Abstract
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.