Nitrogen metabolism and the regulation thereof in Mycobacterium smegmatis

[摘要] ENGLISH ABSTRACT: The nitrogen metabolic pathway is essential for growth and survival of all living organismsincluding prokaryotes. Certain components of the pathway, such as the enzyme glutaminesynthetase (GS), have been studied; however, little information is available regarding thepathway in the mycobacteria. Our in silico studies revealed that many of the components andmechanisms involved in the pathway appear to be conserved between closely relatedActinomycetales. Therefore, we investigated three aspects of nitrogen metabolic control inMycobacterium smegmatis; namely, transcriptional regulation of nitrogen metabolism-relatedgenes, control of enzyme activity and the signalling cascade governing the nitrogen metabolicresponse.At the transcriptional level, it was found that nitrogen metabolism-related genes were regulatedin response to ammonium availability. Two possible transcriptional regulators, AmtR and GlnR,which are the regulators responsible for control of nitrogen-related gene transcription inStreptomyces coelicolor and Corynebacterium glutamicum respectively, were identified in M.smegmatis. Through generation of amtR and glnR deletion mutants, we found that both potentialregulators played a role in the control of nitrogen-related gene expression in M. smegmatis. GlnRacted as both an activator and repressor of gene transcription whilst AmtR appeared to activategene expression which is different to the role its homolog plays in C. glutamicum. On a proteinlevel we found that both GS and glutamate dehydrogenase (GDH) were responsible forammonium assimilation in M. smegmatis and were regulated in response to ammoniumavailability. Two GDH isoforms (NAD+- and NADP+-specific) were identified in M. smegmatisand whereas only an NAD+-GDH was detected in M. tuberculosis. The M. tuberculosis GDHalso played a largely anabolic role with regard to ammonium assimilation which is in contrast tothe belief that ammonium can only be assimilated via GS in this pathogen. The signaling cascadewas investigated through generation of a glnD deletion mutant in M. smegmatis. We were able toshow that this pivotal protein (GlnD) was able to relay the cellular nitrogen status to thetranscriptional machinery as well as to GS.The data presented in this study has advanced our understanding of the nitrogen metabolicpathway in the mycobacteria. Through elucidation of such pathways, our knowledge ofmycobacterial physiology and thus infection and survival improves, which could ultimately leadto the discovery of novel mechanisms to aid in the eradication of the disease.

[发布机构] Stellenbosch University