PUB - Publikationen an der Universität Bielefeld

The sigma H (sigmaEta) and sigma E (sigmaE) subunits of Corynebacterium glutamicum RNA polymerase belong to Group 4 of sigma factors, also called extracytoplasmic function (ECF) sigma factors. Genes of the C. glutamicum sigmaEta regulon that are involved in heat and oxidative stress response have already been defined, whereas the genes of the sigmaE regulon, which is involved in cell surface stress response, have not been explored until now. Using the C. glutamicum RES167 strain and its derivative C. glutamicum DeltacseE with a deletion in the anti-sigmaEpsilon gene, differential gene expression was analyzed by RNA sequencing. We found 296 upregulated and 398 downregulated genes in C. glutamicum DeltacseE compared to C. glutamicum RES167. To confirm the functional link between sigmaEpsilon and the corresponding promoters, we tested selected promoters using the in vivo two-plasmid system with gfpuv as a reporter gene and by in vitro transcription. Analyses with RNAP+sigmaEta and RNAP+sigmaEpsilon, which were previously shown to recognize similar promoters, proved that the sigmaEta and sigmaE regulons significantly overlap. The sigmaE-controlled genes were found to be involved for example in protein quality control (dnaK, dnaJ2, clpB, and clpC), the regulation of Clp proteases (clgR), and membrane integrity maintenance. The single-promoter analyses with sigmaEta and sigmaEpsilon revealed that there are two groups of promoters: those which are exclusively sigmaEta-specific, and the other group of promoters, which are sigmaEta/sigmaE-dependent. No exclusively sigmaE-dependent promoter was detected. We defined the consensus sequences of exclusively sigmaEta-regulated promotors to be -35 GGAAt and-10 GTT and sigmaEta/sigmaE-regulated promoters to be -35 GGAAC and-10 cGTT. Fifteen genes were found to belong to the sigmaEta/sigmaEpsilon regulon. Homology modeling showed that there is a specific interaction between Met170 in sigmaEta and the nucleotides -31 and-30 within the non-coding strand (AT or CT) of the sigmaEta-dependent promoters. In sigmaE, Arg185 was found to interact with the nucleotides GA at the same positions in the sigmaE-dependent promoters. Copyright © 2023 Busche, Dostalova, Rucka, Holatko, Barvik, Stepanek, Patek and Kalinowski.