ABSTRACT
Vibrio parahaemolyticus is a common marine food-borne enteropathogen. In this study, we examined the antioxidative activity, growth, biofilm formation, and cell mobility of an oxyR deletion mutant and its genetically complementary strain of V. parahaemolyticus. oxyR is the regulator of catalase and ahpC genes. Protection against extrinsic H2O2 and against the organic peroxides cumene hydroperoxide and tert-butyl hydroperoxide was weaker in the deletion mutant than in its parent strain. Expression of the major functional antioxidative genes, ahpC1 and VPA1418, was markedly decreased in the oxyR mutant. Growth of this mutant on agar medium was significantly inhibited by autoclaved 0.25% glucose and by 0.25% dipotassium hydrogen phosphate, 0.5% monosaccharides (glucose, galactose, xylose, and arabinose), or 114.8 mM phosphates. The inhibition of the growth of this oxyR mutant by extrinsic peroxides, autoclaved sugars, and phosphates was eliminated by the complementary oxyR gene or by the addition of catalase to the autoclaved medium, while no inhibition of growth was observed when filter-sterilized sugars were used. The formation of biofilm and swimming mobility were significantly inhibited in the oxyR mutant relative to that in the wild-type strain. This investigation demonstrates the antioxidative function of oxyR in V. parahaemolyticus and its possible roles in biofilm formation, cell mobility, and the protection of growth in heated rich medium.
INTRODUCTION
Vibrio parahaemolyticus is a halophilic Gram-negative bacterium that is commonly associated with food-borne gastroenteritis (1), and it has exhibited global significance since the occurrence of pandemic O3:K6 strains in 1996 (2).
The incomplete reduction of oxygen during aerobic metabolism or by exposure to metals, redox-active chemicals, or some environmental stresses produces various reactive oxygen species (ROS) in bacteria (3–5). ROS can damage all cellular components, including protein, DNA, and membrane lipids (6, 7). Therefore, antioxidative activity is required by pathogenic bacteria for their successful growth and survival under environmental stresses and is sometimes associated with their virulence (8), whereas characteristic antioxidative functions have been demonstrated in V. parahaemolyticus (9, 10).
Several common antioxidative factors are used to scavenge ROS, including superoxide dismutases (SOD), catalases, and alkyl hydroperoxide reductase subunit C's (AhpC) (11). The expressions of catalase and ahpC genes are usually regulated by OxyR (12), which is a redox-sensitive transcriptional regulator of the LysR family in Escherichia coli, Salmonella spp., and other bacteria (13–17). OxyR also participates in pathogenesis by promoting biofilm formation, fimbrial expression, and mucosal colonization in pathogenic bacteria (18); nevertheless, the mechanism of oxidative stress defense in these phenomena is not clear (19).
The function of oxyR has been examined in a few Vibrio species but not in V. parahaemolyticus. The effect of oxyR on the survival of bacteria and on their viable but nonculturable state has been investigated in Vibrio vulnificus (20–22) and in Vibrio harveyi (23). The oxyR deletion mutant of Vibrio cholerae is sensitive to H2O2, causes defective growth in a rich medium, and weakens intestinal colonization in zebrafish (24). In this study, the roles of oxyR (VP2752) in the antioxidative function, growth, biofilm formation, and mobility of V. parahaemolyticus were characterized using an oxyR deletion mutant and its genetically complementary strain.
MATERIALS AND METHODS
Bacterial strains and culture conditions.V. parahaemolyticus strain KX-V231 (Kanagawa phenomenon positive, serotype O3:K6), which was isolated in Thailand from a clinical specimen, was used in this work (Table 1). It was stored frozen at −85°C in beads in Microbank cryovials (Pro-Lab Diagnostics, Austin, TX, USA). It was cultured at 37°C on tryptic soy agar or broth (Becton-Dickinson Diagnostic Systems, Sparks, MD, USA) without glucose and phosphate (agar and broth herein abbreviated TSANG and TSBNG, respectively) and was supplemented with 3% sodium chloride, and the pH was adjusted to 7.3. The broth cultures were incubated at 37°C statically or shaken at 160 rpm.
Bacterial strains and plasmids used in this study
Chloramphenicol (final concentration of 6 μg/ml) or chloramphenicol-ampicillin (20 μg/ml and 50 μg/ml, respectively) was added to the medium if required for the cultivation of V. parahaemolyticus or Escherichia coli strains, respectively.
Bacterial growth in the broth medium was monitored by determining the absorbance of the culture at 590 nm using an MRX II microplate reader (Dynex Technologies, Chantilly, VA, USA) or by the standard plate count method using tryptic soy agar (TSA; Becton-Dickinson)-3% NaCl medium, which contained 0.25% glucose and 0.25% dipotassium hydrogen phosphate (K2HPO4), following incubation at 37°C for 18 h.
Construction of deletion mutants.oxyR deletion mutants were constructed by following previously published methods (10, 25). To construct the ΔVPA0305 mutant strain, two DNA fragments were amplified by PCR with V. parahaemolyticus KX-V231 chromosomal DNA as the template—one with the primer pair oxyR-1 sacI and oxyR-2 and the other with the primer pair oxyR-3 and oxyR-4 sphI (Table 2). These two amplified fragments were then used as the templates for a second PCR with the primers oxyR-1 sacI and oxyR-4 sphI, which resulted in the construction of a fragment with a deletion in the oxyR gene. Such a fragment, which contained the deletion, was purified and cloned into the pGEM-T Easy vector and was transformed into E. coli XL1-Blue by following the protocol of the manufacturer (Promega Co., Madison, WI, USA). The inserted sequence was verified by sequencing. 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-resistant gene and the sacB gene, conferring sensitivity to sucrose. The resulting plasmid, pSCA05 (pDS132 with oxyR deleted), was introduced into E. coli SM10-pir and was then mated with V. parahaemolyticus strain KX-V231. Thiosulfate-citrate-bile-sucrose (TCBS) agar, which contained 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; Becton-Dickinson) broth that had been supplemented with 2% NaCl and chloramphenicol. DNA was extracted from these cultures, and the inserted sequence was detected by PCR using the oxyR-1 sacI and oxyR-4 sphI primers. The culture that contained the pDS132-oxyR deletion plasmid was incubated at 37°C for 3 h in the LB broth that contained 2% NaCl and was then plated on an LB agar plate that contained 2% NaCl and 10% sucrose. The isolated colonies that were unable to grow on the LB agar plate that contained chloramphenicol were selected, and the homologous recombination of the deleted fragment was confirmed by PCR using primers oxyR-0 and oxyR-5 (Table 2).
Primers used in the cloning experiments in this study
Sequencing services were 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 oxyR, 1,538 bp, was amplified by PCR with V. parahaemolyticus KX-V231 chromosomal DNA as the template using primer pairs oxyR comp-F and oxyR comp-R (Table 2). The amplicon was ligated to the pGEM-T Easy vector and transformed into the E. coli XL1-Blue strain. The complete gene sequence was cut with SalI and SphI and was ligated to the shuttle vector pSCB01, which had been digested with the same enzymes (10). The plasmid pSCB05, containing the complete sequence of oxyR, was propagated in E. coli SM10 λ-pir and was conjugated to the corresponding oxyR mutant to generate a complementary strain, which was selected by its chloramphenicol resistance (Table 1). The presence of the entire length of the oxyR gene in these strains was verified by PCR (Table 2).
Determination of antioxidative activities.The inhibition by H2O2 (Santoku Chemical Industries, Tokyo, Japan), tert-butyl hydroperoxide (t-BOOH; Tokyo Kasei Chemicals, Tokyo, Japan), and cumene hydroperoxide (cumene; Alfa Aesar, Ward Hill, MA, USA) of the growth of different strains of V. parahaemolyticus was assayed using the disc diffusion method (26). Cultures of different V. parahaemolyticus strains were spread on Mueller-Hinton agar plates (Becton-Dickinson), on which paper discs (6 mm; Creative Media Products, Taiwan) that had absorbed 10 μl of 0.88 M (3%, wt/vol) H2O2, 0.20 M (3%, wt/vol) cumene, or 0.22 M (2%, wt/vol) t-BOOH were placed. These concentrations were modified from those used in other studies (26, 27) in the preliminary experiment. The sizes of the inhibition zones were measured following incubation at 37°C for 18 h.
The growth of V. parahaemolyticus strains against various extrinsic peroxides was also determined in tryptic soy broth (TSB)-3% NaCl that contained 0.25% glucose and 0.25% K2HPO4 and that was incubated statically at 37°C for 8 h.
Effect of sugars and phosphates on bacterial growth.Different bacterial strains were cultured in TSBNG-3% NaCl medium at 37°C until the absorbance at 590 nm was about 0.5; the cultures were then diluted with the same medium, and 10-μl aliquots of the diluted suspensions were dropped on TSANG-3% NaCl, TSA-3% NaCl, and LB-3% NaCl agar media (Becton-Dickinson) and also on TSANG-3% NaCl medium, which contained various monosaccharides (d-glucose, d-galactose, d-xylose, l-arabinose) or phosphates at pH 7.3. Growth was observed following 16 h of incubation at 37°C (28).
The diluted cultures were also inoculated into 200 μl of TSBNG-3% NaCl medium that was supplemented with 0.5% of autoclaved monosaccharides and were incubated statically at 37°C. In control experiments, filter-sterilized (0.22-μm pore size) sugars were added to the TSBNG-3% NaCl medium, or 30 U catalase was added to the broth medium that contained autoclaved sugars. Bacterial growth was determined at different intervals.
Assays of biofilm formation and swimming mobility.Bacterial cultures in TSBNG-3% NaCl were diluted with TSBNG-3% NaCl, TSB-3% NaCl, or minimal mineral salts (MMS)-3% NaCl (29) to an absorbance of 0.06 at 590 nm; 200 μl of this diluted culture was dispensed into the wells of polystyrene microtiter plates. The absorbances of the cultures were measured after incubation at 37°C for 24 h. The spent culture media of these broth cultures were discarded, 220 μl of 0.1% (wt/vol) crystal violet was added to each well, and the plate was incubated for 20 min. The dye solution was removed, and the wells were washed three times with distilled water. The dye remaining in each well was extracted using 200 μl of ethanol-acetone (80:20, vol/vol) for 10 min, and absorbance at 600 nm was measured (30).
To assay their swimming ability, various strains of V. parahaemolyticus were cultured in an LB medium that contained 3% NaCl and 0.3% agar and were incubated at 37°C for 15 h; the sizes of the colonies were then measured (31).
RT-qPCR.The levels of expression of genes (Table 5) were determined in the wild-type and oxyR mutant strains of V. parahaemolyticus using real-time reverse transcription-quantitative PCR (RT-qPCR) as described in our previous publications (9, 10). Briefly, bacterial strains were cultivated statically in TSB-3% NaCl at 37°C, and the cultures in the exponential phase were challenged with 30 μM cumene for 1.5 h. Bacterial cells were harvested by centrifugation and broken by TRIzol reagent (Invitrogen, United Kingdom), and RNA samples were extracted using an RNApure kit (Genesis Biotech Inc., Taipei, Taiwan) by following the manufacturer's instructions. RNA samples were treated with DNase I (TaKaRa Bio Inc., Shiga, Japan) and were then reverse transcribed using a SuperScript III first-strand synthesis supermix (Invitrogen, United Kingdom) according to the instructions of the manufacturer. Primers (Table 3) were designed using the Primer Express Sequence Editor (http://www.fr33.net/seqedit.php) and the Oligo Calculator (http://www.sciencelauncher.com/oligocalc.html), and the 16S rRNA gene was used as the internal control. Real-time PCR was performed using the StepOnePlus real-time PCR system, v.2.0 (Applied Biosystems), with an IQ2 SYBR green fast qPCR system master mix with high ROX (DBU-008) and RT-PCR reagents. All of the data were normalized with the 16S rRNA gene expression levels of the culture at each time point (Applied Biosystems). The expression of each target gene of the experimental group relative to the expression of the corresponding gene of the control (wild-type strain without cumene challenge) is presented. Recombinant plasmids for the ahpC1 and VPA1418 genes were used as calibration standards. The quality of the RNA samples and of the quantification protocols that were adopted for this study was evaluated by previously described methods (9, 10).
Primers used in RT-qPCR experiment
Statistical analysis.Triplicate experiments were performed in this study. The data were analyzed by performing the t test or analysis of variance (ANOVA) with Duncan's multiple-range test and the t test at a significance level (α) of 0.05 using SPSS for Windows v.11.0 (SPSS Inc., Chicago, IL, USA).
RESULTS
Antioxidative activities.The growth of the ΔoxyR mutant strain of V. parahaemolyticus in broth medium (TSB-3% NaCl) that contained 0.25% glucose and 0.25% K2HPO4 in a static culture at 37°C for 8 h did not differ significantly from that of the wild-type KX-V231 strain, and it was not affected by the presence of a cloning vector or complementary oxyR gene (Fig. 1A).
Growth of V. parahaemolyticus strains in TSB-3% NaCl medium under challenge by peroxides in static culture at 37°C. The culture medium contained autoclaved 0.25% glucose and 0.25% K2HPO4, to which different peroxides were added. (A) Control without extrinsic peroxide; (B) 175 μM H2O2; (C) 50 μM cumene; (D) 160 μM t-BOOH.
When these strains were cultured in the same medium with shaking for 8 h, the growth of the cultures reached a maximum absorbance of about 3 after 4 h of incubation, and no significant difference was observed between the strains (see Fig. S1A in the supplemental material). These results indicated that the bacterial growth of this species was higher in shaken culture than that in static culture; nevertheless, mutation of the oxyR gene did not harm its growth in the broth medium. However, plate counting revealed that the ΔoxyR mutant strain did not form a culturable colony on the TSA-3% NaCl agar medium that contained autoclaved 0.25% glucose and 0.25% K2HPO4 (see Fig. S1B in the supplemental material), while the colony-forming ability of this ΔoxyR mutant strain was fully recovered in the presence of the complementary oxyR gene (Fig. S1B).
The antioxidative activities of the KX-V231, the ΔoxyR mutant, KX-V231/V (parent strain containing cloning vector pSCB01), and the ΔoxyR/C mutant (a mutant complemented with the oxyR gene) strains against extrinsic H2O2, cumene, and t-BOOH were assayed in broth medium (TSB-3% NaCl with 0.25% glucose and 0.25% K2HPO4) in static culture at 37°C. In the presence of 175 μM H2O2 or 50 μM cumene, the exponential phase of the ΔoxyR culture was delayed with a lag of about 5 h (Fig. 1B and C). When 160 μM t-BOOH was added to the medium, the growth of the ΔoxyR strain was slower than that of the wild-type strain in the first 2 h of incubation (Fig. 1D). The presence of the complementary oxyR gene eliminated the inhibition of growth by extrinsic peroxides in the ΔoxyR strain (Fig. 1B, C, and D).
The antioxidative activities of the KX-V231, ΔoxyR, and ΔoxyR/C strains against H2O2, cumene, and t-BOOH were also assayed using the disc diffusion method, and the results obtained revealed that antioxidative activities against these peroxides were substantially reduced in the oxyR mutant and were significantly recovered in the complemented ΔoxyR/C strain (Table 4).
Susceptibilities of V. parahaemolyticus strains to different peroxides as determined by the disc diffusion method
Expression of the ahpC1, VPA1418, and oxyR genes.Expression of the ahpC1, VPA1418, and oxyR genes was determined in the wild-type and oxyR mutant strains of V. parahaemolyticus with/without the challenge of cumene (Table 5). The ahpC1 gene (9, 10) and katE-homologous gene VPA1418 (32) are the major functional antioxidative genes in the exponential phase of this pathogen, and their levels of expression were markedly increased in the wild-type strain under the challenge of cumene. In the ΔoxyR deletion mutant strain, levels of expression of these two genes were markedly lower than those in the wild type and were not significantly related to the challenge of cumene (Table 5). Expression of oxyR was not significantly enhanced by the challenge of cumene in the wild-type strain (Table 5). These results demonstrated that expression of these major antioxidative genes, ahpC1 and VPA1418, is probably enhanced by the presence of extrinsic peroxide and regulated by oxyR.
Expression of major antioxidative genes and oxyR in wild-type and oxyR mutant strains of V. parahaemolyticus
Effect of autoclaved sugars on bacterial growth.The growth of the ΔoxyR mutant strain was totally inhibited when the strain was cultured on agar medium (TSA-3% NaCl) that contained autoclaved 0.25% glucose and 0.25% K2HPO4, but such inhibition of growth was not observed in TSANG-3% NaCl or LB-3% NaCl that did not contain autoclaved glucose or K2HPO4 (Fig. 2A). The presence of the complementary oxyR gene eliminated the inhibition of growth in the ΔoxyR mutant on TSA-3% NaCl (Fig. 2A). The presence of 0.5% autoclaved monosaccharides, such as d-glucose, d-galactose, l-arabinose, or d-xylose, inhibited the growth of the ΔoxyR mutant on agar medium (Fig. 2B), but this inhibition was not observed in the ΔoxyR/C strain (Fig. 2B).
Growth of V. parahaemolyticus strains on agar media that contained autoclaved monosaccharides. Different dilutions (on a log scale, starting at about 7 log CFU/ml) of 10-μl bacterial suspensions of the wild type (KX-V231), ΔoxyR mutant, and oxyR complementary strain (ΔoxyR/C) were spotted on various agar media and incubated at 37°C for 16 h. (A) Growth on TSANG-3% NaCl that contained no glucose or phosphate, TSA-3% NaCl that contained 0.25% glucose and 0.25% K2HPO4, and LB-3% NaCl; (B) growth on TSANG-3% NaCl that contained 0.5% of the autoclaved monosaccharides.
The presence of 0.25% autoclaved glucose and phosphate inhibited the colony-forming ability of the ΔoxyR mutant on TSA-3% NaCl medium, but it did not affect the growth of this strain in broth medium in static culture (Fig. 1A). However, when 0.5% autoclaved monosaccharides were added to the broth medium, the growth of the ΔoxyR mutant was significantly inhibited. Glucose and galactose significantly reduced the extent of growth of the ΔoxyR mutant (Fig. 3A and B), and the five-carbon sugars (xylose and arabinose) suppressed it completely (Fig. 3C and D). The application of 0.5% filter-sterilized glucose (Fig. 3E) or the addition of 30 U of catalase to the medium that contained autoclaved glucose eliminated the inhibition of the growth of these sugars (Fig. 3F). The inhibition of growth of the ΔoxyR mutant was also not observed in the medium that contained other filter-sterilized monosaccharides (galactose, xylose, arabinose) or other autoclaved monosaccharides along with 30 U catalase (data not shown).
Growth of different V. parahaemolyticus strains in broth medium that contained autoclaved sugars. TSBNG-3% NaCl that contained 0.5% monosaccharide was inoculated and incubated at 37°C, and absorbance at 590 nm was monitored. (A) Autoclaved glucose; (B) autoclaved galactose; (C) autoclaved xylose; (D) autoclaved arabinose; (E) filtered glucose; (F) autoclaved glucose with 30 U catalase.
Effect of phosphate on bacterial growth.The presence of 57.4 and 114.8 mM autoclaved K2HPO4 and 114.8 mM autoclaved Na2HPO4 significantly inhibited the bacterial growth of the ΔoxyR mutant on agar medium, and this inhibition of growth was eliminated in the presence of the complementary oxyR gene. Adding 229.6 mM KCl to the medium did not inhibit the growth of the ΔoxyR strain (Fig. 4).
Growth of different strains of V. parahaemolyticus on medium that contained autoclaved phosphates. Different dilutions (on a log scale, starting at about 7 log CFU/ml) of 10-μl bacterial suspensions of the wild type (KX-V231), ΔoxyR mutant, and oxyR complementary strain (ΔoxyR/C) were spotted on TSANG-3% NaCl agar that contained autoclaved phosphates or chlorides and were incubated at 37°C for 16 h.
Influence of oxyR on swimming mobility and biofilm formation.The bacterial growth of and biofilm formation by various strains in TSBNG-3% NaCl, TSB-3% NaCl, and minimal mineral salts (MMS)-3% NaCl medium (33) in static or shaken culture were determined. Shaking the culture may have enhanced the significance of the oxidative-stress defense in the oxyR mutant in this study. In the static culture, the growth of the ΔoxyR mutant in rich medium (TSBNG-3% NaCl or TSB-3% NaCl) did not differ significantly from that of the wild-type strain, but it was significantly less than in the synthetic MMS-3% NaCl medium (Table 6). In the shaken 24-h culture, the growth of the ΔoxyR mutant was significantly less than that of the wild-type strain in rich medium (Table 6). The formation of biofilm by the ΔoxyR mutant was significantly less extensive than that by the wild-type strain in the static culture using MMS-3% NaCl medium and in the shaken culture using TSB-3% NaCl or MMS-3% NaCl medium (Table 6). When the formation of biofilm based on the unit bacterial growth (the ratio of the formation of biofilm to the bacterial growth) was calculated, MMS-3% NaCl medium was found to be preferable for the formation of biofilm, whereas biofilm formation by the ΔoxyR mutant in TSB-3% NaCl or MMS-3% NaCl medium in the shaken culture was significantly less than that by the wild-type strain (Table 6).
Growth and biofilm formation by wild-type and oxyR mutant V. parahaemolyticusa
On soft agar medium, the size of the colony of the wild-type strain (with a diameter of 5.3 cm) was significantly larger than that of the ΔoxyR mutant (3.9 cm), revealing that the mutant had a weaker swimming mobility (Fig. 5).
Mobility of V. parahaemolyticus strains on a soft agar plate. The wild-type KX-V231 (WT) and oxyR mutant strains were inoculated into LB-3% NaCl that contained 0.3% agar and were incubated at 37°C for 15 h, and sizes of colonies were measured. Data concerning wild-type and mutant strains were analyzed by t test, and significantly different data at a P of <0.05 are indicated by asterisks.
DISCUSSION
The putative OxyR protein (VP2752) of V. parahaemolyticus RIMD 2210633 comprises 302 amino acid residues and has a calculated molecular mass of 33,081 Da and a pI of 5.72 (34). The entire oxyR gene of V. parahaemolyticus KX-V231 that was sequenced during the construction of the oxyR mutant in this work was identical to VP2752 of V. parahaemolyticus RIMD 2210633 and is expected to have physiological functions that are similar to those of OxyR proteins in other species with high similarity scores (21, 24). This study demonstrated the antioxidative role of oxyR in V. parahaemolyticus (Table 4 and Fig. 1) in regulating the expression of the major functional ahpC1 gene and katE-homologous VPA1418 gene (Table 5) (9, 10). Phenotypic defects associated with the oxyR mutant in this study may be associated with the decreased antioxidative activity.
This study separately verified the growth-inhibiting effect of autoclaved monosaccharides and phosphates (Fig. 3) (35), which is associated with the formation of peroxides that can be scavenged by catalase or with the normal function of oxyR-regulated genes (Fig. 2 to 4).
The growth-inhibiting effect of heated sugars has been demonstrated in Vibrio (36), in Salmonella (37), and in several other bacteria (35, 38). The heating of sugars in an alkaline (pH 8.5) phosphate buffer has a bactericidal effect to which V. parahaemolyticus and other Vibrio species are particularly sensitive (38). However, the toxic products that are generated in a culture medium that contains heated sugars/phosphates have not been confirmed. The heat treatment of d-glucose has been found to yield 5-(hydroxymethyl) furfural, furfural, acetic acid, formic acid, and various other acids in the presence and in the absence of phosphate (39). Acetic acid, formic acid, and other acids are well known to have bactericidal effects (38). Heating glucose-phosphate solutions has been demonstrated to yield peroxides (40), but the toxicity of peroxides may not be significantly antimicrobial and therefore may not account for the toxicity of these heated compounds to E. coli, which is influenced by the rpoS regulon (35). Since rpoS is also responsible for the regulation of the katE gene in E. coli (41), the antioxidative function of bacteria is reasonably inferred to be at least partially responsible for the protection of bacteria from heated sugars/phosphates. Also, growth inhibition of E. coli by the heated galacturonic acids, which are common saccharides generated from pectin, can be restored by adding superoxide dismutase and catalase to the culture medium (42).
In this work, biofilm formation by V. parahaemolyticus was enhanced in basal synthetic medium (MMS), whereas mutation of the oxyR mutant was defective in the formation of biofilm by this pathogen (Table 6). The oxyR mutants of Klebsiella pneumoniae (18), Serratia marcescens (43), and Neisseria gonorrhoeae (44) are also impaired in biofilm formation. Contrarily, the oxyR mutants of E. coli (45) and Burkholderia pseudomallei (46) are biofilm hyper-formers. OxyR has been known to regulate the formation of cell appendages and biofilm in pathogenic bacteria, which is important for their attachment to biotic/abiotic surfaces and for virulence (18, 43, 45); however, the mechanisms that relate oxidative stress defense and biofilm formation are poorly understood at present (19). In V. parahaemolyticus, the rapid biosynthesis of exopolysaccharides, flagella, and other biofilm- or motility-associated factors may accompany the generation of ROS, and thus the lowered expression of catalase/AhpC in the oxyR mutant may impair its biofilm formation (Table 6).
The presence of peroxides and the normal function of OxyR may influence the mobility of bacteria. The oxyR mutant of Pseudomonas aeruginosa cannot swarm on a solid medium, probably owing to its lack of production of rhamnolipid surfactant molecules (47). The oxyR mutant of Serratia marcescens is defective in swarming but exhibits a normal swimming ability, as assayed on media that contain different concentrations of agar (43). Swimming mobility was lowered in the oxyR mutant of V. parahaemolyticus (Fig. 5), but the swarming of this mutant was not significantly affected according to an assay with a 0.6% agar medium at 37°C (31; data not shown). In another study, we revealed decreased swarming mobility in the ahpC mutant of V. parahaemolyticus in agar medium that contained 1.5% agar and that was incubated at 30°C (10), and this result suggests that 0.6% agar medium at 37°C was not suitable for assaying the swarming mobility of this pathogen.
In conclusion, the regulatory function of the oxyR gene in V. parahaemolyticus against a challenge by H2O2 and organic peroxides was demonstrated using the deletion mutation. The inhibition of bacterial growth in the rich culture medium by autoclaved phosphates and monosaccharides and the defective swimming mobility and biofilm formation may be attributed to the decreased antioxidative activity of the oxyR mutant.
ACKNOWLEDGMENTS
We thank Bin Ni for valuable suggestions and Ted Knoy for his editorial assistance.
The Ministry of Science and Technology of the Republic of China provided funding under contracts NSC100-2313-B-031-001-MY3 and MOST103-2313-B-031-001-MY3.
FOOTNOTES
- Received 31 August 2015.
- Accepted 12 November 2015.
- Accepted manuscript posted online 20 November 2015.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.02818-15.
- Copyright © 2016, American Society for Microbiology. All Rights Reserved.
