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Corynebacterium glutamicum  ATCC 13032 harbors five sigma subunits of RNA polymerase belonging to Group IV, also called extracytoplasmic function (ECF) σ factors. These factors σ C , σ D , σ E , σ H , and σ M  are mostly involved in stress responses. The role of σ D  consists in the control of cell wall integrity. The σ D  regulon is involved in the synthesis of components of the mycomembrane which is part of the cell wall in  C. glutamicum . RNA sequencing of the transcriptome from a strain overexpressing the  sigD  gene provided 29 potential σ D -controlled genes and enabled us to precisely localize their transcriptional start sites. Analysis of the respective promoters by both  in vitro  transcription and the  in vivo  two-plasmid assay confirmed that transcription of 11 of the tested genes is directly σ D -dependent. The key sequence elements of all these promoters were found to be identical or closely similar to the motifs -35 GTAAC A / G  and -10 GAT. Surprisingly, nearly all of these σ D -dependent promoters were also active to a much lower extent with σ H   in vivo  and one (P cg0607 ) also  in vitro , although the known highly conserved consensus sequence of the σ H -dependent promoters is different (-35 GGAA T / C  and -10 GTT). In addition to the activity of σ H  at the σ D -controlled promoters, we discovered separated or overlapping σ A - or σ B -regulated or σ H -regulated promoters within the upstream region of 8 genes of the σ D -regulon. We found that phenol in the cultivation medium acts as a stress factor inducing expression of some σ D -dependent genes. Computer modeling revealed that σ H  binds to the promoter DNA in a similar manner as σ D  to the analogous promoter elements. The homology models together with mutational analysis showed that the key amino acids, Ala 60 in σ D  and Lys 53 in σ H , bind to the second nucleotide within the respective -10 promoter elements (G A T and G T T, respectively). The presented data obtained by integrating  in vivo ,  in vitro  and  in silico  approaches demonstrate that most of the σ D -controlled genes also belong to the σ H -regulon and are also transcribed from the overlapping or closely located housekeeping (σ A -regulated) and/or general stress (σ B -regulated) promoters.

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