Transcriptional regulation of the operon encoding stress-responsive ECF sigma factor SigH and its anti-sigma factor RshA, and control of its regulatory network in Corynebacterium glutamicum

Abstract

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.

Expanding the Regulatory Network Governed by the Extracytoplasmic Function Sigma Factor σHin Corynebacterium glutamicum

“…Although no such ORF has been annotated in the genome of C. glutamicum R (42), which was used as the wild-type strain in this study (see Table S1 in the supplemental material), reinspection of the genome revealed a small (267-bp) ORF (cgR_6064) encoding a protein with 100% amino acid sequence identity with the anti-factor RshA, as has been reported previously (25). As H is constitutively active in the rshA mutant, expression of the H regulon is upregulated in the absence of any stress (25). We confirmed that the expression level of mtr (cgR_1832), one of the components of the H regulon in C. glutamicum R (14), was upregulated more than 10-fold by deletion of the rshA gene during normal growth (data not shown).…”

“…In total, 74 genes were found upregulated 2-fold or more in the rshA deletion mutant compared to the level of the wild type. Among them, 34 genes have been previously shown to be upregulated by overexpression of H or deletion of rshA under normal growth conditions (14,25). These results not only confirmed that RshA is the anti-H but also showed that there are H regulon genes yet to be identified.…”

“…1A, C, D, and F). Although upregulation of these genes in the rshA mutant has been previously observed, the putative H -dependent promoter was predicted upstream of only lipA (25). By 5= RACE analysis using RNA extracted from heat-treated cells, a transcriptional start point (TSP) of ilvD was determined to be guanine at 188 nucleotides (nt) up- …”

“…Previously, it was reported that the rshA gene located immediately downstream of the sigH gene encodes an anti-factor of H in C. glutamicum ATCC 13032 (25). Although no such ORF has been annotated in the genome of C. glutamicum R (42), which was used as the wild-type strain in this study (see Table S1 in the supplemental material), reinspection of the genome revealed a small (267-bp) ORF (cgR_6064) encoding a protein with 100% amino acid sequence identity with the anti-factor RshA, as has been reported previously (25). As H is constitutively active in the rshA mutant, expression of the H regulon is upregulated in the absence of any stress (25).…”

“…Thus, H is positioned at the top layer of the transcriptional regulatory network controlling cellular stress responses. Because of the complex hierarchical nature of the regulatory network, genes directly regulated by H are tentatively predicted by the upstream presence of the consensus promoter sequence (G/T)GGAA-N [17][18][19][20] -(T/C)GTT (14,25).…”