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Investigating the role of a novel protein in iron–sulphur cluster biogenesis in mycobacteria
Thesis (PhD)--Stellenbosch University, 2020.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2020
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| _version_ | 1867614051707650048 |
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| access_status_str | Open Access |
| author | Niemand, Nandi |
| author2 | Williams, Monique Joy
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| author_browse | Niemand, Nandi Williams, Monique Joy |
| author_facet | Williams, Monique Joy
Niemand, Nandi |
| author_sort | Niemand, Nandi |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2020. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/109469 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:45:52.267Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2020 |
| publishDateRange | 2020 |
| publishDateSort | 2020 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/109469 Investigating the role of a novel protein in iron–sulphur cluster biogenesis in mycobacteria 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. Iron-sulfur proteins Mycobacterium tuberculosis Succinate dehydrogenase Cluster biogenesis systems UCTD Thesis (PhD)--Stellenbosch University, 2020. 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. AFRIKAANSE OPSOMMING: ʼn Streng gereguleerde proses is verantwoordelik vir die sintese van yster-swawel (Fe–S) groepe, en lewer ko-faktore vir proteïene wat betrokke is by verskillende biologiese prosesse. Die samestelling van Fe–S groepe volg hoogs behoue stappe. Al is die sisteem al breedvoerig bestudeer, is daar steeds onduidelikheid rondom die yster-skenker en die proteïen verantwoordelik vir die vervoer van Fe–S groep. Daar was voorgestel dat A-tipe draer (ATC) proteïene of saamgestelde Fe–S groepe na apo-teiken proteïene kan oor dra, of yster aan steierproteïene kan skenk vir die samestelling van Fe–S groepe. Die Mycobacterium tuberculosis genoom kodeer vir ʼn operon wat ortoloë van die SUF sisteem (SufR-SufB-SufD-SufC-csdNifU-Hyp) bevat, maar hierdie lokus het egter nie ʼn geen wat vir ʼn ATC proteïene kodeer nie. Homologiese soektogte het vir Rv2204c en MSMEG_4272 onderskeidelik identifiseer as twee potensiële ATC proteïene in M. tuberculosis en Mycobacterium smegmatis. Hierdie studie poog om die rol van hierdie proteïene in Fe–S groep sintese en die mikobakteriële metabolisme te verklaar. Die eerste benadering behels die karakterisering van Rv2204c en MSMEG_4272. Verskillende olgimere toestande is waargeneem vir gesuiwerde Rv2204c en MSMEG_4272, wat hoofsaaklik dimere, sowel as tetramere insluit. Dit blyk dat Rv2204c `n 2Fe–2S groep kan koördineer, maar tans kan daar nie onderskei word tussen die koördinasie van `n 2Fe–2S of 4Fe–4S groep nie en benodig verdere bevestiging. Anaërobiese gesuiwerde Rv2204c toon `n spektrum wat lyk soos die koördinasie van `n 2Fe–2S groep, terwyl rekonstitusie `n spektrum opgelewer het wat nie dui op die koördinasie van `n Fe–S groep nie. Verder het die isotermiese titrasie-kalorimetrie aangedui dat Rv2204c Fe(II) bind, maar nie Fe(III) nie. Die drie beware sisteïen residue in Rv2204c dui dat hulle `n mate van betrokkenheid het in Fe–S groep koördinasie. Die mutasie van `n sisteïen residu na `n alanine residu, beïnvloed die sekondêre struktuur van die proteïen, as ook die omgewing moontlik verantwoordelik vir die koördinasie van`n Fe–S groep. As ʼn tweede benadering is gepoog om ʼn M. smegmatis mutant te genereer waarin die MSMEG_4272 proteïen uitdrukking gemoduleer kon word. Verskeie benadering was ondersoek, waarvan dit blyk die CRISPRi sisteem mees doeltreffend MSMEG_4272 vlakke kon verminder. Die onderdrukking van MSMEG_4272 uitdrukking op transkripsionele vlak Stellenbosch University https://scholar.sun.ac.za v het gelei tot ʼn ernstige groei defek onder standaard kultuur en yster-beperkende toestande. Verder was ʼn duidelike afname in intrasellulêre ystervlakke waargeneem. Dit kan moontlik dui dat MSMEG_4272 noodsaaklik is vir die in vitro groei van M. smegmatis, en ʼn rol speel in die regulering van intrasellulêre ystervlakke. Boonop, as MSMEG_4272 vlakke gemoduleer is, kon M. smegmatis nie ʼn volgroeide biofilm vorm nie. Die afname in MSMEG_4272 proteïen vlakke het nie die aktiwiteit van Fe–S bevattende ensieme SDH en akonitase affekteer nie. Dit kan aandui dat MSMEG_4272 nie ʼn rol speel in die aktivering van hierdie Fe–S bevattende ensieme nie, of dat daar oortolligheid in die stelsel bestaan. Afwaartse regulasie van MSMEG_4272 het M. smegmatis se vatbaarheid vir klofasimien, DMNQ en isoniasied verhoog, waar die laasgenoemde die grootste effek getoon het. In geheel dui resultate daarop dat Rv2204c en MSMEG_4272 wel ʼn rol speel in yster homeostase in mikobakterieë, terwyl die presiese rol daarvan in die sintese van Fe–S groepe nog bepaal moet word. Doctorate 2020-12-01T08:24:35Z 2021-02-01T07:56:29Z 2020-12-01T08:24:35Z 2021-02-01T07:56:29Z 2020-11 Thesis http://hdl.handle.net/10019.1/109469 en_ZA Stellenbosch University xv, 142 pages : illustrations. application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Iron-sulfur proteins Mycobacterium tuberculosis Succinate dehydrogenase Cluster biogenesis systems UCTD Niemand, Nandi Investigating the role of a novel protein in iron–sulphur cluster biogenesis in mycobacteria |
| title | Investigating the role of a novel protein in iron–sulphur cluster biogenesis in mycobacteria |
| title_full | Investigating the role of a novel protein in iron–sulphur cluster biogenesis in mycobacteria |
| title_fullStr | Investigating the role of a novel protein in iron–sulphur cluster biogenesis in mycobacteria |
| title_full_unstemmed | Investigating the role of a novel protein in iron–sulphur cluster biogenesis in mycobacteria |
| title_short | Investigating the role of a novel protein in iron–sulphur cluster biogenesis in mycobacteria |
| title_sort | investigating the role of a novel protein in iron sulphur cluster biogenesis in mycobacteria |
| topic | Iron-sulfur proteins Mycobacterium tuberculosis Succinate dehydrogenase Cluster biogenesis systems UCTD |
| url | http://hdl.handle.net/10019.1/109469 |
| work_keys_str_mv | AT niemandnandi investigatingtheroleofanovelproteininironsulphurclusterbiogenesisinmycobacteria |