Regulation of the icl1 Gene Encoding the Major Isocitrate Lyase in Mycobacterium smegmatis

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 coenzyme A (succinyl-CoA) was shown to increase the affinity of binding of RamB to its binding sites and enable RamB to bind to RamBS2, which 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 a similar regulatory mechanism operates for the regulation of icl1 expression in M. tuberculosis.