Constraints on detection of autoinducer-2 (AI-2) signalling molecules using Vibrio harveyi as a reporter

Sigrid C. J. DeKeersmaecker and Jos Vanderleyden

Centre of Microbial and Plant Genetics, K.U. Leuven, Kasteelpark Arenberg 20, 3001 Leuven-Heverlee, Belgium

Correspondence
Jos Vanderleyden
(jozef.vanderleyden{at}agr.kuleuven.ac.be)

Bacteria are capable of regulating gene expression in response to a variety of extracellular signals. When the signal is produced by the bacterium itself, this type of regulation is termed autoinduction or quorum sensing. Through the use of these signals or autoinducers, bacteria can regulate their behaviour according to population density (Winans & Bassler, 2002).

The recently discovered signalling molecule autoinducer-2 (AI-2) is produced by a large number of bacterial species, as judged by the Vibrio harveyi bioassay (see below). AI-2 has been proposed to serve as a ‘universal’ signal for interspecies communication (Surette et al., 1999). The chemical identity of AI-2 of V. harveyi, the first bacterium for which production of AI-2 was reported (Bassler et al., 1994), has recently been discovered to be a cyclic borate diester (furanosyl borate diester) (Chen et al., 2002). LuxS, the AI-2-synthase, occurs in over 40 species of Gram-negative and Gram-positive bacteria (Winans & Bassler, 2002; Winzer et al., 2002). Recent findings strongly suggest that AI-2 molecules from diverse species of bacteria are identical (Schauder et al., 2001). This characteristic was previously hypothesized and exploited to assay AI-2 production by unrelated bacteria (Bassler et al., 1997). The marine bacterium Vibrio harveyi utilizes two distinct quorum-sensing mechanisms to regulate bioluminescence, AI-2 (Bassler et al., 1994) and AI-1 (Cao & Meighen, 1989). A mutant strain of V. harveyi, BB170, which is only able to respond to AI-2 signal molecules for bioluminescence induction, has been commonly used as a bioassay to demonstrate the presence of AI-2-like molecules in conditioned media of other bacterial species (Bassler et al., 1994).

Although luxS homologues have been annotated in the genome sequences of Lactococcus lactis subsp. lactis (GenBank accession no. Q9CIU0), Lactobacillus plantarum WCFS1 (GenBank accession no. NP_784522), Lactobacillus gasseri (GenBank accession no. ZP_00046310) and Bifidobacterium longum NCC2705 (GenBank accession no. NP_696321), production of AI-2-like molecules by lactic acid bacteria has not yet been described. This prompted us to apply the Vibrio bioluminescence assay to test the conditioned medium of different probiotic and non-probiotic lactic acid bacteria, for which the complete genome sequences are not yet available, for the presence of AI-2-type molecules. Our screen included Lactobacillus rhamnosus GG ATCC 53103, L. rhamnosus GG E-96666, L. rhamnosus Lc705, L. rhamnosus LMG 18030, L. rhamnosus LMG 6400, L. rhamnosus NRRL-445, L. rhamnosus 1/6, Lactobacillus casei Immunitas, L. casei Shirota, L. plantarum NCIMB 8826 Int-1, Lactobacillus johnsonii VPI 11088, Lactobacillus casei ATCC 393 and Lactococcus lactis MG1363. In contrast to what has been reported previously (Frias et al., 2001), using cell-free culture fluids of all the tested strains, we were able to detect molecules that can stimulate the quorum-sensing system regulating the expression of the luciferase operon (lux genes) in V. harveyi. This result may seem not so surprising in view of the steadily growing list of bacterial species known to produce AI-2 molecules. However, this finding would have been precluded without the (re)discovery of two important features of the commonly used Vibrio bioassay. We think this could be of interest to the quorum-sensing research community.

Autoinducer bioassays were performed essentially as described previously (Greenberg et al., 1979; Surette & Bassler, 1998). Briefly, the V. harveyi reporter strain was grown for 16 h with aeration (175 r.p.m.) at 30 °C in Autoinducer Bioassay (AB) medium (Greenberg et al., 1979), and then diluted 1 : 5000 in fresh AB medium. Cell-free culture fluids of test strains were then added to the diluted V. harveyi culture at a 10 % (v/v) final concentration, i.e. 10 µl cell-free culture fluid added to 90 µl diluted V. harveyi culture medium in an UV-sterile 96-well microtitre plate (white Cliniplate; Thermo Life Sciences). The microtitre plates were covered with a non-toxic plate sealer (Greiner Labortechnik NV) and incubated at 30 °C with aeration (175 r.p.m.). Light production was measured every hour using a CCD camera (Berthold Night Owl; Perkin Elmer) or a luminescence reader (Fluoroskan Ascent FL; Labsystems). Hourly measurement of light production is required to calculate AI-2 activity carefully since the reporter strain produces and senses its own autoinducer. Based on previous reports (e.g. Ren et al., 2001; Surette & Bassler, 1998) and confirmed in this work, the signal of the reporter strain is minimal between 5 and 5·5 h after inoculation (i.e. interference by endogenous AI-2 of the reporter is <1 % of the bioluminescence signal). So this time point is appropriate for monitoring the effects of exogenous AI-2. Positive control wells contained 10 µl of cell-free conditioned medium from V. harveyi BB152 (AI-1-, AI-2+; Bassler et al., 1993) grown for 16 h at 30 °C with aeration in AB medium, while negative control wells contained 10 µl of sterile growth medium. Cell-free culture fluids were prepared by removing the cells from the growth medium by centrifugation at 12 000 g for 5 min. The cleared supernatants were passed through a 0·2 µm Dynagard (Microgon) filter (Bassler et al., 1997).

AI-2 production has been shown to be growth-medium-dependent (Burgess et al., 2002). Consequently, we prepared cell-free culture fluids of lactobacilli grown in two different media; i.e. the commonly applied Man–Rogosa–Sharpe (MRS) medium and a chemically defined medium (CDM) (Chervaux et al., 2000). Moreover, to evaluate a possible effect of lactic acid (a fermentation product) in AI-2 production, we tested sugar-containing and sugar-free compositions of both growth media. All four different medium compositions were incorporated as negative controls in the Vibrio bioassay. Strikingly, the small amount of background luminescence produced by the reporter strain exposed to sugar-free sterile medium disappeared when its glucose-containing counterpart was assayed. This suggested that the V. harveyi lux genes are subject to catabolite repression by glucose and was confirmed by assaying AI-2-positive Vibrio cell-free culture fluid (BB152; Bassler et al., 1993) supplemented with different concentrations of glucose. Two millimolar (final concentration) glucose added to the cell-free culture fluid of BB152 already completely inhibited light production in the bioassay, while galactose in the same concentrations did not (Fig. 1). Consequently, we replaced glucose by galactose in the growth media of the lactic acid bacteria for preparation of cell-free culture fluids. Indeed, remnants of glucose in cell-free culture fluids could hamper detection of AI-2 activity. This could be important since addition of glucose to the growth medium of many bacteria, e.g. Salmonella typhimurium, is one environmental condition assumed to be required for maximal AI-2 production (Surette & Basler, 1999). The same holds true for L. rhamnosus GG. In medium in which sugar (e.g. glucose) was omitted, although growth of L. rhamnosus GG was not affected, we were unable to observe AI-2-like activity. Moreover, replacing glucose by galactose revealed cross-reaction of medium compounds, still present in the lactobacilli cell-free culture fluid (background signal), i.e. increased light production by the Vibrio reporter strain was not necessarily caused by the presence of AI-2-type molecules in the added cell-free culture fluid. Since the luminescence values in the V. harveyi bioassay are usually reported as the fold induction of luminescence by the reporter strain above the negative control, i.e. medium supplemented with glucose, these values are prone to being overestimated. Indeed, if the medium contains compounds inducing background luminescence, this will stay undetected in the negative control due to the catabolite repression by glucose. However, in the cell-free culture fluid, where glucose is metabolized by the test strain, these medium compounds could contribute to the measured light signal. Although we serendipitously discovered the detrimental effect of glucose on the Vibrio bioassay, it did not imply a new finding. In 1972, Nealson et al. (1972) demonstrated catabolite repression of luminescence by simple batch-culture experiments. Luciferase synthesis in batch cultures of V. harveyi is permanently repressed by D-glucose, and this repression can be overcome by the addition of cAMP to the growth medium. However, until now, this feature of bioluminescence in V. harveyi has been overlooked in studies that use the Vibrio bioassay for AI-2 molecules.



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Fig. 1. Effect of pH and glucose on induction of luminescence in the V. harveyi BB170 bioassay for AI-2. The following cell-free culture fluids were assayed: (1) 2 mM galactose (final concentration) in bioassay of V. harveyi BB152 cell-free culture fluid; (2) 2 mM glucose (final concentration) in bioassay of BB152 cell-free culture fluid; (3) cell-free culture fluid of a 24 h-old L. rhamnosus GG ATCC 53103 modified MRS medium (i.e. glucose replaced by galactose) culture (pH 3·8; final pH in bioassay was 4·3); (4) neutralized cell-free culture fluid of a 24 h-old L. rhamnosus GG ATCC 53103 modified MRS medium culture; (5) neutralized cell-free culture fluid of a 24 h-old L. casei ATCC 393 modified MRS medium culture; (6) BB152 final pH 4·3, i.e. cell-free culture fluid of the positive control V. harveyi BB152 was added at 10 % (final concentration) to the reporter strain BB170 and the pH of the mixture was adjusted to 4·3; (7) BB152 pH 3·8, i.e. cell-free culture fluid of BB152 adjusted to pH 3·8 was added at 10 % (final concentration) to the reporter strain BB170, the mixture reached a final pH of 6·7; (8) BB152 pH 7, i.e. unadjusted cell-free fluid of BB152 (pH 7); (9) neutralized cell-free culture fluid of a 24 h-old L. rhamnosus GG ATCC 53103 modified MRS medium culture supplemented with 1 mM boric acid. Averages of three replicates are reported, with error bars indicating SD values. The data are reported as the increase in light emission by V. harveyi BB170 exposed to the various conditioned media over the level of luminescence obtained from the corresponding sterile growth medium. Assays were performed at least in duplicate due to the inherent variability of the assay (Bassler et al., 1997; Frias et al., 2001).

 
In addition to different growth conditions, a likely explanation as to why AI-2-like molecules produced by L. casei ATCC 393 were not detected previously (Frias et al., 2001) could be the acidic nature of its cell-free culture fluid. Since the supernatant of lactic acid bacteria in media containing sugar reaches a low pH due to lactic acid production (e.g. pH 3·8 after 24 h growth of L. rhamnosus GG in MRS medium), the effect of low pH on the V. harveyi BB170 bioassay was investigated. When cell-free culture fluid prepared from L. rhamnosus GG grown in modified MRS medium (i.e. glucose replaced by galactose) (pH 3·8) was added to the reporter strain BB170 (in AB medium) at a final concentration of 10 %, the mixture reached a final pH of 4·3 and no light production by the reporter strain could be observed (Fig. 1). However, when the cell-free culture fluid of L. rhamnosus GG was neutralized with NaOH prior to addition to the Vibrio reporter strain, we detected bioluminescence (Fig. 1). Also, the neutralized cell-free culture fluid of L. casei ATCC 393 could induce light production by the Vibrio reporter strain (Fig. 1). We included some control experiments to elucidate the role of pH in the Vibrio bioassay. Cell-free culture fluid prepared from the positive control V. harveyi BB152 (AI-2+) was added at a final concentration of 10 % to the reporter strain BB170 and the pH of this mixture was adjusted to 4·3. In addition, cell-free culture fluid of BB152 adjusted to pH 3·8 (final pH in bioassay 6·7) and unadjusted cell-free fluid of BB152 (pH 7) were included in the bioassay. As shown in Fig. 1, a final low pH of the reporter/sample mixture significantly reduces luminescence of BB170. Low pH on its own does not seem to inactivate the AI-2 signalling molecule since cell-free culture fluid of BB152 adjusted to pH 3·8 and unadjusted cell-free fluid of BB152 (pH 7) resulted in approximately the same fold induction of luminescence. It is probable that the presence of lactic acid in the unadjusted cell-free culture fluid of the tested lactic acid bacteria causes the low final pH in the bioassay, hampering light induction. Consequently, the pH of the cell-free culture fluids was always neutralized prior to addition to the Vibrio reporter strain.

In conclusion, our findings call for caution when preparing cell-free culture fluids. Carefully designed control experiments should be included in quorum-sensing research. By taking the catabolite repression of the Vibrio lux genes and the sensitivity towards acidic pH into consideration, we were able to detect AI-2-like molecules in the cell-free culture fluid of several lactic acid bacteria, including L. rhamnosus GG. To examine the kinetics of AI-2-like molecule production, cell-free culture fluids were prepared at time points reflecting all phases of growth. Cell-free culture fluids were prepared from modified MRS medium and CDM, both containing galactose as carbon source. Before assessing the presence of AI-2 molecules in the Vibrio bioassay, the pH of every sample was neutralized with NaOH. Maximal AI-2 activity was detected in cell-free culture fluid after 24 h growth and remained at this level for at least 48 h. These time points correspond to the stationary phase. Cell-free culture fluid prepared from modified MRS medium contained more AI-2-like activity than cell-free culture fluid prepared from CDM with galactose. Chen et al. (2002) reported that the addition of boric acid concentrations as low as 10 µM to the growth medium caused significant induction of luminescence by the cell-free culture fluids of those cultures. This is consistent with the proposed chemical structure of AI-2 (Chen et al., 2002). Addition of 1 mM (final concentration) boric acid to the growth media of L. rhamnosus GG significantly increased the induction of luminescence of BB170 by the corresponding cell-free culture fluids (Fig. 1). The effect was most striking in the cell-free culture fluid prepared from the CDM culture. Addition of boric acid did not influence the growth of L. rhamnosus GG. Assigning a function to the production of AI-2-like molecules by this probiotic bacterium is an exciting challenge to be faced with.

Acknowledgements
We thank the following people for providing strains used in this study: B. Bassler (Department of Molecular Biology, Washington Road, Princeton University, Princeton, NJ 08544-1014, USA) for V. harveyi BB170 and BB152, W. de Vos (Laboratory of Microbiology, Bode 33, Building 341, Hesselink v Suchtelenwg 4, Postbus 8033, 6703 CT Wageningen, The Netherlands) for L. rhamnosus GG ATCC 53103, A. Mercenier (Bioscience Department, Nestlé Research Center, P.O. Box 44, CH-1000 Lausanne 26, Switzerland) for L. plantarum NCIMB 8826 Int-1, Valio Ltd (Meijeritie 6, FIN-00371 Helsinki, P.O. Box 10, 00039 Valio, Helsinki, Finland) for L. rhamnosus 1/6 and Lc705 and I. Nagy (Centre of Microbial and Plant Genetics, Kasteelpark Arenberg 20, B-3001 Heverlee, Belgium) for the isolation of L. casei Shirota and Immunitas. S. C. J. D. K. is a Research Associate of the Belgian Fund for Scientific Research (FWO-Vlaanderen). This work was partially supported by projects STWW-00162 and GBOU-SQUAD-20160.

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