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.

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...