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

Hana Dostálová, Jiří Holátko, Tobias Busche, Lenka Rucká, Andrey Rapoport, Petr Halada, Jan Nešvera, Jörn Kalinowski, Miroslav Pátek

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 (σ(A)), the primary-like σ(B) and five alternative sigma factors (σ(C), σ(D), σ(E), σ(H) and σ(M)). 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 σ(H)) and trxB1 (thioredoxin) genes were found to be σ(H)-dependent, whereas the promoter of the sigB gene (sigma factor σ(B)) was σ(E)- and σ(H)-dependent. It was confirmed that the promoter of the cg2556 gene (iron-regulated membrane protein) is σ(C)-dependent as suggested recently by other authors. The promoter of cmt1 (trehalose corynemycolyl transferase) was found to be clearly σ(D)-dependent. No σ(M)-dependent promoter was identified. The typical housekeeping promoter P2sigA (sigma factor σ(A)) was proven to be σ(A)-dependent but also recognized by σ(B). Similarly, the promoter of fba (fructose-1,6-bisphosphate aldolase) was confirmed to be σ(B)-dependent but also functional with σ(A). The study provided demonstrations of the broad applicability of the developed methods and produced original data on the analyzed promoters.