Construction and phenotypic characterization of Mycobacterium smegmatis mutants deficient in DNA glycosylases

Moolla, Nabiela
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The causative pathogen of tuberculosis, Mycobacterium tuberculosis (Mtb), is equipped with several DNA repair mechanisms for continued survival within the host. One such mechanism is Base Excision Repair (BER) that repairs DNA damage caused by reactive oxygen and nitrogen species (ROS/RNS) generated by the host immune cells during infection. BER is dependent on DNA glycosylases namely: formamidopyrimidine (Fpg/MutM/Fapy), endonucleaseVIII (Nei) and endonucleaseIII (Nth) with Nei being structurally similar to Fpg but functionally similar to Nth. Bioinformatics analysis of the genome sequences of Mtb and its non-pathogenic relative Mycobacterium smegmatis (Msm) identified a unique duplication of Fpg and Nei glycosylases and a single nth gene in the same chromosomal context in both organisms. Previously, it has been shown that the lack of Fpg/Nei glycosylases in Msm display no differences in growth and survival under normal and oxidative stress conditions with no increase in spontaneous mutation rates as compared to the parental strain, suggesting that nth maybe significant for mycobacterial genome maintenance. Hence, in this study the nth gene was site specifically inactivated by homologous recombination in the parental Msm strain and in selected combinatorial mutant strains deficient in the Fpg/Nei glycosylases. Loss of the nth allele in the panel of mutants was genotypically confirmed by PCR and southern blot analyses. Inactivation of the nth gene did not affect the in vitro growth of the mutant strains under normal culture conditions. Interestingly, UV induced DNA damage of the single nth mutant resulted in a dramatic increase in mutation frequency that was not observed in any of the mutants. The progressive loss of fpg, nei and nth genes showed exaggerated reduced survival under oxidative stress. The subsequent deletion of nth in mutants deficient in fpg/nei resulted in a dramatic increase in spontaneous mutation rates and frequencies, implying that nth is integral for the repair of both spontaneous and induced DNA damage. Undoubtedly, these results indicate that Msm nth encoding the Nth glycosylase is involved in DNA repair and has anti-mutator properties. Furthermore, nth together with fpg and nei is part of a robust DNA repair system that maintains the integrity of the mycobacterial genome.
A dissertation submitted to the Faculty of Health science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science in Medicine. Johannesburg 2013