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) 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 (Pcg0607) 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 (GAT and GTT, 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.