The road to clean water: Building collaboration and stakeholder relationships

Abstract

<h3>Abstract</h3> <i>Mycobacterium tuberculosis</i> 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 <i>M. smegmatis</i>, methylcitrate synthase, methylcitrate dehydratase, and methylisocitrate lyase involved in methylcitrate cycle were respectively encoded by <i>prpC</i>, <i>prpD</i>, <i>and prpB</i> in operon <i>prpDBC</i>. In this study, we found that the nitrogen regulator GlnR directly binds to the promoter region of <i>prpDBC</i> 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 <i>prpDBC</i>. The deletion of <i>glnR</i> resulted in poor growth in propionate or cholesterol medium comparing with wild-type strain. The Δ<i>glnR</i> 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 <i>prpDBC</i> operon. The finding reveals an unprecedented link between nitrogen metabolism and methylcitrate pathway, and provides a potential application for controlling populations of pathogenic mycobacteria. <h3>Author Summary</h3> Nutrients are crucial for the survival and pathogenicity of <i>Mycobacterium tuberculosis</i>. 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 β-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 <i>prp</i> operon involved in methylcitrate cycle in <i>M. smegmatis</i>. In this study, we found a typical GlnR binding box in <i>prp</i> 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 <i>glnR</i> 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.