society for Microbiology, Washington, D.C.

14. Eberhard, A., A. L. Burlingame, G. L. Kenyon, K. H. Nealson, and N. J.Oppenheimer. 1981. Structural identification of autoinducer of Photobacte-rium fischeri luciferase. Biochemistry 20:2444–2449.

15. Freeman, J. A., and B. L. Bassier. 1999. A genetic analysis of the function ofLuxO, a two-component response regulator involved in quorum sensing inVibiro harveyi. Mol. Microbiol. 31:665–677.


16. Freeman, J. A., and B. L. Bassler. 1999. Sequence and function of LuxU: atwo-component phosphorelay protein that regulates quorum sensing inVibrio harveyi. J. Bacteriol. 181:899–906.

17. Fuqua, C., S. C. Winans, and E. P. Greenberg. 1996. Census and consensusin bacterial ecosystems: the LuxR-LuxI family of quorum-sensing transcrip-tional regulators. Annu. Rev. Microbiol. 50:727–751.

18. Gilson, L., A. Kuo, and P. V. Dunlap. 1995. AinS and a new family ofautoinducer synthesis proteins. J. Bacteriol. 177:6946–6951.

19. Hanzelka, B. L., and E. P. Greenberg. 1996. Quorum sensing in Vibriofischeri: evidence that S-adenosylmethionine is the amino acid substrate forthe autoinducer synthesis. J. Bacteriol. 178:5291–5294.

20. Hanzelka, B. L., M. R. Parsek, D. L. Val, P. V. Dunlap, J. E. Cronan, Jr., andE. P. Greenberg. 1999. Acylhomoserine lactone synthase activity of the Vibriofischeri AinS protein. J. Bacteriol. 181:5766–5770.

21. Hardman, A. M., G. S. A. B. Stewart, and P. Williams. 1998. Quorum sensingand the cell-cell communication dependent regulation of gene expression inpathogenic and non-pathogenic bacteria. Antonie Leeuwenhoek J. Micro-biol. 74:199–210.

22. Hrabak, E. M., and D. K. Willis. 1992. The lemA gene required for patho-genicity of Pseudomonas syringae pv. syringae on bean is a member of a familyof two-component regulators. J. Bacteriol. 174:3011–3020.

23. Iuchi, S., Z. Matsuda, T. Fujiwara, and E. C. Lin. 1990. The arcB gene ofEscherichia coli encodes a sensor-regulator protein for anaerobic repressionof the arc modulon. Mol. Microbiol. 4:715–727.

24. Jiang, Y., M. Camara, S. R. Chhabra, K. H. Hardie, B. W. Bycroft, A.Lazdunski, G. P. Salmond, G. S. A. B. Stewart, and P. Williams. 1998. Invitro biosynthesis of the Pseudomonas aeruginosa quorum sensing signalmolecule N-butanoyl-L-homoscrine lactone. Mol. Microbiol. 28:193–203.

25. Jobling, M. G., and R. K. Holmes. 1997. Characterization of hapR, a positiveregulator of the Vibrio cholerae HA/protease gene hap, and its identificationas a functional homologue of the Vibrio harveyi luxR gene. Mol. Microbiol.26:1023–1034.

26. Kuo, A. N. V. Blough, and P. V. Dunlap. 1994. Multiple N-acyl-L-homoserinelactone autoinducers of luminescence in the marine symbiotic bacteriumVibrio fischeri. J. Bacteriol. 176:7558–7565.

27. Latifi, A., M. K. Winson, M. Foglino, B. W. Bycroft, G. S. A. B. Stewart, A.Lazdunski, and P. Williams. 1995. Multiple homologues of LuxR and LuxIcontrol expression of virulence determinants and secondary metabolitesthrough quorum sensing in Pseudomonas aeruginosa PAO1. Mol. Microbiol.17:333–343.

28. Lilly, B. N., and B. L. Bassler. 2000. Regulation of quorum sensing in Vibrioharveyi by LuxO and Sigma 54. Mol. Microbiol. 36:940–954.

29. McClean, K. H., M. K. Winson, L. Fish, A. Taylor, S. R. Chhabra, M.Camara, M. Daykin, J. H. Lamb, S. Swift, B. W. Bycroft, G. S. A. B. Stewart,and P. Williams. 1997. Quorum sensing and Chromobacterium violaceum:exploitation of violacein production and inhibition of the detection of N-acylhomoserine lactones. Microbiol. 143:3703–3711.

30. McGee, K., P. Horstedt, and D. L. Milton. 1996. Identification and charac-terization of additional flagellin genes from Vibrio anguillarum. J. Bacteriol.178:5188–5198.

31. Meighen, E. A. 1991. Molecular biology of bacterial bioluminescence. Mi-crobiol. Rev. 55:123–142.

32. Meighen, E. A. 1994. Genetics of bacterial bioluminescence. Annu. Rev.Genet. 28:117–139.

33. Miller, V. L., and J. J. Mekalanos. 1988. A novel suicide vector and its usein construction of insertion mutations: osmoregulation of outer membraneproteins and virulence determinants in Vibrio cholerae requires toxR. J.Bacteriol. 170:2575–2583.

34. Milton, D. L., A. Norqvist, and H. Wolf-Watz. 1992. Cloning of a metallo-protease gene involved in the virulence mechanism of Vibrio anguillarum. J.Bacteriol. 174:7235–7244.

35. Milton, D. L., R. O’Toole, P. Horstedt, and H. Wolf-Watz. 1996. Flagellin Ais essential for the virulence of Vibrio anguillarum. J. Bacteriol. 178:1310–1319.

36. Milton, D. L., A. Hardman, M. Camara, S. R. Chhabra, B. W. Bycroft,G. S. A. B. Stewart, and P. Williams. 1997. Quorum sensing in Vibrioanguillarum: characterization of the vanI/vanR locus and identification of theautoinducer N-(3-oxodecanoyl)-L-homoserine lactone. J. Bacteriol.179:3004–3012.

37. More, M. I., L. D. Finger, J. L. Stryker, C. Fuqua, A Eberhard, and S. C.Winans. 1996. Enzymatic synthesis of a quorum-sensing autoinducer throughuse of defined substrates. Science 272:1655–1658.

38. Nagasawa, S., S. Tokishita, H. Aiba, and T. Mizuno. 1992. A novel sensor-regulator protein that belongs to the homologous family of signal transduc-

tion proteins involved in adaptive responses in Escherichia coli. Mol. Micro-biol. 6:799–807.

39. Norqvist, A., A. Hagstrom, and H. Wolf-Watz. 1989. Protection of rainbowtrout against vibriosis and furunculosis by the use of attenuated strains ofVibrio anguillarum. Appl. Environ. Microbiol. 55:1400–1405.

40. Parkinson, J. S., and E. C. Kofoid. 1992. Communication modules in bac-terial signaling proteins. Annu. Rev. Genet. 26:71–112.

41. Parsek, M. R., D. L. Val, B. L. Hanzelka, J. E. Cronan, Jr., and E. P.Greenberg. 1999. Acyl homoserine lactone quorum-sensing signal genera-tion. Proc. Natl. Acad. Sci. USA 96:4360–4365.

42. Passador, L., K. D. Tucker, K. R. Guertin, M. P. Journet, A. S. Kende, andB. H. Iglewski. 1996. Functional analysis of the Pseudomonas aeruginosaautoinducer PAI. J. Bacteriol. 178:5995–6000.

43. Reed, L. J., and J. Muench. 1938. A simple method of estimating fiftypercent endpoints. Am. J. Hyg. 27:493–497.

44. Salmond, G. P. C., B. W. Bycroft, G. S. A. B. Stewart, and P. Williams. 1995.The bacterial ‘enigma’: cracking the code of cell-cell communication. Mol.Microbiol. 16:615–624.

45. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: alaboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.

46. Schaefer, A. L., B. L. Hanzelka, A. Eberhard, and E. P. Greenberg. 1996.Quorum sensing in Vibrio fischeri: probing autoinducer-LuxR interactionswith autoinducer analogs. J. Bacteriol. 178:2897–2901.

47. Schaefer, A. L., D. L. Val, B. L. Hanzelka, J. E. Cronan, Jr., and E. P.Greenberg. 1996. Generation of cell-to cell signals in quorum sensing: acylhomoserine lactone synthase activity of a purified Vibrio fischeri LuxI pro-tein. Proc. Natl. Acad. Sci. USA 93:9505–9509.

48. Shaw, P. D., G. Ping, S. L. Daly, C. Chung, J. E. Cronan Jr, K. L. Rhinehart,and S. K. Farrand. 1997. Detecting and characterizing N-acylhomoserinelactone signal molecules by thin-layer chromotagraphy. Proc. Nat. Acad. Sci.USA 94:6036–6041.

49. Simon, R., U. Priefer, and A. Puhler. 1983. A broad host range mobilizationsystem for in vivo genetic engineering: transposon mutagenesis in gramnegative bacteria. Bio/Technology 1:787–796.

50. Stout, V., and S. Gottesman. 1990. RcsB and RcsC: A two-componentregulator of capsule synthesis in Escherichia coli. J. Bacteriol. 172:659–669.

51. Surette, M. G., M. B. Miller, and B. L. Bassler. 1999. Quorum sensing inEscherichia coli, Salmonella typhimurium, and Vibrio harveyi: a new family ofgenes responsible for autoinducer production. Proc. Natl. Acad. Sci. USA96:1639–1644.

52. Swift, S., A. V. Karlyshev, E. L. Durant, M. K. Winson, S. R. Chhabra, P.Williams, S. Macintyre, and G. S. A. B. Stewart. 1997. Quorum sensing inAeromonas hydrophila and Aeromonas salmonicida: identification of theLuxRI homologues AhyRI and AsaRI and their cognate signal molecules. J.Bacteriol. 179:5271–5281.

53. Swift, S., M. J. Lynch, L. Fish, D. F. Kirke, J. M. Tomas, G. S. A. B. Stewart,and P. Williams. 1999. Quorum sensing-dependent regulation and blockadeof exoprotease production in Aeromonas hydrophila. Infect. Immun. 67:5192–5199.

54. Tang, J. L., Y. N. Liu, C. E. Barber, J. M. Dow, J. C. Wootton, and M. J.Daniels. 1991. Genetic and molecular analysis of a cluster of rpf genesinvolved in positive regulation of synthesis of extracellular enzymes andpolysaccharide in Xanthomonas campestris pathovar campestris. Mol. Gen.Genet. 226:409–417.

55. Val, D. L., and J. E. Cronan, Jr. 1998. In vivo evidence that S-adenosylme-thionine and fatty acid synthesis intermediates are the substrates for the LuxIfamily of autoinducer substrates. J. Bacteriol. 180:2644–2651.

56. Williams, P., M. Camara, A. Hardman, S. Swift, D. Milton, V. J. Hope, K.Winzer, B. Middleton, D. I. Pritchard, and B. W. Bycroft. 2000. Quorumsensing and the population dependent control of virulence. Philos. Trans. R.Soc. Lond. Sect. B 355:1–14.

57. Winson, M. K., M. Camara, A. Latifi, M. Foglino, S. R. Chhabra, M. Daykin,M. Bally, V. Chapon, G. P. C. Salmond, B. W. Bycroft, A. Lazdunski,G. S. A. B. Stewart, and P. Williams. 1995. Multiple N-acyl-L-homoserinelactone signal molecules regulate production of virulence determinants andsecondary metabolites in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci.USA 92:9427–9431.

58. Winson, M. K., S. Swift, L. Fish, J. P. Throup, F. Jorgensen, S. R. Chhabra,B. W. Bycroft, P. Williams, and G. S. A. B. Stewart. 1998. Construction andanalysis of luxCDABE-based plasmid sensors for investigating N-acylhomo-serine lactone-mediated quorum sensing. FEMS Microbiol. Lett. 163:185–192.


The LuxM Homologue VanM from Vibrio anguillarum Directs the Synthesis of...

Documents

Transcript of The LuxM Homologue VanM from Vibrio anguillarum Directs the Synthesis of...