Minutes-long tracking of single molecule dynamics in live bacteria reveals that molecular motor tug-of-war regulates Bacillus subtilis elongasome dynamics and cell shape

Session

Microbial Imaging

Authors

Dr Séamus Holden (1)

Affiliations

1. University of Warwick

Keywords

Single molecule fluorescence microscopy

Bacteriology

Biophysics

Single molecule tracking

Abstract text

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, and errors in this process lead to cell death, making it an excellent antimicrobial target. Due to the small size of bacteria, cell wall remodelling takes place below the diffraction limit of light microscopy, making observation of elongasome dynamics an intense technical challenge. 

 

We developed a novel single molecule tracking method to track individual elongasomes – the protein complex responsible for cell wall elongation – in live Bacillus subtilis bacteria for minutes. We thereby determined the processivity of the elongasome, which likely determines the length of glycan strands from which the bacterial cell wall is built. 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 between competing cell synthesis complexes. We also found evidence that elongasome processivity and tug-of-war regulate B. subtilis cell size and shape.

 

Our results show that molecular motor tug-of-war, previously thought to be a phenomenon exclusive to eukaryotic molecular motors, plays a key role in regulating elongasome dynamics and cell shape in the model bacterium Bacillus subtilis. This work also pushes the limits of single molecule observation in living cells, delivering entire trajectories of protein complex dynamics in vivo, enabling in vivo determination of complex biomechanical rate kinetics.