Abstract
Corynebacterium diphtheriae and Corynebacterium glutamicum each have one gene (cat) encoding catalase. In-frame Δcat mutants of C. diphtheriae and C. glutamicum were hyper-sensitive to growth inhibition and killing by H2O2. In C. diphtheriae C7(β), both catalase activity and cat transcription decreased ∼2-fold during transition from exponential growth to early stationary phase. Prototypic OxyR in Escherichia coli senses oxidative stress and it activates katG transcription and catalase production in response to H2O2. In contrast, exposure of C. diphtheriae C7(β) to H2O2 did not stimulate transcription of cat. OxyR from C. diphtheriae and C. glutamicum have 52% similarity with E. coli OxyR and contain homologs of the two cysteine residues involved in H2O2 sensing by E. coli OxyR. In-frame ΔoxyR deletion mutants of C. diphtheriae C7(β), C. diphtheriae NCTC13129, and C. glutamicum were much more resistant than their parental wild type strains to growth inhibition by H2O2. In the C. diphtheriae C7(β) ΔoxyR mutant, cat transcripts were about 8-fold more abundant and catalase activity was about 20-fold greater than in the C7(β) wild type strain. The oxyR gene from C. diphtheriae or C. glutamicum, but not from E. coli, complemented the defect in ΔoxyR mutants of C. diphtheriae and C. glutamicum and decreased their H2O2 resistance to the level of their parental strains. Gel-mobility shift, DNaseI footprint, and primer extension assays showed that purified OxyR from C. diphtheriae C7(β) bound, in the presence or absence of DTT, to a sequence in the cat promoter region that extends from nucleotide position −55 to −10 with respect to the +1 nucleotide in the cat ORF. These results demonstrate that OxyR from C. diphtheriae or C. glutamicum functions as a transcriptional repressor of the cat gene by a mechanism that is independent of oxidative stress induced by H2O2.
Highlights
Aerobic organisms have specific mechanisms to protect themselves from reactive oxygen species (ROS) such as superoxide radical (O22) and hydrogen peroxide (H2O2), which can be generated by incomplete reduction of O2 during respiration
As a baseline for studying the role of catalase in resistance of C. diphtheriae to H2O2–induced oxidative stress, we determined the effects of H2O2 on growth (Fig. 1A) and viability (Fig. 1B) of wild type C. diphtheriae C7(b)
When H2O2 was added at 300 mM, the wild-type cells showed complete growth arrest and no viable cells were present after 6 hours exposure
Summary
Aerobic organisms have specific mechanisms to protect themselves from reactive oxygen species (ROS) such as superoxide radical (O22) and hydrogen peroxide (H2O2), which can be generated by incomplete reduction of O2 during respiration. The Fenton reaction, which generates hydroxyl radicals by reduction and oxidation of Fe ions in the presence of ROS, has been proposed as a mechanism for cell damage [1]. The first is a bifunctional catalase hydroperoxidase I (HPI) encoded by katG that has both catalase and peroxidase activities, and the katG gene is transcriptionally activated by the positive regulator OxyR [3]. A monofunctional HPII encoded by katE has catalase activity only, and katE is activated during stationary phase [3] but is not induced by OxyR [4]
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