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.

The respiratory electron transport chain (ETC) of Mycobacterium smegmatis is terminated with two terminal oxidases, the aa3 cytochrome c oxidase and the cytochrome bd quinol oxidase. The bd quinol oxidase with a higher binding affinity for O2 than the aa3 oxidase is known to play an important role in aerobic respiration under oxygen-limiting conditions. Using relevant crp1 (MSMEG_6189) and crp2 (MSMEG_0539) mutant strains of M. smegmatis, we demonstrated that Crp1 plays a predominant role in induction of the cydAB operon under ETC-inhibitory conditions. Two Crp-binding sequences were identified upstream of the cydA gene, both of which are necessary for induction of cydAB expression under ETC-inhibitory conditions. The intracellular level of cAMP in M. smegmatis was found to be increased under ETC-inhibitory conditions. The crp2 gene was found to be negatively regulated by Crp1 and Crp2, which appears to lead to significantly low cellular abundance of Crp2 relative to Crp1 in M. smegmatis. Our RNA sequencing analyses suggest that in addition to the SigF partner switching system, Crp1 is involved in induction of gene expression in M. smegmatis exposed to ETC-inhibitory conditions.

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Mycobacterium smegmatis has two isocitrate lyase (ICL) isozymes (MSMEG_0911 and MSMEG_3706). We demonstrated that ICL1 (MSMEG_0911) is the predominantly expressed ICL in M. smegmatis and plays a major role in growth on acetate or fatty acid as the sole carbon and energy source. Expression of the icl1 gene in M. smegmatis was demonstrated to be strongly upregulated during growth on acetate relative to that in M. smegmatis grown on glucose. Expression of icl1 was shown to be positively regulated by the RamB activator, and three RamB-binding sites (RamBS1, RamBS2, and RamBS3) were identified in the upstream region of icl1 using DNase I footprinting analysis. Succinyl-CoA was shown to increase the binding affinity of RamB to its binding sites and enable RamB to bind to RamBS2 that is the most important site for RamB-mediated induction of icl1 expression. These results suggest that succinyl-CoA serves as a coinducer molecule for RamB. Our study also showed that cAMP receptor protein (Crp1: MSMEG_6189) represses icl1 expression in M. smegmatis grown in the presence of glucose. Therefore, the strong induction of icl1 expression during growth on acetate as the sole carbon source relative to the weak expression of icl1 during growth on glucose is likely to result from combined effects of RamB-mediated induction of icl1 in the presence of acetate and Crp-mediated repression of icl1 in the presence of glucose. IMPORTANCE Carbon flux through the glyoxylate shunt has been suggested to affect virulence, persistence, and antibiotic resistance of Mycobacterium tuberculosis . Therefore, it is important to understand the precise mechanism underlying the regulation of the icl gene encoding the key enzyme of the glyoxylate shunt. Using Mycobacterium smegmatis , this study revealed the regulation mechanism underlying induction of icl1 expression in M. smegmatis when the glyoxylate shunt is required. The conservation of the cis - and trans -acting regulatory elements related to icl1 regulation in both M. smegmatis and M. tuberculosis implies that the similar regulatory mechanism operates for the regulation of icl1 expression in M. tuberculosis .

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