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Nucleoid gene regulation in mycobacteria
Thesis (PhD)--Stellenbosch University, 2017.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2017
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| _version_ | 1867613976010948608 |
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| access_status_str | Open Access |
| author | Steyn, Nastassja Lise |
| author2 | Warren, Robin Mark |
| author_browse | Steyn, Nastassja Lise Warren, Robin Mark |
| author_facet | Warren, Robin Mark Steyn, Nastassja Lise |
| author_sort | Steyn, Nastassja Lise |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhD)--Stellenbosch University, 2017. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/102616 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:41.678Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2017 |
| publishDateRange | 2017 |
| publishDateSort | 2017 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/102616 Nucleoid gene regulation in mycobacteria Steyn, Nastassja Lise Warren, Robin Mark Gey van Pittius, Nico Sampson, Samantha Leigh Williams, Monique Joy Stellenbosch University. Faculty of Health Sciences. Dept. of Biomedical Sciences: Molecular Biology and Human Genetics. Mycobacteria, Nucleoid Gene Regulation Nucleogenesis Tuberculosis -- Treatment UCTD Thesis (PhD)--Stellenbosch University, 2017. ENGLISH ABSTRACT: Mycobacterium tuberculosis, the causative agent of tuberculosis disease, remains one of the leading causes of death worldwide. The ongoing tuberculosis epidemic and poor treatment outcomes can be attributed to the emergence of antibiotic resistant mycobacterial organisms as well as persistent M. tuberculosis infections. It is therefore imperative that we improve our understanding of M. tuberculosis pathogenesis to develop more effective anti-tuberculosis drugs and treatment strategies. M. tuberculosis is known to encounter adverse stress conditions such as nutrient starvation, hypoxia, nitrosative stress and low pH during infection and understanding how this pathogen adapts to its host environment is crucial in finding ways to circumvent tuberculosis disease. Conventional methods of investigating transcriptional responses, such as microarrays and ChIP-seq, are limited to the investigation of a single protein. Although valuable information has been obtained through this approach, adaptation to stress is likely mediated through several regulatory elements. Furthermore, new methodologies are required to identify these proteins. For example, nucleoid-associated proteins (NAPs), a group of global transcriptional regulators which have the unique ability to shape the bacterial chromosome, have been shown to be involved in the mycobacterial stress response. However, due to poor sequence homology with other bacterial organisms and conventional methodologies, only seven nucleoid associated proteins have been identified for mycobacterial organisms compared to 12 NAPs in Escherichia coli. Nucleoprotein - Mass Spectrometry (NP-MS) makes use of affinity purification of formaldehyde cross-linked RNA polymerase transcriptional complexes to identify proteins involved in chromosomal structure, and transcriptional and translational processes using mass spectrometry. This approach successfully identified various DNA and RNA associated proteins as well as numerous proteins associated with energy, carbon, lipid and amino acid metabolism. These results suggested that NP-MS was effective in isolating not only proteins directly involved in transcription and translation but also proteins associated with the RNA polymerase transcriptional complex and DNA. MSMEG_1060, MSMEG_2695, MSMEG_3754, MSMEG_4306 and MSMEG_5512 were identified as possible nucleic acid associated proteins through comparison of conserved proteins domains and were subjected to further investigation. Episomal expression of these proteins as FLAG-tagged fusion proteins in M. smegmatis revealed MSMEG_1060, MSMEG_2695, MSMEG_4306 and MSMEG_5512 to be putative DNA-associated proteins whilst no DNA association was found for MSMEG_3754. NP-MS was applied to investigate differences between DNA-associated proteins in exponential and stationary phase cultures. Investigation into the proteins which make up the RNA polymerase transcriptional complex and its associated proteins in stationary phase M. smegmatis cultures, revealed that NP-MS could effectively be used to identify proteins which are required for adaptation to stress in this organism. These included the dormancy response regulator DevR, the ribosome hibernation promoting factor (hpf), heat shock protein HspX and the universal stress proteins MSMEG_3811, MSMEG_3945 and MSMEG_3950. These results demonstrated the ability of the developed NP-MS method to identify the proteins which mediate DNA structure, and transcriptional and translational changes in M. smegmatis. Furthermore, we propose that NP-MS can be used to investigate the proteins associated with the RNA polymerase complex in not only mycobacterial species but also other bacterial organisms. AFRIKAANSE OPSOMMING: Mycobacterium tuberkulosis, die organisme wat tuberkulose veroorsaak, bly steeds een van die hoofoorsake van dood in mense wêreldwyd. Die heersende tuberkulose epidemie en wanhopige behandelingsuitkomste van die siekte dra by tot die verskyning van antibiotika-weerstandige rasse asook nablywende infeksies. Dit is dus van belang om ons begrip van M. tuberkulose patogenese te verbeter om meer effektiewe anti-tuberkulose middels te ontwikkel. M. tuberkulosis ervaar vele nadelige streskondisies tydens infeksie soos uithongering in nutriënte, suurstof, stikstof en lae pH. Deur te verstaan hoe die patogeen aanpas in die gasheeromgewing is van belang om maniere te vind om die siekte te omseil. Konvensionele maniere om transkripsie response te ondersoek met behulp van “microarrays” en “ChIPseq” is beperk omdat net een proteïen op ‘n slag ondersoek kan word. Alhoewel waardevolle inligting verkry kan word met hierdie benaderings, is verskeie reguleerders betrokke tydens stresaanpassing. Nuwe metodologiese benaderings word dus benodig om hierdie proteïene te identifiseer. As ‘n voorbeeld, nukleoïed-geassosieerde proteïene (NAPs) is ‘n groep transkripsie reguleerders wat die unieke vermoë het om die bakteriële chromosoom te vorm en is al gewys om ‘n rol te speel in die stresrespons van mikobakterieë. Vanweë swak DNS-volgorde homologie met ander bakterieë en konvensionele metodieke is daar nog net sewe NAPS geïdentifiseer vir mikobaterieë in vergelyking met die twaalf NAPs in Escherichia coli. Die tegniek Nukleoproteïen - Massa Spektrometrie (NP-MS) kan proteïene betrokke in die chromosoomstruktuur, transkripsie en translasieprosesse identifiseer deur gebruik te maak van affiniteitsuiwering van formaldehied-kruisbindende RNA polimerase transkripsiekomplekse met behulp van massa spektrometrie. Hierdie benadering kan DNS- en RNS-geassosieerde proteïene sowel as verskeie proteïene betrokke in energie, koolstof, lipied en aminosuurmetabolisme identifiseer. NP-MS is dus effektief in die isolasie van proteïene betrokke in transkripsie en translasie, maar ook proteïene direk geassosieerd met die RNA polimerase kompleks en DNS. Die proteïene MSMEG_1060, MSMEG_2695, MSMEG_3754, MSMEG_4306 en MSMEG_5512 is geïdentifiseer as nukleïensuur-geassosieerde proteïene deur vergelykings te maak met gekonserveerde proteïendomeine en is onderworpe aan verdere navorsing. Episomale uitdrukking van hierdie proteïene as FLAG-gehegte fusieproteïene in Mycobacterium smegmatis wys dat MSMEG_1060, MSMEG_2695, MSMEG_4306 en MSMEG_5512 as vermeende DNSgeassosieerde proteïene optree terwyl geen DNS assosiasie gevind kon word vir die proteïen MSMEG_3754 nie. NP-MS kan toegepas word om verskille te ondersoek tussen DNS-geassosieerde proteïene in eksponensiële en stasionêre fases. ‘n Ondersoek in M. smegmatis kulture in stasionêre fase se proteïene wat bydra tot die RNA polimerase kompleks asook die geassosieerde proteïene wys dat NP-MS gebruik kan word met groot effek om proteïene te identifiseer wat benodig mag word vir die aanpassing tot stres in die organisme. Dit sluit in die dormansierespons reguleerder DevR, die ribosoomhibernasie aanwakkerfaktor hpf, die hitteskokproteïen HspX en die universele stres proteïene MSMEG_3811, MSMEG_3945 en MSMEG_3950. Hierdie resultate toon die vermoë van ‘n NP-MS metodiek om proteïene te identifiseer wat DNS struktuur, transkripsie en translasieveranderinge in M. smegmatis kan fasiliteer. Verder stel ons voor dat NP-MS gebruik kan word om proteïene te identifiseer wat geassosieerd is met die RNA polimerase kompleks in nie net mikobakterieë nie, maar ook in ander bakterieë. Doctoral 2017-10-30T10:29:17Z 2017-12-11T10:32:25Z 2018-06-30T03:00:05Z 2017-12 Thesis http://hdl.handle.net/10019.1/102616 en_ZA Stellenbosch University 250 pages : illustrations application/pdf application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Mycobacteria, Nucleoid Gene Regulation Nucleogenesis Tuberculosis -- Treatment UCTD Steyn, Nastassja Lise Nucleoid gene regulation in mycobacteria |
| title | Nucleoid gene regulation in mycobacteria |
| title_full | Nucleoid gene regulation in mycobacteria |
| title_fullStr | Nucleoid gene regulation in mycobacteria |
| title_full_unstemmed | Nucleoid gene regulation in mycobacteria |
| title_short | Nucleoid gene regulation in mycobacteria |
| title_sort | nucleoid gene regulation in mycobacteria |
| topic | Mycobacteria, Nucleoid Gene Regulation Nucleogenesis Tuberculosis -- Treatment UCTD |
| url | http://hdl.handle.net/10019.1/102616 |
| work_keys_str_mv | AT steynnastassjalise nucleoidgeneregulationinmycobacteria |