Dataset Information

Transcriptional regulation of furA and katG upon oxidative stress in Mycobacterium smegmatis.

ABSTRACT: The DNA region upstream of katG in Mycobacterium smegmatis was cloned and sequenced. The furA gene, highly homologous to Mycobacterium tuberculosis furA, mapped in this region. The furA-katG organization appears to be conserved among several mycobacteria. The transcription pattern of furA and katG in M. smegmatis upon oxidative stress was analyzed by Northern blotting and primer extension. Although transcription of both furA and katG was induced upon oxidative stress, transcripts covering both genes could not be identified either by Northern blotting or by reverse transcriptase PCR. Specific transcripts and 5' ends were identified for furA and katG, respectively. By cloning M. smegmatis and M. tuberculosis DNA regions upstream of a reporter gene, we demonstrated the presence of two promoters, pfurA, located immediately upstream of the furA gene, and pkatG, located within the terminal part of the furA coding sequence. Transcription from pfurA was induced upon oxidative stress. A 23-bp sequence overlapping the pfurA -35 region is highly conserved among mycobacteria and streptomycetes and might be involved in controlling pfurA activity. Transcription from a cloned pkatG, lacking the upstream pfurA region, was not induced upon oxidative stress, suggesting a cis-acting regulatory role of this region.

SUBMITTER: Milano A

PROVIDER: S-EPMC95520 | biostudies-literature | 2001 Dec

REPOSITORIES: biostudies-literature

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Transcriptional regulation of furA and katG upon oxidative stress in Mycobacterium smegmatis.

Milano A A Forti F F Sala C C Riccardi G G Ghisotti D D

Journal of bacteriology 20011201 23

The DNA region upstream of katG in Mycobacterium smegmatis was cloned and sequenced. The furA gene, highly homologous to Mycobacterium tuberculosis furA, mapped in this region. The furA-katG organization appears to be conserved among several mycobacteria. The transcription pattern of furA and katG in M. smegmatis upon oxidative stress was analyzed by Northern blotting and primer extension. Although transcription of both furA and katG was induced upon oxidative stress, transcripts covering both g ...[more]

PMID: 11698368

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Project description:Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized the oxyR-ahpC and furA-katG loci in the nontuberculous pathogen Mycobacterium marinum. In contrast to Mycobacterium smegmatis and like Mycobacterium tuberculosis and Mycobacterium leprae, M. marinum was shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress response oxyR and its subordinate gene ahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to the oxyR-ahpC promoter region from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence. M. marinum AhpC levels detected by immunoblotting, were increased upon treatment with H2O2, in keeping with the presence of a functional OxyR and its binding site within the promoter region of ahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katG expression did not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream of katG in M. marinum. The furA-katG linkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences in ahpC and katG expression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.

Project description:Stringent response is a conserved stress response mechanism in which bacteria employ the second messengers guanosine tetraphosphate and guanosine pentaphosphate [collectively termed (p)ppGpp] to reprogram their cellular processes under stress. In mycobacteria, these alarmones govern a multitude of cellular phenotypes, such as cell division, biofilm formation, antibiotic tolerance, and long-term survival. Mycobacterium smegmatis possesses the bifunctional RelMsm as a (p)ppGpp synthetase and hydrolase. In addition, it contains a short alarmone synthetase MS_RHII-RSD (renamed RelZ), which contains an RNase H domain in tandem with the (p)ppGpp synthetase domain. The physiological functions of RelMsm have been well documented, but there is no clear picture about the cellular functions of RelZ in M. smegmatis RelZ has been implicated in R-loop induced stress response due to its unique domain architecture. In this study, we elucidate the differential substrate utilization pattern of RelZ compared to that of RelMsm We unveil the ability of RelZ to use GMP as a substrate to synthesize pGpp, thereby expanding the repertoire of second messengers known in mycobacteria. We have demonstrated that the pGpp synthesis activity of RelZ is negatively regulated by RNA and pppGpp. Furthermore, we investigated its role in biofilm formation and antibiotic tolerance. Our findings highlight the complex role played by the RelZ in cellular physiology of M. smegmatis and sheds light upon its functions distinct from those of RelMsmIMPORTANCE Bacteria utilize nucleotide messengers to survive the hostile environmental conditions and the onslaught of attacks within the host. The second messengers guanosine tetraphosphate and pentaphosphate [(p)ppGpp] have a profound impact on the long-term survival, biofilm formation, antibiotic tolerance, virulence, and pathogenesis of bacteria. Therefore, understanding the stress response mechanism regulated by (p)ppGpp is essential for discovering inhibitors of stress response and potential drug targets. Mycobacterium smegmatis contains two (p)ppGpp synthetases: RelMsm and RelZ. Our study unravels the novel regulatory mechanisms of RelZ activity and its role in mediating antibiotic tolerance. We further reveal its ability to synthesize novel second messenger pGpp, which may have regulatory roles in mycobacteria.

Dataset Information

Transcriptional regulation of furA and katG upon oxidative stress in Mycobacterium smegmatis.

Publications

Transcriptional regulation of furA and katG upon oxidative stress in Mycobacterium smegmatis.

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