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Expanding the regulatory network governed by the extracytoplasmic function sigma factor σH in Corynebacterium glutamicum.

ABSTRACT: The extracytoplasmic function sigma factor σ(H) is responsible for the heat and oxidative stress response in Corynebacterium glutamicum. Due to the hierarchical nature of the regulatory network, previous transcriptome analyses have not been able to discriminate between direct and indirect targets of σ(H). Here, we determined the direct genome-wide targets of σ(H) using chromatin immunoprecipitation with microarray technology (ChIP-chip) for analysis of a deletion mutant of rshA, encoding an anti-σ factor of σ(H). Seventy-five σ(H)-dependent promoters, including 39 new ones, were identified. σ(H)-dependent, heat-inducible transcripts for several of the new targets, including ilvD encoding a labile Fe-S cluster enzyme, dihydroxy-acid dehydratase, were detected, and their 5' ends were mapped to the σ(H)-dependent promoters identified. Interestingly, functional internal σ(H)-dependent promoters were found in operon-like gene clusters involved in the pentose phosphate pathway, riboflavin biosynthesis, and Zn uptake. Accordingly, deletion of rshA resulted in hyperproduction of riboflavin and affected expression of Zn-responsive genes, possibly through intracellular Zn overload, indicating new physiological roles of σ(H). Furthermore, sigA encoding the primary σ factor was identified as a new target of σ(H). Reporter assays demonstrated that the σ(H)-dependent promoter upstream of sigA was highly heat inducible but much weaker than the known σ(A)-dependent one. Our ChIP-chip analysis also detected the σ(H)-dependent promoters upstream of rshA within the sigH-rshA operon and of sigB encoding a group 2 σ factor, supporting the previous findings of their σ(H)-dependent expression. Taken together, these results reveal an additional layer of the sigma factor regulatory network in C. glutamicum.

SUBMITTER: Toyoda K

PROVIDER: S-EPMC4285989 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe expression of genes in Corynebacterium glutamicum, a Gram-positive non-pathogenic bacterium used mainly for the industrial production of amino acids, is regulated by seven different sigma factors of RNA polymerase, including the stress-responsive ECF-sigma factor SigH. The sigH gene is located in a gene cluster together with the rshA gene, putatively encoding an anti-sigma factor. The aim of this study was to analyze the transcriptional regulation of the sigH and rshA gene cluster and the effects of RshA on the SigH regulon, in order to refine the model describing the role of SigH and RshA during stress response.ResultsTranscription analyses revealed that the sigH gene and rshA gene are cotranscribed from four sigH housekeeping promoters in C. glutamicum. In addition, a SigH-controlled rshA promoter was found to only drive the transcription of the rshA gene. To test the role of the putative anti-sigma factor gene rshA under normal growth conditions, a C. glutamicum rshA deletion strain was constructed and used for genome-wide transcription profiling with DNA microarrays. In total, 83 genes organized in 61 putative transcriptional units, including those previously detected using sigH mutant strains, exhibited increased transcript levels in the rshA deletion mutant compared to its parental strain. The genes encoding proteins related to disulphide stress response, heat stress proteins, components of the SOS-response to DNA damage and proteasome components were the most markedly upregulated gene groups. Altogether six SigH-dependent promoters upstream of the identified genes were determined by primer extension and a refined consensus promoter consisting of 45 original promoter sequences was constructed.ConclusionsThe rshA gene codes for an anti-sigma factor controlling the function of the stress-responsive sigma factor SigH in C. glutamicum. Transcription of rshA from a SigH-dependent promoter may serve to quickly shutdown the SigH-dependent stress response after the cells have overcome the stress condition. Here we propose a model of the regulation of oxidative and heat stress response including redox homeostasis by SigH, RshA and the thioredoxin system.

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Expanding the regulatory network governed by the extracytoplasmic function sigma factor σH in Corynebacterium glutamicum.

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