Thesis (PhD (Med))--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The nitrogen metabolic pathway is essential for growth and survival of all living organisms
including prokaryotes. Certain components of the pathway, such as the enzyme glutamine
synthetase (GS), have been studied; however, little information is available regarding the
pathway in the mycobacteria. Our in silico studies revealed that many of the components and
mechanisms involved in the pathway appear to be conserved between closely related
Actinomycetales. Therefore, we investigated three aspects of nitrogen metabolic control in
Mycobacterium smegmatis; namely, transcriptional regulation of nitrogen metabolism-related
genes, control of enzyme activity and the signalling cascade governing the nitrogen metabolic
response.
At the transcriptional level, it was found that nitrogen metabolism-related genes were regulated
in response to ammonium availability. Two possible transcriptional regulators, AmtR and GlnR,
which are the regulators responsible for control of nitrogen-related gene transcription in
Streptomyces coelicolor and Corynebacterium glutamicum respectively, were identified in M.
smegmatis. Through generation of amtR and glnR deletion mutants, we found that both potential
regulators played a role in the control of nitrogen-related gene expression in M. smegmatis. GlnR
acted as both an activator and repressor of gene transcription whilst AmtR appeared to activate
gene expression which is different to the role its homolog plays in C. glutamicum. On a protein
level we found that both GS and glutamate dehydrogenase (GDH) were responsible for
ammonium assimilation in M. smegmatis and were regulated in response to ammonium
availability. Two GDH isoforms (NAD+- and NADP+-specific) were identified in M. smegmatis
and whereas only an NAD+-GDH was detected in M. tuberculosis. The M. tuberculosis GDH
also played a largely anabolic role with regard to ammonium assimilation which is in contrast to
the belief that ammonium can only be assimilated via GS in this pathogen. The signaling cascade
was investigated through generation of a glnD deletion mutant in M. smegmatis. We were able to
show that this pivotal protein (GlnD) was able to relay the cellular nitrogen status to the
transcriptional machinery as well as to GS.
The data presented in this study has advanced our understanding of the nitrogen metabolic
pathway in the mycobacteria. Through elucidation of such pathways, our knowledge of
mycobacterial physiology and thus infection and survival improves, which could ultimately lead
to the discovery of novel mechanisms to aid in the eradication of the disease. / AFRIKAANSE OPSOMMING: Stikstof metabolisme is noodsaaklik vir die oorlewing en groei van alle organismes, prokariote
ingesluit. Sekere sellulêre komponente, soos die ensiem glutamine sintetase (GS), is al tevore
bestudeer, maar baie min verdere inligting is beskikbaar oor stikstof metabolisme in die
mycobacteria. Ons in silico studies het gewys dat baie van die komponente en meganismes
gekonserveerd gebly het tussen nou-verwante Actinomycetales. Dus het ons drie aspekte in die
beheer van stikstof metabolisme ondersoek; naamlik, die transkriptionele regulering van stikstof
metabolisme-verwante gene, die beheer van ensiem aktiwiteit en die sein-meganisme wat die
reaksie op stikstof konsentrasie reageer.
Op transkripsionele vlak het ons gevind dat stikstof metabolisme-verwante gene gereguleer word
in reaksie op stikstof beskikbaarheid. AmtR en GlnR is twee moontlike transkripsie reguleerders
wat verantwoordelik is vir transkripsionele beheer in onderskeidelik Streptomyces coelicolor en
Corynebacterium glutamicum. Beide hierdie proteïene is geïdentifiseer in M. smegmatis. Deur
die konstruksie van amtR en glnR mutante, het ons gevind dat beide potensiële reguleerders ‘n
rol gespeel het in die beheer van stikstof-verwante transkripsie in M. smegmatis. GlnR het
opgetree as beide ‘n aktiveerder en ‘n onderdrukker van transkripsie terwyl AmtR net ‘n
aktiverende rol gespeel het. Die funksie van AmtR in M. smegmatis is dus verskillend van sy
homoloog in C. glutamicum. Op proteïen-vlak het ons gevind dat beide GS en glutamaat
dehidrogenase (GDH) verantwoordelik was vir die assimilasie van ammonium in M. smegmatis
en albei was gereguleer in reaksie op ammonium beskikbaarheid. Twee vorme van GDH (NAD+-
spesifieke- en NADP+-spesifieke GDH) was geïdentifiseer in M. smegmatis terwyl net ‘n NAD+-
spesifieke GDH in M. tuberculosis gevind is. Die M. tuberculosis GDH het ook ‘n anaboliesie
rol gespeel met betrekking tot ammonium assimilasie wat in teenstelling is met die huidige
opvatting dat ammonium alleenlik deur GS ge-assimileer kan word. Die sein-meganisme is
ondersoek deur ‘n glnD M. smegmatis mutant te konstrueer. Ons het bewys dat hierdie
deurslaggewende proteïen (GlnD) die sellulêre stikstof status aan die transkripsionele masjinerie,
en aan GS kon oordra.
Die data wat in hierdie studie voorgelê word, het ons kennis van stikstof metabolisme in die
mycobacteria gevorder. Sodanige metaboliese studies verbreed ons kennis van mycobacteriële
fisiologie en dus M. tuberculosis infeksie en oorlewing en kan uiteindelik lei tot die ontdekking
van unieke teiken meganismes om te help met die beheer van die siekte en nuwe
middelontwikkeling.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/17991 |
| Date | 12 1900 |
| Creators | Kirsten, Catriona Jane |
| Contributors | Wiid, Ian, Van Helden, Paul, University of Stellenbosch. Faculty of Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics., Harper, Catriona Jane |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 218 p. : ill. |
| Rights | Stellenbosch University |
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