Temperature‐dependent Influence of RpoF Overexpression on the E. coli Extracellular Proteome

Abstract

Biofilm, sessile growth of bacteria surrounded by an extracellular polymeric substance (EPS), is notoriously resistant to antibiotic challenge. While the composition and function of biofilm's extracellular components are still largely under exploration, much is known about the influence of proteins, carbohydrates, and extracellular DNA on biofilm robustness. Here, we examine the effect of overexpressing the rpoF transcription factor, responsible for chemotaxis and motility via late-stage flagellar assembly, on two prominent components of the extracellular proteome: curli and flagellin. With the overexpression of rpoF, we hypothesized that bacterial motility would increase and prevent robust biofilm formation. However, we have previously observed an increase in biofilm formation in rpoF overexpressing E. coli at higher growth temperatures. We are currently harvesting both planktonic and biofilm E. coli grown at several time intervals and temperatures to explore how different environments impact biofilm growth and motility. Using immunoblotting, we ensured rpoF overexpression at the protein level. We are using immunoblotting to quantify csgA, a main component of curli, and the protein flagellin. We are also quantifying curli by absorption experiments using Congo Red Dye. We have observed through immunoblotting that we can induce significant rpoF overexpression in harvested biofilm. We are currently collecting data on csgA and flagellin. Additionally, we are assessing the motility of rpoF overexpressing cells using classical swimming and swarming experiments. Our results will help inform how rpoF overexpression influences biofilm formation and maturation, potentially leading to the development of targeted therapies for biofilm disruption.