The Nitrogen Regulator GlnR Directly Controls Transcription of the Operon Involved in Methylcitrate Cycle in Mycobacterium smegmatis.

utilizes fatty acids of the host as the carbon source. Metabolism of odd-chain fatty acids by produces propionyl coenzyme A (propionyl-CoA). The methylcitrate cycle is essential for mycobacteria to utilize the propionyl-CoA to persist and grow on these fatty acids. In , methylcitrate synthase, methylcitrate dehydratase, and methylisocitrate lyase involved in the methylcitrate cycle are encoded by , , , respectively, in operon In this study, we found that the nitrogen regulator GlnR directly binds to the promoter region of the operon and inhibits its transcription. The binding motif of GlnR was identified by bioinformatic analysis and validated using DNase I footprinting and electrophoretic mobility shift assays. The GlnR-binding motif is separated by a 164-bp sequence from the binding site of PrpR, a pathway-specific transcriptional activator of methylcitrate cycle, but the binding affinity of GlnR to is much stronger than that of PrpR. Deletion of resulted in faster growth in propionate or cholesterol medium compared with the wild-type strain. The Δ mutant strain also showed a higher survival rate in macrophages. These results illustrated that the nitrogen regulator GlnR regulates the methylcitrate cycle through direct repression of the transcription of the operon. This finding not only suggests an unprecedented link between nitrogen metabolism and the methylcitrate pathway but also reveals a potential target for controlling the growth of pathogenic mycobacteria. The success of mycobacteria survival in macrophage depends on its ability to assimilate fatty acids and cholesterol from the host. The cholesterol and fatty acids are catabolized via β-oxidation to generate propionyl coenzyme A (propionyl-CoA), which is then primarily metabolized via the methylcitrate cycle. Here, we found a typical GlnR binding box in the operon, and the affinity is much stronger than that of PrpR, a transcriptional activator of methylcitrate cycle. Furthermore, GlnR repressed the transcription of the operon. Deletion of significantly enhanced the growth of in propionate or cholesterol medium, as well as viability in macrophages. These findings provide new insights into the regulatory mechanisms underlying the cross talk of nitrogen and carbon metabolisms in mycobacteria.