ABSTRACT
Vibrio parahaemolyticus is a halophilic Gram-negative bacterium that causes human gastroenteritis. When the viable but nonculturable (VBNC) state of this bacterium was induced by incubation at 4°C in Morita minimal salt solution containing 0.5% NaCl, the rod-shaped cells became coccoid, and various aberrantly shaped intermediates were formed in the initial stage. This study examined the factors that influence the formation of these aberrantly shaped cells. The proportion of aberrantly shaped cells was not affected in a medium containing d-cycloserine (50 μg/ml) but was lower in a medium containing cephalosporin C (10 μg/ml) than in the control medium without antibiotics. The proportion of aberrantly shaped cells was higher in a culture medium that contained 0.5% NaCl than in culture media containing 1.0 or 1.5% NaCl. The expression of 15 of 17 selected genes associated with cell wall synthesis was enhanced, and the expression of VP2468 (dacB), which encodes d-alanyl-d-alanine carboxypeptidase, was enhanced the most. The proportion of aberrantly shaped cells was significantly lower in the dacB mutant strain than in the parent strain, but the proportion was restored in the presence of the complementary dacB gene. This study suggests that disturbance of the dynamics of cell wall synthesis by enhanced expression of the VP2468 gene is associated with the formation of aberrantly shaped cells in the initial stage of induction of VBNC V. parahaemolyticus cells under specific conditions.
INTRODUCTION
Vibrio parahaemolyticus is a halophilic Gram-negative bacterium that causes food-borne gastroenteritis (1). Most clinical isolates are hemolytic on Wagatsuma agar (Kanagawa phenomenon positive [KP+]) and produce a major virulence factor, thermostable direct hemolysin (TDH) (2). This pathogen has exhibited global significance since the occurrence of the first pandemic O3:K6 strains in 1996 (3). Food-borne V. parahaemolyticus infections occur commonly in the summer, and this pathogen is typically isolated in warm seawater.
A unique physiological state, known as the viable but nonculturable (VBNC) state, is frequently induced in vibrios in seawater or in a nutrient-limited medium kept at a low temperature (4, 5). This phenomenon is generally regarded as a survival strategy of these nonsporulating bacteria (6). Cells in this state are unable to form colonies on common agar medium, but they are viable, and their colony-forming ability can be resuscitated by a temperature upshift treatment (7).
Like that of other vibrios in the marine environment, the VBNC state of V. parahaemolyticus is induced by incubation at a low temperature in a mineral salt starvation medium (8–10). VBNC V. parahaemolyticus cells are resistant to the challenge of heat, low salinity, or acid inactivation (10). Virulence usually decreases considerably as cells enter the VBNC state (11), but virulence genes and pathogenic potential are maintained (12, 13). The stress resistance of VBNC V. parahaemolyticus cells may be associated with the change in cellular morphology in this state (14).
Dramatic morphological and physiological changes have been demonstrated in the VBNC state of V. parahaemolyticus (10, 11). When V. parahaemolyticus was incubated under conditions inducing the VBNC state for 1 to 3 days, many cells changed from rod-like to coccoid, and some exhibited various intermediate, aberrant shapes, such as a club shape, budding, a club shape with budding, a dumbbell shape, and a rabbit ear-like shape (15). Since the shape of a bacterial cell is determined by the functions of the cell wall-synthesizing enzymes/penicillin-binding proteins (PBPs) and cytoskeleton proteins under normal growing conditions (16, 17), the formation of these aberrantly shaped cells during the initial stage of induction of the VBNC state may be associated with the organized relocation of cytoskeleton proteins, such as MreB, MinE, and FtsZ (15).
The formation of these aberrantly shaped cells may be caused by interference with the normal function of the PBPs. Mutation of the genes that encode low-molecular-weight (LMW) PBPs also causes severe morphological aberrations in Escherichia coli, causing major defects to appear in cells that lack PBP 5 and at least two other LMW PBPs (18–20). The LMW PBPs do not synthesize peptidoglycan but modify it by removing the terminal d-alanine residue from the pentapeptide side chains (d-alanyl-d-alanine carboxypeptidase) or by cleaving the peptide cross-links that hold the glycan chains together (endopeptidases) (21). The addition of antibiotics that influence the biosynthesis of bacterial cell walls also induces changes in cell morphology (22, 23).
A physical model reveals that damage to the peptidoglycan of a rod-shaped Gram-negative bacterium manifests as a bulge in the side wall and the consequent bending of the cylindrical cell wall around the bulge (24). Bulging cells and other aberrantly shaped cells that are formed in the initial stage of induction of the VBNC state of V. parahaemolyticus are similar to those that have undergone the morphological changes described in this model. The damage to, or relaxation of, the peptidoglycan cell wall is hypothesized to occur during the initial stage of induction of the VBNC state, resulting in the formation of aberrantly shaped cells. In this study, the formation of aberrantly shaped V. parahaemolyticus cells was investigated, and the association of a d-alanyl-d-alanine carboxypeptidase gene (VP2468) with this phenomenon was demonstrated by gene mutation and complementation.
MATERIALS AND METHODS
Bacterial strains and culture conditions.V. parahaemolyticus strain 1137, a clinical strain (KP+, serotype O3:K6) isolated in 1997 in Taiwan, was used in this study (Table 1). It was stored frozen at −85°C on the surfaces of beads in Microbank cryovials (Pro-Lab Diagnostics, Austin, TX). The strain was cultured at 37°C on tryptic soy agar (Becton Dickinson Diagnostic Systems, Sparks, MD) supplemented with 3% sodium chloride (TSA–3% NaCl), or in tryptic soy broth (TSB)–3% NaCl in a 50-ml tube, which was shaken at 160 rpm. A 50-μl aliquot of the 16-h broth culture was inoculated into 10 ml of fresh TSB–3% NaCl and was incubated at 37°C with shaking for 2 h to enable entry into the early-exponential phase (around 108 CFU/ml). This culture was used as the inoculum in subsequent experiments. Escherichia coli (Table 1) was cultured in Luria broth (Becton Dickinson) at 37°C and was shaken at 160 rpm. Bacterial growth was also determined by measuring the absorbance of the cultures at 600 nm. Chloramphenicol (final concentration, 6 μg/ml) or chloramphenicol (20 μg/ml)-ampicillin (50 μg/ml) was added to the media as required for the cultivation of some of the V. parahaemolyticus or E. coli strains, respectively.
Bacterial strains and plasmids used in this study
To assay the change in cell morphology, the V. parahaemolyticus strain 1137 culture at exponential phase was harvested by centrifugation, washed twice in Morita minimal salt solution (MMS)–0.5% NaCl (15), resuspended to a final cell density of 107 CFU/ml in the same medium, and incubated at 4°C for 12 h. The stressed cells were collected by centrifugation, and their morphology was examined by light and/or electron microscopy. To assay the effects of different osmolarities, the cells were resuspended in MMS medium containing 0.5, 1.0, or 1.5% NaCl. d-Cycloserine (final concentration, 50 μg/ml) or cephalosporin C (10 μg/ml) was added to the cell suspensions in MMS–0.5% NaCl, and the cell suspensions were incubated at 4°C for 12 h in order to assay the influence of antibiotics on cell morphology.
Microscopy.For light microscopy, bacteria in suspension were heat fixed and were observed by phase-contrast microscopy using a Nikon Plan Fluor 100× (numerical aperture, 1.30) oil immersion objective (Nikon, Melville, NY) at room temperature. For each sample, cells of different shapes in 20 randomly selected fields were enumerated. The viability of bacterial cells was examined with a BacLight bacterial viability kit (Molecular Probes, Eugene, OR) as described previously (10).
For the observation of cell size and morphology, phase-contrast images were captured using an Evolution VF charge-coupled device (CCD) camera and the Image-Pro Express software package (both from Media Cybernetics, Inc., Silver Spring, MD). The dimensions of the bacteria were measured using VayTek Image software (VayTek, Inc., Fairfield, IA). For each determination, a total of 200 bacteria from 20 randomly selected fields were examined. The cell shapes were classified into three groups: rod, coccoid, and other (nonrod, noncoccoid) aberrant shapes (15).
For electron microscopy, the bacteria for transmission electron microscopy (TEM) were washed in 0.1 M phosphate buffer, fixed in the same buffer containing 2.5% glutaraldehyde and 4% paraformaldehyde, postfixed in 1% osmium tetroxide, dehydrated, and embedded in epoxy resin. Ultrathin sections were double stained with uranyl acetate and lead citrate and were observed under a Philips CM100 transmission electron microscope (Philips, Eindhoven, the Netherlands) (15).
Construction of deletion mutants.A dacB (VP2468, encoding d-alanyl-d-alanine carboxypeptidase/endopeptidase) (18, 25) deletion mutant (Table 1) was constructed by following the published method (26). PCR-amplified DNA fragments that were used to construct the in-frame deletion mutation of dacB were generated by means of overlap PCR as described previously (26). Two DNA fragments were amplified by PCR with V. parahaemolyticus 1137 chromosomal DNA as the template: one with the primers 1 and 2 and the other with primers 3 and 4 (Table 2). These two amplified fragments were then used as templates for a second PCR with primers 1 and 4, resulting in the construction of a fragment with a deletion in the dacB gene. The fragment containing the deletion was purified, cloned into the pGEM-T Easy vector, and transformed into E. coli XL1-Blue by following the manufacturer's protocol (Promega Co., Madison, WI). The inserted sequence was verified by sequencing using the sequencing primers (Seq 1F, Seq 1R, Seq 2F, and Seq 2R) (Table 2). This fragment was then removed from the pGEM-T Easy vector by digestion using SacI and SphI and was cloned into a suicide vector, pDS132, which contained the chloramphenicol resistance gene and the sacB gene, conferring sensitivity to sucrose. The resulting plasmid, pDS132ΔdacB, was introduced into E. coli SM10λpir, which was then mated with V. parahaemolyticus 1137. Thiosulfate-citrate-bile-sucrose (TCBS) agar containing chloramphenicol was used to screen the V. parahaemolyticus cells containing the inserted plasmid. The V. parahaemolyticus clones were isolated and cultured in Luria-Bertani (LB) broth (Becton Dickinson) supplemented with 2% NaCl and chloramphenicol. The culture containing the pDS132ΔdacB plasmid was incubated at 37°C for 3 h in the LB broth containing 2% NaCl and was then plated on an LB agar plate containing 2% NaCl and 10% sucrose. Isolated colonies that were unable to grow on LB plates containing chloramphenicol were selected, and the homologous recombination of the gene containing the deletion was verified by PCR using primers 1+ and 4+ (Table 2).
Primers used in cloning experiments of this study
The gene with the deletion was demonstrated by PCR using primers 1 and 4 (Table 2), and amplicons of 2,338 and 1,163 bp were detected for the parent strain (strain 1137) and the mutant (strain Δ2468), respectively. The insertion into the chromosome of the fragment containing the VP2468 gene deletion was demonstrated by PCR using primers 1+ and 4+, targeting the flanking sequences of the gene. Amplicons of 3,338 and 2,163 bp were found for the parent and mutant strains, respectively. Sequencing results obtained using primers Seq 1 and Seq 2 also verified the nucleotide sequences of the fragment containing the VP2468 mutant and the native VP2468 gene (27).
Sequencing service was provided by Genomics BioSci & Tech, Inc., Taipei, Taiwan, using Sanger's method with an Applied Biosystems 3730 analyzer.
Construction of complementary strains.The entire length of dacB was amplified by PCR with V. parahaemolyticus 1137 chromosomal DNA as the template by using primers C and 4 (Table 2), cloned into the pGEM-T Easy vector, and transformed into E. coli XL1-Blue. The insert fragment was restricted by SalI and ZraI and was ligated to the pSCB01 shuttle vector, which had been digested with SalI and EcoRV, to form plasmid pSCB01 dacB. Shuttle vector pSCB01 (8,123 bp) was constructed by ligating the mobRP4 fragment recovered from the HindIII-digested fragments of pDS132 to pBR328 that had been digested by HindIII (Table 1). The pSCB01 dacB plasmid, containing the entire length of dacB, was propagated in E. coli SM10λpir and was conjugated to V. parahaemolyticus strain Δ2468 to generate a complementary strain, which was selected by chloramphenicol resistance (Table 1). The presence of dacB in the complementary strain Δ2468C was verified by PCR using primers F and R, targeting flanking sequences of the gene with the deletion (Table 2).
A fragment (2,025 bp) that was amplified by primers C and 4 contained the entire VP2468 gene, which was cloned and used as the complementary gene in strain Δ2468C. The presence of this complementary gene was verified by PCR using primers F and R, and an amplicon of 489 bp was formed only in cells that contained the entire dacB gene in the wild-type strain or the complementary strain containing plasmid pSCB01.
RT-qPCR.The expression of 17 cell wall-associated genes (see Table 3) was determined by real-time quantitative reverse transcription-PCR (RT-qPCR) (26). Briefly, cells were broken by TRIzol reagent (Invitrogen, United Kingdom), and RNA samples were extracted by using an RNApure kit (Genesis Biotech Inc., Taipei, Taiwan) according to the manufacturer's instructions. RNA samples were first treated with DNase I (TaKaRa Bio Inc., Shiga, Japan) and then reverse transcribed by using SuperScript III First-Strand Synthesis SuperMix (Invitrogen, United Kingdom) according to the manufacturer's instructions. Primers (see Table S1 in the supplemental material) were designed using Primer Express software (Perkin-Elmer Applied Biosystems, Foster City, CA), and 16S rRNA was used as the internal control. Real-time PCR was performed using the ABI Prism 7300 sequence detection system (Perkin-Elmer Applied Biosystems) with SYBR green PCR Master Mix and RT-PCR reagents. All the data were normalized to the 16S gene expression levels of the culture at each time point, and the normalized values for each gene were compared. The expression of each gene was compared to its expression in the exponential phase, and the relative values were expressed as the fold change according to the manufacturer's instructions (Perkin-Elmer Applied Biosystems). The quality of the RNA samples and the quantification protocols adopted here were monitored by previously described methods (26).
Statistical analysis.Replicate experiments for the observation of cell morphology and gene expression were performed. The statistical significance of data was assessed by Student's t test or analysis of variance (ANOVA) with Duncan's multiple test at a significance level (α) of 0.05, using SPSS for Windows, version 11.0 (SPSS Inc., Chicago, IL).
RESULTS AND DISCUSSION
Observation of aberrantly shaped cells.The ultrastructures of coccoid VBNC cells of several Vibrio species (4, 9, 28) have been reported, but irregularly shaped intermediates are not often identified. During the induction of the VBNC state of Vibrio cholerae cells in seawater at a low temperature, the curved rods in the exponential phase are transformed to irregular rods within 30 to 60 days; afterwards, they become coccoid cells (29). In our earlier investigation, the morphological changes of V. parahaemolyticus in the first week of induction of the VBNC state were examined, and various aberrantly shaped cells were observed within the first 3 days (15). In the present study, the morphological changes of this pathogen at 0, 6, 12, 18, and 24 h were observed under VBNC-inducing conditions. A large proportion of non-rod-shaped cells were observed in 12 to 24 h, including approximately 5% coccoid cells and 29% cells with other aberrant shapes (Fig. 1A; see also Fig. 3). These aberrantly shaped cells were viable, as determined with the BacLight bacterial viability kit (data not shown). TEM examination revealed bulges (budding) at various stages of formation (Fig. 1B and C). Figure 1C shows the outgrowth of a bulge that was covered by a thin, loosened cell wall. Other aberrantly shaped cells in Fig. 1A may be formed by the outgrowth of bulges at various locations on the cells. Cell bulges have been demonstrated to be covered by loosened, flexible cell walls in the induction of VBNC V. parahaemolyticus cells in modified V-5 minimal (MV-5) medium supplemented with 2% NaCl without the carbon source, mannitol (14).
Formation of aberrantly shaped cells in V. parahaemolyticus 1137 incubated at 4°C in MMS–0.5% NaCl for 12 h. (A) Cells observed under a light microscope. Bar, 5 μm. (B and C) Cells observed under a transmission electron microscope. Arrows indicate the bulging of aberrantly shaped cells. Bars, 0.2 μm.
Influence of antibiotics on cell shapes.PBPs and cytoskeleton proteins determine the shapes of bacterial cells under normal growing conditions (16), and mutation of these PBP genes (30) or inhibition of their activities by particular antibiotics frequently causes the formation of irregularly shaped cells (31, 32). Various β-lactam antibiotics and d-cycloserine are commonly used to interfere with the synthesis of bacterial cell walls. β-Lactam antibiotics, such as cephalexin, ampicillin, penicillin G, or cephalosporin C, have been demonstrated to be sensitive to various PBPs that are involved in the formation of peptidoglycan strands (19). d-Cycloserine is a d-alanine analog that influences the biosynthesis of bacterial peptidoglycan by inhibiting d-Ala-d-Ala ligase and alanine racemase (31).
The inhibition of the growth of V. parahaemolyticus 1137 by antibiotics was assayed in TSB–3% NaCl at 37°C. The results indicated that d-cycloserine and cephalosporin C at 50 and 10 μg/ml, respectively, did not markedly inhibit the growth (see Fig. S1 in the supplemental material) or alter the cell shape (data not shown) of this strain. The effects of these two antibiotics at these predetermined subinhibitory concentrations on the cell morphology of V. parahaemolyticus 1137 cultured in MMS–0.5% NaCl at 4°C for 12 h were examined. The average lengths of 200 cells in the control, d-cycloserine, and cephalosporin C groups and in the exponential phase were 4.06, 4.06, 3.71, and 4.14 μm, respectively, and did not differ significantly from each other. About 200 cells in each group were examined, and the results showed that elongated cells (length, >10 μm) were not found in the exponential phase, while they were found in about 2.1, 2.7, and 0.8% of the control, d-cycloserine, and cephalosporin C groups, respectively. The proportions of coccoid and other aberrantly shaped cells in the d-cycloserine group were similar to those in the control group, while those in the cephalosporin C group were significantly lower (Fig. 2).
Effects of antibiotics on the formation of aberrantly shaped cells in V. parahaemolyticus 1137. Cultures were incubated at 4°C for 12 h in MMS with 0.5% NaCl, either without antibiotics (control) or with d-cycloserine (50 μg/ml) or cephalosporin C (10 μg/ml), and cell shapes were determined by microscopy. The cell shapes were classified into three groups: rod, coccoid, and other (nonrod, noncoccoid) aberrant shapes (15). Open bars, coccoid cells; hatched bars, aberrantly shaped cells; stippled bars, total of coccoid and aberrantly shaped cells. The values for the antibiotic-supplemented groups were compared with the corresponding values for the control group. Asterisks indicate significantly different values (P < 0.05). For each determination, a total of 200 bacteria from 20 randomly selected fields were examined.
The results presented above suggest that the cell walls of a small proportion of V. parahaemolyticus cells under VBNC-inducing conditions are synthesized to form the elongated/filamentous morphology, and that it is not significantly increased by the addition of these antibiotics. The involvement of the cytoskeleton protein MreB in cylindrical cell growth (33) and of FtsZ in cell division (19) may be conserved to form cylindrical cells or altered to form elongated and filamentous cells.
Since the formation of aberrantly shaped V. parahaemolyticus cells under the present conditions was not increased by these antibiotics that influence cell wall synthesis (Fig. 2), it may involve a mechanism different from those of PBPs and cytoskeleton proteins, which normally function to determine the shape of growing cells (31, 32). In E. coli, the cell wall synthesis machinery may not be involved in the initial step of branching of the cells that is induced by their special physiological state (23). E. coli cultured in M9 minimal medium supplemented with sodium acetate and Casamino Acids formed branching cells more frequently than E. coli cultured in a rich medium (Luria broth medium containing 0.2% glucose), and bulges formed randomly along the cell surface under high-branching conditions. The branching of cells is induced by their physiological state rather than by the particular constituents of the medium (23), suggesting that an unknown mechanism in the special physiological state initiates the budding and branching of E. coli cells.
Effect of salinity on aberrantly shaped cells.According to the physical model proposed by Huang et al. (24), damage to peptidoglycan leads to the formation of bulges and the bending of rod-shaped Gram-negative bacteria. In this model, the comparatively unstretchable glycan subunits and the comparatively stretchable peptide cross-links are represented as springs that expand from their relaxed lengths to balance the outward force (turgor pressure) generated by cellular osmotic pressure. Based on this model, increasing the cellular turgor pressure should promote the formation of bulges and cell bending in the presence of localized cell wall damage or relaxation of the peptidoglycan.
To examine the effect of cellular turgor pressure on the formation of aberrantly shaped V. parahaemolyticus cells under the present VBNC-inducing conditions, the cultures were incubated at 4°C for 12 h in MMS medium containing 0.5 to 1.5% NaCl to generate different cellular turgor pressures, and the coccoid and other aberrantly shaped cells were counted. Since V. parahaemolyticus cells are regularly cultured in a medium containing 3% NaCl, the media containing 0.5 to 1.5% NaCl were hypotonic, and the medium containing 0.5% NaCl was expected to yield the highest turgor pressure in the cells. The results show that the proportions of coccoid cells did not differ among the experimental groups, but the number of other aberrantly shaped cells and the total number of coccoid and other aberrantly shaped cells were significantly higher in the medium that contained 0.5% NaCl than in those that contained 1.0 or 1.5% NaCl (P, 0.00) (Fig. 3). This experiment indicates that increasing the cellular turgor pressure promotes the formation of aberrantly shaped cells, and it suggests that localized cell wall damage or relaxation of the peptidoglycan occurs under the VBNC-inducing conditions used here.
Effect of salinity on the formation of aberrantly shaped cells in V. parahaemolyticus 1137. Cultures were suspended in MMS with 0.5, 1.0, or 1.5% NaCl and were incubated at 4°C for 12 h. Coccoid cells (open bars) and aberrantly shaped cells (hatched bars) were counted under a light microscope. Their total was also determined (stippled bars). The values obtained at different salt concentrations were compared. Asterisks indicate significantly different values (P < 0.05). For each determination, a total of 200 bacteria from 20 randomly selected fields were examined.
The peptidoglycan of bacterial cells is in a dynamic state under the influence of polymerizing and hydrolytic enzymes (34), and the damage to, or relaxation of, the cell wall may involve unbalanced hydrolysis of the existing cell wall material and polymerization of the hydrolyzed or nascent synthesized wall materials. The physiological responses of V. parahaemolyticus cells under hypotonic VBNC-inducing conditions (0.5% NaCl) may affect the equilibrium of the dynamic state of peptidoglycan. Mechanical stress in the bacterial cell wall has been demonstrated by a mechanochemical approach to control the dynamic turnover of cell wall material and to determine cell shape in a growing-cell model (35, 36).
Behavior of genes associated with cell wall synthesis in the formation of aberrantly shaped cells.The bacterial cell wall may be damaged or relaxed by disturbing the expression of polymerizing and hydrolytic enzymes. In a search of the genome of V. parahaemolyticus RIMD2210633 (27), 17 genes associated with cell wall synthesis were identified (Table 3). Some of them were annotated as associated with cell wall synthesis, including VP2468, a d-alanyl-d-alanine carboxypeptidase/endopeptidase gene. VP2369 encodes a murein transglycosylase, which is responsible for the synthesis of the bacterial cell wall or plays a role in the recycling of muropeptides during cell elongation and/or cell division (37, 38). VP0457 encodes phospho-N-acetylmuramoyl-pentapeptide transferase, which is responsible for the first step of lipid cycle reactions in the biosynthesis of peptidoglycan (39). VP0564 encodes a non-penicillin-sensitive murein endopeptidase, which is responsible for cleaving the peptide bonds between neighboring strands in mature murein (40). VP2658 encodes a UDP-N-acetylglucosamine 1-carboxyvinyltransferase, which adds enolpyruvyl to UDP-N-acetylglucosamine and participates in the biogenesis/degradation of the cell wall (41). Several PBP genes (VP0454, VP0722, VP2497, VP2751, and VPA0517) in the genome of V. parahaemolyticus RIMD2210633 have been identified and selected in this study (27). BLAST analysis of the MEROPS peptidase database (http://merops.sanger.ac.uk/index.shtml) revealed several other homologous peptidase genes in the RIMD2210633 genome (VP0535, VP0548, VP1385, VP1485, VP2463, VP2471, and VPA1649), and some of these peptidases are membrane bound.
Selected genes associated with cell wall synthesis in V. parahaemolyticus
During the induction of the VBNC state of V. parahaemolyticus, the transcription levels of some cytoskeleton genes remained relatively high initially and declined thereafter (15). In the present study, a large proportion of coccoid and other aberrantly shaped cells were observed in 12 to 24 h (Fig. 1A and 3). We postulated that intensive transcriptional activities of the cell wall-associated genes, including the cytoskeleton genes, occurred during the initial stage (12 to 24 h) of induction of the VBNC state and declined with prolonged incubation. Determination of gene expression by RT-qPCR revealed 0.24- to 18.59-fold increases in the expression of these target genes at 12 h relative to those before incubation at a low temperature. The expression of all of these genes, except for VP0457, VPA0517, and VP2468, was increased 1- to 4-fold. The expression of VP0457 and VPA0517 was increased only about 0.2- to 0.8-fold, respectively, while VP2468 (dacB) was the most highly activated gene, with a ∼18-fold increase (Fig. 4). The genes whose expression was enhanced by VBNC-inducing conditions, except for VP0535, VP2751, and VPA1649, also exhibited enhanced expression in cold-shocked V. parahaemolyticus cells, as determined by a microarray (42). In the VBNC state of V. cholerae, the expression of several cell wall/envelope-associated genes was also enhanced; these were VC0416 (mreC), VC0417 (mreD), VC0602 (mrcB), VC2403 (murD), and VCA0572 (ddlA) (43).
Expression of genes associated with cell wall synthesis in V. parahaemolyticus 1137. Strain 1137 was cultured in MMS–0.5% NaCl and was incubated at 4°C. The expression of target genes at 12 h relative to their expression at 0 h was determined by RT-qPCR.
The VP2468 (dacB) gene encodes a theoretical protein with 473 amino acid residues and a molecular mass of 52.7 kDa, which is functionally a d-alanyl-d-alanine carboxypeptidase (removing C-terminal d-alanyl residues from sugar-peptide cell wall precursors)/endopeptidase (EC 3.4.16.4). This enzyme is one of the LMW PBPs (44). The enhanced expression of this enzyme may disturb the equilibrium of the dynamic state of peptidoglycan, resulting in hydrolysis of the peptide side chains and damage to, and relaxation of, the cell wall. This notion is supported by the fact that addition of cephalosporin C, in order to interfere with the function of this enzyme, reduced the frequency of formation of aberrantly shaped cells (Fig. 2).
Response of the dacB mutant to VBNC-inducing conditions.The role of the VP2468 (dacB) gene was further demonstrated by genetic mutation and complementation. A dacB deletion mutant (strain Δ2468) and its complementary strain (strain Δ2468C) were constructed (Table 1), and the changes in the cell morphologies of these strains under VBNC-inducing conditions were examined.
The deletion and complementation of the VP2468 gene did not significantly influence the growth of strain Δ2468, Δ2468C, or Δ2468V in TSB–3% NaCl at 37°C. The presence of chloramphenicol in the culture medium to maintain the presence of the plasmid did not affect the growth of strains Δ2468C and Δ2468V. Nevertheless, the formation of aberrantly shaped cells was substantially affected (Fig. 5). The proportion of aberrantly shaped cells was reduced from 32.8% in the wild-type strain to 11.7% in strain Δ2468, and this decrease was reversed by the presence of the complementary gene in strain Δ2468C. The presence of cloning vector pSCB01 did not affect the proportion of aberrantly shaped cells (Fig. 5).
Formation of aberrantly shaped cells in the V. parahaemolyticus wild-type strain 1137, Δ2468 (dacB deletion mutant), Δ2468C (the complementary strain with dacB restored), and Δ2468V (dacB deletion mutant containing pSCB01). Cultures were suspended in MMS with 0.5% NaCl and were incubated at 4°C for 12 h, and aberrantly shaped cells were counted under a light microscope. The values for the mutant and complemented strains were compared to that for the wild type. Asterisks indicate significant differences (P < 0.05).
Microscopic observation has revealed that bulges occur in different locations of the variously shaped cells (14, 15), while dramatic changes to, and remodeling of, the MreB cytoskeleton protein may transport the cell wall synthesis machinery to various locations (33, 45). The VP2468 enzyme or other enzymes with increased expression levels may be transported to unspecified locations, and they may function either independently or with the assistance of the mechanical force generated by the hypotonic medium (35, 36) to alter the equilibrium of the stability of the cell wall, damaging or relaxing the cell wall and initiating the formation of bulges.
In conclusion, aberrantly shaped cells were formed in the initial stage of induction of the VBNC state in V. parahaemolyticus in MMS–0.5% NaCl at 4°C, and the association of this phenomenon with the VP2468 (dacB) gene was demonstrated by molecular techniques. Further investigation, including the collection of time course data for gene expression, is needed to clarify the details of the metabolic functioning of this gene in this phenomenon.
ACKNOWLEDGMENTS
We thank the National Science Council of the Republic of China for financial support of this research under contract NSC100-2313-B-031-001-MY3.
Ted Knoy is appreciated for editorial assistance.
FOOTNOTES
- Received 27 May 2013.
- Accepted 9 September 2013.
- Accepted manuscript posted online 20 September 2013.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.01723-13.
- Copyright © 2013, American Society for Microbiology. All Rights Reserved.
