One third of the world's population is infected with Mycobacterium tuberculosis, the bacterium that causes TB. During an infection, bacteria often survive host immune system attacks, which include oxidative stress conditions for bacteria growing inside macrophages. This makes treatment difficult and time-consuming. We hypothesize bacteria can adapt to environmental conditions by changing their mRNA maturation and degradation profiles. Using a model system, Mycobacteruim smegmatis, we focus on how mRNA expression is affected by oxidative stress. After construction and sequencing of RNA expression libraries, preliminary analysis showed that after three hours of H2O2 exposure most upregulated genes were related to DNA repair, while downregulated genes included transport proteins. After six hours of exposure, upregulated genes were similar to three hours and downregulated genes included tRNAs. 5' end mapping libraries were also constructed to access differential cleavage site abundance under oxidative stress conditions. We also investigated the roles RNase J may have in stress response and mRNA processing in Mycobacteria. RNase J and RNase E are thought to be the major RNases in bacteria. While most bacteria only have one of them, mycobacteria encode both in their genome, with RNase J being non-essential. We constructed a set of 4 strains (WT, RNase J overexpression, RNase J deletion, and complemented RNase J deletion) and tested their drug resistance and stress tolerance. Results suggests that RNase J deletion and overexpression alter drug sensitivity. Stress tolerance assays showed that WT is more tolerant to oxidative stress, followed by RNase J deletion strain and overexpression and complemented RNase J deletion strains, with the last two showing no growth when cultured with H2O2. Analysis of the expression profile of these strains was performed to help understand if gene expression differences are responsible for the phenotypes observed. For the complemented RNase J deletion, one operon had almost all its genes upregulated. This operon encodes a hydrogenase (Hyd3), suggesting that redox balance in the strain is perturbed.
Worcester Polytechnic Institute
Biology & Biotechnology
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de Camargo Bertuso, Paula, "Roles of regulation of mRNA cleavage in Mycobacterium smegmatis" (2016). Masters Theses (All Theses, All Years). 776.
oxidative stress, RNase J, Mycobacterium smegmatis, mRNA cleavage