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 H 2 O 2 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. 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 a...

The occurrence of Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae in a total of 72 samples from six aquaculture ponds for groupers, milk fish, and tilapia in southern Taiwan was examined by the membrane filtration and colony hybridization method. The halophilic V. parahaemolyticus was only recovered in seawater ponds, with a high isolation frequency of 86.1% and a mean density of 2.6 log CFU/g. V. cholerae was found in both the seawater and freshwater ponds but preferentially in freshwater ponds, with a frequency of 72.2% and a mean density of 1.65 log CFU/g. V. vulnificus was identified mainly in seawater ponds, with an isolation frequency of 27.8%. The density of V. parahaemolyticus in seawater ponds was positively related to water temperature (Pearson correlation coefficient, r = 0.555) and negatively related to salinity (r = 2 0.333). The density of V. cholerae in all six ponds was positively related to water temperature (r = 0.342) and negatively related to salinity (r = 2 0.432). Two putatively pathogenic tdh(+) V. parahaemolyticus isolates (1.4% of the samples) and no ctx(+) V. cholerae isolates were identified. The experimental results may facilitate assessments of the risk posed by these pathogenic Vibrio species in Taiwan, where aquaculture provides a large part of the seafood supply.

The marine foodborne enteropathogen, Vibrio parahaemolyticus, has four putative catalase genes. Function of the katG-homologous genes, katG1(VPA0768) and katG2(VPA0453), was examined using gene deletion mutants, and compared with those of the katE-homologous genes, katE1(VPA1418) and katE2(VPA0305). Bacterial growth of ΔkatG1 was significantly delayed in the presence of 200-300 μM H2O2, and such inhibition was enhanced when incubation temperature was lowered from 37°C to 22°C. In the stationary phase, the ΔkatG1 strain was more susceptible to the lethal dosage of H2O2 than the ΔkatE1 strain. The minimum inhibitory concentrations and minimum bactericidal concentrations revealed that ΔkatE1/ΔkatE2 strains were more susceptible to H2O2 than the ΔkatG1/ΔkatG2 strains in exponential phase, while ΔkatG1 was more susceptible than the ΔkatE1/ΔkatE2 strains in the starved culture. This study demonstrated the chief antioxidative role of katG1 in the stationary phase and starved culture of V. parahaemolyticus, while katG1 and katG2 were also responsive to H2O2 and cumene hydroperoxide in the exponential phase.

Alkyl hydroperoxide reductase subunit C gene (ahpC) functions were characterized in Vibrio parahaemolyticus, a commonly occurring marine food-borne enteropathogenic bacterium. Two ahpC genes, ahpC1 (VPA1683) and ahpC2 (VP0580), encoded putative two-cysteine peroxiredoxins, which are highly similar to the homologous proteins of Vibrio vulnificus. The responses of deletion mutants of ahpC genes to various peroxides were compared with and without gene complementation and at different incubation temperatures. The growth of the ahpC1 mutant and ahpC1 ahpC2 double mutant in liquid medium was significantly inhibited by organic peroxides, cumene hydroperoxide and tert-butyl hydroperoxide. However, inhibition was higher at 12°C and 22°C than at 37°C. Inhibiting effects were prevented by the complementary ahpC1 gene. Inconsistent detoxification of H 2 O 2 by ahpC genes was demonstrated in an agar medium but not in a liquid medium. Complementation with an ahpC2 gene partially restored the peroxidase effect in the double ahpC1 ahpC2 mutant at 22°C. This investigation reveals that ahpC1 is the chief peroxidase gene that acts against organic peroxides in V. parahaemolyticus and that the function of the ahpC genes is influenced by incubation temperature. Vibrio parahaemolyticus is a halophilic Gram-negative bacterium which frequently causes food-borne gastroenteritis in some Asian countries (1) as well as globally since the isolation of the first pandemic O3:K6 strains in 1996 (2). This bacterium inhabits seawater and is often isolated from seafood (1). Most clinical isolates of this pathogen are hemolytic on Wagatsuma agar (Kanagawa phenomenon-positive, KP ϩ ) and produce the major virulence factor, thermostable direct hemolysin (TDH).Pathogenic bacteria such as V. parahaemolyticus have evolved sophisticated mechanisms to survive oxidative stresses caused by their metabolic activities, host defense systems, or environmental factors. Various reactive oxygen species (ROS) such as superoxide anion (O 2 Ϫ ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical ( ⅐ OH) form in bacteria (3-5), and ROS are known to cause damage in all cellular components, including protein, DNA, and membrane lipids.Several common antioxidative factors are typically used to detoxify ROS. One is alkyl hydroperoxide reductase subunit C (AhpC), which is the catalytic subunit of a family of peroxidases collectively known as peroxyredoxins or thiol peroxidases (TPx family) (6). A genome search of V. parahaemolyticus RIMD2210633 (7) revealed several putative AphC factors, including the VPA1683 and VP0580 genes, which have been designated ahpC1 and ahpC2, respectively (8).The antioxidative activity of pathogenic bacteria is related to their survival, growth, and virulence under some environmental stresses. Our earlier studies revealed enhanced production of AhpC in V. parahaemolyticus cells subjected to concurrent cold stress and starvation (9). Our most recent study demonstrated that, at 4°C, ahpC2 has a stronger protective effect than ahpC1 and that ahpC2 i...

The marine foodborne enteropathogen Vibrio parahaemolyticus has four putative catalase genes. The functions of two katEhomologous genes, katE1 (VPA1418) and katE2 (VPA0305), in the growth of this bacterium were examined using gene deletion mutants with or without complementary genes. The growth of the mutant strains in static or shaken cultures in a rich medium at 37°C or at low temperatures (12 and 4°C), with or without competition from Escherichia coli, did not differ from that of the parent strain. When 175 M extrinsic H 2 O 2 was added to the culture medium, bacterial growth of the ⌬katE1 strain was delayed and growth of the ⌬katE1 ⌬katE2 and ⌬katE1 ⌬ahpC1 double mutant strains was completely inhibited at 37°C for 8 h. The sensitivity of the ⌬katE1 strain to the inhibition of growth by H 2 O 2 was higher at low incubation temperatures (12 and 22°C) than at 37°C. The determined gene expression of these catalase and ahpC genes revealed that katE1 was highly expressed in the wild-type strain at 22°C under H 2 O 2 stress, while the katE2 and ahpC genes may play an alternate or compensatory role in the ⌬katE1 strain. This study demonstrated that katE1 encodes the chief functional catalase for detoxifying extrinsic H 2 O 2 during logarithmic growth and that the function of these genes was influenced by incubation temperature.