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.
Highlights
The multisubunit RNA polymerase (RNAP) holoenzyme in bacteria consists of core enzyme (2α, β, β′ and ω subunits) and a dissociable σ subunit (σ factor) that recognizes specific promoter sequences
We have recently shown that the promoters P1clgR, P2dnaK and P2dnaJ2 are recognized by both σE and σH (Šilar et al 2016)
PrshA sequence elements −35 GGAAGA and −10 GTTAAA (Fig. 1a) conform to the consensus of σH-dependent promoters, the expression of rshA was not found to be up-regulated in C. glutamicum by the microarray analysis (Ehira et al 2009)
Summary
The multisubunit RNA polymerase (RNAP) holoenzyme in bacteria consists of core enzyme (2α, β, β′ and ω subunits) and a dissociable σ subunit (σ factor) that recognizes specific promoter sequences. Sigma factors are key regulatory elements that control different classes of promoters and activate expression of the respective groups of genes (regulons or sigmulons). Dostálová et al AMB Expr (2017) 7:133 the construction of C. glutamicum producers of amino acids, carboxylic acids, alcohols, amines, polymers and biofuels as well as the use of alternative carbon sources like organic acids, pentoses, glycerol, starch and cellulose (Becker and Wittmann 2012). The C. glutamicum genome encodes seven sigma subunits of RNAP: the primary sigma factor σA, the alternative primary-like σB and five other alternative σ factors with extracytoplasmic functions (ECF) (σC, σD, σE, σH and σM)
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