Nitrogen regulator GlnR directly controls transcription of prpDBC operon involved in methylcitrate cycle in Mycobacterium smegmatis

Abstract

Mycobacterium tuberculosis utilizes the fatty acids of the host as the carbon source. While the metabolism of odd chain fatty acids produces propionyl-CoA. Methylcitrate cycle is essential for Mycobacteria to utilize the propionyl-CoA to persist and grow on these fatty acids. In M. smegmatis, methylcitrate synthase, methylcitrate dehydratase, and methylisocitrate lyase involved in methylcitrate cycle were respectively encoded by prpC, prpD, and prpB in operon prpDBC. In this study, we found that the nitrogen regulator GlnR directly binds to the promoter region of prpDBC operon and inhibits its transcription. The typical binding sequence of GlnR was identified by bioinformatics analysis and electrophoretic mobility shift assay. The GlnR-binding motif was seperated by 164 bp with the binding site of PrpR which was a pathway-specific transcriptional activator of methylcitrate cycle. Moreover, the affinity constant of GlnR was much stronger than that of PrpR to prpDBC. The deletion of glnR resulted in poor growth in propionate or cholesterol medium comparing with wild-type strain. The {Delta}glnR mutant strain also showed a higher survival in macrophages. These results illustrated that the nitrogen regulator GlnR regulated methylcitrate cycle through directly repressing the transcription of prpDBC operon. The finding reveals an unprecedented link between nitrogen metabolism and methylcitrate pathway, and provides a potential application for controlling populations of pathogenic mycobacteria.\n\nAuthor SummaryNutrients are crucial for the survival and pathogenicity of Mycobacterium tuberculosis. The success of this pathogen survival in macrophage due to its ability to assimilate fatty acids and cholesterol from host. The cholesterol and fatty acids are catabolized via {beta}-oxidation to generate propionyl-CoA, which is then mainly metabolized via the methylcitrate cycle. The assimilation of propionyl-CoA needs to be tightly regulated to prevent its accumulation and alleviate toxicity in cell. Here, we identified a new regulator GlnR (the nitrogen transcriptional regulator) that repressed the transcription of prp operon involved in methylcitrate cycle in M. smegmatis. In this study, we found a typical GlnR binding box in prp operon, and the affinity is much stronger than that of PrpR which is known as a pathway-specific transcriptional activator of methylcitrate cycle. In addition, deletion of glnR obviously affect the growth of mutant in propionate or cholesterol medium, and show a better viability in macrophage. The findings not only provide the insights into the regulatory mechanism underlying crosstalk of nitrogen metabolism and carbon metabolism, but also reveal a potential application for controlling populations of pathogenic mycobacteria.