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The ISME Journal (2015) 9, 1222–1234 & 2015 International Society for Microbial All rights reserved 1751-7362/15 www.nature.com/ismej ORIGINAL ARTICLE From deep-sea volcanoes to human : a conserved quorum-sensing signal in

Ileana Pe´rez-Rodrı´guez1,2,3, Marie Bolognini1,2, Jessica Ricci1,2,4, Elisabetta Bini1,5 and Costantino Vetriani1,2 1Department of Biochemistry and Microbiology, Rutgers University, New Brunswick, NJ, USA and 2Institute of Marine and Coastal Sciences, Rutgers University, New Brunswick, NJ, USA

Chemosynthetic Epsilonproteobacteria from deep- hydrothermal vents colonize substrates exposed to steep thermal and redox gradients. In many , substrate attachment, biofilm formation, expression of genes and host colonization are partly controlled via a density-dependent mechanism involving signal molecules, known as quorum sensing. Within the Epsilonproteobacteria, quorum sensing has been investigated only in human pathogens that use the luxS/autoinducer-2 (AI-2) mechanism to control the expression of some of these functions. In this study we showed that luxS is conserved in Epsilonproteobacteria and that pathogenic and mesophilic members of this class inherited this gene from a thermophilic ancestor. Furthermore, we provide evidence that the luxS gene is expressed—and a quorum-sensing signal is produced—during growth of Sulfurovum lithotrophicum and mediatlanticus,twoEpsilonproteobacteria from deep-sea hydrothermal vents. Finally, we detected luxS transcripts in Epsilonproteobacteria- dominated biofilm communities collected from deep-sea hydrothermal vents. Taken together, our findings indicate that the epsiloproteobacterial lineage of the LuxS originated in high-temperature geothermal environments and that, in vent Epsilonproteobacteria, luxS expression is linked to the production of AI-2 signals, which are likely produced in situ at deep-sea vents. We conclude that the luxS gene is part of the ancestral epsilonproteobacterial and represents an evolutionary link that connects to human pathogens. The ISME Journal (2015) 9, 1222–1234; doi:10.1038/ismej.2014.214; published online 14 November 2014

Introduction The metabolic versatility observed in pure cultures of vent Epsilonproteobacteria (Campbell et al., 2006; Epsilonproteobacteria have been recognized as an Sievert and Vetriani, 2012), along with their high ecologically significant group of bacteria in deep-sea growth rates (Campbell et al., 2006) and ability to geothermal environments as they are highly repre- attach to substrates and form biofilms (Alain et al., 1 sented in moderately warm (25–35 C) and oxidized 2004), is likely to contribute to the ecological 1 as well as in relatively hot (40–70 C) and anoxic success of these bacteria in environments character- vents (Longnecker and Reysenbach, 2001; Nakagawa ized by high turbulence and steep thermal and redox et al., 2005; Campbell et al., 2006; Moussard et al., gradients. Epsilonproteobacteria also include 2006; Huber et al., 2007; Sievert and Vetriani, 2012). human pathogens (for example, and At deep-sea hydrothermal vents, chemosynthetic spp., Supplementary Figure S1) Epsilonproteobacteria mediate the transfer of carbon (Nakagawa et al., 2007), whose ability to colonize and energy from the geothermal source to higher their hosts is related to high growth rates, attach- trophic levels, functioning as primary producers. ment and biofilm formation (Stark et al., 1999; Joshua et al., 2006). Correspondence: C Vetriani, Institute of Marine and Coastal Quorum sensing (QS), a cell-to-cell communica- Sciences, Rutgers University, 71 Dudley Road, New Brunswick, tion mechanism that relies on cell density and the NJ 08901, USA. production of extracellular signaling molecules, is E-mail: [email protected] 3Current address: Geophysical Laboratory, Carnegie Institution of used by bacteria to control coordinated gene Washington, Washington, DC, USA. expression, including virulence genes and functions 4Current address: Department of Biology, California Institute of associated with biofilm formation and host coloni- Technology, Pasadena, CA, USA. zation (Bassler et al., 1994; Keller and Surette, 2006). 5Current address: Department of Pharmacy Practice and Admin- istration, Rutgers University, Piscataway, NJ, USA. One QS system that appears to be widespread across Received 27 May 2014; revised 27 September 2014; accepted the bacterial (Figure 1a), and that has been 1 October 2014; published online 14 November 2014 proposed to function as the universal language for Quorum sensing in deep-sea vent Epsilonproteobacteria IPe´rez-Rodrı´guez et al 1223 interspecies communication, is based on a furanone lithotrophicum and Caminibacter mediatlanticus as derivative, known as autoinducer-2 (AI-2), model strains for mesophilic and thermophilic vent synthesized by the LuxS enzyme (Kaper et al., Epsilonproteobacteria, respectively, to investigate 1999; Rader et al., 2007). The LuxS enzyme is an the expression of the luxS gene and the production integral component of the activated methyl cycle of a QS signal during growth. Finally, we identified (AMC), the major methyl donor in the cell luxS transcripts in Epsilonproteobacteria-dominated (Vendeville et al., 2005; Turovskiy et al., 2007). In chemosynthetic biofilms collected from active addition to its role as AI-2 synthase, the main deep-sea hydrothermal vents. function of the LuxS enzyme in the AMC is the conversion of S-ribosylhomocysteine to homocys- teine, with the production of 4,5-dihydroxyl-2,3- Materials and methods pentanedione (DPD) as a by-product of this reaction (Chen et al., 2002; Winzer et al., 2003). Through and growth conditions T processes of dehydration and cyclization, DPD is S. lithotrophicum (DSM 23290 ; Inagaki et al., 2004), rearranged into a mixture of chemically related a microaerobic, facultative anaerobic thiosulfate- molecules that function as AI-2 chemical signals in oxidizing mesophile, was obtained from the German QS. These molecules include 4-hydroxy-5-methyl-3 Collection of and Cell Cultures (2H) furanone (MHF), (2R, 4S)-2-methyl-2,3,3,4- (Braunschweig, Germany). Caminibacter mediatlanti- T tetrahydroxy-tetrhydrofuran (R-THMF) and furanosyl cus strain TB-2 (DSM 16658 ), a strictly anaerobic, borate diester (S-THMF borate; (Schauder et al., hydrogen-oxidizing was isolated and 2001; Winzer et al., 2002; Chen et al., 2002; Winzer described previously in our laboratory (Voordeckers et al., 2003; Vendeville et al., 2005; Rader et al., et al., 2005). The two reporter strains, Vibrio harveyi 2007; Shen et al., 2010). strains BB170 and BB120 (Bassler et al., 1993), were The importance of luxS/AI-2 for the coordinated kindly provided to us by Dr Chikindas (Rutgers expression of virulence factors, biofilm formation University, New Brunswick, NJ, USA) and Dr Bassler and has been confirmed in a (Princeton University, Princeton, NJ, USA). S. litho- variety of microorganisms (Bassler et al., 1994; trophicum was grown anaerobically (N2/CO2, 80%: Forsyth and Cover, 2000; Wright et al., 2000; Elvers 20%)at301C in 356 MJ basal medium (Inagaki et al., and Park, 2002; Shimizu et al., 2002; Stevenson and 2004) supplemented with 10 mM Na2S2O3  5H2Oand Babb, 2002; Winzer et al., 2002; McNab et al., 2003; 14 mM KNO3 as electron donor and acceptor, respec- Rhee et al., 2003). In Epsilonproteobacteria, the role tively. C. mediatlanticus was grown at 55 1Cin of AI-2-mediated QS has been investigated exclu- modified SME medium (Stetter et al., 1983) supple- sively in the human pathogens, Helicobacter and mented with 14 mM KNO3 under a phase of H2/CO2 Campylobacter spp., where AI-2 secretion into the (20%: 80%). Strains of V. h a r v e y i were grown in extracellular environment has been shown to occur modified Autoinducer Bioassay (AB) medium by a LuxS-dependent mechanism (Forsyth and (Turovskiy and Chikindas, 2006). Time course experi- Cover, 2000; Joyce et al., 2000; Elvers and Park, ments for S. lithotrophicum and C. mediatlanticus were 2002; Rader et al., 2007). In ,AI-2 performed in 1 liter of culture medium in triplicate. modulates flagellar morphogenesis and gene expres- sion in a dose-dependent manner (Rader et al., 2007; Shen et al., 2010; Rader et al., 2011). Similarly, in Autoinducer-2 bioassay , luxS mutants showed The AI-2 detection assay utilizes the luxN-null reduced motility and attachment during host colo- mutant, V. harveyi BB170, as a reporter strain that nization (Plummer et al., 2012). LuxS-dependent produces bioluminescence only in response to AI-2 motility/positioning patterns have been shown to be QS signals (Bassler et al., 1994). The assay was important virulence strategies required for coloniza- initiated by growing V. harveyi BB170 cultures for tion and dispersal of both Helicobacter and Campy- 16 h in modified Autoinducer-2 Bioassay (AB) lobacter spp. in several models (Eaton et al., medium (Turovskiy and Chikindas, 2006) with 1996; O’Toole et al., 2000; Ottemann and Lowenthal, aeration at 30 1C. The cultures were diluted 1:5000 2002; Blaser and Atherton, 2004; Rader et al., 2007; in fresh AB medium to obtain B105 CFUs per ml. Plummer et al., 2012). Cell-free supernatant (CFS) preparations of As LuxS has been linked to the regulation S. lithotrophicum and C. mediatlanticus were added of motility/colonization functions in pathogenic to the diluted V. harveyi to a 10% v/v final Epsilonproteobacteria (Eaton et al., 1996; Forsyth concentration, and these cultures were shaken (140 and Cover, 2000; Joyce et al., 2000; O’Toole et al., r.p.m.) at 30 1C for 5 and 6 h, respectively. Biolumi- 2000; Wright et al., 2000; Elvers and Park, 2002; nescence measurements were taken every 30 min Rader et al., 2007; Shen et al., 2010; Plummer et al., with a Luminoscan TL plus luminometer (Thermo- 2012), we investigated the distribution of the luxS Fisher Scientific, Waltham, MA, USA). Uninocu- gene in Epsilonproteobacteria and we reconstructed lated S. lithotrophicum and C. mediatlanticus media the evolutionary history of the epsilonproteobacter- were used as negative controls, whereas the CFS from ial LuxS lineage. Furthermore, we used Sulfurovum the wild-type V. h a r v e y i strain BB120 was used as a

The ISME Journal Quorum sensing in deep-sea vent Epsilonproteobacteria IPe´rez-Rodrı´guez et al 1224 positive control. All bioluminescence induction The activity of AI-2 is expressed in relative values obtained from the three independent growth bioluminescence units. The relative bioluminescence experiments for each bacterium are averages of units were normalized by subtracting the light duplicate AI-2 bioassays. intensity values of the negative control from the light

The ISME Journal Quorum sensing in deep-sea vent Epsilonproteobacteria IPe´rez-Rodrı´guez et al 1225 intensity values of all samples (including the positive each in triplicate, were carried out according to the controls) in the assays. The relative bioluminescence manufacturer’s instructions, using 0.15 mg of total unit values were expressed for both S. lithotrophicum RNA for S. lithotrophicum and 0.3 mg of total and C. mediatlanticus as percentages of biolumines- RNA for C. mediatlanticus in 15 ml reactions. cence induction, where each measured time point Primers were manually designed and analyzed was divided by the maximum bioluminescence using the Oligo Analyzer 3.1 from IDT (Integrated induction value obtained from each bacterium, DNA Technologies, Coralville, IA, USA) to have including the positive controls. a composition suitable for use in qRT-PCR The CFSs of S. lithotrophicum and C. mediatlanticus (Supplementary Table S3). Specificity of each pair were prepared from 10 ml of cell suspensions at of primers was confirmed by PCR amplification different stages during the time course growth experi- using the genomic DNA of the model organisms ments. The CFS of V. h a r v e y i BB120 (positive control) followed by sequencing. The efficiency of the PCR was prepared from cell suspensions of approximately amplifications was determined from the slopes of OD600 of 0.5–0.6. All cell suspensions were filtered the dilution curves of the target RNA. through a 0.2 mm-pore-size filter, centrifuged at 6000 Â g Standard curves for the luxS and 16S rRNA genes for 30 min (Nalgene Nunc International, Rochester, NY, were constructed using dilutions (range of stan- USA) and transferred into new tubes. Samples were dards: 103–107 copies per ml for S. lithotrophicum stored at À 20 1C until further processing. and C. mediatlanticus in a 15 ml reaction) of the corresponding PCR amplicon (Chini et al., 2007; Microscopy and cell counts Cuebas et al., 2011) using the iQ SYBR Green Cells were routinely stained with 0.1% acridine Supermix (Bio-Rad). The cycle threshold (Ct) values orange and visualized with an Olympus BX 60 obtained were used to calculate the absolute number microscope (Center Valley, PA, USA) with an oil of transcript copies per ng of RNA for both strains immersion objective (UPlanF1 100/1.3). Samples for (Supplementary Tables S4 and S5). The number of direct cell counts were collected to monitor cell 16S rRNA transcripts was monitored as the internal growth in the time course experiments and at standard to ensure that equal amounts of RNA were different time points during the AI-2 bioassay to used in each reaction and to normalize the levels of determine the number of V. h a r v e y i BB170 cells per luxS transcripts across samples (Supplementary ml (Supplementary Table S2). Figure S2). Values for 16S rRNA and luxS transcript copies per ng presented here are an average of RNA extraction and quantitative reverse transcription triplicate qRT-PCR experiments for the three inde- PCR (qRT-PCR) analyses of luxS transcripts in model pendent growth experiments carried out for each organisms model . Negative controls omitting the Total RNA was isolated from S. lithotrophicum templates were included. and C. mediatlanticus cultures at different phases during growth. Then, 75 ml of S. lithotrophicum cultures and 50 ml of C. mediatlanticus cultures were Sampling of natural biofilms from deep-sea preserved in a 1:1 volume of RNAlater (Ambion, hydrothermal vents ThermoFisher Scientific) and stored at À 80 1C until Samples of microbial biofilms were collected during further processing. RNA was extracted from cell R/V Atlantis cruises AT 15-15 (January–February pellets using the RNAeasy Mini kit (Qiagen, Valencia, 2007) and AT 15–28 (December 2007–January 2008) CA, USA) and treated with TURBO DNase (Ambion), using the Deep-Submergence Vehicle Alvin. Both as recommended by the manufacturer. The quantity biofilms used in this study were collected using and quality of the RNA obtained was evaluated both experimental microbial colonization devices made spectrophotometrically on a NanoDrop ND-1000 of stainless steel mesh. The first biofilm (designated spectrophotometer (ThermoFisher Scientific) and by as low T biofilm) was collected from a diffuse flow agarose gel electrophoresis. vent with no associated megafauna (‘Mk 33’, fluid Absolute quantification of luxS and 16S rRNA temperature: 10–28 1C, depth 2506 m, East Pacific transcripts was carried out by qRT-PCR using the Rise, 91 50 N, 1041 18 W). The second biofilm iScript One-Step RT-PCR kit with SYBR Green (designated as high T biofilm) was collected from (Bio-Rad, Hercules, CA, USA) and the real-time the wall of a structure colonized by Alvinella detection system iCycler iQ (Bio-Rad). Reactions, pompejana tubeworms venting 20–50 1C fluids

Figure 1 (a) Distribution of the luxS gene among phyla of the domain Bacteria. The tree was constructed using the neighbor-joining method from the 16S ribosomal RNA genes of 469 representative bacterial . The luxS homologs were indentified in 167 Beta-, Delta-, Gamma- and Epsilonproteobacteria, , , , /, and /Chlorobi (branches shown in bold face). The number of luxS genes identified in each phylum is indicated in parenthesis, followed by the total number of surveyed. The bar represents 0.10 base substitutions per nucleotide. The asterisks indicate branches that include Firmicutes.(b) Phylogenetic tree of LuxS sequences of Epsilonproteobacteria. The tree was constructed with Bayesian inference and maximum likelihood methods. The genera of the organisms within each collapsed branch are shown. Supplementary Table S6 lists all the organisms used in this analysis. The ancestral nodes A, B and C are shown as solid circles. Posterior probability values that support each topological element are shown at the branch nodes. Bar, 20% estimated substitutions.

The ISME Journal Quorum sensing in deep-sea vent Epsilonproteobacteria IPe´rez-Rodrı´guez et al 1226 (‘Jumeaux’ site, depth 2621 m, , 121 the amplification of partial luxS genes using the luxS 48 N, 1031 56 W). primers CM/NP and SL/SD (Caminibacter/Nautilia and Sulfurovum/ spp., respectively; Vent natural biofilms: RNA extraction, RT-PCR Supplementary Table S3) and the touchdown PCR detection of luxS transcripts and library construction conditions described above. Long-term stocks of Samples of the low T and high T biofilms were these reference strains, prepared by adding 50 mlof resuspended in RNAlater (Ambion) and stored at dimethyl sulfoxide to 1 ml of cultures, À 80 1C. Total RNA was extracted from the biofilm are stored at À 80 1C. Live cultures are available samples using the RNAeasy kit (Qiagen) and the upon request. DNase-treated RNA (0.5 mg) was reversed transcribed in 25 ml reactions using SuperScript III One-Step RT- PCR System with Platinum Taq DNA Polymerase Phylogenetic analyses (Invitrogen, Carlsbad, CA, USA). Two sets of The distribution of the luxS gene within the Bacteria primers, the first based on the luxS genes of S. was investigated in 465 representative taxa within lithotrophicum and Sulfurimons denitrificans (SL/ the bacterial domain by genome search or PCR SD luxS primers; Supplementary Table S3) and the amplification in reference strains. A phylogenetic second based on the luxS genes of C. mediatlanticus tree based on the 16S ribosomal RNA genes of the and Nautilia profundicola (CM/NP luxS primers: taxa surveyed was then constructed using the ARB Supplementary Table S3), were designed and used neighbor-joining method (http://www.arb-home.de/ in RT-PCR reactions to amplify the luxS gene home.html; Figure 1a). transcripts from the low T and high T biofilms. Both The phylogenetic tree in Figure 1b was con- primer sets were used to amplify luxS transcripts in structed by aligning trimmed LuxS amino acid the biofilm samples. sequences from GenBank, using Muscle (Edgar, Touchdown PCR conditions for all luxS amplifi- 2004) and Gblocks (Castresana, 2000). Trees were cations were as follow: an initial denaturation for reconstructed with Bayesian inference and max- 5 min at 94 1C, followed by 10 cycles of denaturation imum likelihood methods. The maximum likeli- at 94 1C for 30 s, annealing at 44 1C for 1 min, hood phylogeny was inferred from the alignment extension at 72 1C for 1 min and 30 s, followed by using PHYML (Guindon and Gascuel, 2003) with the 30 cycles of denaturation at 94 1C for 30 s, annealing CpREV substitution model and 1000 bootstrap at 40 1C for 1 min, extension at 72 1C for 1 min and resamplings. The substitution model was selected 30 s and a final extension of 5 min at 72 1C. based on AIC values using ProTest3 (Darriba et al., The amplified luxS transcripts were gel-purified 2011). Bayesian phylogeny was inferred using using the MinElute Gel Extraction Kit (Qiagen), MrBayes (Ronquist and Huelsenbeck, 2003) per- cloned into the pCR4-TOPO plasmid vector and the forming 500 000 generations with two parallel ligation products were transformed into searches with the CpREV amino acid matrix model OneShotTOP10CompetentCellusingtheTOPO (selected using forward selection among all possible TA Cloning Kit for Sequencing (Invitrogen). In all, substitution models) and a burn-in of 125 000 52 and 46 randomly chosen clones from the two generations. The tree was rooted using the mid- luxS environmental libraries were analyzed, point convergence method and manually using respectively, for insert-containing plasmids by LuxS from Gram-positive bacteria as the outgroup. direct PCR followed by gel electrophoresis of the Both rooting methodologies gave the same results. amplified products. The resulting PCR fragments The organisms used in the phylogenetic tree were screened by restriction fragment length reconstruction shown in Figure 1b are listed in polymorphism analysis using the RsaI restriction Supplementary Table S6. Ancestral state reconstruc- endonuclease (Boehringer Mannheim, Mannheim, tion of the LuxS sequence was performed using the Germany). The restriction fragment length poly- MEGA package (Tamura et al., 2013) using the morphism reactions were analyzed on a 3% maximum likelihood option with the CpREV amino MetaPhor Agarose (Lonza, Rockland, ME, USA) acid matrix model of substitution. gel. Representative luxS genes with unique restric- For the construction of the phylogenetic tree tion fragment length polymorphism patterns were shown in Figure 3, the luxS sequences obtained by purified using the MiniPrep Plasmid Purification PCR from reference strains and natural vent biofilm Kit (Qiagen) and sequenced. communities were translated into amino acid sequences using EMBOSS Transeq (http://www. PCR amplification of the luxS gene from reference ebi.ac.uk/emboss/transeq/), aligned using ClustalX strains v 1.8 (Thompson et al., 1997) and manually adjusted Genomic DNA extracted from 10 ml cultures of using Seaview (Galtier et al., 1996). Phylogenetic Caminibacter sp. strain TB1 (Voordeckers et al., distances were calculated using the CpREV sub- 2005), Nautilia spp. strains MT3 and MT4 stitution model and the maximum likelihood (Voordeckers et al., 2008), Nautilia nitratireducens method to evaluate tree topologies and their robust- strain MB-1 (Perez-Rodriguez et al., 2010) and ness was tested by bootstrap analysis with 100 S. lithotrophicum (Inagaki et al., 2004) was used for resamplings.

The ISME Journal Quorum sensing in deep-sea vent Epsilonproteobacteria IPe´rez-Rodrı´guez et al 1227 Nucleotide sequence accession numbers nested within a group of Campylobacter sequences The luxS sequences from this study are available (Campylobacter group 1 in Figure 1b). Finally, through GenBank under the following accession our analysis showed that the LuxS enzyme numbers: KF876791-KF876794 for luxS genes in of H. pylori was related to the Gram þ lineage Caminibacter sp. strain TB1, Nautilia sp. strain (Figure 1b). MT3, Nautilia sp. strain MT4 and N. nitratireducens strain MB1; KF876760-KF876777 for luxS tran- scripts obtained from the low T biofilm; and Quantitative detection of luxS transcripts and AI-2 KF876778-KF876790 for luxS transcripts obtained activity in S. lithotrophicum and C. mediatlanticus from high T biofilm. We carried out a time course experiment and quantified luxS transcripts at different phases during the growth cycle of these bacteria (Figures 2a and d). Results We found that in both S. lithotrophicum and C. mediatlanticus, the expression of the luxS gene Distribution of the LuxS enzyme in the bacterial was highest during the mid-exponential phase of domain and reconstruction of the evolutionary history growth and gradually dropped to baseline levels during of the epsilonproteobacterial LuxS lineage the stationary phase (Figures 2b and e). The drop in In this study we investigated the distribution of the luxS transcripts was B336-fold in S. lithotrophicum luxS gene by searching representative genomes from 30–100 h of growth and B59-fold in C. media- within the bacterial domain and we identified luxS tlanticus from 8 to 40 h of growth (Figures 2b and e). gene homologs in 167 out of 469 bacterial taxa To establish whether S. lithotrophicum and (Supplementary Tables S7 and S8). Representative C. mediatlanticus produced an AI-2-like QS signal species coding for luxS were found within the Beta- during the time course experiment, we carried out (5%), Delta- (9%), Gamma- (55%) and Epsilonpro- an AI-2 detection assay using V. harveyi strain teobacteria (100%), Firmicutes (63%), Fusobacteria BB170, a reporter strain that produces biolumines- (60%), Actinobacteria (24%), Deinococcus/Thermus cence in response to AI-2 QS signals (Bassler et al., (100%), Spirochaetes (40%) and Bacteroidetes/ 1994; Turovskiy et al., 2007). Indeed, supplementing Chlorobi (13%) (Figure 1a and Supplementary cultures of V. harveyi BB170 with CFSs of Table S7). S. lithotrophicum and C. mediatlanticus induced Bayesian phylogenetic analyses of the LuxS a bioluminescence response (Figures 2c and f). In enzyme of Epsilonproteobacteria indicated that the both microorganisms, the observed bioluminescence lineage found in pathogenic, commensal and meso- induction increased with cell densities during philic Epsilonproteobacteria shared a common growth (Figures 2c and f). Maximum biolumines- ancestor with members of the order Nautiliales cence induction (expressed in relative biolumines- (node A in Figure 1b; 100% posterior probability) cence units) was observed during the stationary and with sp. (node B in Figure 1b). All phase of growth, at 68.7±8.1 h for S. lithotrophicum members of the Nautiliales and Nitratiruptor sp. are and at 26±3.5 h for C. mediatlanticus deep-sea vent thermophilic chemolithoautotrophs (Supplementary Table S1). In fact, bioluminescence (Campbell et al., 2006; Sievert and Vetriani, 2012) induction in V. harveyi BB170 was significantly (Figure 1b and Supplementary Table S6). correlated to cell density in both S. lithotrophicum A reconstruction of the amino acid sequence of the (r ¼ 0.60, Po0.01, n ¼ 25) and C. mediatlanticus LuxS ancestral state indicated that the sequence (r ¼ 0.63, Po0.01, n ¼ 33). Bioluminescence induc- corresponding to node A (Figure 1b) was 83.1% and tion using the CFSs of V. harveyi strain BB120 (wild 72.3% identical to the LuxS of C. mediatlanticus type) as a positive control was 2.5- and 2.2-fold and , respectively. higher than that measured when the CFSs from All the mesophilic Epsilonproteobacteria, which S. lithotrophicum and C. mediatlanticus were used, include environmental species of the genera respectively (Supplementary Table S2). The net Sulfurovum, Sulfurimonas, and positive bioluminescence induction measured in Sulfurospirillum, the commensal organism V. harveyi BB170 in response to the CFSs of succinogenes and the pathogenic species, S. lithotrophicum and C. mediatlanticus indicated Campylobacter and Helicobacter, shared a common that growth of these organisms was necessary for the ancestor (node C in Figure 1b) nested within the synthesis of AI-2-like molecules and to elicit a thermophilic lineage. However, the topology of this bioluminescence response in the reporter strain. section of the LuxS tree showed a polytomy; hence When changes in luxS transcription and biolumines- the evolutionary relationships between mesophilic, cence induction at different times during growth were commensal and pathogenic Epsilonproteobacteria compared, a negative correlation was established could not be resolved. Interestingly, a group of LuxS between the two variables. Expression of the luxS gene sequences found in members of the Gammaproteo- (Figures 2b and e) in both S. lithotrophicum (r (24) ¼ bacteria (including species of the genera Vibrio, À 0.68, Po0.01) and C. mediatlanticus (r (31) ¼À0.54, Photobacterium, Salmonella, Escherichia and Enter- Po0.01) is inversely correlated to bioluminescence obacter) clustered with the Arcobacter lineage and induction in V. h a r v e y i (Figures 2c and f).

The ISME Journal Quorum sensing in deep-sea vent Epsilonproteobacteria IPe´rez-Rodrı´guez et al 1228

Figure 2 Growth curves for S. lithotrophicum (a) and C. mediatlanticus (d) cultured under nitrate-reducing conditions. Expression of the luxS gene (filled symbols) and induction of bioluminescence (open symbols) in S. lithotrophicum (b, c) and C. mediatlanticus (e, f). Exponential (E), stationary (S) and death (D) growth phases are labeled within each graph. All experiments were carried out in triplicates.

Detection of luxS transcripts in biofilm communities old) from two vents located on the East Pacific Rise from deep-sea hydrothermal vents at 91 and 131 North, respectively, that were char- Following our finding that laboratory strains of vent acterized by different thermal and biological Epsilonproteobacteria express the luxS gene during regimes. Both the low T and high T biofilms were growth and elicit a QS response in a V. harveyi dominated by Epsilonproteobacteria (93.1% and reporter strain, we hypothesized that the luxS gene 86.6%, respectively; data not shown). The luxS was expressed in situ at deep-sea vents during the transcripts related to Epsilonproteobacteria were formation of microbial biofilms. To this end, we amplified by RT-PCR from RNA extracted from the collected recently established biofilms (3 to 7 days biofilm communities. However, luxS transcripts

The ISME Journal Quorum sensing in deep-sea vent Epsilonproteobacteria IPe´rez-Rodrı´guez et al 1229 from the low T biofilm were detected only when the (Figure 1b). This finding is in SL/SD primers were used, whereas luxS transcripts line with previous studies that showed that the from the high T biofilm were obtained only with the LuxS from H. pylori was related to the enzyme CM/NP primers. Phylogenetic analysis of the amino found in members of the Firmicutes (Lerat and acid sequences of the LuxS enzyme deduced from Moran, 2004; Doherty et al., 2010). Moreover, RT-PCR-amplified transcripts showed that the high T marine of the genera Photo- biofilm expressed related to C. mediatlanticus, bacterium and Vibrio, along with E. coli and other Caminibacter sp. strain TB-1 and N. nitratireducens human-associated bacteria, appear to share a (Figure 3; 98–99% amino acid identity to LuxS of C. common luxS ancestor with Arcobacter nitrofigilis mediatlanticus). In contrast, the LuxS retrieved from and A. butzleri, a marine organism and a the low T biofilm was distributed in three clusters. human , respectively (Figure 1b and The majority of the sequences (cluster 1 in Figure 3) Supplementary Table S6). These findings indicate were grouped in three subclusters with 82–83% that bacteria inhabiting similar ecological niches, amino acid identity to the LuxS of S. lithotrophi- regardless of their taxonomic distance, share closely cum. Two sequences were related to the enzyme of related forms of the luxS gene. Because and S. lithotrophicum and Sulfurovum sp. strain NBC37- niche play an important role in horizontal gene 1 (low T biofilm A6 and B8; 88% and 87% identity transfer (Polz et al., 2013), we suggest that inter- to the enzyme of S. lithotrophicum, respectively), species QS within the same habitat is an important whereas a single sequence (low T biofilm C12) was driver in the evolution of luxS. placed in a discrete cluster (83% identity to the LuxS of S. lithotrophicum). luxS expression and AI-2 activity in laboratory cultures of vent Epsilonproteobacteria Discussion Here we present one of the few studies on how the expression of the luxS gene relates to AI-2 activity. Thermophilic ancestry of the LuxS lineage and luxS In Salmonella typhimurium, the constitutive expres- gene flow in Epsilonproteobacteria sion of luxS suggests that AI-2 production is Bayesian reconstruction of the LuxS evolutionary regulated by the availability of the LuxS substrate, history indicated a thermophilic origin of the S-ribosylhomocysteine (Beeston and Surette, 2002). epsilonproteobacterial lineage (Figure 1b). This In contrast, the expression of luxS in E. coli and finding is supported by the observation that the Edwardsiella spp. is under the control of catabolite LuxS sequences from mesophilic, commensal and repression, and it is correlated with growth and with pathogenic Epsilonproteobacteria are nested within AI-2 activity levels (Wang et al., 2005; Zhang et al., the thermophilic clades and that the ancestral state 2008; Zhang and , 2012). In comparison, the reconstruction of the LuxS sequence (corresponding number of luxS transcripts and AI-2 extracellular to node A in Figure 1b) showed higher amino acid activity levels in S. lithotrophicum and C. media- identity to the enzymes found in modern thermo- tlanticus showed highest luxS expression during the philes than those found in modern mesophiles. As mid and late exponential phases of growth, whereas all members of the Nautiliales and Nitratiruptor sp. maximum AI-2 activity levels occurred during the are thermophiles, the most parsimonious interpreta- stationary phase (Figures 2b–f). This temporal tion of the data is that the ancestral LuxS proteins uncoupling between luxS expression and AI-2 represented by nodes A and B were in thermophilic activity levels is conserved in both vent strains Epsilonproteobacteria, and that a more recent and may be indicative of a mechanism of feedback ancestor was transferred to mesophilic Epsilonpro- inhibition of luxS expression, perhaps controlled by teobacteria between nodes B and C (Figure 1b). This the concentration of AI-2. interpretation is in line with phylogenomic and 16S rRNA gene-based reconstructions of the epsilonpro- teobacterial tree that showed the Nautiliales as the LuxS function in vent Epsilonproteobacteria: QS vs first line of divergence (Zhang and Sievert, 2014). AMC Overall, our data suggest that the epsilonproteobac- The dual function of LuxS as AI-2 synthase and in terial luxS lineage originated in geothermal environ- recycling methionine via the homocysteine precur- ments. However, the direction of the luxS gene flow sor in the AMC cannot be ignored when investigat- among mesophilic Epsilonproteobacteria (for example, ing its role in new species. However, in geothermal from environmental strains to commensals/pathogens environments, where reduced is not limiting, of warm-blooded or vice versa) was less the de novo synthesis of methionine is thought to be defined and remains unresolved (Figure 1b). energetically inexpensive; hence, the function of Our phylogenetic analysis revealed two events of LuxS in recycling methionine may not be as critical luxS horizontal gene transfer within the Epsilonpro- as in other (Winzer et al., 2003). This teobacteria. H. pylori appears to have acquired the consideration suggests that luxS expression in vent gene from Enterococcus faecium, a Gram-positive Epsilonproteobacteria may be more directly bacterium that, like H. pylori, inhabits the human involved in AI-2 production rather than in recycling

The ISME Journal Quorum sensing in deep-sea vent Epsilonproteobacteria IPe´rez-Rodrı´guez et al 1230 98 Nautilia sp. strain MT3 90 Nautilia sp. strain MT4 Nautilia profundicola High T biofilm clone B12 High T biofilm clone B7 High T biofilm clone B2 High T biofilm clone A11 High T biofilm clone 10 High T biofilm clone A4 High T biofilm clone A2 High T biofilm clone A1 C. mediatlanticus cluster High T biofilm clone B1 High T biofilm clone A5 High T biofilm clone B11 High T biofilm clone B8 100 92 Nautilia nitratireducens Caminibacter sp.strain TB1 Caminibacter mediatlanticus High T biofilm clone C10 Nitratiruptor sp. strain SB155-2

68 Wolinella succinogenes Sulfurospirillum deleyianum Sulfurimonas denitrificans 88 72 Campylobacter jejuni Nitratifractor salsuginis 100 Arcobacter nitrofigilis Sulfurimonas autotrophica 100 Sulfurovum sp. strain NBC37-1 Sulfurovum lithotrophicum 56 S. lithotrophicum cluster Low T biofilm clone A6 90 Low T biofilm clone B8 Low T biofilm clone C12 Low T biofilm clone A10 50 62 Low T biofilm clone D11 58Low T biofilm clone D5 78 Low T biofilm clone A1 80 60 100 Low T biofilm clone B5 Low T biofilm clone A11 Low T biofilm clone B9 Low T biofilm clone A2 Cluster 1 Low T biofilm clone A3 98 76 Low T biofilm clone A4

0.1 Low T biofilm clone A5 100 Low T biofilm clone D3 Low T biofilm clone D8 76 Low T biofilm clone D9 Low T biofilm clone D4 Figure 3 Maximum likelihood phylogenetic tree inferred from the amino acid sequence deduced from a fragment of the luxS gene transcripts obtained from the high and low T biofilm communities collected from deep-sea hydrothermal vents on the East Pacific Rise. The tree shows the position of the biofilm-derived LuxS sequences relative to that of pure cultures of Epsilonproteobacteria. The model organisms, Caminibacter mediatlanticus and Sulfurovum lithotrophicum, are shown in bold face. Percentages 450% of 100 bootstrap resampling that support each topological element are shown at the branch nodes. Bar, 10% estimated substitutions.

methionine. Moreover, the ability of H. pylori to previous studies showed that the precursor of synthesize cysteine under an incomplete AMC via AI-2, DPD, spontaneously cyclizes into different the reverse transsulfuration pathway further sug- isomers, including S-THMF borate with AI-2 activ- gests the importance of LuxS in AI-2 production in ity in V. harveyi, R-THMF with AI-2 activity in Epsilonproteobacteria (Doherty et al., 2010; Shen S. typhimurium and the less active metabolic et al., 2010). by-product, MHF (Feather, 1981; Nedvidek et al., Although we have not identified the specific 1992). In particular, MHF has been shown to exhibit furanone derivative responsible for the signaling at least a 10-fold reduction in activity compared activity that we measured in our experiments, with S-THMF borate when tested as a QS-inducing

The ISME Journal Quorum sensing in deep-sea vent Epsilonproteobacteria IPe´rez-Rodrı´guez et al 1231 molecule in V. harveyi reporter strains (Feather, to a QS response in V. harveyi, the detection of 1981; Nedvidek et al., 1992; Schauder et al., 2001; epsilonproteobacterial luxS transcripts in vent Winzer et al., 2003). The bioluminescence induced microbial biofilms implies that AI-2 signals are by S. lithotrophicum and C. mediatlanticus was very produced in situ. It is reasonable to hypothesize similar (2.5- and 2.2-fold lower, respectively) to that that, similar to what has been reported in induced by the CFSs of the wild-type V. harveyi H. pylori and C. jejuni, AI-2 activity in vent strain BB120, suggesting that the CFSs of the vent Epsilonproteobacteria might be involved in strains contained an active form of AI-2-like mole- controlling the genes involved in flagellar motility, cule with a much higher activity than MHF. adhesion and other functions associated with the Furthermore, as bioluminescence induction by formation of biofilms. S. lithotrophicum and C. mediatlanticus was measured using similar concentration of V. harveyi cells in all the assays (Supplementary Table S2), Concluding remarks our results indicate that the gradual increase in Epsilonproteobacteria are a versatile group of bioluminescence induction during growth (Figures bacteria that include chemolithoautotrophic 2a and d) reflected the increasing production of thermophiles and mesophiles inhabiting marine AI-2-like molecules in the CFS of the two strains geothermal environments, symbionts of inverte- (Figures 2c and f). We argue that the significant brates, commensals of and human patho- correlation between AI-2 activity levels and cell gens (Dubilier et al., 2008; Ruehland and Dubilier, density in our model organisms could, in principle, 2010). Identifying the genes encoded by the core—or provide a measure of population density. ancestral—genome shared by Epsilonproteobacteria As no AI-2 receptors have yet been identified in may reveal how these microorganisms evolved to Epsilonproteobacteria, the complete luxS/AI-2 QS colonize very diverse habitats. The luxS gene is part circuit remains unclear. Two main classes of AI-2 of the epsilonproteobacterial core genome and repre- receptors have been characterized: the LuxP family sents an evolutionary link that connects vent thermo- receptors, found in Vibrio spp. (Chen et al., 2002), philes to human pathogens. Why is luxS/AI-2, rather and the LrsB family, found in S. typhimurium and than other QS mechanisms, conserved in Epsilon- other enteric bacteria (Miller et al., 2004). In ? These bacteria live in environments, addition, the AI-2-binding capability has been such as the deep-sea vents, the rumen of cows and demonstrated in RbsB-like receptors of the ribose the human , characterized by harsh physico- binding protein family (Armbruster et al., 2011). chemical conditions. DPD, the precursor of AI-2, is However, no homologs to these receptors have been very stable over a broad pH range, whereas other QS found in Epsilonproteobacteria. Hence, other recep- signaling molecules, such as acylhomoserine lac- tors must exist, as responses to AI-2 have been tones and autoinducing peptides, appear to have reported in bacteria lacking LuxP, LsrB and RbsB inherent susceptibility to hydrolytic degradation receptors. For instance, the established role of luxS/ (Globisch et al., 2012). In line with this hypothesis, AI-2 in cell–cell signaling in H. pylori in the absence a study where the vent of LuxP, LsrB or RbsB homologs suggests the and were existence of yet unidentified receptors (Doherty found to produce AI-2 via a luxS-independent biotic/ et al., 2010). This is further supported by the small abiotic reaction pathway also evidenced the stability sequence similarity between LuxP, LrsB and RbsB of AI-2 at elevated temperature (Nichols et al., 2009). (Pereira et al., 2009) that likely evolved indepen- Hence, it is possible that the chemical stability of dently while converging toward the same function. DPD has played a role in the selection of AI-2 as a chemical signal in Epsilonproteobacteria. Although it is known that luxS/AI-2 regulates Vent Epsilonproteobacteria express the luxS gene functions associated with biofilm formation in in situ during the formation of biofilms pathogenic Epsilonproteobacteria (Eaton et al., We demonstrated that the luxS gene was expressed 1996; O’Toole et al., 2000; Ottemann and in situ in two biofilms from active vents character- Lowenthal, 2002; Blaser and Atherton, 2004; Rader ized by different thermal regimes. The luxS gene et al., 2007; Plummer et al., 2012), its involvement transcripts that we identified in these biofilms in attachment and colonization of substrates in reflected the distribution of specific taxa of Epsilon- geothermal environments remains unknown. Future proteobacteria along the thermal gradient. Tran- efforts leading to the development of a genetic scripts related to Sulfurovum and Sulfurimonas system in vent Epsilonproteobacteria will provide spp., two sulfur-oxidizing mesophiles, were detected the necessary tools to further understand the luxS/ in the low T biofilm, whereas transcripts related to AI-2 QS system in geothermal environments. Caminibacter and Nautilia spp., two hydrogen- oxidizing thermophiles, were identified in the high T biofilm (Figure 3). In light of our finding that in Conflict of Interest S. lithotrophicum and C. mediatlanticus the expression the luxS gene is correlated to AI-2-like activity and The authors declare no conflict of interest.

The ISME Journal Quorum sensing in deep-sea vent Epsilonproteobacteria IPe´rez-Rodrı´guez et al 1232 Acknowledgements Doherty NC, Shen F, Halliday NM, Barrett DA, Hardie KR, Winzer K et al. (2010). In Helicobacter pylori, LuxS is We express our appreciation to Dr Michael Chikindas and a key enzyme in cysteine provision through a reverse Dr Bonnie Bassler for making reporter strains and transsulfuration pathway. J Bacteriol 192: 1184–1192. laboratory equipment for the AI-2 assays available to us. Dubilier N, Bergin C, Lott C. (2008). Symbiotic diversity in We thank Dr Stefan Sievert for inviting us on cruise AT marine animals: the art of harnessing . 15-28 and Dr Donato Giovannelli for his assistance with Nat Rev Microbiol 6: 725–740. phylogenetic analyses. We also thank the crew of R/V Eaton KA, Suerbaum S, Josenhans C, Krakowka S. (1996). Atlantis and the crew and pilots of the DSV Alvin for their Colonization of gnotobiotic piglets by Helicobacter skilled operations at sea. This work was supported by NSF pylori deficient in two flagellin genes. Infect Immun Grants MCB 04-56676, MCB 08-43678 and OCE 11-36451 64: 2445–2448. to CV and by a PEO scholar award and a Carnegie Edgar RC. (2004). MUSCLE: multiple sequence alignment Postdoctoral Fellowship to IP-R. This paper is C-DEBI with high accuracy and high throughput. Nucleic contribution 232. Acids Res 32: 1792–1797. Elvers KT, Park SF. (2002). Quorum sensing in Campylo- bacter jejuni: detection of a luxS encoded signalling References molecule. Microbiology 148: 1475–1481. Feather MS. (1981). Amine-assisted sugar dehydration Alain K, Zbinden M, Le Bris N, Lesongeur F, Querellou J, reactions. Prog Food Nutr Sci 5: 37–45. Gaill F et al. (2004). Early steps in microbial Forsyth MH, Cover TL. (2000). Intercellular communica- colonization processes at deep-sea hydrothermal tion in Helicobacter pylori: luxS is essential for the vents. Environ Microbiol 6: 227–241. production of an extracellular signaling molecule. Armbruster CE, Pang B, Murrah K, Juneau RA, Perez AC, Infect Immun 68: 3193–3199. Weimer KED et al. (2011). RbsB (NTHI_0632) mediates Galtier N, Gouy M, Gautier C. (1996). SEAVIEW and quorum signal uptake in nontypeable PHYLO_WIN: two graphic tools for sequence align- influenzae strain 86-028NP. Mol Microbiol 82: ment and molecular phylogeny. Comput Appl Biosci 836–850. 12: 543–548. Bassler BL, Wright M, Showalter RE, Silverman MR. Globisch D, Lowery CA, McCague KC, Janda K. (2012). (1993). Intercellular signalling in Vibrio harveyi: Uncharacterized 4,5-dihydroxy-2,3-pentanedione (DPD) sequence and function of genes regulating expression molecules revealed through NMR spectroscopy: of luminescence. Mol Microbiol 9: 773–786. implications for a greater signaling diversity in Bassler BL, Wright M, Silverman MR. (1994). Multiple bacterial species. Angew Chem Int Ed 51: 4204–4208. signalling systems controlling expression of lumines- Guindon S, Gascuel O. (2003). A simple, fast, and accurate cence in Vibrio harveyi: sequence and function of algorithm to estimate large phylogenies by maximum genes encoding a second sensory pathway. Mol likelihood. Syst Biol 52: 696–704. Microbiol 13: 273–286. Huber JA, Welch DB, Morrison HG, Huse SM, Neal PR, Beeston AL, Surette MG. (2002). pfs-dependent regulation Butterfield DA et al. (2007). Microbial population of autoinducer 2 production in Salmonella structures in the deep marine . Science 318: enterica serovar Typhimurium. J Bacteriol 184: 97–100. 3450–3456. Inagaki F, Takai K, Nealson KH, Horikoshi K. (2004). Blaser MJ, Atherton JC. (2004). Helicobacter pylori Sulfurovum lithotrophicum gen. nov., sp. nov., a novel persistence: biology and disease. J Clin Invest 113: sulfur-oxidizing chemolithoautotroph within the 321–333. epsilon-Proteobacteria isolated from Okinawa Trough Campbell BJ, Engel AS, Porter ML, Takai K. (2006). hydrothermal sediments. Int J Syst Evol Microbiol 54: The versatile epsilon-proteobacteria: key 1477–1482. players in sulphidic habitats. Nat Rev Microbiol 4: Joshua GW, Guthrie- C, Karlyshev AV, Wren BW. 458–468. (2006). Biofilm formation in Campylobacter jejuni. Castresana J. (2000). Selection of conserved blocks from Microbiology 152: 387–396. multiple alignments for their use in phylogenetic Joyce EA, Bassler BL, Wright A. (2000). Evidence for a analysis. Mol Biol Evol 17: 540–552. signaling system in Helicobacter pylori: detection of a Chen X, Schauder S, Potier N, Van Dorsselaer A, Pelczer I, luxS-Encoded autoinducer. JBacteriol182: 3638–3643. Bassler BL et al. (2002). Structural identification of a Kaper JB, Sperandio V, Mellies JL, Nguyen W, Shin S. bacterial quorum-sensing signal containing boron. (1999). Quorum sensing controls expression of the type Nature 415: 545–549. III secretion gene transcription and protein secretion in Chini V, Foka A, Dimitracopoulos G, Spiliopoulou I. enterohemorrhagic and enteropathogenic Escherichia (2007). Absolute and relative real-time PCR in the coli. Proc Natl Acad Sci USA 96: 15196–15201. quantification of tst gene expression among methicil- Keller L, Surette MG. (2006). Communication in bacteria: lin-resistant Staphylococcus aureus: evaluation by an ecological and evolutionary perspective. Nat Rev two mathematical models. Lett Appl Microbiol 45: Microbiol 4: 249–258. 479–484. Lerat E, Moran NA. (2004). The evolutionary history of Cuebas M, Villafane A, McBride M, Yee N, Bini E. (2011). quorum-sensing systems in bacteria. Mol Biol Evol 21: Arsenate reduction and expression of multiple 903–913. chromosomal ars operons in Geobacillus kaustophilus Longnecker K, Reysenbach AL. (2001). Expansion of A1. Microbiology 157: 2004–2011. the geographic distribution of a novel lineage of Darriba D, Taboada GL, Doallo R, Posada D. (2011). epsilon-Proteobacteria to a site on ProtTest 3: fast selection of best-fit models of protein the Southern East Pacific Rise. FEMS Microbiol Ecol evolution. Bioinformatics 27: 1164–1165. 35: 287–293.

The ISME Journal Quorum sensing in deep-sea vent Epsilonproteobacteria IPe´rez-Rodrı´guez et al 1233 McNab R, Ford SK, El-Sabaeny A, Barbieri B, Cook GS, Ronquist F, Huelsenbeck JP. (2003). MrBayes 3: Bayesian Lamont RJ. (2003). LuxS-based signaling in Strepto- phylogenetic inference under mixed models. Bioinfor- coccus gordonii: autoinducer 2 controls carbohydrate matics 19: 1572–1574. metabolism and biofilm formation with Porphyromo- Ruehland C, Dubilier N. (2010). Gamma- and epsilonpro- nas gingivalis. J Bacteriol 185: 274–284. teobacterial ectosymbionts of a shallow- marine Miller ST, Xavier KB, Campagna SR, Taga ME, worm are related to deep-sea hydrothermal vent Semmelhack MF, Bassler BL et al. (2004). Salmonella ectosymbionts. Environ Microbiol 12: 2312–2326. typhimurium recognizes a chemically distinct form of Schauder S, Shokat K, Surette MG, Bassler BL. (2001). the bacterial quorum-sensing signal Al-2. Mol Cell 15: The LuxS family of bacterial autoinducers: biosynthesis 677–687. of a novel quorum-sensing signal molecule. Mol Moussard H, Corre E, Cambon-Bonavita MA, Fouquet Y, Microbiol 41: 463–476. Jeanthon C. (2006). Novel uncultured Epsilonproteo- Shen F, Hobley L, Doherty N, Loh JT, Cover TL, Sockett RE bacteria dominate filamentous sulphur mat from et al. (2010). In Helicobacter pylori auto-inducer-2, but the 131N hydrothermal vent field, East Pacific Rise. not LuxS/MccAB catalysed reverse transsulphuration, FEMS Microbiol Ecol 58: 449–463. regulates motility through modulation of flagellar gene Nakagawa S, Takai K, Inagaki F, Hirayama H, Nunoura T, transcription. BMC Microbiol 10: 210. Horikoshi K et al. (2005). Distribution, phylogenetic Shimizu T, Ohtani K, Hayashi H. (2002). The luxS gene is diversity and physiological characteristics of epsilon- involved in cell-cell signalling for toxin production in Proteobacteria in a deep-sea hydrothermal field. . Mol Microbiol 44: 171–179. Environ Microbiol 7: 1619–1632. Sievert SM, Vetriani C. (2012). Chemoautotrophy at Nakagawa S, Takaki Y, Shimamura S, Reysenbach AL, deep-sea vents: past, present, and future. Takai K, Horikoshi K. (2007). Deep-sea vent 25: 218–233. e-proteobacterial genomes provide insights into emer- Stark RM, Gerwig GJ, Pitman RS, Potts LF, Williams NA, gence of pathogens. Proc Natl Acad Sci USA 104: Greenman J et al. (1999). Biofilm formation by 12146–12150. Helicobacter pylori. Lett Appl Microbiol 28: 121–126. Nedvidek W, Ledl F, Fischer P. (1992). Detection of Stetter KO, Ko¨nig H, Stackebrandt E. (1983). Pyrodictium, 5-hydroxymethyl-2-methyl-3(2H)-furanone and of a new of submarine disc-shaped sulfur reducing a-dicarbonyl compounds in reaction mixtures archaebacteria growing optimally at 1051C. Syst Appl of hexoses and pentoses with different amines. Microbiol 4: 535–551. Z Lebensm Unters Forsch 194: 222–228. Stevenson B, Babb K. (2002). LuxS-mediated quorum Nichols JD, Johnson MR, Chou CJ, Kelly RM. (2009). sensing in Borrelia burgdorferi, the Lyme disease Temperature, not LuxS, mediates AI-2 formation in spirochete. Infect Immun 70: 4099–4105. hydrothermal habitats. FEMS Microbiol Ecol 68: 173–181. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. O’Toole PW, Lane MC, Porwollik S. (2000). Helicobacter (2013). MEGA6: molecular evolutionary genetics pylori motility. Microbes Infect 2: 1207–1214. analysis version 6.0. MolBiol Evol 30: 2725–2729. Ottemann KM, Lowenthal AC. (2002). Helicobacter pylori Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, uses motility for initial colonization and to attain Higgins DG. (1997). The CLUSTAL_X windows robust infection. Infect Immun 70: 1984–1990. interface: flexible strategies for multiple sequence Pereira CS, de Regt AK, Brito PH, Miller ST, Xavier KB. alignment aided by quality analysis tools. Nucleic (2009). Identification of functional LsrB-like Acids Res 25: 4876–4882. autoinducer-2 receptors. J Bacteriol 191: 6975–6987. Turovskiy Y, Chikindas ML. (2006). Autoinducer Perez-Rodriguez I, Ricci J, Voordeckers JW, Starovoytov V, 2 bioassay is a qualitative, not quantitative method Vetriani C. (2010). Nautilia nitratireducens sp nov., influenced by glucose. J Microbiol Methods 66: a thermophilic, anaerobic, chemosynthetic, nitrate- 497–503. ammonifying bacterium isolated from a deep-sea Turovskiy Y, Kashtanov D, Paskhover B, Chikindas ML. hydrothermal vent. Int J Syst Evol Microbiol 60: (2007). Quorum sensing: fact, fiction, and everything 1182–1186. in between. Adv Appl Microbiol 62: 191–234. Plummer P, Sahin O, Burrough E, Sippy R, Mou K, Vendeville A, Winzer K, Heurlier K, Tang CM, Hardie KR. Rabenold J et al. (2012). Critical role of LuxS in the (2005). Making ‘sense’ of metabolism: autoinducer-2, virulence of Campylobacter jejuni in a guinea pig LuxS and . Nat Rev Microbiol 3: model of abortion. Infect Immun 80: 585–593. 383–396. Polz MF, Alm EJ, Hanage WP. (2013). Horizontal gene Voordeckers J, Starovoytov V, Vetriani C. (2005). Camini- transfer and the evolution of bacterial and archaeal bacter mediatlanticus sp. nov., a thermophilic, population structure. Trends Genet 29: 170–175. chemolithoautotrophic, nitrate ammonifying Rader BA, Campagna SR, Semmelhack MF, Bassler BL, bacterium isolated from a deep-sea hydrothermal vent Guillemin K. (2007). The quorum-sensing molecule on the Mid-Atlantic Ridge. Int J Syst Evol Microbiol 55: autoinducer 2 regulates motility and flagellar morphogen- 773–779. esis in Helicobacter pylori. J Bacteriol 189: 6109–6117. Voordeckers JW, Do M, Hu¨ gler M, Ko V, Sievert SM, Rader BA, Wreden C, Hicks KG, Sweeney EG, Ottemann KM, Vetriani C. (2008). Culture dependent and indepen- Guillemin K. (2011). Helicobacter pylori perceives the dent analyses of 16S rRNA and ATP citrate lyase quorum-sensing molecule AI-2 as a chemorepellent genes: a comparison of microbial communities from via the chemoreceptor TlpB. Microbiology 157: different black smoker chimneys on the Mid-Atlantic 2445–2455. Ridge. 12: 627–640. Rhee JH, Kim SY, Lee SE, Kim YR, Kim CM, Ryu PY et al. Wang L, Li J, March JC, Valdes JJ, Bentley WE. (2005). (2003). Regulation of virulence by luxS-Dependent gene regulation in Escherichia coli the LuxS quorum-sensing system. Mol Microbiol 48: K-12 revealed by genomic expression profiling. 1647–1664. J Bacteriol 187: 8350–8360.

The ISME Journal Quorum sensing in deep-sea vent Epsilonproteobacteria IPe´rez-Rodrı´guez et al 1234 Winzer K, Hardie KR, Williams P. (2003). LuxS and Zhang M, Sun K, Sun L. (2008). Regulation of autoinducer autoinducer-2: their contribution to quorum sensing 2 production and luxS expression in a pathogenic and metabolism in bacteria. Adv Appl Microbiol 53: Edwardsiella tarda strain. Microbiology 154: 291–396. 2060–2069. Winzer K, Sun Y, Green A, Delory M, Blackley D, Hardie KR Zhang M, Sun L. (2012). Edwardsiella ictaluri LuxS: et al. (2002). Role of luxS in activity, expression, and involvement in patho- cell to cell signaling and bacteremic infection. Infect genicity. Pol J Microbiol 61: 263–271. Immun 70: 2245–2248. Zhang Y, Sievert SM. (2014). Pan-genome analyses Wright A, Joyce EA, Bassler BL. (2000). Evidence for a identify lineage- and niche-specific markers of signaling system in Helicobacter pylori: detection of a evolution and adaptation in Epsilonproteobacteria. luxS-Encoded autoinducer. J Bacteriol 182: 3638–3643. Front Microbiol 5: 110.

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