Overlapping SigH and SigE sigma factor regulons in Corynebacterium glutamicum

Tobias Busche; Hana Dostálová; Lenka Rucká; Jiří Holátko; Ivan Barvík; Václav Štěpánek; Miroslav Pátek; Jörn Kalinowski

doi:10.3389/fmicb.2022.1059649

Overlapping SigH and SigE sigma factor regulons in Corynebacterium glutamicum

Front Microbiol. 2023 Feb 28:13:1059649. doi: 10.3389/fmicb.2022.1059649. eCollection 2022.

Authors

Tobias Busche^{1

2}, Hana Dostálová³, Lenka Rucká³, Jiří Holátko³, Ivan Barvík⁴, Václav Štěpánek³, Miroslav Pátek³, Jörn Kalinowski¹

Affiliations

¹ Microbial Genomics and Biotechnology, Center for Biotechnology, Bielefeld University, Bielefeld, Germany.
² Medical School East Westphalia-Lippe, Bielefeld University, Bielefeld, Germany.
³ Institute of Microbiology, Academy of Sciences of the Czech Republic (ASCR), Prague, Czechia.
⁴ Faculty of Mathematics and Physics, Institute of Physics, Charles University, Prague, Czechia.

Abstract

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

Keywords: Corynebacterium; RNA-seq; consensus sequence; promoter; regulon; sigma factor; stress; transcriptional start site.