AUTHOR=Dostálová Hana , Busche Tobias , Holátko Jiří , Rucká Lenka , Štěpánek Václav , Barvík Ivan , Nešvera Jan , Kalinowski Jörn , Pátek Miroslav TITLE=Overlap of Promoter Recognition Specificity of Stress Response Sigma Factors SigD and SigH in Corynebacterium glutamicum ATCC 13032 JOURNAL=Frontiers in Microbiology VOLUME=9 YEAR=2019 URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.03287 DOI=10.3389/fmicb.2018.03287 ISSN=1664-302X ABSTRACT=

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^Hin 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.