Identification of the Clostridium perfringens genes involved in the adaptive response to oxidative stress

Abstract

Clostridium perfringens is a ubiquitous gram-positive pathogen that is present in the air, soil, animals, and humans. Although C. perfringens is strictly anaerobic, vegetative and stationary cells can survive in a growth-arrested stage in the presence of oxygen and/or low concentrations of superoxide and hydroxyl radicals. Indeed, it possesses an adaptive response to oxidative stress, which can be activated in both aerobic and anaerobic conditions. To identify the genes involved in this oxidative stress response, C. perfringens strain 13 mutants were generated by Tn916 insertional mutagenesis and screened for resistance or sensitivity to various oxidative stresses. Three of the 12 sensitive mutants examined harbored an independently inserted single copy of the transposon in the same operon as two genes orthologous to the ydaD and ycdF genes of Bacillus subtilis, which encode a putative NADPH dehydrogenase. Complementation experiments and knockout experiments demonstrated that these genes are both required for efficient resistance to oxidative stress in C. perfringens and are probably responsible for the production of NADPH, which is required for maintenance of the intracellular redox balance in growth-arrested cells. Other Tn916 disrupted genes were also shown to play important roles in the oxidative stress response. This is the first time that some of these genes (e.g., a gene encoding an ATP-dependent RNA helicase, the beta-glucuronidase gene, and the gene encoding the atypical iron sulfur prismane protein) have been shown to be involved in the oxidative response.

FIG. 1.

Sensitivities of the resistant mutants and wild-type C. perfringens strain 13R to air (a), PL (b), H₂O₂ (c), and t-TB (d). The results are means based on two experiments. Symbols: ○, strain 13R; •, mutant 13052; ⧫, mutant 13055; ▴, mutant 13095; ▪, mutant 13133.

FIG. 2.

Sensitivities of the sensitive mutants and wild-type strain 13R of C. perfringens to air (a), PL (b), H₂O₂, (c), and t-TB (d). The results are means based on two experiments. Symbols: ○, strain 13R; •, mutant 13050; ⧫, mutant 13096; ▪, mutant 13543; ▴, mutant 13521.

FIG. 3.

Partial restriction map of pKNTE1280 and pKNTS1160 showing the location of Tn916 in mutants 13052, 13050, 13096, 13543, and 13133. The solid arrows indicate the locations of the various ORFs identified by sequence analysis. The scale is in kilobases.

FIG. 4.

Partial restriction map of pKNTE1300 and pKNTS1030 showing the location of Tn916 in mutant 13521. The solid arrows indicate the locations of the various ORFs identified by sequence analysis. The scale is in kilobases.

FIG. 5.

Sensitivities of 13R, 13096, and 13133 containing plasmid pKNTE1280 (solid symbols) or pKNT19 (open symbols) to H₂O₂ and t-TB. The results are means based on three experiments. Symbols: ○, strain 13R/pKNT19; •, strain 13R/pKNTE1280; □, strain 13096/pKNT19; ▪, strain 13096/pKNT1280; ◊, strain 13133/pKNT19; ⧫, strain 13133/pKNTE1280.

FIG. 6.

Survival curves for C. perfringens strain 13R and for the knockout mutants 13R ycdF ortholog::catP and 13R ydaD ortholog::ermc′, treated with H₂O₂ in anaerobic conditions. The data are means based on two experiments. Symbols: ○, strain 13R; ▪, strain 13R ycdF ortholog::catP; ⧫, strain 13R ydaD ortholog::ermc′. (a) Exponentially growing cells; (b) stationary-phase cells.

FIG. 7.

(a) β-Glucuronidase activities of 13R, 13521, and 13R uidA::ermc′. (b) Sensitivities of 13R, 13521, and 13R uidA::ermc′ to various concentrations of PL. Symbols: ○, strain 13R; ▴, strain 13521; ◊, strain 13R uidA::catP.

FIG. 8.

Survival of strain 13R Prismane::ermc′ and the same strain complemented with a wild-type prismane gene cloned onto pJIR418, treated with H₂O₂ in anaerobic conditions (a) and with PL (b). Symbols: ○, strain 13R; ▪, strain 13R Prismane::ermc′; □, strain 13R Prismane::ermc′ complemented with a wild-type prismane gene cloned onto pJIR418.