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The opportunistic pathogen
Vibrio vulnificus is a common isolate of marine and
estuarine waters. At water temperatures below 10°C, V. vulnificus enters a viable but nonculturable (VBNC) state (20, 25). Starvation of V. vulnificus prior
to shifting cells to low temperatures induces the
starvation-induced maintenance of culturability (SIMC) response which
delays the induction of the VBNC response (24, 27),
suggesting that starvation-induced or stationary-phase genes are
important for the adaptation of this organism to low-temperature
survival, in addition to other stress conditions.
In many bacteria, the regulation of phenotypes is controlled via
signaling pathways where extracellular factors are used to coordinate
the expression of phenotypes at the population level, many of which are
induced during stationary phase. For example, Rhizobium
leguminosarum uses signal molecules to induce stationary phase
(11), and conditioned supernatants have been shown to induce carbon starvation proteins in Vibrio angustum
(32). Signaling molecules have also been demonstrated or
suggested to regulate the expression of virulence factors in a variety
of organisms (4, 23, 28, 31), several of which are induced
during stationary phase in V. vulnificus.
Vibrio harveyi possesses genes (luxR and
luxS) which encode regulatory proteins that are members of a
signaling system recently identified in a broad range of organisms
(2); however, the genes regulated by this system are
generally unknown. We report here the characterization of a V. vulnificus smcR mutant, which is a homologue of the
V. harveyi luxR gene (21). This regulatory gene
appears to play an important role in starvation adaptation and in the
regulation of many stationary-phase-regulated genes, including some
virulence factors. Furthermore, we report that V. vulnificus
produces extracellular signals. The role of signals in the expression
of these stationary-phase proteases and in the development of
starvation adaptation is supported by the inhibition of these
phenotypes upon addition of a signal antagonist that represses
autoinducer system 2 (AI-2) phenotypes.
The plasmids and bacterial strains used in this study and their
genotypes are listed in Table 1. The
V. harveyi strains were a gift from Bonnie Bassler. Where
specified, glucose was added to a final concentration of 0.5% for
Luria-Bertani medium (LB) and 0.4% for 2M minimal medium
(27). The antibiotics ampicillin, streptomycin,
chloramphenicol, and colistin were used at concentrations of 50, 200, 34, and 100 µg ml
1, respectively. General chemicals
were purchased from Sigma Chemical Co., St. Louis, Mo. Genomic DNA was
isolated by the method of Tillett and Neilan (37).
Restriction enzymes, molecular weight markers, shrimp alkaline
phosphatase, ligase, Pwo polymerase, and T4 DNA ligase were
purchased from Boehringer Mannheim (Indianapolis, Ind.).
V. vulnificus produces compounds that induce
luminescence in V. harveyi.
The ability of V. vulnificus supernatants to induce bioluminescence in the V. harveyi AI-2 reporter strain BB170 was determined as previously
described (33). Cell-free supernatants were prepared from
late-exponential-phase cells of V. vulnificus C7814 and
UTHS-1 (optical dencity at 610 nm [OD610] = 0.796) grown
in LB with aeration at 37°C and added to the reporter strain at a
concentration of 10%. Induction by the V. vulnificus strain
C7184 or UTHS-1 was 215 or 350%, respectively, of the positive control
activity (data not shown). Maximal signal production in V. vulnificus occurred as cells enter the stationary phase of growth
(Fig. 1). To assess the effect of
nutrient starvation conditions on the induction of AI-2 activity in
V. vulnificus, cells were grown to mid-exponential phase in
LB containing NaCl (20 g liter1), collected by
centrifugation (8,000 × g, 10 min, 24°C), and washed
and resuspended in 0.5× NSS (27). Cell-free supernatants taken immediately after the shift to starvation conditions (time zero)
induced 0.4% of the luminescence observed in the V. harveyi reporter strain in the presence of the positive control
supernatant. Induction of luminescence increased to 849% for
supernatants taken from cells after 4 h of starvation; by 9 h
of starvation, luminescence had dropped to 245% (data not
shown). These data indicate that signal production is growth phase
regulated and that starvation conditions are able to stimulate the
production of AI-2-like activity in V. vulnificus.
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FIG. 1.
Effect of growth phase on the production of
substances able to induce luminescence in V. harveyi
BB170. V. vulnificus C7184 was grown with aeration in
2M at 37°C, and OD610 was determined (squares). Cell-free
supernatants were prepared at various times and assayed for the ability
to induce luminescence in the V. harveyi reporter strain
(bars). The activity of supernatants is presented as the percentage of
activity obtained when V. harveyi BB152 cell-free spent
supernatant is added to the reporter strain. Data presented are
representative of results obtained in at least three independent
experiments.
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In contrast to Salmonella enterica serovar Typhimurium and
Escherichia coli (34), the addition of 0.5%
glucose to LB inhibits the production of AI-2 activity in V. vulnificus. Furthermore, AI-2 activity was produced by V. vulnificus cells grown in LB or the minimal medium, 2M
(27), at room temperature and at 37°C (data not shown).
Supernatants collected from the smcR mutant (see below) were
also able to induce luminescence in V. harveyi to similar
levels as the wild type (data not shown), indicating smcR is
not required for signal production. The autoinducer activity of
cell-free supernatants heated to 80°C for 10 min was reduced by
52.7%, while heat treatment at 100°C for 10 min abolished activity, indicating that the V. vulnificus AI-2-stimulating
factor is a heat-labile compound.
V. vulnificus possesses the AI-2 synthase gene,
luxS.
The presence of a putative luxS homologue
in V. vulnificus was previously suggested by Southern
hybridization (21). A 320-bp fragment was amplified and
cloned from V. vulnificus using primers based on the AI-2
synthase gene, luxS, of V. harveyi.
Sequence analysis determined that the fragment,
luxSVv, showed >80, 79, and 68%
nucleotide identities to the luxS gene from V. harveyi, a putative luxS in the Vibrio
cholerae genome database, and the ygaG gene of E. coli (data not shown). The high degree of nucleotide identity and
the presence of AI-2 activity in the supernatants of V. vulnificus confirms the presence of a luxS gene in
V. vulnificus.
Characterization of a mutant in V. vulnificus of the
luxR transcriptional regulator homologue.
A potential
rho-independent terminator lies 19 nucleotides downstream of
the smcR stop codon, and the smcR coding region
is followed by a convergently transcribed homologue of lpd
(dihydrolipoamide dehydrogenase) (21). pUC19
(40) and pBluescript II SK were used as shuttle
vectors for the cloning of smcR. A null mutation in
smcR was generated by disruption with the insertion of a
streptomycin resistance cassette from pCAM140 (38)
(pSmcR.SM) 183 bp downstream from the ATG codon. The vector used for
delivery and homologous recombination, pMacSB, was constructed by the
insertion of the sacB gene derived from pCVD442
(7) into pLG401 (constructed by Lynn Gilson, University of
Hawaii). The smcR gene, containing the streptomycin
disruption was inserted into pMacSB to generate pMacSmcRK. The null
mutant was generated by conjugation of E. coli
BW20767(pMacSmcRK) with V. vulnificus C7184 and
selection for streptomycin and sucrose resistance. This
disruption was confirmed by Southern hybridization and PCR.
SmcR is involved in the regulation of starvation survival and the
SIMC response.
The effect of smcR on stationary-phase
survival was determined by growth of cells of V. vulnificus
C7184 and the smcR mutant (DM7) to early exponential phase
in LB with 20 g of NaCl per liter (OD610 nm = 0.22; 4.0 × 108 CFU ml1), followed by
resuspension in 2 M lacking glucose (2M-C) (27) at 1:100
dilution. During room temperature starvation, there was an initial
decrease of 76% in the CFU for the mutant strain and no decrease for
the wild type after 1 day (Fig. 2A).
After 14 days, the smcR mutant strain exhibited a decrease
of 91% of CFU whereas the wild type had a loss of 70%. The loss in
CFU for the two strains was not significantly different after the first
14 to 20 days of starvation.
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FIG. 2.
SmcR affects starvation survival and SIMC at low
temperature. V. vulnificus C7184 (filled symbols) and DM7
(smcR::Sm) (open symbols) were grown to mid-exponential
phase in LB with NaCl (20 g liter1), the cells were
collected by centrifugation (10,000 × g, 10 min),
washed in 2M-C and resuspended in 2M-C. Cultures were held statically
at 24°C (A) or were allowed to starve for 0 (B;  ,  ) or 4 (B;
 ,  ) h before being shifted to 4°C. (C) C7184 starved in the
presence (open symbols) or absence (closed symbols) of C2.
Determination of CFU was performed on DVNSS agar plates. Data are
presented as percentages of the initial count (1.1 × 105 to 2.9 × 105 CFU
ml11) and are representative of three independent
experiments. Error bars represent the 95% confidence interval.
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The defect in survival during the first 2 weeks of starvation exhibited
by DM7 prompted us to investigate the effect of the smcR
null mutation on the SIMC response. This SIMC effect possibly allows
cells to synthesize proteins that will be important in survival and
recovery when conditions are again favorable. Cells of C7184 and DM7
prepared as above were starved for 0 or 4 h at 24°C and then
shifted to 4°C. The results in Fig. 2B clearly indicate that the
smcR mutant strain is defective in mounting the SIMC response. The cultures that were shifted to 4°C without prestarvation showed very little difference in the rate of loss of culturability, in
contrast to those starved at room temperature before cold incubation (Fig. 2B). By the third day of cold incubation, DM7 had lost 77% of
total CFU, while the wild-type strain showed a decrease of only 28%.
This trend continued throughout the cold incubation.
These data indicate that SmcR affects the prestarvation response. Given
that signals regulate starvation in some bacteria (14, 16, 32,
36), we tested the effect of a signal antagonist on the
starvation response. The marine red alga Delisea pulchra has
been shown to produce a range of halogenated furanones that specifically inhibit signaling phenotypes regulated by the acylated homoserine lactone and AI-2 systems in bacterial species (9, 10,
17, 18, 32). Cells of C7184 were collected during early
exponential phase, washed and resuspended in 2M-C with or without
furanone compound 2 (C2; 2 µg ml1) and shifted to 4°C
at time zero and after 4 h of starvation at room temperature (Fig. 2C).
Room temperature starvation of cells in the presence of C2 does not
allow V. vulnificus to mount the SIMC response which occurs
in the absence of C2. C2 was added at 10 µg ml1 to
growing cultures of V. vulnificus C7184 during exponential phase (OD610 = 0.4) to assess whether signal
transduction is required for the production of autoinducer activity.
Supernatants collected from V. vulnificus during growth with
C2 induced V. harveyi 152%.
DM7 exhibits increased exoenzyme production.
Typical results
for exoprotease expression of cells grown in LB at 37°C as determined
by HPA (1), azocasein (35), and elastin-Congo
red (8) substrate degradation by cell-free supernatants are represented in Fig. 3. Similar trends
in exoenzyme activity were obtained from supernatants collected from
cultures grown at 24°C (data not shown) and for expression of
alkaline phosphatase activity (Table 2).
In all cases, exoenzyme expression of the smcR mutant
occurred earlier and the final activity was higher than for the
wild-type strain. Growth of the wild-type strain in the presence of C2
inhibited protease production (Table 2), indicating that the signaling
pathway is important for protease production. In V. cholerae, a mutation in the luxR homologue, hapR,
resulted in a loss of expression of the hemagglutinin/protease metalloenzyme (15). Interestingly, our results indicate
that unlike hapR in V. cholerae, smcR
is involved in the repression of protease expression during exponential
growth rather than its induction.
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FIG. 3.
Exoprotease activity of V. vulnificus C7814
and DM7 (smcR::Sm). Cultures of V. vulnificus
C7814 ( ,  ) and DM7 (, ) were grown in LB at 37°C with
shaking at 200 rpm on a rotary shaker. At various time points, aliquots
were removed and cell-free supernatants were prepared by centrifugation
(10,000 × g, 10 min). The supernatant was then fi
through 0.2-pore-size µm filters. Exoprotease activity (closed
symbols) was assayed by degradation of HPA (A), azocasein (B), and
elastin-Congo red (C) at 37°C. Results are presented as the
exoprotease activity per cell and are representative of at least three
independent experiments.
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Recent intriguing discoveries suggest that signal molecules may
regulate phenotypes that are not density dependent but are regulated in
relation to growth phase or in response to local environmental
conditions. For example, signal molecules have been shown to regulate
the induction of stationary phase in R. leguminosarum (36) and Pseudomonas aeruginosa
(41) and to induce the carbon starvation response in
V. angustum (32). These reports suggest there
are density-independent signaling systems in some bacteria that
regulate starvation and/or stationary-phase phenotypes.
SmcR is important for starvation survival and SIMC. While the general
features of the AI-2 systems appear to be highly conserved across a
broad range of genera and species, some of the specific features of the
system clearly differ and may reflect individual adaptation of the AI-2
system to the specific needs of particular bacteria. In V. vulnificus, the SmcR appears to act as an activator as well as a
repressor, in contrast to data presented for similar phenotypes on
other bacteria (15, 19).
Indeed, it has been previously suggested that LuxR may
function as a repressor. For example, LuxR binds independently
to two sites upstream of its own open reading frame (6)
and represses transcription from the luxR promoter as a
result of possibly interfering with and displacing RNA polymerase from
the promoter (6). LuxR is a member of the TetR family of
transcriptional regulators, which act as repressors (13).
Taken together, these data indicate that the primary function of the
LuxR regulator, at least in some organisms, may in fact be the
repression, rather than activation, of gene transcription. We propose
that SmcR in V. vulnificus appears to act as both an
activator and a repressor, similarly to TyR (29) and
nitrogen regulator I (30). It seems likely that signal production and recognition, which occurs at the transition into stationary phase or shortly after entry into starvation, when these
phenotypes are normally expressed, may be the mediator of this relief
of repression.
The loss of a functional smcR impairs starvation survival
and prevents V. vulnificus from exhibiting the SIMC response
upon starvation prior to low-temperature incubation. In addition to an
increase in exoenzyme production, the mutant strain also exhibited increases in motility, fimbria production (data not shown), and biofilm
formation (Table 2). We propose that the defect in starvation survival
by the smcR mutant may be reflected by the altered
regulation of the many stationary-phase phenotypes reported here. To
our knowledge, this is the first report of the regulation of starvation adaptation by a V. harveyi luxR homologue. This discovery
expands the role of signaling systems to include global regulation of nongrowth physiology.
Funding for this project was provided by Centre for Marine Biofouling
and Bio-Innovation.
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