Abstrakt
The sigma H (sigma(Eta)) and sigma E (sigma(E)) 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 sigma(Eta) regulon that are involved in heat and oxidative stress response have already been defined, whereas the genes of the sigma(E) 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 Delta cseE with a deletion in the anti-sigma(Epsilon) gene, differential gene expression was analyzed by RNA sequencing. We found 296 upregulated and 398 downregulated genes in C. glutamicum Delta cseE compared to C. glutamicum RES167.
To confirm the functional link between sigma(Epsilon) 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+sigma(Eta) and RNAP+sigma(Epsilon), which were previously shown to recognize similar promoters, proved that the sigma(Eta) and sigma(E) regulons significantly overlap.
The sigma(E)-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 sigma(Eta) and sigma(Epsilon) revealed that there are two groups of promoters: those which are exclusively sigma(Eta)-specific, and the other group of promoters, which are sigma(Eta)/sigma(E)-dependent.
No exclusively sigma(E)-dependent promoter was detected. We defined the consensus sequences of exclusively sigma(Eta)-regulated promotors to be -35 GGAAt and - 10 GTT and sigma(Eta)/sigma(E)-regulated promoters to be -35 GGAAC and - 10 cGTT.
Fifteen genes were found to belong to the sigma(Eta)/sigma(Epsilon) regulon. Homology modeling showed that there is a specific interaction between Met170 in sigma(Eta) and the nucleotides -31 and - 30 within the non-coding strand (AT or CT) of the sigma(Eta)-dependent promoters.
In sigma(E), Arg185 was found to interact with the nucleotides GA at the same positions in the sigma(E)-dependent promoters.