RESUMEN
The partner switching system (PSS) of the SigF regulatory pathway in Mycobacterium smegmatis has been previously demonstrated to include the anti-sigma factor RsbW (MSMEG_1803) and two anti-sigma factor antagonists RsfA and RsfB. In this study, we further characterized two additional RsbW homologs and revealed the distinct roles of three RsbW homologs [RsbW1 (MSMEG_1803), RsbW2 (MSMEG_6129), and RsbW3 (MSMEG_1787)] in the SigF PSS. RsbW1 and RsbW2 serve as the anti-sigma factor of SigF and the protein kinase phosphorylating RsfB, respectively, while RsbW3 functions as an anti-SigF antagonist through its protein interaction with RsbW1. Using relevant mutant strains, RsfB was demonstrated to be the major anti-SigF antagonist in M. smegmatis. The phosphorylation state of Ser-63 was shown to determine the functionality of RsfB as an anti-SigF antagonist. RsbW2 was demonstrated to be the only protein kinase that phosphorylates RsfB in M. smegmatis. Phosphorylation of Ser-63 inactivates RsfB to render it unable to interact with RsbW1. Our comparative RNA sequencing analysis of the wild-type strain of M. smegmatis and its isogenic Δaa 3 mutant strain lacking the aa 3 cytochrome c oxidase of the respiratory electron transport chain revealed that expression of the SigF regulon is strongly induced under respiration-inhibitory conditions in an RsfB-dependent way.
RESUMEN
The glpD (MSMEG_6761) gene encoding glycerol-3-phosphate dehydrogenase was shown to be crucial for M. smegmatis to utilize glycerol as the sole carbon source. The glpD gene likely forms the glpFKD operon together with glpF and glpK, encoding a glycerol facilitator and glycerol kinase, respectively. The gylR (MSMEG_6757) gene, whose product belongs to the IclR family of transcriptional regulators, was identified 182 bp upstream of glpF It was demonstrated that GylR serves as a transcriptional activator and is involved in the induction of glpFKD expression in the presence of glycerol. Three GylR-binding sites with the consensus sequence (GKTCGRC-N3-GYCGAMC) were identified in the upstream region of glpF by DNase I footprinting analysis. The presence of glycerol-3-phosphate was shown to decrease the binding affinity of GylR to the glpF upstream region with changes in the quaternary structure of GylR from tetramer to dimer. Besides GylR, cAMP receptor protein (Crp) and an alternative sigma factor, SigF, are also implicated in the regulation of glpFKD expression. Crp functions as a repressor, while SigF induces expression of glpFKD under energy-limiting conditions. In conclusion, we suggest here that the glpFKD operon is under the tripartite control of GylR, SigF, and Crp, which enables M. smegmatis to integrate the availability of glycerol, cellular energy state, and cellular levels of cAMP to exquisitely control expression of the glpFKD operon involved in glycerol metabolism.IMPORTANCE Using genetic approaches, we first revealed that glycerol is catabolized through the glycolytic pathway after conversion to dihydroxyacetone phosphate in two sequential reactions catalyzed by glycerol kinase (GlpK) and flavin adenine dinucleotide (FAD)-containing glycerol-3-phosphate dehydrogenase (GlpD) in M. smegmatis Our study also revealed that in addition to the GylR transcriptional activator that mediates the induction of the glpFKD operon by glycerol, the operon is regulated by SigF and Crp, which reflect the cellular energy state and cAMP level, respectively.