DNA metabolism in mycobacteria

dc.contributor.authorWarner, Digby Francis
dc.descriptionPhD - Scienceen
dc.description.abstractSpecialised mechanisms have evolved in pathogenic bacteria to enable adaptation to hostile and fluctuating host environments. Inducible mutagenesis, in particular, has been implicated in the emergence of antibiotic- and stress-resistant mutants. This thesis examined mycobacterial DNA metabolism with specific emphasis on the roles of multiple Y-family polymerases in the evolution of inter-strain variation and drug resistance in M. tuberculosis. The contribution of the nrdZ-encoded class II ribonucleotide reductase (RNR) to the maintenance of dNTP pools for replication and repair under hypoxic conditions was also explored. In addition, the co-factor requirement of NrdZ prompted an investigation into the biosynthesis and transport of adenosylcobalamin (AdoCbl) in M. tuberculosis. The data suggest that the mycobacterial Y-polymerases are tightly regulated and restricted to specialised damage-free repair or replication restart. Disruptions in individual M. smegmatis mc2155 DinB (pol IV) homologues resulted in novel antibiotic-resistance polymorphisms that were suggestive of non-redundant function. In contrast, abrogation of all error-prone polymerase activity failed to impair long-term competitive survival of mc2155 in vitro. Similarly, heterologous overexpression of M. tuberculosis pol IV homologues did not increase spontaneous mutation rates in wild-type mc2155, or complement damage hypersensitivity. However, treatment of M. smegmatis with gyrase inhibitors confirmed the differential induction of pol IV homologues in response to replication stalling and demonstrated elevated rates of spontaneous mutagenesis as a result of GyrB inhibition. The class II RNR does not appear to play a significant role in mycobacterial pathogenesis. Specifically, NrdZ was unable to substitute for the class I RNR under aerobic conditions in vitro, and a M. tuberculosis ÄnrdZ deletion mutant was not impaired in its ability to adapt to hypoxia in vitro. Similarly, infection of immunocompetent mice suggested that nrdZ is not required for the survival or virulence of M. tuberculosis in vivo. Disruptions in genes required for AdoCbl and methionine biosynthesis revealed that complex regulatory functions govern mycobacterial methionine and AdoCbl homeostasis. Loss of early (cobK) or late (cobU) stage AdoCbl biosynthetic enzymes had no effect on the growth of M. tuberculosis H37Rv in vitro. In contrast, deletion of the B12-independent methionine synthase (metE) resulted in impaired growth on solid media that could be rescued by vitamin B12 but not Lmethionine supplementation, simultaneously demonstrating the ability of M. tuberculosis to transport exogenous vitamin B12. Significantly, double ÄcobU/ÄmetE and ÄcobK/ÄmetE deletion mutants in which all predicted methionine synthase activity was eliminated, were not impaired for growth in liquid minimal media, suggesting that M. tuberculosis H37Rv possesses alternative mechanisms for methionine generation. Finally, the attenuated virulence of the ÄcobU and ÄmetE deletion mutants in vivo in immunocompetent mice indicated the relevance of AdoCbl biosynthesis to mycobacterial pathogenesis.en
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dc.titleDNA metabolism in mycobacteriaen
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