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Isolation and analysis of the Xanthomonas alkyl hydroperoxide reductase gene and the peroxide sensor regulator genes ahpC and ahpF-oxyR-orfX.

ABSTRACT: From Xanthomonas campestris pv. phaseoli, we have isolated by two independent methods genes involved in peroxide detoxification (ahpC and ahpF), a gene involved in peroxide sensing and transcription regulation (oxyR), and a gene of unknown function (orfX). Amino acid sequence analysis of AhpC, AhpF, and OxyR showed high identity with bacterial homologs. OrfX was a small cysteine-rich protein with no significant homology to known proteins. The genes ahpC, ahpF, oxyR, and orfX were arranged in a head-to-tail fashion. This unique arrangement was conserved in all of the Xanthomonas strains tested. The functionalities of both the ahpC and oxyR genes were demonstrated. In X. campestris pv. phaseoli, increased expression of ahpC alone conferred partial protection against growth retardation and killing by organic hydroperoxides but not by H2O2 or superoxide generators. These genes are likely to have important physiological roles in protection against peroxide toxicity in Xanthomonas.

SUBMITTER: Loprasert S

PROVIDER: S-EPMC179203 | biostudies-other | 1997 Jun

REPOSITORIES: biostudies-other

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Isolation and analysis of the Xanthomonas alkyl hydroperoxide reductase gene and the peroxide sensor regulator genes ahpC and ahpF-oxyR-orfX.

Loprasert S S Atichartpongkun S S Whangsuk W W Mongkolsuk S S

Journal of bacteriology 19970601 12

From Xanthomonas campestris pv. phaseoli, we have isolated by two independent methods genes involved in peroxide detoxification (ahpC and ahpF), a gene involved in peroxide sensing and transcription regulation (oxyR), and a gene of unknown function (orfX). Amino acid sequence analysis of AhpC, AhpF, and OxyR showed high identity with bacterial homologs. OrfX was a small cysteine-rich protein with no significant homology to known proteins. The genes ahpC, ahpF, oxyR, and orfX were arranged in a h ...[more]

PMID: 9190810

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Project description:AIM:To identify alkyl hydroperoxide reductase subunit C (AhpC) homologs in Bacillus subtilis (B. subtilis) and to characterize their structural and biochemical properties. AhpC is responsible for the detoxification of reactive oxygen species in bacteria. METHODS:Two AhpC homologs (AhpC_H1 and AhpC_H2) were identified by searching the B. subtilis database; these were then cloned and expressed in Escherichia coli. AhpC mutants carrying substitutions of catalytically important Cys residues (C37S, C47S, C166S, C37/47S, C37/166S, C47/166S, and C37/47/166S for AhpC_H1; C52S, C169S, and C52/169S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahpC genes were disrupted by the short flanking homology method, and the phenotypes of the resulting AhpC-deficient bacteria were examined. RESULTS:Comparative characterization of AhpC homologs indicates that AhpC_H1 contains an extra C37, which forms a disulfide bond with the peroxidatic C47, and behaves like an atypical 2-Cys AhpC, while AhpC_H2 functions like a typical 2-Cys AhpC. Tryptic digestion analysis demonstrated the presence of intramolecular Cys37-Cys47 linkage, which could be reduced by thioredoxin, resulting in the association of the dimer into higher-molecular-mass complexes. Peroxidase activity analysis of Cys→Ser mutants indicated that three Cys residues were involved in the catalysis. AhpC_H1 was resistant to inactivation by peroxide substrates, but had lower activity at physiological H2O2 concentrations compared to AhpC_H2, suggesting that in B. subtilis, the enzymes may be physiologically functional at different substrate concentrations. The exposure to organic peroxides induced AhpC_H1 expression, while AhpC_H1-deficient mutants exhibited growth retardation in the stationary phase, suggesting the role of AhpC_H1 as an antioxidant scavenger of lipid hydroperoxides and a stress-response factor in B. subtilis. CONCLUSION:AhpC_H1, a novel atypical 2-Cys AhpC, is functionally distinct from AhpC_H2, a typical 2-Cys AhpC.

Project description:Mutations to the regulatory region of the ahpC gene, resulting in overproduction of alkyl hydroperoxide reductase, were encountered frequently in a large collection of isoniazid (INH)-resistant clinical isolates of Mycobacterium tuberculosis but not in INH-susceptible strains. Overexpression of ahpC did not seem to be important for INH resistance, however, as most of these strains were already defective for catalase-peroxidase, KatG, the enzyme required for activation of INH. Transformation of the INH-susceptible reference strain, M. tuberculosis H37Rv, with plasmids bearing the ahpC genes of M. tuberculosis or M. leprae did not result in a significant increase in the MIC. Two highly INH-resistant mutants of H37Rv, BH3 and BH8, were isolated in vitro and shown to produce no or little KatG activity and, in the case of BH3, to overproduce alkyl hydroperoxide reductase as the result of an ahpC regulatory mutation that was also found in some clinical isolates. The virulence of H37Rv, BH3, and BH8 was studied intensively in three mouse models: fully immunocompetent BALB/c and Black 6 mice, BALB/c major histocompatibility complex class II-knockout mice with abnormally low levels of CD4 T cells and athymic mice producing no cellular immune response. The results indicated that M. tuberculosis strains producing catalase-peroxidase were considerably more virulent in immunocompetent mice than the isogenic KatG-deficient mutants but that loss of catalase-peroxidase was less important when immunodeficient mice, unable to produce activated macrophages, were infected. Restoration of virulence was not seen in an INH-resistant M. tuberculosis strain that overexpressed ahpC, and this finding was confirmed by experiments performed with appropriate M. bovis strains in guinea pigs. Thus, in contrast to catalase-peroxidase, alkyl hydroperoxide reductase does not appear to act as a virulence factor in rodent infections or to play a direct role in INH resistance, although it may be important in maintaining peroxide homeostasis of the organism when KatG activity is low or absent.

Dataset Information

Isolation and analysis of the Xanthomonas alkyl hydroperoxide reductase gene and the peroxide sensor regulator genes ahpC and ahpF-oxyR-orfX.

Publications

Isolation and analysis of the Xanthomonas alkyl hydroperoxide reductase gene and the peroxide sensor regulator genes ahpC and ahpF-oxyR-orfX.

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