Browsing by Author "Niemand, Nandi"

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    Investigating the role of a novel protein in iron–sulphur cluster biogenesis in mycobacteria
    (Stellenbosch : Stellenbosch University, 2020-11) Niemand, Nandi; Williams, Monique Joy ; Warren, Robin; Weber, Brandon; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Molecular Biology and Human Genetics.
    ENGLISH ABSTRACT: Iron–sulphur (Fe–S) cluster biogenesis is a tightly regulated process, which produces co-factors for proteins involved in a diversity of biological processes. Fe–S cluster assembly occurs by highly conserved steps, and although the system has been studied extensively, controversy around the iron donor and Fe–S cluster transporter proteins still exists. A-type carrier (ATC) proteins have been suggested to convey assembled Fe–S clusters to the apo-target protein, or donate iron to scaffold proteins for Fe–S cluster assembly. The genome of Mycobacterium tuberculosis encodes an operon that contains orthologues of the sulphur mobilization (SUF) system (SufR-SufB-SufD-SufC-csd-NifU-Hyp), however this locus lacks a gene encoding for an ATC protein. Homology searches identified Rv2204c or MSMEG_4272 as a potential ATC protein within M. tuberculosis and Mycobacterium smegmatis respectively. This study aimed to investigate the role of these proteins in Fe–S cluster biogenesis and mycobacterial metabolism. The first approach involved characterization of recombinantly expressed and purified Rv2204c and MSMEG_4272. Recombinant Rv2204c and MSMEG_4272 were observed in various oligomeric states, which included predominantly dimers and tetramers. Rv2204c seems to be able to coordinate a 2Fe–2S cluster but further confirmation is required to be able to distinguish between the coordination of a 2Fe–2S or 4Fe–4S cluster. The anaerobic purification of Rv2204c indicated a signal resembling the coordination of a 2Fe–2S, but reconstitution did not produce a spectrum indicative of an Fe–S cluster. Furthermore, isothermal titration calorimetry revealed that Rv2204c binds Fe(II) but not Fe(III). The three conserved cysteine residues in Rv2204c’s indicated some involvement Fe–S cluster coordination, as changing the cysteine residue to alanine impacted protein secondary structure and ultimately the environment where an Fe–S cluster can possibly be coordinated. The second approach involved generating a strain of M. smegmatis in which the level of MSMEG_4272 protein could be modulated. Testing of various knock-down approaches revealed the CRISPRi was most efficient to deplete MSMEG_4272 levels. Silencing MSMEG_4272 expression on a transcriptional level resulted in a severe growth defect under standard culture and iron limiting conditions, with a significant decrease in intracellular iron Stellenbosch University https://scholar.sun.ac.za iii levels, suggesting that MSMEG_4272 might be essential for in vitro growth of M. smegmatis and play a role in the regulation of intracellular iron. In addition, when MSMEG_4272 levels were modulated, M. smegmatis was unable to form a mature biofilm. Decreasing MSMEG_4272 protein levels did not affect the activity of Fe–S containing enzymes SDH and aconitase, suggesting that it did not play a role in the maturation of these Fe–S containing enzymes, or that redundancy exists in the system. The downregulation of MSMEG_4272 in M. smegmatis increased its susceptibility to clofazimine, DMNQ and isoniazid, where the latter was most significant. Overall, this suggests that Rv2204c and MSMEG_4272 plays a role in iron homeostasis in mycobacteria, while their exact role in Fe-S cluster biogenesis remains to be determined.