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1 School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney 2052, Australia
2 Centre for Marine Biofouling and Bio-Innovation, University of New South Wales, Sydney 2052, Australia
Correspondence
Staffan Kjelleberg
s.kjelleberg{at}unsw.edu.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Present address: Department of Microbiology, University of Washington, Seattle, WA, USA.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). It appears that quorum sensing systems regulate the expression of phenotypes that are unproductive when expressed by an individual bacterium, but not when expressed by a group of cells. It should be noted that this density most likely reflects the number of cells within a particular environment and it is theoretically possible for a single cell to represent a quorum under specific conditions. Hence, it can be argued that the density of signalling molecules could be increased by limiting the space around the cells or by altering diffusion of the autoinducer, rather than by increasing the cell number per se. Thus, a complementary explanation for these systems proposed in the literature is that the autoinducer molecules serve as a means for sensing the diffusion potential of the surrounding environment rather than acting as census-taking machinery (Redfield, 2002). For example, bacteria utilize the secretion of enzymes for the breakdown of nutrients, an action that will only be productive when the exoenzymes are not able to diffuse away from the cell surface. In many cases, these secreted substances are regulated by autoinducers (e.g. proteases, siderophores and virulence factors). Redfield (2002) has proposed that autoinducer molecules represent a more efficient and less costly way for bacterial cells to sense diffusion. Both explanations suggest that these cells are surveying their environment as quorum sensing would be dependent on signal accumulation, governed by factors such as diffusion processes, signal stability and the presence of other cells using the same signal (Redfield, 2002). The results of this survey are interpreted by the bacteria to induce a specific phenotype. As mediators of adaptive responses, two quorum sensing systems have received particular attention of late.
Some Gram-negative bacterial species utilize the production and secretion of N-acylated homoserine lactone (AHL) molecules to regulate diverse phenotypes such as plasmid conjugal transfer in Agrobacterium tumefaciens (Piper et al., 1993), swarming motility in Serratia liquefaciens (Eberl et al., 1996), production of virulence factors in Pseudomonas aeruginosa (Gambello & Iglewski, 1991; Passador et al., 1993) and Yersinia enterocolitica (Throup et al., 1995), and antibiotic production and pathogenicity in Erwinia (Bainton et al., 1992; Beck von Bodman & Farrand, 1995). Many of these phenotypes facilitate the association of bacteria with higher organisms or surfaces. The paradigm for the AHL quorum sensing system is the symbiotic marine bacterium Vibrio fischeri (Eberhard et al., 1981; Engelbrecht et al., 1983). Bioluminescence is regulated by an AHL autoinducer synthesized by the luxI gene product and detected by the luxR-encoded receptor/transcriptional activator.
The free-living marine bacterium, Vibrio harveyi possesses two signal systems that function to control luminescence, siderophore production and colony morphology (Bassler et al., 1994). Signal system 1 is an AHL system composed of an autoinducer synthase (LuxM) (Cao & Meighen, 1989) and sensor 1 (LuxN) (Bassler & Silverman, 1995). The second system consists of the AI-2 autoinducer, a furanosyl borate diester (Chen et al., 2002), whose synthesis is dependent on the LuxS enzyme (Bassler et al., 1994). The periplasmic protein, LuxP, is the AI-2 binding protein and initiates signal transduction. Information from sensor 1 and sensor 2 is relayed to the LuxO response regulator (Freeman & Bassler, 1999a, b). LuxO, together with RpoN, activates a repressor of the bioluminescence operon. The LuxO protein is inactivated at high AI-2 concentrations, allowing the transcriptional regulator, LuxR, to initiate transcription of the bioluminescence operon. It should be noted that the autoinducer synthase, the signal receptors and the response regulators share no homology to the genes that mediate AHL-based quorum sensing in V. fischeri. Based on the identification of AI-2 signal activity or luxS in a wide range of Gram-negative and Gram-positive bacteria, it has been proposed that the AHL system in V. harveyi allows for intraspecies communication while the AI-2 system acts as a universal interspecies communication system (Bassler, 2002).
In Vibrio cholerae, three parallel quorum-sensing systems have been demonstrated to control virulence (Miller et al., 2002). System 1 involves the production of a CqsA-dependent signal, CAI-1, and detection by the sensor CqsS. System 2 resembles the AI-2 system described in V. harveyi and is composed of the LuxS synthase and LuxPQ sensor. The third proposed signalling system involves an intracellular signal in contrast to the extracellular signals of systems 1 and 2, and thus this system would not be involved in extracellular quorum signalling. The components of this recently proposed third quorum sensing system have not yet been identified. All three of these signalling systems are shown to be integrated at the level of the LuxO response regulator.
Vibrio vulnificus has been demonstrated to possess the AI-2 system (McDougald et al., 2000, 2001). We have identified and cloned the luxS signal synthase and the luxR response regulator homologue, smcR. To determine the role of signalling systems in the adaptive responses of marine vibrios, we have previously constructed a null mutation in the luxR homologue, smcR (McDougald et al., 2001). This should ensure that we are able to assess the effect of any signalling systems present, as all those systems identified to date in Vibrio strains are integrated at the level of LuxO, and LuxR is under regulation of LuxO. We have previously reported the regulation of a metalloprotease, fimbriae production, motility, biofilm formation and starvation adaptation by SmcR, the V. harveyi LuxR homologue in V. vulnificus (McDougald et al., 2001). Thus, the SmcR appears to function both as an activator and a repressor with its primary role being the repression of stationary-phase phenotypes, including many virulence factors, in exponential growth (McDougald et al., 2001). Similar phenotypes were subsequently identified to be controlled by the LuxR homologue, HapR, in V. cholerae (Zhu et al., 2002).
The regulation of relevant environmental phenotypes, such as stress adaptation in the marine environment, by signalling systems would further demonstrate if these systems truly function as cell communication systems in these organisms. In this report, we further investigated the role of signalling in the regulation of the environmentally relevant phenotypes of starvation adaptation and oxidative stress survival, and examined the potential for ‘cross-talk’ among members of the Vibrio genus. We define ‘cross-talk’ as the ability of native signal molecules to induce phenotypes in other Vibrio species. While supernatants from numerous species have been shown to induce the V. harveyi system (Bassler et al., 1997), it has not been determined if species which are likely to occur together in the environment are able to cross-talk. Here, we demonstrate the cross-talk potential of two free-living marine vibrios, ‘Vibrio angustum’ S14 and V. vulnificus, and the regulation of environmentally relevant phenotypes by the signalling system(s).
‘V. angustum’ S14 is a model organism that has been used by our laboratory to study programmed starvation and stress adaptation (Srinivasan & Kjelleberg, 1998). It has been demonstrated that a signal antagonist, furanone-2, prevents the highly organized development of starvation adaptation in this bacterium (Srinivasan & Kjelleberg, 1998; Srinivasan et al., 1998) and prevents the expression of proteins induced upon carbon starvation (Srinivasan et al., 1998). The addition of ‘V. angustum’ S14 stationary-phase supernatant extract (SSE) to furanone-2-treated ‘V. angustum’ S14 cultures can override the inhibitory effects of furanone-2 (Srinivasan et al., 1998). Likewise, V. vulnificus has been used extensively to investigate starvation adaptation and the formation of viable but non-culturable cells in response to adverse conditions (Oliver, 1993b, 1995; Paludan-Müller et al., 1996). V. vulnificus enters a viable but non-culturable state in response to low temperatures (Oliver, 1993a, b, 1995). It has been shown that exposing the cells to carbon or multiple-nutrient starvation, prior to incubation in the cold, prolongs culturability at 4 °C (Oliver et al., 1991; Paludan-Müller et al., 1996). This adaptive response has been referred to as starvation-induced maintenance of culturability (SIMC) and has been shown to require specific carbon starvation proteins (Paludan-Müller et al., 1996). It has been demonstrated previously that V. vulnificus produces a signal molecule (McDougald et al., 2001) which is induced by starvation. Similar to the prevention of starvation adaptation seen in furanone-treated ‘V. angustum’ S14 cells, we have shown that furanone-2 can inhibit the SIMC response in V. vulnificus (McDougald et al., 2001). In this study, we present data showing that cell-to-cell signalling is not only involved in these important adaptation processes, but that the bacteria are able to cross-talk by responding to non-native signals as part of their adaptation strategies.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. ‘V. angustum’ S14 was grown in Marine Minimal Medium (3M) (Östling et al., 1991) with a final glucose concentration of 0·1 % (w/v) in all experiments unless otherwise specified. Culture flasks were inoculated with fresh colonies of ‘V. angustum’ S14 from a Luria–Bertani agar plate made with 20 g NaCl l-1 (LB20) and the culture was kept in exponential growth at 25–28 °C by recurrent dilutions until maximal growth rate was achieved. Growth was monitored as a function of OD610. Carbon starvation conditions were attained by pelleting cells grown to mid-exponential phase by centrifugation at 15 000 g for 10 min at 20 °C. The cells were washed once with 3M lacking carbon (3M-C) and resuspended in 3M-C to an OD610 (1 cm path length; Novaspec II; Pharmacia Biotech) of 0·1 (approx. 1x108 c.f.u.). The cultures were statically incubated in the dark at 24 °C. Plate counts were measured as c.f.u. by the drop plate method (Hoben & Somasegaran, 1982) on LB20 agar plates.
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) was grown in 2M medium (Paludan-Müller et al., 1996) with a final glucose concentration of 0·4 % (w/v). 2M medium is identical to 3M, but contains only 50 % of the salts (0·5x NSS). For starvation and cold incubation experiments with V. vulnificus, the cells were grown in Luria–Bertani broth made with 10 g NaCl l-1 (LB10) overnight at room temperature and transferred to fresh 2M medium at a dilution of 1 : 50 and grown overnight. The cells were transferred again to fresh medium at a dilution of 1 : 100, grown to mid-exponential phase and harvested by centrifugation (10 000 g for 10 min). Cells were washed once in 2M without a carbon source (2M-C), resuspended in 2M-C and incubated statically in the dark to avoid oxidative stress caused by light (Gong et al., 2002). For cold incubation experiments, cells that were washed and resuspended in 2M-C were starved at room temperature for the times indicated and subsequently transferred to 4 °C. Culturability was assessed as c.f.u. on half-strength (containing 50 % of the salts required for VNSS) VNSS agar plates (Östling et al., 1991; V-medium modified from Väätänen, 1976). ‘V. angustum’ S14 supernatants were prepared by maintaining the bacteria in stationary phase for 5 h in 3M. The cells were removed by centrifugation at 15 000 g for 20 min followed by filtration (Millipore or Pall Gelman; pore size 0·2 µm) to remove any residual cells. Two litres of stationary-phase ‘V. angustum’ S14 culture (OD610=0·751) was used to prepare the SSE. Two volumes of supernatant were extracted with 1 vol. dichloromethane three times. The organic solvent was removed by rotary evaporation at 30 °C. Extracts were dissolved in 96 % ethanol at a concentration of 5 mg ml-1, which was used as the stock solution.
V. cholerae, V. fischeri, V. harveyi 47-6661, V. harveyi 642, V. vulnificus, Vibrio alginolyticus and Vibrio anguillarum were also maintained for 5 h in stationary phase in 2M. The cells were removed by centrifugation (15 000 g) and filtration through a 0·2 µm filter (Pall Gelman). The supernatants were extracted three times with dichloromethane and the solvent was removed by rotary evaporation at 30 °C. The extracts were dissolved in 96 % ethanol to a stock concentration of 5 mg ml-1.
Competition experiments.
Exponentially growing ‘V. angustum’ S14 cells were subjected to carbon starvation conditions as described above. The competition assay was performed when the cells had been simultaneously exposed to 5 µg furanone-2 (C2) ml-1 and appropriate concentrations of SSE. The concentrations of furanone-2 used in these and all subsequent assays were non-growth inhibitory. Viable counts were measured as c.f.u. on LB20 or half-strength LB20 agar by the drop plate method. The results were expressed as percentage c.f.u. ml-1 by comparing the c.f.u. of ‘V. angustum’ S14 at different periods of starvation (0–5 h) to time 0 of starvation. Control cultures were treated the same way without the addition of furanone-2. For V. vulnificus experiments, the cultures were shifted to 4 °C after starving the cells for the times indicated. Culturability was monitored as c.f.u. on LB20 agar (‘V. angustum’ S14) or half-strength VNSS agar (V. vulnificus). The results were reported as percentage c.f.u. ml-1, whereby c.f.u. at different times into the starvation period were normalized to the c.f.u. at time 0 of starvation.
V. harveyi bioluminescence bioassay.
The V. harveyi monitor strains, BB170 (sensor 1- sensor 2+, responds via signal pathway 2) and BB886 (sensor 1+ sensor 2-, responds via signal pathway 1) were used to assess if interaction with the signalling system in V. harveyi was occurring via signal pathway 1 or signal pathway 2 (Bassler et al., 1993, 1994). The monitor strains were grown in LB20 broth overnight and diluted 1 : 5000 in fresh LB20 broth. ‘V. angustum’ S14 supernatants were prepared from cultures grown for 5 h into the stationary phase in 3M. Cells were removed by centrifugation at 15 000 g followed by filtration through a 0·2 µm filter (Pall Gelman). Supernatants were stored at -20 °C prior to use for up to 2 weeks. ‘V. angustum’ S14 SSE was added at a concentration of 50 µg ml-1 when tested for activity in the AI-2 pathway. The aqueous sample consisted of 50 % (v/v) water-phase solution remaining after extraction of the supernatants with dichloromethane and 50 % (v/v) fresh LB20. For BB170 experiments, supernatant from V. harveyi BB152 (AI-1- AI-2+) was used as a positive control. For BB886 experiments, 10 µM N-(3-hydroxybutanoyl)-L-homoserine lactone (HBHL; Fluka) was used as a positive control. Extracted sterile media served as negative controls. The results were reported as a relative value by dividing the counts per second (c.p.s.) of the sample by the c.p.s. of the corresponding negative control at the time when the difference in luminescence between the sample and the control was maximal (Bassler et al., 1993).
Oxidative stress experiments.
For the oxidative stress experiments, cells were grown at 37 °C to early exponential phase (OD610=0·25–0·3) or stationary phase (OD610=0·8) in LB10 and were exposed to 0·5 or 2·5 mM H2O2, respectively, for 30 min. Control samples were incubated in the presence of phosphate buffer, which served as the diluent for the H2O2 stocks. The results are presented as the percentage survival compared to the untreated control and are representative of at least three experiments. For experiments with signal-containing supernatant addition, supernatants from 5 h starved cultures of V. vulnificus were added to a final concentration of 10 % (v/v). Furanone-2 was added at a concentration of 2 µg ml-1, which is a non-growth-inhibitory concentration.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). V. harveyi reporter strains with mutations in the sensors of each system (BB170 responds via the AI-2 pathway and BB886 responds via the HBHL pathway) were employed to ascertain the presence of signals in ‘V. angustum’ S14 cell-free supernatants (Fig. 1). The supernatant obtained from ‘V. angustum’ S14 cultures grown for 5 h into the stationary phase exhibited a 111-fold increase in bioluminescence in the AI-2 reporter strain. This is comparable to the 149-fold increase in bioluminescence shown by the positive control, supernatants from V. harveyi BB152 that produces only AI-2 and not HBHL. The ‘V. angustum’ S14 supernatants did not induce bioluminescence via the HBHL pathway (Fig. 1E and F), suggesting that ‘V. angustum’ S14 does not produce HBHL.
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). The SSE exhibited a 58-fold increase in bioluminescence while the aqueous phase showed 65-fold activity. This suggests that the putative signal molecule in ‘V. angustum’ S14 does not partition efficiently between the solvent and aqueous phases, and has both polar and non-polar solubility properties; however, the presence of more than one signal in the supernatant cannot be ruled out. Extracts were utilized for further experiments to minimize any effects of metabolic by-products and medium components.
Non-native SSE can override the effects of a signal antagonist on starvation adaptation
It has been demonstrated that halogenated furanones, produced by a red marine alga, Delisea pulchra, can act as inhibitors of AHL quorum sensing systems (Manefield et al., 1999) by accelerating the turnover of the transcriptional regulator, the LuxR protein (Manefield et al., 2002). To investigate the effect of these compounds on the signal system 2, furanone-2 was added to the V. harveyi AI-2 reporter strain BB170 and was shown to inhibit bioluminescence (Fig. 2). Thus, the furanone compounds target the AI-2 system as well as the AHL system, although the mode of action of inhibition in the AI-2 pathway is at this time unknown. Here furanone-2 was utilized as a tool for the investigation of the ability of non-native signal molecules from ‘V. angustum’ S14 and V. vulnificus to rescue cells from the effects of the furanone on starvation adaptation. It was hypothesized that V. vulnificus SSE could cross-talk with ‘V. angustum’ S14 and have a similar effect as ‘V. angustum’ S14 SSE. This was examined by determining if the addition of 50 µg V. vulnificus SSE ml-1 can rescue carbon-starved ‘V. angustum’ S14 cells exposed to furanone-2 from loss of culturability (Fig. 3). As presented in Fig. 3, V. vulnificus SSE provides protection against the inhibitory effect on c.f.u. caused by furanone-2. While the native signal molecules (‘V. angustum’ S14 SSE) have the capacity to prevent a greater loss in culturability, these data show that these two vibrios can cross-talk. Cells exposed to the medium extracts at the same concentration did not exhibit increases in culturability when exposed to furanones.
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). Exponentially grown V. vulnificus cells were resuspended either in 2M-C with furanone-2 (5 µg ml-1) or with both furanone-2 and ‘V. angustum’ S14 SSE (50 µg ml-1). Cultures were incubated at room temperature for 4 h prior to being shifted to 4 °C and culturability was monitored. The results indicate that ‘V. angustum’ S14 extract can also provide protection against the negative effect of furanone-2 on SIMC, and hence starvation, in V. vulnificus (Fig. 4). Cells exposed to the medium extract at the same concentration did not exhibit induction of SIMC responses (data not shown). This suggests that extracts from ‘V. angustum’ S14 not only counteract the effect of furanone-2 on itself, but also cross-talk with V. vulnificus to achieve the same effect as the native signal from V. vulnificus. We have made significant efforts to construct luxS mutants of both V. vulnificus and ‘V. angustum’ S14 to use as negative supernatants in these survival assays, but have as yet been unsuccessful.
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). Cells were exposed to 5 µg furanone-2 ml-1, or both furanone-2 and 50 µg Vibrio SSE ml-1 or medium extract and incubated at room temperature. While all the vibrios tested had some ability to protect ‘V. angustum’ S14 cells against the negative effect of furanone-2, V. cholerae SSE (59 % activity of the positive ‘V. angustum’ S14 SSE activity after 24 h of starvation) had the most effect after the native ‘V. angustum’ S14 SSE while V. harveyi 642 exhibited the least signal activity after 24 h (25 % activity of the positive ‘V. angustum’ S14 SSE activity after 24 h of starvation). The medium extracts did not elicit any significant protection against loss in culturability. These data indicate that there is cross-species interaction among a range of Vibrio species using extracellular signals that can mediate the starvation survival strategies of ‘V. angustum’ S14.
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). As a major effect of starvation on non-growing cells is oxidative stress (Dukan & Nyström, 1999), we utilized this strain for the investigation of the role of signalling in response to oxidative stress. Cells of V. vulnificus were grown at 37 °C to either early exponential phase (OD610=0·25) or stationary phase (OD610=0·8) and then were exposed to 0·5 or 2·5 mM H2O2, respectively, for 30 min (Fig. 6a). Control samples were incubated in the presence of phosphate buffer. As can be seen in Fig. 6(a), the wild-type strain (C7184O) is much more resistant to oxidative stress than the mutant strain (DM7), especially in exponential phase. Complementation of the DM7 mutant strain containing the plasmid pLS6 carrying smcR was performed in LB10 at 37 °C on early exponential-phase cells (OD610=0·26–0·3) exposed to 0·5 mM H2O2 for 30 min (Fig. 6b). As can be seen, the complemented strain survived as well as the wild-type strain.
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). Early exponential-phase cultures of C7184O were exposed to 0·5 mM H2O2 for 30 min in LB10 broth (control) or in the presence of 2 µg furanone-2 ml-1, LB with 10 % supernatant from 5 h starved cultures of V. vulnificus or LB with furanone-2 and supernatant. Wild-type cells incubated in the presence of furanone-2 were more sensitive to oxidative stress than the control cells and the addition of signal-containing supernatant to the furanone-2-incubated cells rescued the cells from the effects of the furanone. Interestingly, the incubation of the smcR mutant strain in the presence of furanone-2 and/or supernatants had no effect on oxidative stress survival (data not shown), further indicating a role for signalling in the response of V. vulnificus to oxidative stress.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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We utilized SSE rather than spent cell-free supernatants to limit the addition of other metabolites and nutrients found in the spent supernatant. Furthermore, due to the potential for disruption of the activated methyl cycle of luxS mutants and the possibility of more than one signalling system being present, we investigated the effect of the V. vulnificus SmcR (luxR homologue) on survival during oxidative stress. It appears that the LuxR homologues, in at least some of the Vibrio species studied (Jobling & Holmes, 1997; McCarter, 1998; Zhu et al., 2002), have global effects on gene regulation and are most likely regulated by other factors and/or other signalling systems in addition to the AI-2 autoinducer. It follows that these LuxR homologues may in fact be global regulators with the AI-2 signal system being just one of their inducers. We have identified components of the AI-2 system in ‘V. angustum’ S14 and V. vulnificus and have demonstrated the ability of the supernatants from these strains to induce the V. harveyi AI-2 reporter strain. We have been unable to identify any homologues of the AHL systems found in V. cholerae and V. harveyi in the Vibrio species investigated here. Likewise, we have demonstrated that supernatants of these organisms are unable to induce or repress AHL signalling phenotypes in other AHL monitors available (data not shown). Furthermore, repeated attempts to identify AHL molecules in the supernatants of both ‘V. angustum’ S14 and V. vulnificus by GC/MS and TLC indicated that no AHLs are present in these organisms, at least at the level of detection offered by these methods. However, we cannot altogether rule out the possibility that there are other signal systems present in these species.
‘V. angustum’ S14 produces extracellular signalling metabolites during carbon and energy starvation that play an important role in the expression of proteins crucial to the development of starvation- and stress-resistant phenotypes (Srinivasan et al., 1998). The ‘V. angustum’ S14 signal molecules induce the AI-2 signalling system in V. harveyi (Fig. 1) and a signal antagonist from a marine red alga, furanone-2, inhibits the V. harveyi AI-2 signalling pathway (Fig. 2). Moreover, V. vulnificus has been shown to have a functional V. harveyi luxS (AI-2 synthase) homologue and can induce bioluminescence in V. harveyi via the second signalling pathway (McDougald et al., 2000). In light of these observations, cross-talk between V. vulnificus and ‘V. angustum’ S14 was investigated.
Evidence of cross-species communication was provided by the ability of SSEs from V. vulnificus to rescue the loss in culturability caused by the signal antagonist, furanone-2, during carbon starvation in ‘V. angustum’ S14 (Fig. 3). Furanone-2 also has been shown to affect the starvation response in V. vulnificus by blocking the SIMC response when cells are shifted to low temperatures after starvation at room temperature (McDougald et al., 2001). SSEs from ‘V. angustum’ S14 were also capable of protecting V. vulnificus cells from the inhibitory effect of furanone-2 (Fig. 4).
We have previously demonstrated the presence of V. harveyi luxR homologues in seven Vibrio strains, V. cholerae, V. harveyi 642, V. harveyi 47-6661, V. vulnificus, ‘V. angustum’ S14, V. anguillarum and V. alginolyticus (McDougald et al., 2000). The V. harveyi luxR-like gene is highly conserved in all the Vibrio species tested (V. harveyi, V. cholerae, V. parahaemolyticus and V. vulnificus), suggesting that this gene is widely dispersed in marine Vibrio species and was inherited from a common ancestor. Given that several Vibrio species can induce the second signal system in V. harveyi and have also been shown to have a luxR homologue, the ability of six Vibrio strains to cross-talk with ‘V. angustum’ S14 was investigated. The results revealed that V. vulnificus, V. cholerae and V. alginolyticus were capable of rescuing ‘V. angustum’ S14 cells counteracting the effect of furanone-2 on culturability during carbon starvation (Fig. 5). The other Vibrio strains, V. anguillarum, V. harveyi 47-6661 and V. harveyi 642, were less efficient in protecting the furanone-2-affected cells from loss of culturable counts. This suggests that while some aspects of the signalling systems are conserved between species, there are specific features in each system, particularly with respect to the production and activity of the signal molecules. For example, there are reports that in Escherichia coli and Salmonella typhimurium, a heat-labile molecule produced in the mid-exponential phase only in LB medium in the presence of glucose, has the ability to substitute for AI-2 in V. harveyi (Surette & Bassler, 1998). On the other hand, the extracellular molecule produced by V. vulnificus is repressed by glucose addition and is produced during the late exponential and early stationary phases (McDougald et al., 2000), indicating the differences in the characteristics of AI-2-like signal molecules which can contribute to variations in the physical and chemical properties of the signal molecules.
Oxidative stress is a situation likely to be encountered in the marine environment and we have found a significant overlap in the global regulators utilized by a cell to withstand both starvation and oxidative stress (Gong et al., 2002; Hild et al., 2000). Bacteria adapt to non-growth conditions (starvation or other stresses) primarily by defending against increased levels of intracellular oxidative stress (Dukan & Nyström, 1999) due to the accumulation of oxidative species in the non-growing cell (Dukan & Nyström, 1998, 1999) and extracellular oxidative stress due to exposure to UV irradiation and reactive chemical species. Given that signalling is involved in adaptation to starvation stress, and that a major component of starvation stress is oxidative stress, we investigated the role of signalling in resistance to oxidative stress in V. vulnificus.
The addition of a signal antagonist was able to prevent survival of wild-type V. vulnificus cells during oxidative stress, while the addition of signal-containing supernatant was able to rescue the cells from the effects of the furanones (Fig. 7). To determine the role of signalling systems during survival under conditions of oxidative stress, we utilized an smcR mutant strain. An smcR mutant strain was shown to be more sensitive to oxidative stress induced by H2O2 than the wild-type strain, especially in the exponential phase (Fig. 6a). Therefore, there are likely to be multiple regulators involved in the oxidative stress response in stationary phase. This is not surprising given that protection against oxidative stress is an important feature of the adaptation strategy. Furthermore, the smcR mutant strain was unaffected by the addition of furanones, indicating that furanone-2 is acting through the signalling pathway to prevent resistance to oxidative stress. We have also shown that the signal antagonist furanone-2 has a negative effect on the capacity of ‘V. angustum’ S14 cells to develop stress resistance against oxidative stress (UV and H2O2) (Srinivasan & Kjelleberg, 1998). Additionally, in previous studies, we have shown that starved ‘V. angustum’ S14 cells exposed to furanone-2 are unable to mount resistance against oxidative stress, a critical feature of the starvation-survival programme (Srinivasan & Kjelleberg, 1998).
During the course of these studies, the structure of V. harveyi AI-2 has been determined and shown to be a furanosyl borate diester (Chen et al., 2002). The signal antagonist from D. pulchra, furanone-2, has structural similarities to the V. harveyi AI-2 molecule and, as we have shown that furanone-2 has an inhibitory effect on bioluminescence in the V. harveyi AI-2 signal system (Fig. 2), it is possible that furanone-2 acts as a competitor of the AI-2 for its receptor. Based on our hypothesis that AI-2-like molecules play a role in starvation and stress phenotypes in ‘V. angustum’ S14 and V. vulnificus, any inhibitor of this system should also have effects on starvation and stress phenotypes in these two bacteria, which is consistent with the effects of the furanones on starvation and stress. The demonstration that ‘V. angustum’ S14 and V. vulnificus produce exogenous molecules that interact with the AI-2 system in V. harveyi, and that these molecules are able to counteract the effects of furanone-2, suggests that both the extracellular molecules and furanone-2 are acting on the same pathway. Indeed, in an earlier analysis we have shown that the extracellular molecules produced during starvation and furanone-2 act on the same carbon starvation pathway by 2D-PAGE analysis of carbon starvation proteins (Srinivasan et al., 1998).
The data presented in this report indicate that the signal molecules produced by these Vibrio species are indeed true signalling molecules regulating the environmentally relevant phenotypes of starvation and resistance to oxidative stress. In addition to the cross-talk between ‘V. angustum’ S14 and V. vulnificus, a variety of other Vibrio species produced molecules that were able to elicit a starvation protection response in ‘V. angustum’ S14. The data also support the suggestion that the AI-2 system can be used as an interspecies communication system to either detect or respond to conditions sensed by other bacteria in the same environment, at least in Vibrio species (Bassler, 2002). In contrast, it has been proposed that the AI-2 autoinducer is not a true signal, but rather a metabolite that can be excreted from the cell in early growth and can be metabolized in the later stages of growth (Winzer et al., 2002a, b). Furthermore, it was suggested that the main function of LuxS is a metabolic one as it is required for the activated methyl cycle, converting S-ribosylhomocysteine (RH) to homocysteine and AI-2 (Winzer et al., 2002b). Thus, the effects of mutations in luxS would largely impact on the metabolic status of the cell and may not necessarily be due to loss of a ‘quorum sensing’ autoinducer. Winzer et al. (2002a) propose that a cell-to-cell signalling molecule must be produced during specific growth conditions, accumulate extracellularly, induce a concerted response upon reaching a critical threshold and must regulate functions beyond those of metabolism or detoxification of the autoinducer. The AI-2 system of V. harveyi fulfils all of these requirements as the signal molecule is produced only during certain growth phases and induces luminescence. Furthermore, searches of sequence databases indicate that most, if not all members of the genus Vibrio, possess the AI-2 receptor protein, LuxP, and other proteins in the signal transduction cascade. Thus, the data presented here and searches of the available genomes indicate that the AI-2 receptor, LuxP, is widespread in Vibrio species, which supports AI-2 as being a true signal in marine Vibrio species for the regulation of adaptive phenotypes.
We see this as a particularly exciting concept whereby stress adaptation is not only controlled as an individual's response to its environment, but that bacteria may also be capable of determining the status of other cells within mixed species communities and thus respond, perhaps serving as an early response mechanism to increase the bacterium's chances of mounting a successful defensive response. Moreover, there is interference by eukaryotic signal molecules which have probably evolved as anti-fouling tactics employed by marine plants. This highlights the intricacies of signalling systems which appear to have evolved as a result of several inter-dependencies. This is clearly an emerging field of research and future work in this area is likely to have significant implications for our understanding of the adaptation and survival of bacteria in the natural environment.
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Received 27 February 2003;
revised 28 March 2003;
accepted 4 April 2003.
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