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Characterization of OxyR as a negative transcriptional regulator that represses catalase production in Corynebacterium diphtheriae.

Kim JS, Holmes RK - PLoS ONE (2012)

Bottom Line: In contrast, exposure of C. diphtheriae C7(β) to H(2)O(2) did not stimulate transcription of cat.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 H(2)O(2) resistance to the level of their parental strains.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 H(2)O(2).

View Article: PubMed Central - PubMed

Affiliation: Dept of Microbiology, University of Colorado School of Medicine, Aurora, Colorado, United States of America.

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 H(2)O(2). 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 H(2)O(2). In contrast, exposure of C. diphtheriae C7(β) to H(2)O(2) 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 H(2)O(2) 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 H(2)O(2). 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 H(2)O(2) 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 H(2)O(2).

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Structure of cat::lacZ transcriptional fusions and assessment of cat promoter activity and catalase activity in C. diphtheriae C7(β).A: Organization of the sigC-cat locus and schematic representation of cat::lacZ reporter plasmids. B: Histogram comparing β-galactosidase reporter activities for wild-type C. diphtheriae C7(β) carrying either the pPLΩ control plasmid, or the pPL-cat100, pPL-cat200 or pPL-cat300 reporter plasmid. Cultures grown in high-iron PGT medium were harvested either during exponential phase growth (shaded bars) or at early stationary phase (open bars). Error bars show standard deviations for β-galactosidase activities. C: Cultures of wild-type C. diphtheriae C7(β) or the C7(β) Δcat mutant were grown to exponential phase (labeled L) or early stationary phase (labeled ES) in low-iron PGT (labeled PGT) or high-iron PGT (labeled PGT-iron). Samples were analyzed by native PAGE followed by in gel assays for catalase activity, which appears as the single bright band in each lane containing lysate from wild type C7(β).
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pone-0031709-g002: Structure of cat::lacZ transcriptional fusions and assessment of cat promoter activity and catalase activity in C. diphtheriae C7(β).A: Organization of the sigC-cat locus and schematic representation of cat::lacZ reporter plasmids. B: Histogram comparing β-galactosidase reporter activities for wild-type C. diphtheriae C7(β) carrying either the pPLΩ control plasmid, or the pPL-cat100, pPL-cat200 or pPL-cat300 reporter plasmid. Cultures grown in high-iron PGT medium were harvested either during exponential phase growth (shaded bars) or at early stationary phase (open bars). Error bars show standard deviations for β-galactosidase activities. C: Cultures of wild-type C. diphtheriae C7(β) or the C7(β) Δcat mutant were grown to exponential phase (labeled L) or early stationary phase (labeled ES) in low-iron PGT (labeled PGT) or high-iron PGT (labeled PGT-iron). Samples were analyzed by native PAGE followed by in gel assays for catalase activity, which appears as the single bright band in each lane containing lysate from wild type C7(β).

Mentions: The most obvious candidate enzymes for detoxifying H2O2 are catalases and peroxidases. A search of the published C. diphtheriae NCTC13129 genome sequence for homologs of catalase identified a single gene annotated as cat (DIP0281) (http://www.ncbi.nlm.nih.gov/gene/2649075). The cat gene ORF is separated by a 127-bp intervening sequence from the divergently oriented sigC ORF, which encodes an RNA polymerase sigma factor presumed to be involved in responses to environmental stress (Fig. 2A). We constructed an in-frame deletion in the cat gene of C. diphtheriae C7(β) by allelic exchange, and we used this Δcat mutant strain to examine the role of catalase in the resistance C. diphtheriae to H2O2. In contrast with the results described previously with wild type C7(β), addition of H2O2 at 10 mM, 100 mM or 300 mM to exponential phase cultures of the C7(β) Δcat mutant resulted in immediate cessation of growth and complete loss of viability after 6 hours of exposure (Figs. 1A and 1B). When methyl viologen (MV, a superoxide radical generating agent) was spotted on agar plates inoculated with sufficient numbers of wild type C. diphtheriae C7(β) or the C7(β) Δcat mutant to produce confluent lawns during subsequent incubation, no significant differences were seen in the diameters of the zones of inhibition that developed (data not shown). These results indicate that catalase confers resistance to H2O2-induced stress, but not to MV-induced stress, in C. diphtheriae.


Characterization of OxyR as a negative transcriptional regulator that represses catalase production in Corynebacterium diphtheriae.

Kim JS, Holmes RK - PLoS ONE (2012)

Structure of cat::lacZ transcriptional fusions and assessment of cat promoter activity and catalase activity in C. diphtheriae C7(β).A: Organization of the sigC-cat locus and schematic representation of cat::lacZ reporter plasmids. B: Histogram comparing β-galactosidase reporter activities for wild-type C. diphtheriae C7(β) carrying either the pPLΩ control plasmid, or the pPL-cat100, pPL-cat200 or pPL-cat300 reporter plasmid. Cultures grown in high-iron PGT medium were harvested either during exponential phase growth (shaded bars) or at early stationary phase (open bars). Error bars show standard deviations for β-galactosidase activities. C: Cultures of wild-type C. diphtheriae C7(β) or the C7(β) Δcat mutant were grown to exponential phase (labeled L) or early stationary phase (labeled ES) in low-iron PGT (labeled PGT) or high-iron PGT (labeled PGT-iron). Samples were analyzed by native PAGE followed by in gel assays for catalase activity, which appears as the single bright band in each lane containing lysate from wild type C7(β).
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Related In: Results  -  Collection

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pone-0031709-g002: Structure of cat::lacZ transcriptional fusions and assessment of cat promoter activity and catalase activity in C. diphtheriae C7(β).A: Organization of the sigC-cat locus and schematic representation of cat::lacZ reporter plasmids. B: Histogram comparing β-galactosidase reporter activities for wild-type C. diphtheriae C7(β) carrying either the pPLΩ control plasmid, or the pPL-cat100, pPL-cat200 or pPL-cat300 reporter plasmid. Cultures grown in high-iron PGT medium were harvested either during exponential phase growth (shaded bars) or at early stationary phase (open bars). Error bars show standard deviations for β-galactosidase activities. C: Cultures of wild-type C. diphtheriae C7(β) or the C7(β) Δcat mutant were grown to exponential phase (labeled L) or early stationary phase (labeled ES) in low-iron PGT (labeled PGT) or high-iron PGT (labeled PGT-iron). Samples were analyzed by native PAGE followed by in gel assays for catalase activity, which appears as the single bright band in each lane containing lysate from wild type C7(β).
Mentions: The most obvious candidate enzymes for detoxifying H2O2 are catalases and peroxidases. A search of the published C. diphtheriae NCTC13129 genome sequence for homologs of catalase identified a single gene annotated as cat (DIP0281) (http://www.ncbi.nlm.nih.gov/gene/2649075). The cat gene ORF is separated by a 127-bp intervening sequence from the divergently oriented sigC ORF, which encodes an RNA polymerase sigma factor presumed to be involved in responses to environmental stress (Fig. 2A). We constructed an in-frame deletion in the cat gene of C. diphtheriae C7(β) by allelic exchange, and we used this Δcat mutant strain to examine the role of catalase in the resistance C. diphtheriae to H2O2. In contrast with the results described previously with wild type C7(β), addition of H2O2 at 10 mM, 100 mM or 300 mM to exponential phase cultures of the C7(β) Δcat mutant resulted in immediate cessation of growth and complete loss of viability after 6 hours of exposure (Figs. 1A and 1B). When methyl viologen (MV, a superoxide radical generating agent) was spotted on agar plates inoculated with sufficient numbers of wild type C. diphtheriae C7(β) or the C7(β) Δcat mutant to produce confluent lawns during subsequent incubation, no significant differences were seen in the diameters of the zones of inhibition that developed (data not shown). These results indicate that catalase confers resistance to H2O2-induced stress, but not to MV-induced stress, in C. diphtheriae.

Bottom Line: In contrast, exposure of C. diphtheriae C7(β) to H(2)O(2) did not stimulate transcription of cat.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 H(2)O(2) resistance to the level of their parental strains.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 H(2)O(2).

View Article: PubMed Central - PubMed

Affiliation: Dept of Microbiology, University of Colorado School of Medicine, Aurora, Colorado, United States of America.

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 H(2)O(2). 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 H(2)O(2). In contrast, exposure of C. diphtheriae C7(β) to H(2)O(2) 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 H(2)O(2) 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 H(2)O(2). 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 H(2)O(2) 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 H(2)O(2).

Show MeSH
Related in: MedlinePlus