Browsing by Author "Tamuhla, Tsaone"

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    Functional characterisation of a SufT homologue in mycobacterium smegmatis
    (Stellenbosch : Stellenbosch University, 2018-12) Tamuhla, Tsaone; Williams, Monique Joy; Willemse, Danicke; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Mycobacterium tuberculosis is one of the leading causes of death globally and with drug resistant tuberculosis (TB) on the rise, there is an urgent need to find new anti-TB drugs and drug targets. Increasing our understanding of the physiology of M. tuberculosis can aid in elucidating novel essential pathways which can be used as new drug targets. One such pathway is the iron-sulphur (Fe-S) biogenesis pathway, which is encoded by the sufR-sufBsufD-sufC-csd-sufU-sufT operon (suf operon) in mycobacteria. Fe-S biogenesis is a vital process in cellular physiology yet the functioning of the Fe-S biogenesis machinery in mycobacteria is not fully understood. The last gene in the suf operon, sufT, encodes the only protein in the genome that contains a DUF59 domain. This study used targeted gene deletion and phenotypic characterisation of the resultant mutant to investigate the role of SufT in the physiology of mycobacteria, using Mycobacterium smegmatis as a model organism. An M. smegmatis ΔsufT knockout mutant harbouring an unmarked deletion in sufT was generated using allelic exchange mutagenesis. SufT was confirmed to be dispensable for growth in standard aerobic culture. Loss of SufT significantly decreased the activity of the Fe-S containing enzyme succinate dehydrogenase (SDH) and is therefore proposed to be a putative Fe-S maturation protein. No decrease in aconitase (ACN) activity was observed, suggesting that its role could be limited to certain Fe-S cluster proteins. Loss of SufT did not impact the survival of M. smegmatis after exposure to oxidative stress induced by the redox cycler 2,3- dimethoxy-1,4-naphthoquinone (DMNQ), or the sensitivity of M. smegmatis to the anti-TB drugs isoniazid, clofazimine or rifampicin. The M. smegmatis ΔsufT mutant displayed a growth defect during planktonic growth under iron limiting conditions. This defect was characterised by an extended lag phase, which was observed for all iron concentrations below 2 µM. This suggests that SufT is needed for adaptation to growth under iron limitation. The exponential growth and final cell density achieved by the M. smegmatis ΔsufT mutant under iron limiting conditions was comparable to wild-type, suggesting that induction of a protein that compensates for the loss of sufT occurs. The study also confirmed that the cellular demand for iron during biofilm formation far exceeds that for planktonic growth, particularly during the maturation of the biofilms to form an extracellular matrix. This is the first study to functionally characterise SufT in mycobacteria providing a basis for further mechanistic studies.