Novel mechanisms of DNA repair and cell cycle regulation in bacteria

Project Summary: The long-term goal of this research is to understand the contribution of unstudied genes and novel mechanisms to genome maintenance in bacteria. A major challenge in medicine today is addressing the emergence and persistence of antibiotic resistant bacteria. Bacterial species are able to adapt and cope with unfavorable conditions by regulating growth and their response to DNA damage. Bacterial cells are constantly exposed to a broad spectrum of DNA damage caused by intracellular sources, environmental stressors, antibiotic treatments, and disinfectants applied in domestic and hospital settings. Although genome maintenance has been studied in some bacteria, far less is known about these processes in Gram-positive bacteria. One major challenge is that even for the most well studied Gram-positive bacterium, Bacillus subtilis, >40% of the genes are undefined with the vast majority composed of domains of unknown function. This lack of knowledge represents a major fundamental gap in our understanding of how bacteria mitigate stress that affects survival, proliferation, and the acquisition of mutations. While B. subtilis does not cause disease, its genome maintenance pathways are closely related to a number of important human pathogens, including Staphylococcus, Enterococcus, and Streptococcus species which cause hospital-acquired infections imposing a significant economic burden on our healthcare system. We have employed several large-scale genome- wide screens to identify numerous uncharacterized genes that are highly conserved among bacteria and are critical for DNA repair and the regulation of the response to DNA damage. We have also identified new enzymatic activities for existing proteins that will strongly impact our understanding of survival to genotoxic agents. We expect that these studies will result in the complete mechanistic characterization of proteins involved in excision repair, transcriptional response to DNA damage, and DNA break repair. All the genes we propose to study cause severe growth defects, or lead to antibiotic resistance when impaired. The strong phenotypes associated with the deletion mutants underscores these candidates as important targets for the development of effective antimicrobial intervention efforts.