Dissertation (PhD)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: Sequence analysis showed that the essential glnA1 gene of Mycobacterium tuberculosis might
be closely related to an actinomycetes progenitor sequence and that this sequence may have
undergone duplication into other non-essential GS encoding genes in some Actinobateria,
notably the mycobacteria. Also, the M. tuberculosis glnA1 sequence remains conserved
throughout the evolution of M. tuberculosis. It was also shown that glnA1 is widely expressed in
M. tuberculosis infected human pulmonary tissue, where M. tuberculosis might be present in
altered phenotypes. These results imply that glnA1 is under selective pressure against
evolutionary change.
At transcriptional level it was shown that M. tuberculosis glnA1 might be expressed from two
alternate promoter sites, but that these promoter sites may not be controlled by environmental
nitrogen (in the form of ammonium) variation. We also showed that M. tuberculosis GS is
effectively exported by M. smegmatis to the cell wall, but that GS secretion into the exogenous
environment does not occur. Also, evidence has been presented which suggested that M.
tuberculosis GS might be modified at the C-terminus, probably as part of a mechanism that
retains GS in the cytosol. This hypothesis was further substantiated where it was demonstrated
that two GS isoforms of different size (short isoform in cytosol, longer isoform in cell wall) are
present in M. bovis BCG. It is unknown what causes this modification, since it couldn’t be
observed in M. smegmatis, but it was suggested that it might be through the action of a cis- or
trans-acting element present in proximity of the M. tuberculosis glnA1 gene. It was also shown
that a cluster of genes found immediately downstream of the M. tuberculosis glnA1 sequence
might be regulated in an operonic fashion under conditions of elevated environmental nitrogen
concentrations. Two of the genes (glnE and glnA2) in this operon arrangement have been
previously shown to be involved in nitrogen and glutamine metabolism. The function of the other
gene, Rv2223c, is unknown. It was shown that Rv2223c homologs are mostly found in the
mycobacteria and that it may encode an exported protease. It was hypothesised that this
sequence and its adjacently located progenitor sequence, Rv2224c, might be involved in M.
tuberculosis GS mediated metabolism. It was showed that over-expression of Rv2223c and
Rv2224c may be toxic to E. coli and mycobacterial hosts, such as M. smegmatis, but that
inhibition of transcription of these genes may be fatal to M. bovis BCG and M. tuberculosis
H37Rv. It was also shown that Rv2223c is widely expressed in M. tuberculosis infected human
tissue, which was comparable to that of glnA1. The results presented in this study shed more light on the distribution and transcriptional
regulation of GS in mycobacteria and has identified new genes that might be involved in GS
regulation. These results may present new approaches to tuberculosis control and thereby
contribute to alleviate the burden of the disease. / AFRIKAANSE OPSOMMING: Genetiese en proteien volgorde analise het aangedui dat die glnA1 (kodeer vir glutamien
sintetase (GS), ‘n essentiele protein) geen van Mycobacterium tuberculosis die naaste verwant
is aan ‘n actinomycetes voorloper volgorde wat duplikasie ondergaan het om die ander nieessensiele
GS koderende gene in sommige Actinobakterieë te vorm, veral in die mikobakterieë.
Voords het die glnA1 geen geneties gekonserveerd gebly gedurende die evolusie van M.
tuberculosis. Dit is ook aangetoon dat volop transkribasie van die glnA1 geen voorkom in die M.
tuberculosis geïnfekteerde pulmonêre weefsel waar M. tuberculosis moontlik mag voorkom.
Op transkriptionele vlak is dit aangetoon dat die M. tuberculosis glnA1 geen vanaf twee
onderskeie promotors uitgedruk mag word, maar dat hierdie twee promotors nie deur variasies
in die konsentrasie van stikstof (in die vorm van ammonium) in die omgewing beïnvloed word
nie. Daar is ook aangedui dat M. tuberculosis GS effektief deur M. smegmatis oor die
selmembraan na die selwand vervoer word, maar dat daar nie GS sekresie na die ekstrasellulêre
omgewing geskied nie. Ook is bewyse gevind dat M. tuberculosis GS modifikasie aan
die C-terminus mag ondergaan wat waarskynlik dien om GS beweging uit die sitosol te verhoed.
Hierdie hipotese is versterk deurdat twee isoforms van verskillende grootte (klein in sitosol en
groter in die selwand) gevind is in M. bovis BCG. Dit modifikasie meganisme is onbekend, maar
vind moontlik nie plaas in nie-patogeniese mikobakterieë soos M. smegmatis nie en mag
moontlik deur cis- of trans-werkende elemente gefasiliteer word. Daar is aangedui dat ‘n
groepering van vier gene lanksaan die glnA1 lokus in ‘n operoniese meganisme gereguleer mag
word onder variërende konsentrasies van stikstof in die omgewing. Dit is bekend dat twee van
die gene in die operon (glnE en glnA2) betrokke in stikstof en gultamien metabolisme is.
Die funksie van die ander twee gene (Rv2223c en Rv2224c) is onbekend. Daar is aangetoon
dat volgordes soortgelyk aan Rv2223c beperk is tot die mikobakterieë en dat die geen ‘n
protease, wat moontlik gesekreteer word vanuit die sitosol, kodeer. Daar is aangetoon dat die
oor-produksie van die Rv2223c en Rv2224c proteine toksies is vir E. coli en mikobakterieë, soos M. smegmatis, maar dat transkripsie inhibisie hierdie gene dodelik is vir M. tuberculosis
H37Rv en M. bovis BCG. Daar is ook angedui dat die ekspresie van hierdie gene volop
verspreid is in M. tuberculosis geïnfekteerde menslike weefsel, soortgelyk aan diè van glnA1.
Die resultate vervat in hierdie studie werp meer lig op die verspreiding en transkiptionele
regulasie van GS in mikobakteriee en nuwe gene is ontdek wat betrokke by GS regulasie mag
wees. Hierdie resultate mag bydra tot nuwe maniere om tuberkulose te bekamp en daardeur die
voorkoms van die siekte te beperk.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/21883 |
| Date | 12 1900 |
| Creators | Hayward, Don |
| Contributors | Wiid, I.J.F., Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | Unknown |
| Type | Thesis |
| Format | xv, 214 leaves : ill. |
| Rights | Stellenbosch University |
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