Doctor of Philosophy (PhD)
First Committee Member
Second Committee Member
Third Committee Member
Fourth Committee Member
Fifth Committee Member
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The process of stationary-phase mutagenesis, also known as adaptative or stress-induced mutagenesis, is a phenomenon where bacterial cells accumulate mutations in non-replicative conditions. This process has mainly been studied in Escherichia coli and Bacillus subtilis; however, the underlying mechanisms found in each of these systems differ. Here, I use B.msubtilis to study previously understudied aspects of stationary-phase mutagenesis. In this dissertation, I describe work that has led to three major discoveries which are described below.
First in B. subtilis, Mfd is important for stationary-phase mutagenesis and its mutagenic function at regions of the genome that are transcriptionally upregulated has been reported. However, the mechanisms by which Mfd promotes the accumulation of mutations in stationary phase remain elusive. Observations presented here suggest Mfd promotes mutagenesis in stressed B. subtilis cells by synchronizing error-prone nucleotide excision repair and base excision repair mediated by UvrA, MutY, and PolI. These processes would likely cause genetic diversity in highly transcribed genomic regions.
Second, having established that Mfd and MutY cooperate, we next chose to further study their involvement in the tolerance of oxidative damage. I used a genetic approach to determine the involvement of Mfd in the tolerance of cells when exposed to different oxidants. I also examined the involvement of these factors in stationary-phase mutagenesis. My results suggest that Mfd protects cells from all types of oxidation tested including protein oxidation. Further, the results suggest that oxidative stress and transcription potentiate the cooperation of Mfd and MutY to generate stationary-phase mutations, thus limiting the mutagenic process to highly transcribed regions.
Finally, stressed B. subtilis cells may limit mutagenesis in space by another mechanism – the development of a hyper-mutable sub-population. Previous work has shown that ComK, the transcriptional regulator of the development of competence, also known as the K-state, influences the development of mutations in stressed B. subtilis cells. By subjecting strains differing only in ComEA, a protein essential to binding and uptake of DNA during the K-state, to a point‐mutation reversion system, we found that stationary‐phase revertants were more likely to be K-cells, the uptake of DNA did not influence the accumulation of mutations, and exogenous oxidants enhanced mutagenesis in K‐cells. Therefore, the observations presented here provide insight into how non-replicative cells limit mutagenesis to highly transcribed regions and a subpopulation. Interestingly, both mutagenic processes are potentiated by oxidative damage. Furthermore, this research helps improve our understanding of bacterial evolution by illuminating mutagenic mechanisms used by cells under nutritionally stressful conditions.
Competence; DNA repair; Transcription-coupled repair
Genetics | Microbiology
University of Nevada, Las Vegas
Martin, Holly Anne, "The Significance of MFD and Oxidative Damage in Stationary-Phase Mutagenesis in Bacillus subtilis" (2020). UNLV Theses, Dissertations, Professional Papers, and Capstones. 3925.
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