Role of OxyR as a peroxide-sensing positive regulator in Streptomyces coelicolor A3(2) - PubMed (original) (raw)

Role of OxyR as a peroxide-sensing positive regulator in Streptomyces coelicolor A3(2)

Ji-Sook Hahn et al. J Bacteriol. 2002 Oct.

Abstract

Genes encoding a homolog of Escherichia coli OxyR (oxyR) and an alkyl hydroperoxide reductase system (ahpC and ahpD) have been isolated from Streptomyces coelicolor A3(2). The ahpC and ahpD genes constitute an operon transcribed divergently from the oxyR gene. Expression of both ahpCD and oxyR genes was maximal at early exponential phase and decreased rapidly as cells entered mid-exponential phase. Overproduction of OxyR in Streptomyces lividans conferred resistance against cumene hydroperoxide and H2O2. The oxyR mutant produced fewer ahpCD and oxyR transcripts than the wild type, suggesting that OxyR acts as a positive regulator for their expression. Both oxyR and ahpCD transcripts increased more than fivefold within 10 min of H2O2 treatment and decreased to the normal level in 50 min, with kinetics similar to those of the CatR-mediated induction of the catalase A gene (catA) by H2O2. The oxyR mutant failed to induce oxyR and ahpCD genes in response to H2O2, indicating that OxyR is the modulator for the H2O2-dependent induction of these genes. Purified OxyR protein bound specifically to the intergenic region between ahpC and oxyR, suggesting its direct role in regulating these genes. These results demonstrate that in S. coelicolor OxyR mediates H2O2 induction of its own gene and genes for alkyl hydroperoxide reductase system, but not the catalase gene (catA), unlike in Escherichia coli and Salmonella enterica serovar Typhimurium.

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Figures

FIG. 1.

FIG. 1.

(A) Restriction map and organization of the ahpC, ahpD, and oxyR genes. A restriction map of the 3.8-kb _Pst_I fragment containing the ahpCD and oxyR genes is presented. Thick arrows indicate the positions and directions of the three genes. The two divergent transcripts are shown as dashed arrows. Abbreviations: B, _Bam_HI; Pt, _Pst_I; Pv, _Pvu_II; SI, _Sal_I; Sm, _Sma_I. (B) Comparison of the predicted amino acid sequence of OxyR with its homologues. The amino acid sequence of OxyR from S. coelicolor (Sco) (AF186371) was aligned with those from M. leprae (Mle) (Al035300), E. coli (Eco) (J04553), and X. campestris (Xca) (U94336). The position of two cysteine residues involved in disulfide bond formation and activation of OxyR in E. coli are shaded and presented in boldface type.

FIG. 2.

FIG. 2.

Transcription of the ahpCD and oxyR genes. (A) Northern blot analysis of the _ahpC_-ahpD transcript. RNA was isolated from S. coelicolor M145 cells grown in YEME for 24 h. Fifty micrograms of RNA was loaded on 1.2% agarose gel containing formamide, and Northern blot analysis was carried out using a 0.6-kb ahpC DNA probe as described in Materials and Methods (lane 2). Lane 1 shows 23S and 16S rRNA bands stained with ethidium bromide. (B and C) High-resolution S1 mapping of the 5′ ends of ahpCD (B) and oxyR (C) mRNA. RNAs prepared from M145 cells grown in YEME at 30°C for 24, 28, and 32 h, were subjected to S1 mapping analysis as described in Materials and Methods. The protected fragments were analyzed on sequencing gels with sequencing ladders generated from the same primers and templates used for the preparation of the probes. The transcription start sites are shown in boldface type and designated by arrows on the sense sequence of each transcript. (D) Nucleotide sequence of the intergenic region of oxyR and ahpCD. The oxyR sense strand sequence is presented, except in the center line, where both strands are presented to show divergent promoter elements. Transcription start sites of the ahpCD (ahpCp) and oxyR (oxyRp) are indicated by bent arrows. The putative −10 and −35 elements of ahpCD and oxyR promoters are in boldface type and underlined. Primers used to generate S1 probes (ACS1 and OXYS1) are indicated by arrows.

FIG. 3.

FIG. 3.

Growth-dependent expression of AhpC and AhpD proteins. M145 cells were grown in YEME. At various time points aliquots were taken for measurement of optical density at 640 nm and lysed to prepare cell extracts. (A) The AhpC and AhpD proteins were detected by Western blot analysis as described in Materials and Methods. The positions of the monomer (M) and dimer (D) of AhpC are shown. (B) The growth curve and the relative band intensity of the densitometric tracing data in panel A are presented. The densitometric value was set to 1 for AhpC at 40 h (closed square) and for AhpD at 70 h (open triangle).

FIG. 4.

FIG. 4.

Overproduction of AhpC, AhpD, and OxyR and their contribution to resistance against oxidants. (A) Overproduction of AhpC and AhpD proteins. S. lividans cells containing oxyR (pJHOxyR), ahpCD (pJHAhpCD), or ahpC (pJHAhpC) genes on multicopy plasmid pIJ702 were grown on NA plates containing thiostrepton (50 μg/ml) until they formed substrate mycelium (24 h) or aerial mycelium (36 h). The amount of CatA, CatC, AhpC, and AhpD proteins in cell extracts was determined by Western blot analysis. (B) Effect of overproduction on resistance against hydrogen peroxide and cumene hydroperoxide. About 106 spores of cells harboring plasmids were transferred to plates of NA medium containing 200 μM hydrogen peroxide, 150 to 200 μM cumene hydroperoxide (CHP), or no oxidants (control) with thiostrepton (50 μg/ml). Cells were incubated at 30°C for 3 days.

FIG. 5.

FIG. 5.

Effect of oxyR mutation on the production of ahpCD and oxyR transcripts. M145 (WT) and JH10 (oxyR mutant) cells were grown in YEME for 22, 27, and 32 h. The amounts of ahpCD and oxyR transcripts were determined by S1 mapping as described in the text.

FIG. 6.

FIG. 6.

OxyR-dependent induction of the ahpCD and oxyR genes by H2O2. Induction of ahpCD and oxyR by H2O2 in wild-type (A) and oxyR mutant (B) cells. S. coelicolor M145 and JH10 cells were grown in YEME to the early exponential phase and treated with 200 μM H2O2. Samples were taken at 10-min intervals over 1 h, and S1 mapping analysis was carried out for oxyR and ahpCD transcripts.

FIG. 7.

FIG. 7.

Binding of OxyR to ahpCD-oxyR intergenic region. A gel mobility shift assay of OxyR binding to different promoter fragments was performed. Two hundred nanograms of purified OxyR (6 pmol) was incubated with 32P-labeled promoter fragments of _oxyR_-ahpCD, catA, or furA-catC as described in Materials and Methods. OxyR binding was detected by electrophoresis on 4% polyacrylamide gel and autoradiography. Lanes 1, 5, and 7 contain only the radiolabeled probes. Probes were incubated with OxyR without any competitors except poly(dI-dC) (lanes 2, 6, and 8) or with a 200-fold molar excess of unlabeled probe DNA (S; lane 3) or nonspecific DNA fragments (N; lane 4).

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