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LuxS distribution and AI-2 activity of Campylobacter spp.

2011; Oxford University Press; Volume: 112; Issue: 3 Linguagem: Inglês

10.1111/j.1365-2672.2011.05221.x

1365-2672

Greta Gölz, Linda Adler, Stephan Huehn, Thomas Alter,

Listeria monocytogenes in Food Safety

Journal of Applied MicrobiologyVolume 112, Issue 3 p. 571-578 ORIGINAL ARTICLEFree Access LuxS distribution and AI-2 activity of Campylobacter spp. G. Gölz, G. Gölz Institute of Food Hygiene, Free University Berlin, Berlin, GermanySearch for more papers by this authorL. Adler, L. Adler Department of Biology, Chemistry, and Pharmacy, Free University Berlin, Berlin, GermanySearch for more papers by this authorS. Huehn, S. Huehn Institute of Food Hygiene, Free University Berlin, Berlin, GermanySearch for more papers by this authorT. Alter, T. Alter Institute of Food Hygiene, Free University Berlin, Berlin, GermanySearch for more papers by this author G. Gölz, G. Gölz Institute of Food Hygiene, Free University Berlin, Berlin, GermanySearch for more papers by this authorL. Adler, L. Adler Department of Biology, Chemistry, and Pharmacy, Free University Berlin, Berlin, GermanySearch for more papers by this authorS. Huehn, S. Huehn Institute of Food Hygiene, Free University Berlin, Berlin, GermanySearch for more papers by this authorT. Alter, T. Alter Institute of Food Hygiene, Free University Berlin, Berlin, GermanySearch for more papers by this author First published: 21 December 2011 https://doi.org/10.1111/j.1365-2672.2011.05221.xCitations: 9 Greta Gölz, Free University Berlin, Institute of Food Hygiene, Königsweg 69, 14163 Berlin, Germany. E-mail: goelz.greta@vetmed.fu-berlin.de AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Aims: This study investigates the distribution of LuxS within Campylobacter (Camp.) species and Autoinducer (AI)-2 activity of Camp. jejuni NCTC 11168 in food matrices. Methods and Results: LuxS (S-ribosylhomocysteinase) sequences of different Campylobacter spp. were compared, and AI-2 activity was measured with an AI-2 reporter assay. Highest LuxS homologies were shared by Camp. jejuni, Camp. coli and Camp. upsaliensis, and their LuxS sequences had more similarities to the analysed Arcobacter and Vibrio harveyi strains than to all other analysed Campylobacter species. Of 15 analysed species only Camp. lari, Camp. peloridis and Camp. insulaenigrae did not produce AI-2 molecules. Cultivation of Camp. jejuni NCTC 11168 in chicken juice reduced AI-2 activity, and this reduction is not because of lower luxS expression or functionality. Conclusion: Not all Campylobacter species encode luxS. Food matrices can reduce AI-2 activity in a LuxS-independent manner. Significance and Impact of the Study: Besides, Camp. lari, Camp. peloridis and Camp. insulaenigrae do not show AI-2 activity. Further investigations should clarify the function of AI-2 in Campylobacter spp. and how species lacking luxS could overcome this alteration. Furthermore, the impact of food matrices on these functions needs to be determined as we could show that chicken juice reduced AI-2 activity. Introduction Despite the low tenacity of Campylobacter (Camp.) spp., the thermotolerant species Camp. jejuni and Camp. coli belong to the most prevalent bacterial foodborne pathogens in the industrialized world, causing gastroenteritis in humans (Newell et al. 2010). The major source for human Campylobacter infections is consumption or handling of contaminated food products. At cold temperature, Camp. jejuni survived for longer times in chicken juice than in culture medium (Birk et al. 2004) and one of four genes that were up-regulated after cultivation in chicken juice was luxS (Ligowska et al. 2010). The AI-2 synthase LuxS (S-ribosylhomocysteinase) catalyses the cleavage of S-ribosylhomocysteine (SRH) to homocysteine and 4,5-dihydroxyl-2,3-pentanedione (DPD), which is the precursor of the quorum-sensing signalling molecule Autoinducer (AI)-2 (He et al. 2008). It is also a part of the methionine cycle as it has an important function in the S-adenosylhomocysteine (SAH) recycling to homocysteine (Winzer et al. 2002), which can be converted to methionine. Methionine participates in protein synthesis or is condensed by the S-adenosylmethionine synthetase to produce the ubiquitous nucleotide S-adenosyl-l-methionine (SAM) (LaMonte and Hughes 2006). By transferring the methyl residue to another product, SAM is converted to SAH. The presence of the luxS has been reported in several subgroups of the bacterial kingdom, being widely found in Bacteroidetes, Actinobacteria, and β-, γ- and ε-Proteobacteria, but not in Archaea or Eukarya (Winzer et al. 2003). Relatively less is known about the mode and role of luxS expression or AI-2 production in Campylobacter spp. The AI-2-synthase LuxS and AI-2 production in Campylobacter were described first in Camp. jejuni NCTC 11168 (Elvers and Park 2002) and until now also for some other Camp. jejuni, Camp. coli (Cloak et al. 2002; Jeon et al. 2003; Quinones et al. 2009), Camp. fetus and Camp. upsaliensis strains (Tazumi et al. 2011). But LuxS was not detectable in Camp. lari strains (Tazumi et al. 2011). To our knowledge, no AI-2 receptor is described for Campylobacter spp. so far. Neither homologues for the AI-2 receptors LuxP of Vibrionales nor homologues for LsrB of Escherichia coli and Salmonella spp. could be detected in Campylobacter spp. (Rezzonico and Duffy 2008). These authors speculate that the ubiquitous ribose ABC-transporter RbsB could function as AI-2 receptor. In Helicobacter (H.) pylori, the chemoreceptor TlpB has recently been described as AI-2 receptor (Rader et al. 2011), but none of them shares high sequence homologies to any Campylobacter protein. It has been shown previously that Camp. jejuni luxS mutants have reduced motility at 37°C, decreased flaA expression (Jeon et al. 2003), lower biofilm formation (Reeser et al. 2007) and a lower viability in chicken juice (Ligowska et al. 2010), the colonization ability is reduced in chicken (Quinones et al. 2009) and such mutants are more sensitive to oxidative stress (He et al. 2008). It is not clear whether these effects are because of a lack of AI-2 or because of the disrupted methionine cycle. As luxS expression increases after cultivation of Camp. jejuni in chicken juice and AI-2 is produced by Camp. jejuni and Camp. coli cultivated in milk and chicken broth (Cloak et al. 2002), it seems likely that quorum-sensing mechanisms are active within food matrices. In this study, we wanted to analyse the LuxS distribution in Campylobacter spp. and investigate AI-2 activity in different food matrices. Material and methods Bacterial strains Campylobacter strains were cultured at 37°C in Brucella broth (BB) (BD, Heidelberg, Germany) or on Mueller-Hinton blood agar (Oxoid, Wesel, Germany) under microaerobic conditions (5% O2, 10% CO2). Vibrio (V.) harveyi BB152 and V. harveyi BB170 strains were cultured at 30°C under aerobic conditions in Autoinducer Bioassay (AB) media (Greenberg et al. 1979) containing 0·33 mol l−1 NaCl, 50 mmol l−1 MgSO4 (both Merck, Darmstadt, Germany), 0·2% casamino acid (BD), 10 mol l−1 potassium phosphate (pH 7·0), 1 mmol l−1l-arginine and 1% glycerol (all Roth, Karlsruhe, Germany) and on Luria–Bertani (LB) agar plates (Merck). All strains used in the assays are listed in Table S1. LuxS sequence analysis Cells of each strain were washed in 0·1× TE buffer, and pellets were resuspended in 5% Chelex Resin 100 (Bio-Rad, Munich, Germany). A 1-h incubation at 56°C was followed by 10 min at 95°C, and supernatants were used for amplification of luxS. PCRs were performed in a total volume of 50 μl containing 45 μl Reddy Mix (ABgene, Epsom, UK), 2 μl DNA and 2 μmol l−1 of each primer. Primers and the PCR protocol for Camp. jejuni strains are described elsewhere (Cloak et al. 2002). For Camp. coli strains, the primers CCluxSF (5′-ATATTACACATCTGGCACATC-3′) and CCluxSR (5′-GTCCTATAACTACTTCAAACAT-3′) were designed [primers are flanking the luxS coding DNA sequence (cds) amplifying a 748-bp fragment] and used with the same protocol but 55°C annealing temperature. PCR products were purified by a clean-up kit (GE Healthcare, Freiburg, Germany) and sequenced (GATC Biotech, Konstanz, Germany) with the same primers used for PCR. Sequences were trimmed and cds translated with standard genetic code by EditSeq (DNASTAR Lasergene v8, Madison, WI, USA). The LuxS phylogenetic tree was calculated with BioNumerics v6.1 (Applied Maths, Sint-Martens-Latem, Belgium) by cluster analysis with pairwise alignment fast algorithm and unweighted pair group method using arithmetic means analysis (UPGMA) with our sequence data and LuxS amino acid sequences blasted with YP_002344589.1 (Table S2) in the NCBI protein database. Differences in Camp. jejuni LuxS consensus sequence were analysed by ClustalW with MegAlign (DNASTAR Lasergene v8). Conditioned cell-free supernatant (CFS) Overnight cultures were inoculated in 5 ml BB (OD600 = 0·001), respectively, AB and incubated for 24 h or until they reached an OD600 of c. 0·1. Conditioned CFS was generated by centrifugation of bacterial cultures at 8000 g for 10 min. The supernatants were sterilized by passing through a 22-μm filter (VWR, Darmstadt, Germany) and stored at −20°C until assayed. AI-2 activity assay To detect AI-2 activity produced by Campylobacter spp., a reporter assay described previously (Bassler et al. 1997) was applied with V. harveyi BB 170 as reporter strain. Vibrio harveyi BB 152 was used as positive control and uninoculated BB, respectively, AB as negative control for AI-2 activity. To determine AI-2 activity, V. harveyi BB170 was grown overnight in AB and diluted (1 : 5000) into fresh AB containing 10% (v/v) of either Campylobacter or V. harveyi BB152-conditioned CFS or sterile medium. Vibrio harveyi BB170 reporter cultures with CFS were grown at 30°C for 4 h, and luminescence was measured for 10 s per well using a FLUOstar OPTIMA luminometer (BMG Labtech, Offenburg, Germany). n-fold luminescence induction values were calculated from relative light units (RLU) obtained with conditioned CFS vs RLU obtained with sterile medium. The analysis was performed in triplicate with conditioned CFS prepared on three different occasions. To evaluate AI-2 activity in different food matrices, conditioned CFS from Camp. jejuni NCTC 11168 and V. harveyi BB152 grown in milk (1·5 or 3·5% fat content), chicken juice (described by Birk et al. 2004) or BB (respectively AB) was generated and tested with the reporter assay as described above. As negative control, uninoculated milk, chicken juice, BB or AB were used. Influence of chicken juice on AI-2 activity Conditioned CFS from Camp. jejuni NCTC11168 grown in BB was incubated for 3 min and 3 h with chicken juice (1 : 2) and tested with the reporter assay as described above. As negative control, conditioned CFS was incubated with BB. Expression analysis of Campylobacter jejuni NCTC 11168 in chicken juice To prepare total cellular RNA, Camp. jejuni NCTC 11168 was harvested from BB and chicken juice after 24 h incubation under microaerobic conditions. Cell pellets were lysed using TRIzol reagent (Invitrogen, Karlsruhe, Germany) and treated with RNase-free DNase (DNA-free Kit, Applied Biosystems, Foster City, CA, USA). cDNA was synthesized with the SuperScriptII-Kit and Random Hexamer primers (Invitrogen) from 1 μg RNA. The expression of luxS, flaA (reduced expression in luxS mutants) and selected virulence-associated genes (cdtB, ciaB and cadF) were analysed. Prior to real-time PCR analysis (SYBR Green assays on ABI Prism 7500), all assays were optimized. Quantitative real-time PCR assays were performed in a total volume of 25 μl with QuantiTect SYBR Green PCR Mastermix (Qiagen, Hilden, Germany), 100 ng cDNA and 900 nmol l−1 of each primer for rpoA, flaA, cadF (all described by Ritz et al. 2009), cdtB (cdtBF 5′-TTTACCAAGAACAGCCACTCCA-3′; cdtBR 5′-TATCAGGCCTTGAAAGAGTTCCTAA-3′), ciaB (Malik-Kale et al. 2008) and 300 nmol l−1 of each primer for luxS (luxS1 5′-GCGTAAGCGATCAAAGCAAAA-3′; luxS2 5′-AAGAATGCATTGCGCAAGTTC-3′). Amplification protocol was an initial denaturation at 95°C (15 min) followed by 40 cycles of denaturation at 95°C (15 s), annealing at 60°C (30 s) and elongation at 72°C (60 s). The melting curve was obtained from 60 to 95°C. Relative quantification was carried out with the method (Livak and Schmittgen 2001) with rpoA as reference gene whose expression was not affected by our cultivation conditions (data not shown). Results LuxS comparisons We compared the LuxS amino acid (aa) sequence from 34 Camp. jejuni and 20 Camp. coli strains isolated from different sources (Table S1). LuxS sequences within the species Camp. jejuni as well as within Camp. coli showed high similarity (Fig. S1a). The consensus sequence was determined for all analysed sequences and used for further comparisons. Campylobacter jejuni ssp. doylei 269·97 and Camp. jejuni 414 showed the highest deviation from the Camp. jejuni LuxS consensus sequence (12·2 and 6·71%), while the others exhibited 97·6–99·4% similarity (Fig. S1a). Based on Camp. coli LuxS sequences, two clusters were calculated, but both share >95% similarity to the consensus sequence and seven of 20 sequences have no aa exchange when compared with the calculated consensus sequence. The deviations were mostly located within the last 70 aa (Fig. S1b). The LuxS sequence of the species Camp. coli and Camp. upsaliensis shares the highest homology to LuxS of Camp. jejuni (90·2–92·1%) (Fig. 1). The LuxS sequences of all three species (Camp. jejuni, Camp. coli and Camp. upsaliensis) showed more similarities to Arcobacter (A.) butzleri, A. nitrofigilis and V. harveyi (71·9–76·2%) than to other Campylobacter spp. analysed in this study (Fig. 1). The Campylobacter species Camp. curvus, Camp. fetus, Camp. rectus, Camp. showae, Camp. concisus, Camp. hominis and C. gracilis (with similarities from 68·9 to 76·8% to the Camp. jejuni sequence) belong to a second cluster. The third cluster is composed of Helicobacter hepaticus and Wolinella (W.) succinogenes with c. 70% homology to Camp. jejuni LuxS (Fig. 1). Figure 1Open in figure viewerPowerPoint Dendrogram of LuxS aa sequences from different species of Campylobacterales and Vibrio harveyi. Cluster analysis of LuxS aa sequences was performed by pairwise alignment using UPGMA, and deviations of aa as well as % homology to Campylobacter jejuni consensus sequences are shown. The consensus sequences for Campylobacteracea, Camp. jejuni and Camp. coli are shown below the dendrogram with grey background for homologues aa in all analysed sequences and bold letters for the LuxS catalytically active site as well as for aa G86, essential for activity. LuxS sequence length differs among the analysed Campylobacterales. LuxS protein from Camp. jejuni, Camp. coli, Camp. upsaliensis and Camp. hominis is composed of 164 aa, whereas W. succinogenes sequence is shorter. LuxS of H. hepaticus, Camp. fetus ssp. fetus, Camp. gracilis, A. butzleri and A. nitrofigilis, Camp. concisus, Camp. rectus and Camp. curvus as well as Camp. showae and V. harveyi are composed of longer sequences (Table S2). Overall 80 of 164 aa were conserved in all analysed species (Fig. 1) including the catalytically active site (Fig. S1 boxI) as well as glycine at position 86 (Fig. S1 boxII). With specific primers for Camp. jejuni and Camp. coli, no amplification of luxS PCR products from Camp. helveticus, Camp. hyoilei, Camp. sputorum, Camp. hyointestinales, Camp. mucosalis, Camp. peloridis, Camp. sputorum or Camp. insulaenigrae (for which we found no published LuxS sequence in the NCBI protein database) were possible. Although LuxS sequence differs between the analysed Campylobacterales species, they are highly conserved among strains of the species Camp. jejuni and Camp. coli, respectively. AI-2 activity For AI-2 detection, the V. harveyi reporter assay described by Bassler et al. (1997) was used and tested on reference strains of different Campylobacter spp. Only CFS containing AI-2 molecules can induce bioluminescence in the V. harveyi BB170 reporter strain. CFS from Camp. coli, Camp. curvus, Camp. fetus ssp. fetus, Camp. gracilis, Camp. helveticus, Camp. hyoilei, Camp. hyointestinales, Camp. jejuni, Camp. mucosalis, Camp. rectus, Camp. sputorum and Camp. upsaliensis could induce bioluminescence in the reporter strain V. harveyi BB170 (Fig. 2a). However, Camp. insulaenigrae, Camp. peloridis as well as Camp. lari did not induce bioluminescence in this reporter assay (Fig. 2a). Figure 2Open in figure viewerPowerPoint Production of Autoinducer-2 (AI-2) signalling molecules by different Campylobacter spp. AI-2 production of cell-free supernatant from different Campylobacter species (listed in Table S1) were determined with an AI-2 reporter assay. AI-2 signalling molecule production is shown as mean + SD of n-fold induction of luminescence. AI-2 activity in food matrices As Campylobacter infections are often associated with contaminated food, we analysed the AI-2 activity of Camp. jejuni NCTC 11168 and V. harveyi BB152 (as control) after 24 h incubation in milk (with different fat content) and chicken juice. The incubation of the reporter strain V. harveyi BB170 with CFS from food matrices did not alter their plate count compared to CFS from BB during the reporter assay (data not shown). AI-2 activity was 10-fold reduced after incubation in food matrices by both organisms (Fig. 3). Figure 3Open in figure viewerPowerPoint Autoinducer-2 (AI-2) activity by Campylobacter jejuni NCTC 11168 and Vibrio harveyi BB152 cultivated in different food matrices. Cell-free supernatant (CFS) from Camp. jejuni NCTC 11168 and V. harveyi BB152 were harvested after 24 h incubation in milk or chicken juice, and AI-2 production was determined. AI-2 signalling molecule production is shown as mean + SD of n-fold induction of luminescence. CFS from V. harveyi BB152 and Camp. jejuni NCTC 11168 cultured in AB medium, respectively, BB served as positive control. () V. harveyi BB152 and () Camp. jejuni NCTC 11168. To investigate whether AI-2 activity was decreased because of reduced luxS expression, we analysed the gene expression of luxS and some virulence-associated genes by RT-qPCR. Expression levels of luxS (1·13 ± 0·25) and ciaB (1·62 ± 0·3) were not altered, whereas expression of cadF (3·35 ± 0·79) was up-regulated and flaA (0·34 ± 0·06) down-regulated (Fig. 4). CdtB (0·48 ± 0·05) expression seems to be down-regulated albeit the reduced expression level is close to a value where biological relevance is questionable. Figure 4Open in figure viewerPowerPoint Relative gene expression of selected Campylobacter jejuni NCTC 11168 genes in chicken juice. Expression of selected genes of Camp. jejuni NCTC 11168 after incubation in BB vs chicken juice was determined by real-time PCR, and the expression level relatively quantified compared to rpoA gene expression level by the method. As decreased AI-2 activity after cultivation in chicken juice was not because of reduced luxS expression, it could be postulated that components of the chicken juice interact directly with AI-2 molecules or with the AI-2 receptor of the reporter strain, respectively. Therefore, only the CFS from Camp. jejuni NCTC 11168 grown in BB was incubated with chicken juice. After 3 min incubation of CFS with chicken juice, the AI-2 activity was already reduced compared to medium control and did not change over time of 3 h incubation (Fig. 5). Figure 5Open in figure viewerPowerPoint Influence of chicken juice on AI-2 activity in cell-free supernatant (CFS) from Campylobacter jejuni NCTC 11168. CFS of Camp. jejuni NCTC 11168 grown in BB was incubated with chicken juice or BB for 3 min, respectively, for 3 h (1 : 2) and AI-2 activity was determined. AI-2 activity is shown as n-fold induction of luminescence. () cfs + BB and () cfs + chicken juice. Discussion LuxS sequences and AI-2 activity LuxS aa sequences within the species Camp. jejuni, respectively, Camp. coli showed high conservation among strains of different sources and countries. The homology within analysed Camp. jejuni strains ranges from 87·8 to 99·39% and within Camp. coli strains from 95·12 to 100%. There is no indication of a correlation between LuxS sequence and source of strains. Although LuxS sequences differ between analysed species, conserved regions were found. The aa G at position 86 (essential for functionality) as well as the catalytically active site is conserved in all analysed strains (De Keersmaecker et al. 2006; Plummer et al. 2007). Highest LuxS homologies were shared among Camp. jejuni, Camp. coli and Camp. upsaliensis. This cluster had more sequence similarities to analysed Arcobacter spp. and V. harveyi than to all other analysed Campylobacter species. Cell-free supernatant from analysed Camp. coli, Camp. curvus, Camp. fetus ssp. fetus, Camp. gracilis, Camp. jejuni, Camp. rectus and Camp. upsaliensis strains, known to encode luxS, showed induction of bioluminescence in the reporter strain V. harveyi BB170. Also CFS from Camp. helveticus, Camp. hyoilei, Camp. hyointestinales, Camp. mucosalis and Camp. sputorum, for which to our knowledge no sequence of luxS is published to date, could induce AI-2-dependent bioluminescence in the reporter assay but for these strains, no luxS PCR products were obtained with primers used for luxS detection in Camp. jejuni or Camp. coli. This could be due to variation of the luxS gene sequence or different location of luxS within the genome (the used primers are partially flanking the cds of luxS). Only Camp. lari, Camp. peloridis and Camp. insulaenigrae showed no AI-2 activity at all. For Camp. insulaenigrae and Camp. peloridis, no entry in the NCBI database was found. In the whole genome sequenced Camp. lari strain RM2100, no luxS homologous gene or protein could be identified (Miller et al. 2008). Campylobacter peloridis belongs to the former Camp. lari -like group (Debruyne et al. 2009) and Camp. insulaenigrae is closely related to Camp. lari within the thermotolerant taxa (Miller et al. 2008). As the common niches of these three are distinct to other Campylobacter species, for example, marine mammals, seawater, shellfish and shorebirds, it could be possible that they all have lost or never took up the luxS gene according to different requirements in these habitats. However, as the conversion of SAH to homocystein by Pfs and LuxS is a commonly used mechanism by widespread subgroups of the bacterial kingdom (Rezzonico and Duffy 2008), it is surprising that some Campylobacter species lack LuxS. The conversion of SAH to homocystein could also be catalysed in a one-step reaction by SAH hydrolase (Winzer et al. 2002), but we were not able to find a similar protein in the genome sequenced Camp. lari RM2100 or any other Campylobacter species in the NCBI databases. As genes coding for LuxS and SAH hydrolase are missing in Camp. lari, this species seems to be unable to recycle methionine from SAH. Also important genes for methionine biosynthesis (metABEF) are missing in Camp. lari RM2100. Therefore, Camp. lari has to take up methionine as free aa or obtain it via protein degradation (Miller et al. 2008). In all analysed genome sequenced Campylobacter strains, even for Camp. lari, we found homologous proteins of the Pfs from Camp. jejuni NCTC 11168 (blastp with YP_002343577.1), which converts the toxic SAH to SRH (data not shown). As the Camp. lari genome contains genes for SAM-synthase (metK) and Pfs, they could produce SAM and convert it to SRH. The biological function of AI-2 molecules for Campylobacter spp. is still not clear. For H. pylori it has been shown that AI-2 regulates flagellar gene expression (Shen et al. 2010), acts as chemorepellent and thereby might coordinate its distribution and motility within the stomach (Rader et al. 2011). Also luxS mutants of Camp. jejuni showed changed chemotactic behaviour towards organic and amino acids (Quinones et al. 2009). Although the overall sequence homology from TlpB to any Campylobacter proteins is <40%, the homology within the methyl-accepting chemotaxis domain is much higher. As Camp. jejuni has up to ten integral membrane and three soluble chemoreceptors, it has to be investigated whether any of these sense AI-2 as chemotactic signal. Effect of food matrices on AI-2 activity AI-2 activity of Camp. jejuni NCTC 11168 and V. harveyi BB152 cultivated in milk and chicken juice was c. 10-fold reduced. This reduction was not because of lesser luxS gene expression after cultivation in chicken juice but rather by a direct interaction of components from chicken juice with AI-2 molecules or with the V. harveyi BB170 reporter strain. Likewise, other authors showed reduced AI-2 activity after cultivation of Campylobacter, E. coli and Salmonella enterica ssp. enterica serovar Typhimurium in different food matrices (Cloak et al. 2002; Widmer et al. 2007a; Soni et al. 2008). Widmer et al. (2007b) demonstrated an inhibition of AI-2 activity by fatty acid components of poultry meat wash suspension. They speculated that the fatty acids might inactivate LuxS or interfere with transporter systems needed for AI-2 uptake by the bacteria from the environment (Widmer et al. 2007b). As our data showed a reduced AI-2 activity even when AI-2 containing CFS were incubated with chicken juice, the hypothesized LuxS inactivation seems not to be the reason for reduced AI-2 activity. Our results commend for a direct interaction of chicken juice components with AI-2 molecules or the AI-2 signalling cascade from the reporter strain V. harveyi BB170, which has to be investigated by further analysis. Supporting Information Figure S1 LuxS aa-sequence alignment. Table S1 Bacterial strains used in this study. Table S2 Accession no. of LuxS used in this study. Filename Description JAM_5221_sm_FigS1a.pdf17.3 KB Supporting info item JAM_5221_sm_FigS1b.pdf20.5 KB Supporting info item JAM_5221_sm_FigS1c.pdf20.9 KB Supporting info item JAM_5221_sm_TableS1.pdf13.9 KB Supporting info item JAM_5221_sm_TableS2.pdf13.4 KB Supporting info item Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. References Bassler, B.L., Greenberg, E.P. and Stevens, A.M. (1997) Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi. J Bacteriol 179, 4043– 4045. CrossrefCASPubMedWeb of Science®Google Scholar Birk, T., Ingmer, H., Andersen, M.T., Jorgensen, K. and Brondsted, L. (2004) Chicken juice, a food-based model system suitable to study survival of Campylobacter jejuni. Lett Appl Microbiol 38, 66– 71. 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