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

Corynebacterium glutamicum ATCC 13032 harbors five sigma subunits of RNA polymerase belonging to Group IV, also called extracytoplasmic function (ECF) sigma factors. These factors sigma(C), sigma(D), sigma(E), sigma(H), and sigma(M) are mostly involved in stress responses. The role of sigma(D) consists in the control of cell wall integrity. The sigma(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 sigma(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 sigma(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 sigma(D)-dependent promoters were also active to a much lower extent with sigma(H) in vivo and one (Pcg0607) also in vitro, although the known highly conserved consensus sequence of the sigma(H)-dependent promoters is different (-35 GGAA(T)/C and -10 GTT). In addition to the activity of sigma(H) at the sigma(D)-controlled promoters, we discovered separated or overlapping sigma(A) - or sigma(B)-regulated or sigma(H)-regulated promoters within the upstream region of 8 genes of the sigma(D)-regulon. We found that phenol in the cultivation medium acts as a stress factor inducing expression of some sigma(D)-dependent genes. Computer modeling revealed that sigma(H) binds to the promoter DNA in a similar manner as sigma(D) to the analogous promoter elements. The homology models together with mutational analysis showed that the key amino acids, Ala 60 in sigma(D) and Lys 53 in sigma(H), bind to the second nucleotide within the respective -10 promoter elements (GAT and GTT, respectively). The presented data obtained by integrating in vivo, in vitro and in silico approaches demonstrate that most of the sigma(D)-controlled genes also belong to the sigma(H)-regulon and are also transcribed from the overlapping or closely located housekeeping (sigma(A)-regulated) and/or general stress (sigma(B)-regulated) promoters.