Abstract

Background: Although little is known about the location and movement of enzymes within bacterial cells, this information is crucial for comprehending how metabolism is regulated spatially. The heavy RF synthase, a sizable protein complex with a capsid structure formed by RibH and an encapsulated RibE homotrimer, which regulates substratechanneling, has been the focus of intensive Research on the four key enzymes (Rib enzymes) in the riboflavin (RF) production pathway in vitro using Bacillus subtilis, a Gram-positive model bacteria. Unfortunately, little is understood about these enzymes’ activity and mobility in vivo.

Results: We looked at where the Rib enzymes were located and how they were moving around in the cytoplasm of B. subtilis. We present evidence for limited diffusion at the cell poles and otherwise Brownian motion by employing single particle tracking to characterise the diffusion of rib enzymes in living cells. Most RibH particles exhibited evident nucleoid blockage and a high level of restricted motion, which are largely eliminated following Rifampicin treatment, demonstrating that confinement is reliant on ongoing transcription. In contrast, RibE is primarily diffusive within the cell and only exhibits RibH nanocompartment encapsulation. In single cells, we localise several diffusive populations and discover that fast diffusion mostly crosses nucleoids in the cell centres whereas slower, constrained subdiffusion takes place at the congested cell poles.

Conclusions: Our findings demonstrate that active enzyme mobility varies locally within the bacterial cytoplasm, establishing metabolic compartmentalization primarily at the cell poles.