Title

Role of DNA repair and oxidative stress defenses in {\it Bacillus subtilis\/} spore resistance

Date of Completion

January 1998

Keywords

Biology, Genetics|Biology, Microbiology|Agriculture, Plant Pathology

Degree

Ph.D.

Abstract

Bacillus subtilis spores are extremely resistant to different environmental insults. Although several factors contribute to spore resistance, not much is known about the role that enzymes responsible for DNA repair and oxidative stress protection in B. subtilis cells might play. To obtain this information, we generated mutations in genes coding for proteins involved in DNA repair and oxidative stress defenses in cells, and measured the resistance of the various mutant spores to different treatments. Loss of genes involved in DNA repair, including those coding for apurinic endonucleases (nfo and xth), a mismatch repair enzyme (mutS) and two glycosylases (ung and nth), had no effect on spore killing by UV and wet heat treatments when compared to wild type (WT) spores. However, the uvrB mutant was more sensitive to UV than WT spores, indicating the importance of excision repair in the repair of UV damage during spore germination. We also measured the resistance to oxidizing agents of spores lacking various oxidative stress protective enzymes including mutations to the vegetative catalases (KatA and KatB), a spore catalase KatX, superoxide dismutase (SodA), a DNA protective protein (MrgA) and two alkyl hydroperoxide reductases (AhpA and AhpC). Mutations in these genes had no effect on spore resistance to oxidizing agents, although some mutations sensitized the mutant cells to oxidizing agents. Since we found that a number of enzymes involved in oxidative stress resistance are present in spores, we suggest that these enzymes do not work inside spores, presumably because the environment inside the spore inhibits enzyme activity. However, some of these enzymes do play a role during spore outgrowth if oxidizing agents are added, as mutations in katX and sodA resulted in a block in spore outgrowth in the presence of oxidizing agents. We also studied if a mutation in a transcriptional regulator perR that causes overproduction of oxidative stress protective enzymes in B. subtilis cells, affects spore resistance. The levels of oxidative stress related proteins in perR spores were similar to those of WT spores, suggesting that PerR does not play a significant role in the composition or the resistance of B. subtilis spores. ^