Tripartite Regulation of the glpFKD Operon Involved in Glycerol Catabolism by GylR, Crp, and SigF in Mycobacterium smegmatis

Abstract

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.

Induced expression of the zwf gene in the presence of glucose contributes to lowering of glucose 6-phosphate level and consequently reduction of growth rate of Mycobacterium smegmatis

“…We found high GlpK activity in glycerol-grown Msm cells and induced expression from the glpFKD operon that codes for the glycerol utilizing enzymes, including GlpK. We expected that glycerol would induce expression of the genes belonging to the glpFKD operon as in an earlier study [21]; the authors had demonstrated using reporter assays that this operon’s promoter is glycerol inducible. But the novel observation that we have made here is that Fbp activity increases significantly during growth in glycerol.…”

“…The genes that code for the glycerol utilizing enzymes, glpK (glycerol kinase), glpD (glycerol-3-phosphate dehydrogenase), and glpF (glycerol uptake facilitator), are organized in an operon, the glpFKD operon. The operon's expression is controlled in a tripartite manner by GlpR, the operon regulator, cyclic AMP receptor protein (Crp), and SigF [21]. We sought to investigate the expression of the genes encoded by the glpFKD operon using qRT-PCR assays.…”

Energy extraction from air: structural basis of atmospheric hydrogen oxidation

“…For Huc production, protein expression was performed using the M. smegmatis mc 2 155 strain PRC1, which lacks the glycerol response regulator gylR (gylR Leu154->Frameshift) and the Hhy hydrogenase (hhyS inactivation) [33, 75]. To facilitate Huc purification, a 2×StrepII tag was inserted at the N-terminal end of the small subunit (MSMEG_2262, HucS) through allelic exchange mutagenesis as described previously [76].…”

Molecular mechanism of GylR-mediated regulation of glycerol metabolism in Streptomyces clavuligerus NRRL 3585

“…Thus it can be seen that glycerol can serve as a common precursor to biosynthesize various products. Usually, the genes encoding the enzymes for conversion of glycerol to DHAP are tightly controlled by different types of transcriptional regulators (Lin, 1976;Hindle and Smith, 1994;Bong et al, 2019;Poblete-Castro et al, 2020). Understanding the molecular regulatory mechanism of glycerol metabolism is critical for better use of glycerol to produce valuable products.…”

“…In vivo studies have revealed that the expression of glp operon can be induced by the addition of glycerol during fermentation (Baños et al, 2009;Guo et al, 2013). Studies in non-Streptomyces strains, such as Mycolicibacterium smegmatis (previously known as Mycobacterium smegmatis), indicated that G3P rather than glycerol might bind to GylR-like proteins, and, as a result, regulate the glp operon (Bong et al, 2019). Despite the aforementioned studies, the exact binding sequences and the small molecule effectors of GylR-like proteins remain elusive in Streptomyces.…”