Assignment of sigma factors of RNA polymerase to promoters in Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum is an important industrial producer of various amino acids and other metabolites. The C. glutamicum genome encodes seven sigma subunits (factors) of RNA polymerase: the primary sigma factor SigA (σA), the primary-like σB and five alternative sigma factors (σC, σD, σE, σH and σM). We have developed in vitro and in vivo methods to assign particular sigma factors to individual promoters of different classes. In vitro transcription assays and measurements of promoter activity using the overexpression of a single sigma factor gene and the transcriptional fusion of the promoter to the gfpuv reporter gene enabled us to reliably define the sigma factor dependency of promoters. To document the strengths of these methods, we tested examples of respective promoters for each C. glutamicum sigma factor. Promoters of the rshA (anti-sigma for σH) and trxB1 (thioredoxin) genes were found to be σH-dependent, whereas the promoter of the sigB gene (sigma factor σB) was σE- and σH-dependent. It was confirmed that the promoter of the cg2556 gene (iron-regulated membrane protein) is σC-dependent as suggested recently by other authors. The promoter of cmt1 (trehalose corynemycolyl transferase) was found to be clearly σD-dependent. No σM-dependent promoter was identified. The typical housekeeping promoter P2sigA (sigma factor σA) was proven to be σA-dependent but also recognized by σB. Similarly, the promoter of fba (fructose-1,6-bisphosphate aldolase) was confirmed to be σB-dependent but also functional with σA. The study provided demonstrations of the broad applicability of the developed methods and produced original data on the analyzed promoters.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0436-8) contains supplementary material, which is available to authorized users.

Overlap of Promoter Recognition Specificity of Stress Response Sigma Factors SigD and SigH in Corynebacterium glutamicum ATCC 13032

“…ClgR controls expression of several genes involved in protein quality control, and the function of ECF σ factors and the transcriptional regulator is thus integrated into a network. The same σ H /σ E overlap was found in the transcription initiation of the C. glutamicum sigB gene, encoding the general stress response sigma factor σ B (Dostálová et al, 2017). The possible activity of both σ H and σ M (involved in oxidative stress response) at the same C. glutamicum promoters was suggested, but has not yet been proven (Nakunst et al, 2007; Šilar et al, 2016; Dostálová et al, 2017), although the respective promoter consensus sequences seem very similar.…”

“…The same σ H /σ E overlap was found in the transcription initiation of the C. glutamicum sigB gene, encoding the general stress response sigma factor σ B (Dostálová et al, 2017). The possible activity of both σ H and σ M (involved in oxidative stress response) at the same C. glutamicum promoters was suggested, but has not yet been proven (Nakunst et al, 2007; Šilar et al, 2016; Dostálová et al, 2017), although the respective promoter consensus sequences seem very similar. Promoters of σ C -regulated genes in C. glutamicum were found to be different from other promoters recognized by ECF σ factors, and σ C regulon, which includes genes involved in the aerobic respiratory chain in C. glutamicum , seems to be separated from the other σ factor regulons (Toyoda and Inui, 2016).…”

“…Sigma factors were assigned to individual promoters in vivo using the two-plasmid system for C. glutamicum described recently (Dostálová et al, 2017) and similar that developed for the identification of σ E -dependent promoters in E. coli (Rezuchova and Kormanec, 2001). In principle, the sigma factors overproduced using the expression vector pEC-XT99A drove transcription from the individual promoters transcriptionally fused to the gfpuv reporter gene in the other plasmid present in the cell, the promoter-test vector pEPR1.…”

“…Promoters were cloned to pEPR1 as approximately 75-nt fragments using the restriction sites Pst I and Bam HI. The same pEC-XT99A constructs as described previously (Dostálová et al, 2017) were used to overexpress the sig genes after the addition of isopropyl-β-thiogalactopyranoside (IPTG). Two-plasmid strains were cultivated in 2xYT medium (containing Km and Tc) for 24 h and the cell samples were collected.…”

Ribosomal binding site sequences and promoters for expressing glutamate decarboxylase and producing γ-aminobutyrate in Corynebacterium glutamicum

“…P dnaK is recognized by σ A , σ E and σ H , while P clgR be recognized by σ E and σ H (Šilar et al 2016 ). P clpB is depended on σ M and σ H (Ehira et al 2009 ), while P trxB1 and P sufR are σ H -specific (Dostálová et al 2017 ). Even with prolonged length and bicistronic architecture, the strength of P dnaK did not enhance (Figs.…”