My lab uses advanced light microscopy to study the biophysics of bacterial growth and division. We study how proteins function as nanoscale machines that build complex microscale structures. We particularly focus on proteins that build and remodel the bacterial cell wall, which is one of the most important targets for antibiotics. To achieve this we use super-resolution microscopy to follow the activity of individual cell wall synthesis proteins at nanoscale resolution inside live bacteria.

Abstract

Almost all bacteria are surrounded by a mesh-like peptidoglycan cell wall essential for their survival. Defects in cell wall structure cause bacteria to burst and die due to the cell’s high internal osmotic pressure. Many rod-shaped bacteria, including major antibiotic resistant pathogens, elongate by adding new material to the sides of their cell wall. In these organisms, an essential multi-protein synthesis complex called the elongasome inserts glycan strands around the circumference of the cell, elongating and reinforcing the cell wall and giving cells their rod shape. How cells regulate the length of circumferential glycan strands and the effect of circumferential glycan strand length on cell size and shape is largely unknown.

We used single molecule tracking method to measure the processivity of the Bacillus subtilis elongasome, which determines the initial length of new glycan strands. We found that elongasomes are highly processive, and that B. subtilis elongasome dynamics and processivity are determined by a balance between processive synthesis and molecular motor tug-of-war, where multiple synthases pull individual MreB filaments in opposite directions. We also found evidence that elongasome processivity and tug-of-war regulate B. subtilis cell size and shape.

In unpublished work, we have been further investigating the molecular mechanisms of elongasome tug-of-war using a combination of bacterial genetics, single molecule tracking and super-resolution microscopy. We have also applied MINFLUX single particle tracking to this problem, giving measurements of elongasome dynamics with at least an order of magnitude improvement in spatiotemporal resolution than obtained previously. I will present recent progress in this area.