Abstract 1623 Understanding the role of autophosphorylation of tyrosine kinases in Bacillus subtilis biofilm formation

Biofilms are communities of bacteria that form on surfaces and are held together by an extracellular matrix of self-produced biomolecules. The ability of bacteria to form biofilms can be dangerous as they infiltrate hospitals and cause bacteria to have increased resistance to antibiotics. However, biofilms can also have beneficial roles, including in the environment and industrial processes such as wastewater treatment. Despite their importance, the cellular and environmental conditions which drive the formation of biofilms are not fully understood. To study why and when biofilms grow, we used a non-pathogenic, model organism known as Bacillus subtilis. This bacteria has two known tyrosine kinases: EpsB and PtkA. The former has a known target in B. subtilis biofilm formation, while the latter has primarily been studied in cellular conditions unrelated to biofilm formation. This research combines genetic and protein biochemistry techniques to examine the role of the tyrosine kinases in the regulation of bacterial biofilms. As bacterial tyrosine kinase proteins are known to undergo autophosphorylation at their C-terminus, we examined phosphomimetic mutants of these proteins, with a focus on the less well understood PtkA. We find that the phosphomimetic mutants have dramatic effects on biofilm formation. Using Western Blot analyses, we examined total phosphorylation of proteins in three separate conditions that produce biofilms. Interestingly, although there are dramatic changes in the ability to form biofilms, phosphorylation patterns associated with the phosphomimetic mutants were more subtle. In order to better understand whether biofilm conditions affect autophosphorylation of PtkA, we conducted pull-downs of PtkA under different genetic conditions that drive or prevent B. subtilis biofilms. While more experimentation is needed to understand the downstream targets of PtkA and how it is regulated by autophosphorylation, these findings are a first step in elucidating the role of PtkA in biofilm formation. This research was supported by an NIH R15 grant.