Browsing by Author "Williams, Monique J"

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    Conditions affecting ergothioneine levels in Mycobacterium smegmatis & the attempted isolation of α-N, N, N-Histidine methyltransferase, the first enzyme in ergothioneine biosynthesis
    (2007) Williams, Monique J; Steyn, Lafras M; Steenkamp, D J
    Ergothioneine and mycothiol are the two major low molecular weight thiols present in mycobacteria. The generation of mycothiol-deficient mutants has demonstrated its role in protecting M tuberculosis against oxidative and nitrosative stress. To date, no ergothioneine-deficient mutants have been identified and the role of ergothioneine in mycobacteria remains unknown. The work in this thesis was performed with the aim of better understanding the function of ergothioneine in mycobacteria, by studying its biosynthesis and the conditions affecting its production.
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    Identifying nucleic acid-associated proteins in Mycobacterium smegmatis by mass spectrometry-based proteomics
    (2020-03-23) Kriel, Nastassja L; Heunis, Tiaan; Sampson, Samantha L; Gey van Pittius, Nico C; Williams, Monique J; Warren, Robin M
    Abstract Background Transcriptional responses required to maintain cellular homeostasis or to adapt to environmental stress, is in part mediated by several nucleic-acid associated proteins. In this study, we sought to establish an affinity purification-mass spectrometry (AP-MS) approach that would enable the collective identification of nucleic acid-associated proteins in mycobacteria. We hypothesized that targeting the RNA polymerase complex through affinity purification would allow for the identification of RNA- and DNA-associated proteins that not only maintain the bacterial chromosome but also enable transcription and translation. Results AP-MS analysis of the RNA polymerase β-subunit cross-linked to nucleic acids identified 275 putative nucleic acid-associated proteins in the model organism Mycobacterium smegmatis under standard culturing conditions. The AP-MS approach successfully identified proteins that are known to make up the RNA polymerase complex, as well as several other known RNA polymerase complex-associated proteins such as a DNA polymerase, sigma factors, transcriptional regulators, and helicases. Gene ontology enrichment analysis of the identified proteins revealed that this approach selected for proteins with GO terms associated with nucleic acids and cellular metabolism. Importantly, we identified several proteins of unknown function not previously known to be associated with nucleic acids. Validation of several candidate nucleic acid-associated proteins demonstrated for the first time DNA association of ectopically expressed MSMEG_1060, MSMEG_2695 and MSMEG_4306 through affinity purification. Conclusions Effective identification of nucleic acid-associated proteins, which make up the RNA polymerase complex as well as other DNA- and RNA-associated proteins, was facilitated by affinity purification of the RNA polymerase β-subunit in M. smegmatis. The successful identification of several transcriptional regulators suggest that our approach could be sensitive enough to investigate the nucleic acid-associated proteins that maintain cellular functions and mediate transcriptional and translational change in response to environmental stress.