The Nitrogen Regulator GlnR Directly Controls Transcription of the <i>prpDBC</i> Operon Involved in Methylcitrate Cycle in Mycobacterium smegmatis

Abstract

<i>Mycobacterium tuberculosis</i> utilizes fatty acids of the host as the carbon source. Metabolism of odd-chain fatty acids by <i>Mycobacterium tuberculosis</i> 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 <i>M. smegmatis</i>, methylcitrate synthase, methylcitrate dehydratase, and methylisocitrate lyase involved in the methylcitrate cycle are encoded by <i>prpC</i>, <i>prpD</i>, <i>and prpB</i>, respectively, in operon <i>prpDBC</i> In this study, we found that the nitrogen regulator GlnR directly binds to the promoter region of the <i>prpDBC</i> 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 <i>prpDBC</i> is much stronger than that of PrpR. Deletion of <i>glnR</i> resulted in faster growth in propionate or cholesterol medium compared with the wild-type strain. The Δ<i>glnR</i> 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 <i>prpDBC</i> 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.<b>IMPORTANCE</b> 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 <i>prp</i> operon, and the affinity is much stronger than that of PrpR, a transcriptional activator of methylcitrate cycle. Furthermore, GlnR repressed the transcription of the <i>prp</i> operon. Deletion of <i>glnR</i> significantly enhanced the growth of <i>Mycobacterium tuberculosis</i> 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.