Characterization of OxyR as a Negative Transcriptional Regulator That Represses Catalase Production in Corynebacterium diphtheriae

Corynebacterium diphtheriae and Corynebacterium glutamicum each have one gene (cat) encoding catalase. In-frame Delta cat mutants of C. diphtheriae and C. glutamicum were hyper-sensitive to growth inhibition and killing by H2O2. In C. diphtheriae C7(beta), both catalase activity and cat transcription decreased similar to 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(beta) 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 Delta oxyR deletion mutants of C. diphtheriae C7(beta), 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(beta) Delta oxyR mutant, cat transcripts were about 8-fold more abundant and catalase activity was about 20-fold greater than in the C7(beta) wild type strain. The oxyR gene from C. diphtheriae or C. glutamicum, but not from E. coli, complemented the defect in Delta oxyR 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(beta) bound, in the presence or absence of DTT, to a sequence in the cat promoter region that extends from nucleotide position 255 to 210 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.