Mechanistic Insights into MinD Regulation and Pattern Formation in Bacillus subtilis

Abstract

Bacteria precisely regulate the place and timing of their cell division. One of the best-understood systems for division site selection is the Min system in Escherichia coli . In E. coli , the Min system displays remarkable pole-to-pole oscillation, creating a time-averaged minimum at the cell’s geometric center, which marks the future division site. Interestingly, the Gram-positive model species Bacillus subtilis also encodes homologous proteins: the cell division inhibitor MinC and the Walker-ATPase MinD. However, B. subtilis lacks the activating protein MinE, which is essential for Min dynamics in E. coli . We have shown before that the B. subtilis Min system is highly dynamic and quickly relocalizes to active sites of division. This raised questions about how Min protein dynamics are regulated on a molecular level in B. subtilis . Here, we show with a combination of in vitro experiments and in vivo single-molecule imaging that the ATPase activity of B. subtilis MinD is activated solely by membrane binding. Additionally, both monomeric and dimeric MinD bind to the membrane, and binding of ATP to MinD is a prerequisite for fast membrane detachment. Single-molecule localization microscopy data confirm membrane binding of monomeric MinD variants. However, only wild type MinD enriches at cell poles and sites of ongoing division, likely due to interaction with MinJ. Monomeric MinD variants and locked dimers remain distributed along the membrane and lack the characteristic pattern formation. Single-molecule tracking data further support that MinD has a freely diffusive population, which is increased in the monomeric variants and a membrane binding defective mutant. Thus, MinD dynamics in B. subtilis do not require any unknown protein component and can be fully explained by MinD’s binding and unbinding kinetics with the membrane. The generation of MinD patterns relies on the short-lived temporal residence of MinD dimers at the membrane.