Functions of VPA1418 and VPA0305 Catalase Genes in Growth of Vibrio parahaemolyticus under Oxidative Stress.

The marine foodborne enteropathogen Vibrio parahaemolyticus has four putative catalase genes. The functions of two katE-homologous 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 µM extrinsic H2O2 was added to the culture medium, bacterial growth of the ΔkatE1 strain was delayed and growth of the ΔkatE1 ΔkatE2 and ΔkatE1 ΔahpC1 double mutant strains was completely inhibited at 37°C for 8 h. The sensitivity of the ΔkatE1 strain to the inhibition of growth by H2O2 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 H2O2 stress, while the katE2 and ahpC genes may play an alternate or compensatory role in the ΔkatE1 strain. This study demonstrated that katE1 encodes the chief functional catalase for detoxifying extrinsic H2O2 during logarithmic growth and that the function of these genes was influenced by incubation temperature.

Influence of oxyR on Growth, Biofilm Formation, and Mobility of Vibrio parahaemolyticus.

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.

Activities of Alkyl Hydroperoxide Reductase Subunits C1 and C2 of Vibrio parahaemolyticus against Different Peroxides.

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 H2O2 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.

Roles of alkyl hydroperoxide reductase subunit C (AhpC) in viable but nonculturable Vibrio parahaemolyticus.

Alkyl hydroperoxide reductase subunit C (AhpC) is the catalytic subunit responsible for the detoxification of reactive oxygen species that form in bacterial cells or are derived from the host; thus, AhpC facilitates the survival of pathogenic bacteria under environmental stresses or during infection. This study investigates the role of AhpC in the induction and maintenance of a viable but nonculturable (VBNC) state in Vibrio parahaemolyticus. In this investigation, ahpC1 (VPA1683) and ahpC2 (VP0580) were identified in chromosomes II and I of this pathogen, respectively. Mutants with deletions of these two ahpC genes and their complementary strains were constructed from the parent strain KX-V231. The growth of these strains was monitored on tryptic soy agar-3% NaCl in the presence of the extrinsic peroxides H(2)O(2) and tert-butyl hydroperoxide (t-BOOH) at different incubation temperatures. The results revealed that both ahpC genes were protective against t-BOOH, while ahpC1 was protective against H(2)O(2). The protective function of ahpC2 at 4°C was higher than that of ahpC1. The times required to induce the VBNC state (4.7 weeks) at 4°C in a modified Morita mineral salt solution with 0.5% NaCl and then to maintain the VBNC state (4.7 weeks) in an ahpC2 mutant and an ahpC1 ahpC2 double mutant were significantly shorter than those for the parent strain (for induction, 6.2 weeks; for maintenance, 7.8 weeks) and the ahpC1 mutant (for induction, 6.0 weeks; for maintenance, 8.0 weeks) (P < 0.03). Complementation with an ahpC2 gene reversed the effects of the ahpC2 mutation in shortening the times for induction and maintenance of the VBNC state. This investigation identified the different functions of the two ahpC genes and confirmed the particular role of ahpC2 in the VBNC state of V. parahaemolyticus.