Nitrogen metabolism in Mycobacterium smegmatis

Nitrogen is an essential component of the bacterial cell and bacteria have developed elaborate regulatory mechanisms used to control the uptake, assimilation and metabolism of nitrogen. In this thesis, I have developed a nitrogen-limited continuous culture system to gain further insights into nitrogen metabolism in mycobacteria and their response to nitrogen limitation. I identified 357 differentially expressed genes in response to nitrogen limitation in continuous culture, including changes in amino acid metabolic pathways. I found 26 transcriptional regulators that mediated the global transcriptomic response of Mycobacterium smegmatis to nitrogen limitation and I identified several non-coding RNAs that might be involved in the regulation of nitrogen- regulated gene expression. Subsequently, I characterised two differentially expressed transcriptional regulators, AmtR (MSMEG_4300) and CadC (MSMEG_3297) using a combination of genome-wide expression profiling, physiological, biochemical and biophysical analyses. I identified the AmtR regulon and showed that AmtR was a transcriptional repressor of an urea degradation pathway. I identified xanthine and allantoin as ligands of mycobacterial AmtR and showed that addition of these metabolites had no effect on the release of AmtR from DNA. In further work, I demonstrated that deletion of the gene cadC in M. smegmatis resulted in a severe growth defect manifested as cell lysis during growth on rich medium. Subsequently, I identified the CadC regulon that included genes involved in the diaminopimelate (DAP) and lysine biosynthesis pathway. Supplementation with DAP or lysine could not rescue the growth defect of the ∆cadC mutant. Furthermore, I showed that M. smegmatis has a high-affinity lysine uptake system that exhibited high rates of lysine transport during growth in minimal medium, which was significantly reduced during growth in rich medium. My data suggest that a ∆cadC mutant is defective in the generation or replenishment of intracellular lysine and DAP levels that are essential for growth and survival in mycobacteria. I conclude that M. smegmatis has a broad network of regulatory systems that together enable M. smegmatis to adapt its nitrogen metabolism to rapidly changing environments