Quantifying the Influence of rpoF Overexpression on Extracellular Biomolecules in PHL628 E. coli Biofilms by Confocal Microscopy

Abstract

Biofilms form when planktonic, or freely swimming, bacteria adhere to a surface. Once adhered, connections form with other bacteria through extracellular polymeric substances (EPS). Biofilms can form on medical devices, causing persistent infections in patients, and on industrial equipment, which is known as biofouling. Biofilms present a unique challenge, as they can exhibit a higher tolerance for antibiotics than their planktonic counterparts. Curli are extracellular quaternary protein structures associated with robust biofilm formation in E. coli. They have been shown to aid in initial adhesion of the bacterial cells to a surface.Here, we induced the overexpression of rpoF, also known as fliA and σ28, which codes for a flagellar transcription factor important in late-stage flagellar assembly, in PHL628 E. coli, which overexpress curli at growth temperatures less than 30 °C. Because rpoF increases cell motility, we originally predicted that rpoF overexpression would decrease the amount of biofilm formation. However, we observed that rpoFoverexpression increased the amount of biofilm formed. We now suspect that the nature of rpoF overexpression's effects on biofilm formation may be through changing the composition of the EPS. To test this hypothesis, we grew biofilm under different conditions, and then analyzed the biomolecule composition of the EPS, as well as the overall biofilm volume using confocal laser scanning microscopy (CLSM). We used the dyes Thioflavin T and Calcofluor White to quantify extracellular amyloid proteins and carbohydrates, respectively. We found that rpoF overexpression increases the amount of extracellular carbohydrates. We also observed more curli formation when rpoF was overexpressed at lower temperatures, but not at higher growth temperatures. This suggests that curli does not account for increased biofilm formation at higher temperatures. In future experiments, we will continue similar imaging and analysis methods with Sypro Ruby (extracellular protein concentration), propidium iodide (eDNA), and DiD’ oil (lipid concentration) to obtain a more complete picture of how rpoF changes the composition of the biofilm EPS.