# The hanR/hanI quorum-sensing system of Halomonas anticariensis, a moderately halophilic bacterium

Ali Tahrioui, E. Quesada, I. Llamas

► To cite this version:

Ali Tahrioui, E. Quesada, I. Llamas. The hanR/hanI quorum-sensing system of Halomonas anticariensis, a moderately halophilic bacterium. Microbiology, 2011, 157 (12), pp.3378 - 3387. 10.1099/mic.0.052167-0. hal-01937954

HAL Id: hal-01937954

https://normandie-univ.hal.science/hal-01937954v1

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# The *hanR/hanl* quorum-sensing system of *Halomonas anticariensis*, a moderately halophilic bacterium

Ali Tahrioui, Emilia Quesada and Inmaculada Llamas

Microbial Exopolysaccharide Research Group, Department of Microbiology, Faculty of Pharmacy, Cartuja Campus, University of Granada, 18071 Granada, Spain

Quorum sensing is a cell density-dependent gene expression mechanism found in many Gram-negative bacteria which involves the production of signal molecules such as *N*-acylhomoserine lactones (AHLs). One significant group of micro-organisms in which quorum sensing has not been previously studied, however, are the moderate halophiles. We describe here the results of our studies of the quorum-sensing system in *Halomonas anticariensis* FP35<sup>T</sup>, which is composed of *luxR luxI* homologues: *hanR* (the putative transcriptional regulator gene) and *hanl* (the autoinducer synthase gene). To understand how the *hanR/hanl* system is organized and regulated we conducted RT-PCR and quantitative real-time PCR assays. Transcriptional analysis indicated that the *hanR* and *hanl* genes are on the same transcript and that their transcription is growth phase-dependent. Hanl seems to be the only autoinducer synthase responsible for the synthesis of AHLs by the bacterium, since the inactivation of *hanl* resulted in the complete loss of its AHLs. We also found that the *hanl* gene appears to be transcribed from its own promoter and that its expression does not depend upon HanR. This finding was supported by the fact that the FP35*hanR* mutant showed AHL-producing activity and *hanl* expression similar to that of the wild-type strain, the latter being measured by RT-PCR. Moreover, *hanR* is expressed from its own promoter and appears to be independent of the AHL signalling molecules produced by Hanl.

Correspondence
Inmaculada Llamas
illamas@ugr.es

Received 21 June 2011

Revised 11 October 2011

Accepted 13 October 2011

## INTRODUCTION

Over the past few years it has become increasingly evident that numerous bacteria can establish cell-to-cell communication within their communities by producing small signal molecules that either diffuse freely across the cell membranes or are actively transported out of the cell. These signals, known as autoinducers, control gene expression in response to bacterial cell density in a process called quorum sensing (reviewed by González & Marketon, 2003; Ng & Bassler, 2009; Parker & Sperandio, 2009).

The most thoroughly characterized Gram-negative, bacterial, intraspecific autoinducers are *N*-acylhomoserine lactones (AHLs), which have been reported to be synthesized by a member of the LuxI protein family in over 70 genera belonging to the phylum Proteobacteria. AHL molecules

differ in the length and substitution of their respective acyl side chains, conferring upon them signal specificity. As the density of the population increases AHL molecules accumulate in the growth medium, and on reaching a critical concentration threshold they bind and activate an AHL receptor protein belonging to the LuxR family of transcriptional regulators. The activated LuxR/AHL complex then binds to a specific palindromic sequence on the DNA, known as the ‘lux box’, located upstream of the genes regulated by quorum sensing. This results in the activation or repression of target genes, including in many cases the activation of the autoinducer synthase, which leads in turn to the production of more AHLs and consequently a positive feedback loop (Eberhard et al., 1991; Fuqua et al., 1994).

This phenomenon has usually been found in bacteria that live in pathogenic or symbiotic associations in which quorum-sensing systems regulate genes involved in the expression of virulence factors and exoenzymes (de Kievit & Iglewski, 2000; Song et al., 2005; von Bodman et al., 2003), conjugal DNA transfer and plasmid copy number control (Farrand, 1998; Fuqua et al., 2001, 1994; Marketon & González, 2002), production of and susceptibility to antibiotics (Horng et al., 2002; McClean et al., 1997; Pumbwe et al., 2008; Van Houdt et al., 2007), biofilm formation (Davies et al., 1998; McNab et al., 2003; Niu

**Abbreviations:** AHL, *N*-acylhomoserine lactone; *C*₄-HL, *N*-butanoyl homoserine lactone; *C*₆-HL, *N*-hexanoyl homoserine lactone; *C*₈-HL, *N*-octanoyl homoserine lactone; *C*₁₂-HL, *N*-dodecanoyl homoserine lactone; 3-oxo-*C*₆-HL, *N*-(*β*-ketocaproyl)-*L*-homoserine lactone; RT-qPCR, reverse transcription quantitative real-time PCR.

The GenBank/EMBL/DDBJ accession number for the *hanR-hanl* DNA sequence of *H. anticariensis* is HM236058.

Three supplementary figures and two supplementary tables are available with the online version of this paper.
et al., 2008) and exopolysaccharide production (Marketon et al., 2003; von Bodman et al., 1998).

In addition to AHLs, other quorum-sensing autoinducer molecules recently identified in Gram-negative bacteria are autoinducer 2 (AI-2) (Chen et al., 2002), which has been described as a global signal molecule for interspecific communication, 2-alkyl-4-quinolones (AQs), such as PQS in *Pseudomonas* (Diggle et al., 2007), long-chain fatty acids, such as the ‘diffusible signal factor’ (DSF) in *Xanthomonas campestris* (Wang et al., 2004), *p*-coumaroyl-HSL in *Rhodopseudomonas palustris* (Schaefer et al., 2008) and others (González & Keshavan, 2006; Ng & Bassler, 2009).

It has been reported that in complex environments bacterial signalling does not accumulate at a constant rate (Boyer & Wisniewski-Dyé, 2009). The diffusion and perception of AHLs in and around cells can be influenced by abiotic environmental factors such as pH, temperature and medium composition, and by other members of the bacterial community in a process known as ‘quorum quenching’ (Defoirdt et al., 2008; Taga & Bassler, 2003). A variety of bacterial biosensor strains have been constructed to detect autoinducer molecules (Llamas et al., 2004; McClean et al., 1997; Shaw et al., 1997; Winson et al., 1998). These strains do not produce any AHL signal molecules but can detect exogenous AHLs by the activation of a reporter gene such as *lacZ* or *lux* or, in *Chromobacterium violaceum*, by the production or inhibition of a purple pigment. The use of these biosensors in a previous study led us to observe that the exopolysaccharide-producing *Halomonas anticariensis* FP35<sup>T</sup> synthesizes quorum-sensing molecules in a growth phase-dependent manner and that its maximum activity is reached by the end of the exponential phase. In addition, using electrospray ionization MS (ESI MS/MS), we have identified some of the AHL molecular structures in this bacterium, such as *N*-butanoyl homoserine lactone (*C*<sub>4</sub>-HL), *N*-hexanoyl homoserine lactone (*C*<sub>6</sub>-HL), *N*-octanoyl homoserine lactone (*C*<sub>8</sub>-HL) and *N*-dodecanoyl homoserine lactone (*C*<sub>12</sub>-HL) (Llamas et al., 2005).

Cell density-dependent gene regulation has not been studied to date in extreme environments such as those inhabited by *Halomonas* species. The genus *Halomonas* currently comprises more than 70 species of halophilic bacteria, most of which have been isolated from saline environments such as salterns, saline soils, seawater and marshes (Euzéby, 2010). Its members are extremely versatile from the ecological, physiological and metabolic point of view. The interest in *Halomonas* species has centred on their ability to degrade aromatic compounds and produce compatible solutes, halophilic enzymes and exopolysaccharides with potential applications in biotechnology (Sutherland, 2002).

In this study we have genetically characterized the quorum-sensing system involved in the production of AHLs in *H. anticariensis* FP35<sup>T</sup>, which is composed of a putative transcriptional regulator HanR (LuxR homologue) and an AHL synthase HanI (LuxI homologue).

Gene expression analysis demonstrates that the *hanR/hanI* genes are organized in the same operon and that their expression is cell density-dependent. The expression of *hanR* appears to be independent of the AHL signalling molecules produced by HanI, an unusual characteristic compared with other LuxRI-like systems already characterized (González & Marketon, 2003).

## METHODS

**Bacterial strains, plasmids, media and culture conditions.** The bacterial strains and plasmids used are listed in Table 1. *H. anticariensis* strains were routinely cultured at 32 °C in SWYE (Nieto et al., 1989) or MY medium (Moraine & Rogovin, 1966) modified with a balanced mixture of sea salts (Rodríguez-Valera et al., 1981). *Escherichia coli* strains were grown at 37 °C in LB medium (Sambrook & Russell, 2001). Antibiotics were added at the following final concentrations: rifampicin, 50 µg ml⁻¹; kanamycin, 50 µg ml⁻¹; gentamicin, 25 µg ml⁻¹; tetracycline, 15 µg ml⁻¹; and ampicillin, 100 µg ml⁻¹. *Agrobacterium tumefaciens* NTL4 (pZLR4) was cultured at 30 °C in LB medium supplemented with 2.5 mM CaCl₂ and 2.5 mM MgSO₄ (LB/MC) and in MGM minimal medium (per litre: 11 g Na₂HPO₄, 3 g KH₂PO₄, 0.5 g NaCl, 1 g glutamate, 10 g mannitol, 1 mg biotin, 27.8 mg CaCl₂, 246 mg MgSO₄) containing 50 µg gentamicin ml⁻¹. *C. violaceum* CV026 was grown at 30 °C in LB medium.

**Transposon mutagenesis and isolation of the FP35-11I (FP35::hanR::mini-Tn5 Km2) mutant.** Transposon mutagenesis was carried out via conjugation by biparental mating, as described in a previous publication (Llamas et al., 2000). The donor strain was *E. coli* S17-1 λpir, harbouring the suicide vector pUT mini-Tn5 Km2 (de Lorenzo et al., 1990). The recipient strain was *H. anticariensis* FP35-R, a spontaneous rifampicin-resistant mutant of *H. anticariensis* FP35<sup>T</sup>. The transconjugants were isolated on SWYE medium containing 2% (w/v) salts, rifampicin (50 µg ml⁻¹) and kanamycin (50 µg ml⁻¹). Selected clones were replicated onto both the same isolating medium and trypticase soy agar (TSA) (per litre: 15 g trypticase peptone, 5 g phytone peptones, 5 g NaCl, 15 g agar) and incubated overnight at 32 °C. The halophilic clones that did not grow in TSA medium due to its low salt content were then streaked onto LB/MC or MGM media, where the indicator strains [C. violaceum CV026 or A. tumefaciens NTL4 (pZLR4)] had been previously streaked in order to isolate AHL-deficient strains. They were incubated overnight at 32 °C to allow the indicator organisms to grow and surround the clone growths with either purple or blue haloes.

**Cloning of the hanRI genes.** To isolate the DNA fragment carrying the mini-Tn5 insertion, which encodes the kanamycin-resistance gene, genomic DNA from *H. anticariensis* FP35-11I was completely digested with enzymes that do not cleave within the minitransposon region. DNA fragments were separated on an agarose gel and transferred onto a nylon filter by standard techniques (Sambrook & Russell, 2001). Fragments containing the mini-Tn5 Km2 insertion were analysed by Southern hybridization using a digoxigenin DNA labelling and detection kit (Boehringer Mannheim) as described in previous publications (Llamas et al., 1997, 2003). Chromosomal DNA fragments digested with *Pst*I were selected and ligated into pGEM-T to create pP11I. The cloned fragments were sequenced directly by primer walking using a BigDye Terminator Cycle Sequencing kit in an ABI 3100 DNA sequencer (Applied Biosystems). The genomic sequence located upstream of the known nucleotide sequence was obtained by inverse PCR (Ochman et al., 1988; Llamas et al., 2003). One plasmid, pBFP35, was isolated and sequenced. The DNA
<caption>**Table 1.** Bacterial strains and plasmids used in this study</caption>

Abbreviations: Ap<sup>r</sup>, ampicillin resistance; Gm<sup>r</sup>, gentamicin resistance; Km<sup>r</sup>, kanamycin resistance; Rif<sup>r</sup>, rifampicin resistance; Tc<sup>r</sup>, tetracycline resistance.

<table><thead><tr><th>Strain or plasmid</th><th>Genotype or description</th><th>Reference or source</th></tr></thead><tbody><tr><td colspan="3"><strong>H. anticariensis</strong> strains:</td></tr><tr><td>FP35<sup>T</sup> (=CECT 5854<sup>T</sup>)</td><td>Wild-type isolate, mucoid</td><td>Martínez-Cánovas et al. (2004)</td></tr><tr><td>FP35-R</td><td>Spontaneous rifampicin-resistant mutant of FP35, Rif<sup>r</sup></td><td>This work</td></tr><tr><td>FP35-11I</td><td>FP35-R containing <i>hanR</i>::mini-Tn5 Km2, Rif<sup>r</sup>, Km<sup>r</sup></td><td>This work</td></tr><tr><td>FP35<sub>hanI</sub></td><td>FP35-R containing <i>hanI</i>::<i>lacZ</i>, Rif<sup>r</sup>, Km<sup>r</sup></td><td>This work</td></tr><tr><td>FP35<sub>hanR</sub></td><td>FP35-R containing <i>hanR</i>::<i>lacZ</i>, Rif<sup>r</sup>, Km<sup>r</sup></td><td>This work</td></tr><tr><td>FP35(pMP220)</td><td>FP35-R containing pMP220, Rif<sup>r</sup>, Tc<sup>r</sup></td><td>This work</td></tr><tr><td>FP35(pMP-phanR)</td><td>FP35-R containing pMP-phanR, Rif<sup>r</sup>, Tc<sup>r</sup></td><td>This work</td></tr><tr><td>FP35(pMP-phanI)</td><td>FP35-R containing pMP-phanR, Rif<sup>r</sup>, Tc<sup>r</sup></td><td>This work</td></tr><tr><td>FP35-11I(pJN-HanR)</td><td>FP35-R containing <i>hanR</i>::mini-Tn5 Km2 and pJN-HanR, Rif<sup>r</sup>, Km<sup>r</sup>, Gm<sup>r</sup></td><td>This work</td></tr><tr><td>FP35<sub>hanI</sub>(pJN-HanI)</td><td>FP35-R containing <i>hanI</i>::<i>lacZ</i> and pJN-HanI, Rif<sup>r</sup>, Km<sup>r</sup>, Gm<sup>r</sup></td><td>This work</td></tr><tr><td><strong>A. tumefaciens</strong> NTL4(pZLR4)</td><td>NT1 derivative carrying a <i>traG</i>::<i>lacZ</i> reporter fusion, Gm<sup>r</sup></td><td>Shaw et al. (1997)</td></tr><tr><td><strong>C. violaceum</strong> CV026</td><td>CV017 derivative containing <i>cviI</i>::Tn5xyI<sup>E</sup>, Km<sup>r</sup></td><td>McClean et al. (1997)</td></tr><tr><td colspan="3"><strong>E. coli</strong> strains:</td></tr><tr><td>DH5α</td><td>F<sup>-</sup> φ80d <i>lacZ</i>ΔM15 Δ(<i>lacZY-argF</i>)U169 <i>deoR recA1 endA1 hsdR17(r<sub>K</sub> m<sub>K</sub>) supE44 λ<sup>-</sup> thi-1 gira96 relA1</sup></td><td>Laboratory collection</td></tr><tr><td>S17-1 λ<sub>pir</sub></td><td>RK2 <i>tra</i> regulon, <i>pir</i>, host for <i>pir</i>-dependent plasmids</td><td>Miller & Mekalanos (1988)</td></tr><tr><td colspan="3"><strong>Plasmids:</strong></td></tr><tr><td>pGEM-T</td><td>High-copy-number cloning vector, Ap<sup>r</sup>, <i>bla</i>, <i>lacZ</i></td><td>Promega</td></tr><tr><td>pUT mini-Tn5 Km2</td><td>Ap<sup>r</sup>, Km<sup>r</sup>, <i>ori</i> R6K, <i>oriT</i> RP4</td><td>de Lorenzo et al. (1990)</td></tr><tr><td>pP11I</td><td>pGEM-T with a 5 kb <i>PstI</i> fragment carrying mini-Tn5 insertion, Ap<sup>r</sup>, Km<sup>r</sup></td><td>This work</td></tr><tr><td>pBFP35</td><td>pGEM-T with a 1.6 kb inverse PCR product amplified from a 2.2 kb fragment from strain FP35 genome, Ap<sup>r</sup></td><td>This work</td></tr><tr><td>pVIK112</td><td><i>lacZY</i> for disruption and transcriptional fusion, Km<sup>r</sup></td><td>Kalogeraki & Winans (1997)</td></tr><tr><td>pVIKHanI</td><td>pVIK112 containing an internal <i>hanI</i> fragment, transcriptional fusion, Km<sup>r</sup></td><td>This work</td></tr><tr><td>pVIKHanR</td><td>pVIK112 containing an internal <i>hanR</i> fragment, transcriptional fusion, Km<sup>r</sup></td><td>This work</td></tr><tr><td>pMP220</td><td>Promoter-probe vector, <i>'lacZ</i>; IncP; Tc<sup>r</sup></td><td>Spaink et al. (1987)</td></tr><tr><td>pMP-phanI</td><td>pMP220 containing <i>hanI</i> promoter</td><td>This work</td></tr><tr><td>pMP-phanR</td><td>pMP220 containing <i>hanR</i> promoter</td><td>This work</td></tr><tr><td>pJN105</td><td><i>araC-PBAD</i> cassette cloned in pBBR1MCS5</td><td>Newman & Fuqua (1999)</td></tr><tr><td>pJN-HanI</td><td>pJN105 containing intact <i>hanI</i>, Gm<sup>r</sup></td><td>This work</td></tr><tr><td>pJN-HanR</td><td>pJN105 containing intact <i>hanR</i>, Gm<sup>r</sup></td><td>This work</td></tr></tbody></table>

sequences thus obtained were analysed using a BLAST search of the GenBank database (National Center for Biotechnology Information; NCBI; http://www.ncbi.nlm.nih.gov). Promoter regions were predicted with the BPROM software (Softberry; http://linux1.softberry.com/berry.phtml). The conserved domains of the LuxR-type transcriptional regulator were identified using the SMART program (Letunic et al., 2006).

**DNA manipulations.** All molecular techniques were performed using standard protocols (Sambrook & Russell, 2001). The primers used in this study are listed in Supplementary Table S1.

**Construction of the *hanR* and *hanl* gene mutations.** An internal fragment lacking both the 5' and 3' ends of the *hanR* and *hanl* genes was cloned separately into the suicide plasmid pVIK112, containing the promoterless *lacZ* gene. Chromosomal gene disruptions were then effected in a single step by Campbell-type integration of the entire plasmid by homologous recombination (Kalogeraki & Winans, 1997).

Internal segments of 301 and 356 bp of the *hanR* and *hanl* genes, respectively, were amplified from *H. anticariensis* FP35<sup>T</sup> chromosomal DNA by using the primers hanRsub-EcoRI-F and hanRsub-Xbal-R for *hanR*, and hanIsub-EcoRI-F and hanIsub-Xbal-R for *hanl*,

which contain EcoRI and XbaI restriction sites (underlined) at their respective 5' ends (Supplementary Table S1). PCR entailed 30 cycles of 30 s at 95 °C, 30 s at 66 °C and 30 s at 72 °C. The PCR fragments were purified, cloned into pGEM-T, and digested with EcoRI and XbaI. The purified fragments were then cloned into the suicide plasmid pVIK112. The resulting plasmids, pVIKHanR and pVIKHanI, were subsequently transformed separately into *E. coli* S17-1 λpir and transferred into a rifampicin-resistant FP35 derivative (FP35-R) by biparental mating, where the promoterless reporter *lacZ* gene simultaneously disrupted and created fusions to the *hanR* and *hanl* genes by single recombination. The *H. anticariensis* transconjugants, FP35*hanI* and FP35*hanR*, were selected by plating them on SWYE medium containing 2% (w/v) salts, rifampicin (50 µg ml<sup>-1</sup>) and kanamycin (50 µg ml<sup>-1</sup>) (Kalogeraki & Winans, 1997; Llamas et al., 2004) (Table 1).

**Cloning of the *hanR* and *hanl* genes in the expression vector pJN105.** The ORFs of *hanR* and *hanl* were amplified from *H. anticariensis* FP35<sup>T</sup> chromosomal DNA by using the primers hanR-EcoRI-F and hanR-XbaI-R, and hanI-EcoRI-F and hanI-XbaI-R, respectively, which contain EcoRI and XbaI restriction sites at their respective 5' ends (Supplementary Table S1). PCR entailed 30 cycles
of 30 s at 95 °C, 30 s at 60 °C and 1 min at 72 °C. The PCR fragment was purified, digested with EcoRI and XbaI, and cloned into the broad-host-range expression vector pJN105, which carries the L-arabinose-inducible *E. coli araBAD* promoter and the *araC* regulator (*araC*-P<sub>BAD</sub>) (Newman & Fuqua, 1999) to create pJN-HanR and pJN-HanI. The pJN-HanR and pJN-HanI plasmids were then transformed separately into *E. coli* S17-1 λ pir and transferred by biparental mating into the AHL-deficient strains FP35-11I and FP35hanI, respectively, to be used in complementation analysis. To confirm whether AHL production was restored in strains FP35-11I (pJN-HanR) and FP35hanI (pJN-HanI), signal molecules were extracted and analysed following the technique described in previous studies (Llamas *et al.*, 2004, 2005; Marketon *et al.*, 2002). All constructs were confirmed by PCR.

**RNA extraction and RT-PCR assays.** Total bacterial RNA was isolated using the RNA II Nucleospin kit (Macherey-Nagel), followed by rigorous treatment with Turbo DNA-free RNase (Ambion) according to the manufacturer's protocol. All RNA samples were visualized on the gel and the concentrations were measured with a NanoDrop ND-2000c spectrophotometer (Thermo Scientific). RT-PCR was conducted with 0.05-0.5 µg total RNA at a final volume of 50 µl using the SuperScript III One-Step RT-PCR System with Platinum Taq according to the manufacturer's instructions (Invitrogen). cDNA was synthesized at 55 °C for 30 min. Denaturation was performed for 2 min at 94 °C, followed by 35 cycles of PCR, as suggested by the manufacturer. A final elongation step was conducted at 68 °C for 7 min. The annealing temperature was calculated for each reaction on the basis of the melting temperatures of the pairs of primers used (Supplementary Table S1). Positive and negative controls were included in all the assays. 16S rRNA was used as a control for normalization. RT-PCR products were visualized by electrophoresis.

**Reverse transcription quantitative real-time PCR analysis (RT-qPCR).** Total RNA from bacterial cultures growing at the early exponential and stationary phases (OD<sub>600</sub> values of 0.4 and 1.4, respectively) was extracted as described above. All RNA samples were prepared from two independent cultures. RNA preparations (1 µg) treated with Turbo DNA-free RNase (Ambion) were reverse-transcribed using the iScript cDNA Synthesis kit (Bio-Rad). RT-qPCR was done with an iCycler iQ system (Bio-Rad). Each reaction mixture contained 1 µl of the target cDNA or a dilution (1:10 000, for amplification of the 16S rRNA gene), 100 nM of each of the forward and reverse primers, 10 µl 2x iQ SYBR Green Supermix (Bio-Rad) and nuclease-free water to a final volume of 20 µl. All reactions were repeated in triplicate. PCRs were also carried out with the RNA samples untreated with reverse transcriptase to confirm the absence of contaminating genomic DNA. The oligonucleotide sequences used for RT-qPCR analysis are listed in Supplementary Table S1. The amplification conditions consisted of an initial 3 min denaturation step at 95 °C, followed by 40 cycles each of denaturation at 95 °C for 10 s, annealing at 62 °C for 30 s, and extension at 72 °C for 30 s. Negative controls without cDNA for each primer set were included in each run. At the end of the real-time PCR cycles, a melting curve was generated and analysed to confirm product specificity. For all genes, melting-curve analysis showed clear melting peaks without non-specific products or artefacts. Standard curves were obtained using recombinant plasmids containing a fragment of the gene of interest. The relative expression of each gene was normalized to that of 16S rRNA and evaluated during the early exponential phase versus the stationary phase using the Relative Expression Software Tool (REST2009) (Pfaffl *et al.*, 2002).

**Analysis of hanR and hanI promoter activity.** The promoter regions of the *hanR* and *hanI* genes (phanR and phanI respectively) were amplified by PCR with primers incorporating BglII and XbaI linkers for phanR, and EcoRI and XbaI linkers for phanI, respectively

(Supplementary Table S1). The promoter region of each gene was fused to the lacZ gene in the promoterless low-copy-number pMP220 vector (Spaink *et al.*, 1987). Upon amplification, DNA was cloned into the pGEM-T vector, digested with the appropriate restriction enzymes, and ligated to pMP220 for the construction of pMP-phanR and pMP-phanI. The constructions were confirmed by DNA sequencing. The pMP220, pMP-pHanR and pMP-pHanI plasmids were then transformed separately into *E. coli* S17-1 λ pir and transferred by biparental mating into *H. anticariensis* FP35-R (Table 1). Promoter activity was analysed by measuring β-galactosidase activity (Miller, 1972).

**Extraction, detection and TLC analysis of crude AHL extracts.** AHL molecules were extracted following the technique described in our previous studies (Llamas *et al.*, 2004; Marketon *et al.*, 2002). To detect AHLs, an overnight culture of the AHL indicator strain C. violaceum CV026 was diluted 1:100 in 5 ml LB medium and poured onto LB agar. Once the plates were dried, paper disks 5 mm in diameter were placed on an agar plate and the AHL samples were applied. The assay plates were incubated overnight at 32 °C and the appearance of pigment around the filter was determined. To characterize the AHLs, the samples were subjected to analytical and preparative TLC. AHL samples and standards were spotted onto a TLC plate and developed with 70% (v/v) methanol in water. The plate was air-dried and overlaid with top agar containing the *A. tumefaciens* NTL4 (pZLR4) indicator strain before being incubated at 32 °C (Llamas *et al.*, 2005).

AHL standards were obtained from Sigma [N-(β-ketocaproyl)-L-homoserine lactone (3-oxo-C<sub>6</sub>-HL)] and Fluka (C<sub>6</sub>-HL and C<sub>8</sub>-HL).

# RESULTS AND DISCUSSION

## Isolation and characterization of the AHL-deficient strain FP35-11I

In a previous paper we reported that the exopolysaccharide-producing *H. anticariensis* FP35<sup>T</sup> synthesizes at least four AHLs (C<sub>4</sub>-HL, C<sub>6</sub>-HL, C<sub>8</sub>-HL and C<sub>12</sub>-HL) (Llamas *et al.*, 2005). In an effort to identify the genes involved in their production, one of the four partially AHL-deficient *H. anticariensis* clones obtained by transposon mutagenesis, the FP35-11I mutant, was chosen for this study because it activated the two indicator strains to a lesser extent than did the wild-type strain. To further confirm the lower AHL production exhibited by this mutant, 5 ml culture extracts from both the *H. anticariensis* wild-type and FP35-11I mutant strains were analysed on an agar plate and by TLC. Analysis with the CV026 and NTL4 (pZLR4) indicator organisms revealed that the FP35-11I mutant (Fig. 1a, panel 2 and b, lane 2) produced lower levels of AHLs than the wild-type strain (Fig. 1a, panel 1 and b, lane 1).

## Cloning and sequencing of *luxR* and *luxI* homologues from *H. anticariensis* FP35<sup>T</sup>

The nucleotide sequences of the chromosomal DNA in the two recombinant plasmids, pP11I and pBFP35 (see Methods), revealed the presence of two complete ORFs (Fig. 2a). The transposon insertion site within the FP35-11I genome was located after base pair 394 from the translation
<figure><img src="image_2.png" /><figcaption>**Fig. 1.** AHL production in *H. anticariensis* FP35<sup>T</sup> (1), strain FP35-11I (2), strain FP35-11I harbouring pJN-HanR (3), strain FP35*hanl* (4), strain FP35*hanl* harbouring pJN-Hanl (5) and strain FP35*hanR* (6). AHLs extracted from cultures were visualized on an agar plate (a) by means of the *C. violaceum* CV026 biosensor, and by TLC (b) overlaid with the *A. tumefaciens* NTL4 (pZLR4) indicator strain. Synthetic AHL compounds were used as references: 3-oxo-C<sub>6</sub>-HL (4.7 pmol); C<sub>6</sub>-HL (80.4 pmol for CV026 and 804 pmol for NTL4); C<sub>8</sub>-HL (31.6 pmol).</figcaption></figure>

start of ORF1. A Southern blot analysis confirmed that a single copy of the transposable element had been integrated into the chromosome of this mutant (data not shown).

The results of a homology search for the nucleotide sequence of the two ORFs in the NCBI database showed 26 to 31% similarity to the *luxR* and *luxI* family genes. ORF1, called *hanR* (for *H. anticariensis luxR*), contained 759 bp and encoded a putative protein of 252 aa with a predicted molecular mass of 28.9 kDa. HanR shared about 31% identity with LuxR from *Burkholderia glumae* (Devecovi *et al.*, 2007) and 30% identity with PhzR from *Pseudomonas chlororaphis* strain 30-84 (Khan *et al.*, 2007). Genetic and biochemical studies have identified two conserved domains in LuxR-type proteins: the autoinducer-binding N-terminal domain and the DNA-binding C-terminal domain (Whitehead *et al.*, 2001). Analysis of the putative protein HanR in *H. anticariensis* FP35<sup>T</sup> with the SMART program revealed that it contained both domains: an autoinducer-binding domain (amino acids 30–178) and a DNA-binding domain with a helix-turn-helix motif, which is characteristic of the LuxR family (amino acids 189–246) (Letunic *et al.*, 2006) (Supplementary Fig. S1). The second ORF, named *hanI* (for *H. anticariensis luxI*), was located downstream of *hanR* and was transcribed in the same direction (Fig. 2a). The *hanI* gene (645 bp) encoded a 24.4 kDa protein homologous to members of the LuxI family of proteins, which are responsible for AHL synthesis. HanI shared 27% identity with ExpI from *Erwinia carotovora* (Pirhonen *et al.*, 1993)

and 26% identity with EanI from *Pantoea ananatis* (Morohoshi *et al.*, 2007). The HanI protein has similar conserved amino acids at the N terminus to those found in other related AHL synthases (Supplementary Fig. S1).

The quorum-sensing *hanR* and *hanI* genes identified are organized in tandem. A similar genetic arrangement has recently been reported in other micro-organisms, such as *R. palustris* (Schaefer *et al.*, 2008), *Gluconacetobacter intermedius* (Iida *et al.*, 2008), *Pseudomonas corrugata* (Licciardello *et al.*, 2007), *Mesorhizobium tianshanense* (Zheng *et al.*, 2006) and *Burkholderia cenocepacia* (Malott *et al.*, 2005). Nevertheless, in other bacteria, such as *Aliivibrio fischeri* (Urbanczyk *et al.*, 2007) (formerly *Vibrio fischeri*), the *luxI* and *luxR* genes are transcribed divergently (Engebrecht & Silverman, 1984).

## The *hanR*1 genes are co-transcribed

Given that the *hanR* and the *hanI* genes are aligned in the same direction and only 76 bp of intergenic region separates them (Fig. 2a), they may well constitute an operon. To ascertain whether these two genes were part of the same transcriptional unit we carried out RT-PCR analysis using primers based on their intergenic region sequence. Fig. 2(b) shows the predicted fragment corresponding to the intergenic region.

Two possible $\sigma^{70}$ promoters were identified upstream of the *hanR* and *hanI* regions (Fig. 2c) using the promoter
<figure><img src="image_3.png" /><figcaption>**Fig. 2.** Transcriptional organization of the *hanR/hanI* locus of *H. anticariensis* FP35T. (a) Schematic drawing of the *hanR* and *hanI* genetic arrangements in *H. anticariensis* FP35T. Arrows indicate the proposed direction of transcription. Plasmids used for DNA sequencing, pP11I and pBFP35, containing *PstI* and *BamHI* restriction fragments, are indicated on the physical map. The mini-Tn5 Km2 transposon insertion site is indicated by a black triangle. (b) Transcriptional analysis of the *hanR-hanI* intergenic region by RT-PCR. Lanes: M, molecular mass marker (100 bp–1 kb); 1, amplification of DNA sample (positive control); 2, amplification of RNA sample without reverse transcriptase (negative control); 3, amplification of RNA sample using reverse transcriptase (expected size of the product, 351 bp). The location of the primers and span of the intergenic region used in the RT-PCR experiment are shown. (c) Sequence of the DNA region upstream of *hanR* and *hanI*, showing the start codon (M) and stop codon (asterisk). A putative *lux* box-like sequence is present in the region upstream of the *hanR* gene.</figcaption></figure>

prediction software BPROM. We then constructed fusions of the putative promoter regions of each gene to a 'lacZ gene in the low-copy-number pMP220 vector and transferred it into *H. anticariensis* (see Methods and Table 1). In both cases, promoter activity was detected and measured by β-galactosidase assays: the FP35(pMP-phanR) strain gave a value of 451.49 ± 21.61 Miller units, whereas the FP35 (pMP-phanI) strain gave a value of 72.40 ± 8.21 Miller units [the FP35(pMP220) strain gave a value of 6.94 ± 0.98 Miller units]. Thus our results suggest that the *hanR* and *hanI* genes constitute the same transcriptional unit, although there are promoter activities upstream of each gene.

A putative *lux* box-like sequence, a 20 bp inverted repeat sequence, was found between positions –78 and –98 upstream of the *hanR* start translational site (Fig. 2c). This sequence is homologous to other known *lux*

box sequences (Supplementary Table S2). The *lux* box sequences are thought to represent binding sites of LuxR homologues (Gray *et al.*, 1994) and are located within the promoter regions of many genes regulated by the quorum-sensing system.

## The expression of *hanR* genes is growth phase-dependent

In a previous work we demonstrated that the synthesis of AHLs in *H. anticariensis* FP35T is growth phase-dependent and that maximum production is reached during the late exponential to stationary phases (Llamas *et al.*, 2005). The relative expression of the *hanR* and *hanI* genes, which was measured by using RT-qPCR, increased 83.6- and 45.1-fold, respectively, by the end of the exponential growth phase. This finding was to be expected and it confirms that
the expression of the *hanR/hanI* system is cell density-
dependent, as it is in most quorum-sensing systems
(González & Marketon, 2003).

hanl gene function

AHL production by the *FP35hanI* mutant was revealed on
an agar plate (Fig. 1a, panel 4) and its pattern was analysed
by TLC (Fig. 1b, lane 4), which indicated that it did not
produce any detectable levels of AHLs. The slight induction
of the NTL4 (pZLR4) indicator strain corresponding to
this strain, as shown by TLC, is the same as the signal
obtained from a sample prepared from 5 ml uninoculated
MY broth medium (Llamas *et al.*, 2005). Therefore,
according to these results, HanI would seem to be the sole
autoinducer synthase responsible for the production of
AHLs in *H. anticariensis* FP35T. The fact that *H. anticariensis*
produces at least four different AHLs is not surprising, as the
same has been reported in other bacteria such as *M.
tianshanense* (Zheng *et al.*, 2006). It has been suggested that
the variability in the acyl chain length of AHLs is not only a
function of acyl chain specificity but may also be influenced
by the cellular pool of acyl ACPs available (Watson *et al.*,
2002). Moreover, AHL production was almost completely
restored in the *FP35hanI* mutant when it was complemented
with the *hanI* gene cloned into the pJN105 expression vector
(Fig. 1a, panel 5 and b, lane 5). Although the pJN105
plasmid is a broad-host-range expression vector, it has not
been used before in studies with moderate halophiles and so
it may be that AHLs were not fully produced because the
vector was not properly expressed.

The transcription of *hanI* synthase from the *hanI* promoter was measured in the *FP35hanI* mutant by β-galactosidase assays, which indicated that it was unaffected by adding exogenous AHLs to the culture (Supplementary Fig. S2). Since we were unable to detect autoinduction in the synthesis of AHLs in *H. anticariensis* FP35T, we suggest that the classic *A. fischeri* autoinduction of the *hanI* gene does not take place in this strain in the same way as has been described in other bacteria (Andersson *et al.*, 2000; Christensen *et al.*, 2003; Nasser *et al.*, 1998; Throup *et al.*, 1995). Moreover, No *lux* box-like regulatory element could be identified within the region upstream of the translational start codon of the *hanI* gene.

hanR gene function

The *FP35hanR* mutant produced quantities of signal
molecules similar to those of the wild-type (Fig. 1a, panel
6 and b, lane 6). This result was unexpected, since the
majority of *luxR* mutants described in other bacteria
produce fewer or no AHLs. Since the *FP35hanR* mutant
was completely defective in *hanR* expression (Supple-
mentary Fig. S3a) and the level of expression of the *hanI*
gene was similar to that of the wild-type strain (Supple-
mentary Fig. S3a), it would seem that the HanR protein is
not essential for AHL production in *H. anticariensis* FP35T.

In contrast, the *FP35hanR* mutant synthesized higher
quantities of AHLs than *FP35-11I* (mini-Tn5::*hanR*) (Fig.
1b, lanes 2 and 6), another mutant in which the *hanR* gene
is not transcribed (Supplementary Fig. S3a). This result
may well be due to a polar effect caused by the transposon
on the *hanI* gene downstream, since the *hanI* gene
expression level is lower than that of *FP35T* or *FP35hanR*
(Supplementary Fig. S3a). In fact, AHL production was not
restored in the *FP35-11I* mutant when it was complemen-
ted with the pJN-HanR plasmid (Fig. 1a, panel 3 and b,
lane 3).

The presence of a putative *lux* box-like sequence upstream
of the *hanR* start codon suggested that the expression of
*hanR* was AHL-dependent. Nevertheless, we found that
transcription of the *hanR* gene was induced in the AHL-
deficient strain *FP35hanI* to a degree similar to that of the
wild-type strain even when crude extracts of AHLs were
added to the bacterial culture (Supplementary Fig. S3b).
This result suggests that *hanR* is expressed from its own
promoter but does not seem to be modulated by the AHL
signalling molecules under our assay conditions. One
explanation is that HanR might have the capacity to act as
a regulator of its own expression even without binding to
the AHLs. Another explanation is that there might be one
or more additional transcriptional *LuxR* regulators in *H.
anticariensis* FP35T. These unpaired *LuxR* family proteins
have been well characterized and shown to be integrated
with the resident complete AHL quorum-sensing regula-
tory network (Patankar & González, 2009; Subramoni &
Venturi, 2009). This has been reported in the SinRI
quorum-sensing system of *Sinorhizobium meliloti*, where
the *ExpR* regulator, an unpaired *LuxR* homologue, binds to
a site in the *sinR–sinI* intergenic region, which affects the
transcription of the *sinI* synthase gene (Hoang *et al.*, 2004;
McIntosh *et al.*, 2009). More studies must be undertaken in
*H. anticarisensis* FP35T to test these hypotheses.

Having identified and characterized the quorum-sensing
*hanR/hanI* system involved in the synthesis of AHLs in *H.
anticariensis* FP35T, we are now turning our attention to
the role of this type of cell density-dependent gene
regulation in the extreme environments in which
*Halomonas* species live and in which they are ubiquitous.
Within this context, *Halomonas* species are in general
considered to be free-living bacteria, and many of them
produce exopolysaccharides that allow them to form
biofilms; cell-to-cell communication may then be necessary
to regulate the bacterial community in any given niche. To
our knowledge this is the first report describing genetically
the quorum-sensing system in a moderately halophilic
bacterium, and it confirms previous evidence for cell-to-
cell communication amongst micro-organisms in an
extreme environment (Llamas *et al.*, 2005).

ACKNOWLEDGEMENTS

This research was supported by grants from the Spanish Ministerio de Educación y Ciencia (CGL2008-02399/BOS; AGL2009-07656), the
Consejería de Educación Ciencia y Empresa, of the Andalucian Regional Government (P06-CVI-01850) and from the Plan Andaluz de Investigación. We thank Dr Nuria Ferrol for her help with the real-time PCR assays. We also thank Dr Berenguer's laboratory for their helpful discussions and critical reading of the manuscript. We thank our colleague Dr J. Trout for revising our English text. A.T. was supported by a postgraduate grant from the Junta de Andalucía.

REFERENCES

Andersson, R. A., Eriksson, A. R., Heikinheimo, R., Mäe, A., Pirhonen, M., Kõiv, V., Hyytiäinen, H., Tuikkala, A. & Palva, E. T. (2000). Quorum sensing in the plant pathogen *Erwinia carotovora* subsp. *carotovora*: the role of *expR(Ecc)*. *Mol Plant Microbe Interact* **13**, 384–393.

Boyer, M. & Wisniewski-Dyé, F. (2009). Cell-cell signalling in bacteria: not simply a matter of quorum. *FEMS Microbiol Ecol* **70**, 1–19.

Chen, X., Schauder, S., Potier, N., Van Dorsselaer, A., Pelczer, I., Bassler, B. L. & Hughson, F. M. (2002). Structural identification of a bacterial quorum-sensing signal containing boron. *Nature* **415**, 545–549.

Christensen, A. B., Riedel, K., Eberl, L., Flodgaard, L. R., Molin, S., Gram, L. & Givskov, M. (2003). Quorum-sensing-directed protein expression in *Serratia proteamaculans* B5a. *Microbiology* **149**, 471–483.

Davies, D. G., Parsek, M. R., Pearson, J. P., Iglewski, B. H., Costerton, J. W. & Greenberg, E. P. (1998). The involvement of cell-to-cell signals in the development of a bacterial biofilm. *Science* **280**, 295–298.

de Kievit, T. R. & Iglewski, B. H. (2000). Bacterial quorum sensing in pathogenic relationships. *Infect Immun* **68**, 4839–4849.

de Lorenzo, V., Herrero, M., Jakubzik, U. & Timmis, K. N. (1990). Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in Gram-negative eubacteria. *J Bacteriol* **172**, 6568–6572.

Defoirdt, T., Boon, N., Sorgeloos, P., Verstraete, W. & Bossier, P. (2008). Quorum sensing and quorum quenching in *Vibrio harveyi*: lessons learned from in vivo work. *ISME J* **2**, 19–26.

Devescovi, G., Bigirimana, J., Degrassi, G., Cabrio, L., LiPuma, J. J., Kim, J., Hwang, I. & Venturi, V. (2007). Involvement of a quorum-sensing-regulated lipase secreted by a clinical isolate of *Burkholderia glumae* in severe disease symptoms in rice. *Appl Environ Microbiol* **73**, 4950–4958.

Diggle, S. P., Griffin, A. S., Campbell, G. S. & West, S. A. (2007). Cooperation and conflict in quorum-sensing bacterial populations. *Nature* **450**, 411–414.

Eberhard, A., Longin, T., Widrig, C. A. & Stranick, S. J. (1991). Synthesis of the *lux* gene autoinducer in *Vibrio fischeri* is positively autoregulated. *Arch Microbiol* **155**, 294–297.

Engebrecht, J. & Silverman, M. (1984). Identification of genes and gene products necessary for bacterial bioluminescence. *Proc Natl Acad Sci U S A* **81**, 4154–4158.

Euzéby, J. P. (2010). List of Prokaryotic Names with Standing in Nomenclature. http://www.bacterio.cict.fr

Farrand, S. K. (1998). Conjugation in *Rhizobiaceae*. In *The Rhizobiaceae, Molecular Biology of Model Plant-Associated Bacteria*, pp. 199–233. Edited by H. P. Spaink, A. Kondorosi & P. J. J. Hooykaas. Dordrecht: Kluwer Academic.

Fuqua, W. C., Winans, S. C. & Greenberg, E. P. (1994). Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators. *J Bacteriol* **176**, 269–275.

Fuqua, C., Parsek, M. R. & Greenberg, E. P. (2001). Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing. *Annu Rev Genet* **35**, 439–468.

González, J. E. & Keshavan, N. D. (2006). Messing with bacterial quorum sensing. *Microbiol Mol Biol Rev* **70**, 859–875.

González, J. E. & Marketon, M. M. (2003). Quorum sensing in nitrogen-fixing rhizobia. *Microbiol Mol Biol Rev* **67**, 574–592.

Gray, K. M., Passador, L., Iglewski, B. H. & Greenberg, E. P. (1994). Interchangeability and specificity of components from the quorum-sensing regulatory systems of *Vibrio fischeri* and *Pseudomonas aeruginosa*. *J Bacteriol* **176**, 3076–3080.

Hoang, H. H., Becker, A. & González, J. E. (2004). The LuxR homolog ExpR, in combination with the Sin quorum sensing system, plays a central role in *Sinorhizobium meliloti* gene expression. *J Bacteriol* **186**, 5460–5472.

Horng, Y. T., Deng, S. C., Daykin, M., Soo, P. C., Wei, J. R., Luh, K. T., Ho, S. W., Swift, S., Lai, H. C. & Williams, P. (2002). The LuxR family protein SpnR functions as a negative regulator of N-acylhomoserine lactone-dependent quorum sensing in *Serratia marcescens*. *Mol Microbiol* **45**, 1655–1671.

Iida, A., Ohnishi, Y. & Horinouchi, S. (2008). Control of acetic acid fermentation by quorum sensing via N-acylhomoserine lactones in *Gluconacetobacter intermedius*. *J Bacteriol* **190**, 2546–2555.

Kalogeraki, V. S. & Winans, S. C. (1997). Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria. *Gene* **188**, 69–75.

Khan, S. R., Herman, J., Krank, J., Serkova, N. J., Churchill, M. E. A., Suga, H. & Farrand, S. K. (2007). N-(3-hydroxyhexanoyl)-L-homoserine lactone is the biologically relevant quormone that regulates the *phz* operon of *Pseudomonas chlororaphis* strain 30-84. *Appl Environ Microbiol* **73**, 7443–7455.

Letunic, I., Copley, R. R., Pils, B., Pinkert, S., Schultz, J. & Bork, P. (2006). SMART 5: domains in the context of genomes and networks. *Nucleic Acids Res* **34** (Database issue), D257–D260.

Licciardello, G., Bertani, I., Steindler, L., Bella, P., Venturi, V. & Catara, V. (2007). *Pseudomonas corrugata* contains a conserved N-acyl homoserine lactone quorum sensing system; its role in tomato pathogenicity and tobacco hypersensitivity response. *FEMS Microbiol Ecol* **61**, 222–234.

Llamas, I., del Moral, A., Béjar, V., Girón, M. D., Salto, R. & Quesada, E. (1997). Plasmids from *Halomonas eurihalina*, a microorganism which produces an exopolysaccharide of biotechnological interest. *FEMS Microbiol Lett* **156**, 251–257.

Llamas, I., Argandoña, M., Quesada, E. & del Moral, A. (2000). Transposon mutagenesis in *Halomonas eurihalina*. *Res Microbiol* **151**, 13–18.

Llamas, I., Suárez, A., Quesada, E., Béjar, V. & del Moral, A. (2003). Identification and characterization of the *carAB* genes responsible for encoding carbamoylphosphate synthetase in *Halomonas eurihalina*. *Extremophiles* **7**, 205–211.

Llamas, I., Keshavan, N. & González, J. E. (2004). Use of *Sinorhizobium meliloti* as an indicator for specific detection of long-chain N-acyl homoserine lactones. *Appl Environ Microbiol* **70**, 3715–3723.

Llamas, I., Quesada, E., Martínez-Cánovas, M. J., Gronquist, M., Eberhard, A. & González, J. E. (2005). Quorum sensing in halophilic bacteria: detection of N-acyl-homoserine lactones in the exopolysaccharide-producing species of *Halomonas*. *Extremophiles* **9**, 333–341.

Malott, R. J., Baldwin, A., Mahenthiralingam, E. & Sokol, P. A. (2005). Characterization of the *cciR* quorum-sensing system in *Burkholderia cenocepacia*. *Infect Immun* **73**, 4982–4992.
Marketon, M. M. & González, J. E. (2002). Identification of two quorum-sensing systems in *Sinorhizobium meliloti*. *J Bacteriol* **184**, 3466-3475.

Marketon, M. M., Gronquist, M. R., Eberhard, A. & González, J. E. (2002). Characterization of the *Sinorhizobium meliloti sinR/sinI* locus and the production of novel N-acyl homoserine lactones. *J Bacteriol* **184**, 5686-5695.

Marketon, M. M., Glenn, S. A., Eberhard, A. & González, J. E. (2003). Quorum sensing controls exopolysaccharide production in *Sinorhizobium meliloti*. *J Bacteriol* **185**, 325-331.

Martínez-Cánovas, M. J., Béjar, V., Martínez-Checa, F. & Quesada, E. (2004). *Halomonas anticariensis* sp. nov., from Fuente de Piedra, a saline-wetland wildfowl reserve in Malaga, southern Spain. *Int J Syst Evol Microbiol* **54**, 1329-1332.

McClean, K. H., Winson, M. K., Fish, L., Taylor, A., Chhabra, S. R., Camara, M., Daykin, M., Lamb, J. H., Swift, S. & other authors (1997). Quorum sensing and *Chromobacterium violaceum*: exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. *Microbiology* **143**, 3703-3711.

McIntosh, M., Meyer, S. & Becker, A. (2009). Novel *Sinorhizobium meliloti* quorum sensing positive and negative regulatory feedback mechanisms respond to phosphate availability. *Mol Microbiol* **74**, 1238-1256.

McNab, R., Ford, S. K., El-Sabaeny, A., Barbieri, B., Cook, G. S. & Lamont, R. J. (2003). LuxS-based signaling in *Streptococcus gordonii*: autoinducer 2 controls carbohydrate metabolism and biofilm formation with *Porphyromonas gingivalis*. *J Bacteriol* **185**, 274-284.

Miller, J. (1972). *Experiments in Molecular Genetics*. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Miller, V. L. & Mekalanos, J. J. (1988). A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in *Vibrio cholerae* requires *toxR*. *J Bacteriol* **170**, 2575-2583.

Moraine, R. A. & Rogovin, P. (1966). Kinetics of polysaccharide B-1459 fermentation. *Biotechnol Bioeng* **8**, 511-524.

Morohoshi, T., Nakamura, Y., Yamazaki, G., Ishida, A., Kato, N. & Ikeda, T. (2007). The plant pathogen *Pantoea ananatis* produces N-acylhomoserine lactone and causes center rot disease of onion by quorum sensing. *J Bacteriol* **189**, 8333-8338.

Nasser, W., Bouillant, M. L., Salmond, G. & Reverchon, S. (1998). Characterization of the *Erwinia chrysanthemi* *expI*-expR locus directing the synthesis of two N-acyl-homoserine lactone signal molecules. *Mol Microbiol* **29**, 1391-1405.

Newman, J. R. & Fuqua, C. (1999). Broad-host-range expression vectors that carry the L-arabinose-inducible *Escherichia coli* araBAD promoter and the *araC* regulator. *Gene* **227**, 197-203.

Ng, W. L. & Bassler, B. L. (2009). Bacterial quorum-sensing network architectures. *Annu Rev Genet* **43**, 197-222.

Nieto, J. J., Fernández-Castillo, R., Márquez, M. C., Ventosa, A., Quesada, E. & Ruíz-Berraquero, F. (1989). Survey of metal tolerance in moderately halophilic eubacteria. *Appl Environ Microbiol* **55**, 2385-2390.

Niu, C., Clemmer, K. M., Bonomo, R. A. & Rather, P. N. (2008). Isolation and characterization of an autoinducer synthase from *Acinetobacter baumannii*. *J Bacteriol* **190**, 3386-3392.

Ochman, H., Gerber, A. S. & Hartl, D. L. (1988). Genetic applications of an inverse polymerase chain reaction. *Genetics* **120**, 621-623.

Parker, C. T. & Sperandio, V. (2009). Cell-to-cell signalling during pathogenesis. *Cell Microbiol* **11**, 363-369.

Patankar, A. V. & González, J. E. (2009). Orphan LuxR regulators of quorum sensing. *FEMS Microbiol Rev* **33**, 739-756.

Pfaffl, M. W., Horgan, G. W. & Dempfle, L. (2002). Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. *Nucleic Acids Res* **30**, e36.

Pirhonen, M., Flego, D., Heikinheimo, R. & Palva, E. T. (1993). A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen *Erwinia carotovora*. *EMBO J* **12**, 2467-2476.

Pumbwe, L., Skilbeck, C. A. & Wexler, H. M. (2008). Presence of quorum-sensing systems associated with multidrug resistance and biofilm formation in *Bacteroides fragilis*. *Microb Ecol* **56**, 412-419.

Rodríguez-Valera, F., Ruiz-Berraquero, F. & Ramos-Cormenzana, A. (1981). Characteristics of the heterotrophic bacterial populations in hypersaline environments of different salt concentrations. *Microb Ecol* **7**, 235-243.

Sambrook, J. & Russell, D. W. (2001). *Molecular Cloning: a Laboratory Manual*. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Schaefer, A. L., Greenberg, E. P., Oliver, C. M., Oda, Y., Huang, J. J., Bittan-Banin, G., Peres, C. M., Schmidt, S., Juhaszova, K. & other authors (2008). A new class of homoserine lactone quorum-sensing signals. *Nature* **454**, 595-599.

Shaw, P. D., Ping, G., Daly, S. L., Cha, C., Cronan, J. E., Jr, Rinehart, K. L. & Farrand, S. K. (1997). Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography. *Proc Natl Acad Sci U S A* **94**, 6036-6041.

Song, Y., Xie, C., Ong, Y.-M., Gan, Y.-H. & Chua, K.-L. (2005). The BpsIR quorum-sensing system of *Burkholderia pseudomallei*. *J Bacteriol* **187**, 785-790.

Spaink, H. P., Wijffelman, C. A., Pees, E., Okker, R. J. H. & Lugtenberg, B. J. J. (1987). *Rhizobium* nodulation gene *nodD* as a determinant of host specificity. *Nature* **328**, 337-340.

Subramoni, S. & Venturi, V. (2009). LuxR-family 'solos': bachelor sensors/regulators of signalling molecules. *Microbiology* **155**, 1377-1385.

Sutherland, I. W. (2002). Polysaccharides from prokaryotes. In *Biopolymers. Polysaccharides I*, pp. 1-19. Edited by E. J. Vandamme, S. De Baets & A. Steinbüchel. Weinheim: Wiley-VCH.

Taga, M. E. & Bassler, B. L. (2003). Chemical communication among bacteria. *Proc Natl Acad Sci U S A* **100** (Suppl 2), 14549-14554.

Throup, J. P., Câmera, M., Briggs, G., Winson, M. K., Bycroft, B. W., Williams, P. & Stewart, G. S. (1995). Characterisation of the *yenI/yenR* locus from *Yersinia enterocolitica* mediating the synthesis of two N-acylhomoserine lactone signal molecules. *Mol Microbiol* **17**, 345-356.

Urbanczyk, H., Ast, J. C., Higgins, M. J., Carson, J. & Dunlap, P. V. (2007). Reclassification of *Vibrio fischeri*, *Vibrio logei*, *Vibrio salmonicida* and *Vibrio wodanis* as *Aliivibrio fischeri* gen. nov., comb. nov., *Aliivibrio logei* comb. nov., *Aliivibrio salmonicida* comb. nov. and *Aliivibrio wodanis* comb. nov. *Int J Syst Evol Microbiol* **57**, 2823-2829.

Van Houdt, R., Moons, P., Aertsen, A., Jansen, A., Vanoirbeek, K., Daykin, M., Williams, P. & Michiels, C. W. (2007). Characterization of a *luxI/luxR*-type quorum sensing system and N-acyl-homoserine lactone-dependent regulation of exo-enzyme and antibacterial component production in *Serratia plymuthica* RVH1. *Res Microbiol* **158**, 150-158.

von Bodman, S. B., Majerczak, D. R. & Coplin, D. L. (1998). A negative regulator mediates quorum-sensing control of exopolysaccharide production in *Pantoea stewartii* subsp. *stewartii*. *Proc Natl Acad Sci U S A* **95**, 7687-7692.

von Bodman, S. B., Bauer, W. D. & Coplin, D. L. (2003). Quorum sensing in plant-pathogenic bacteria. *Annu Rev Phytopathol* **41**, 455-482.
Wang, L. H., He, Y., Gao, Y., Wu, J. E., Dong, Y. H., He, C., Wang, S. X., Weng, L. X., Xu, J. L. & other authors (2004). A bacterial cell–cell communication signal with cross-kingdom structural analogues. *Mol Microbiol* **51**, 903–912.

Watson, W. T., Minogue, T. D., Val, D. L., von Bodman, S. B. & Churchill, M. E. (2002). Structural basis and specificity of acyl-homoserine lactone signal production in bacterial quorum sensing. *Mol Cell* **9**, 685–694.

Whitehead, N. A., Barnard, A. M., Slater, H., Simpson, N. J. & Salmond, G. P. (2001). Quorum-sensing in Gram-negative bacteria. *FEMS Microbiol Rev* **25**, 365–404.

Winson, M. K., Swift, S., Fish, L., Throup, J. P., Jørgensen, F., Chhabra, S. R., Bycroft, B. W., Williams, P. & Stewart, G. S. (1998). Construction and analysis of *luxCDABE*-based plasmid sensors for investigating *N*-acyl homoserine lactone-mediated quorum sensing. *FEMS Microbiol Lett* **163**, 185–192.

Zheng, H., Zhong, Z., Lai, X., Chen, W.-X., Li, S. & Zhu, J. (2006). A LuxR/LuxI-type quorum-sensing system in a plant bacterium, *Mesorhizobium tianshanense*, controls symbiotic nodulation. *J Bacteriol* **188**, 1943–1949.

Edited by: D. Demuth