# Localization and Single Molecule Dynamics of Bacillus subtilis Penicillin-Binding Proteins Depend on Substrate Availability and Are Affected by Stress Conditions

## TL;DR

This study uses single molecule tracking to show how penicillin-binding proteins in Bacillus subtilis change their movement and location based on substrate availability and stress conditions.

## Contribution

The study reveals distinct mobility states and localization changes of PBPs under stress and substrate conditions, suggesting functional flexibility in cell wall synthesis.

## Key findings

- PBPs show two mobility states: slow (cell wall synthesis) and fast (free diffusion), with about 50% in the slow state for most PBPs.
- Osmotic stress alters Pbp2a and Pbp4a localization to include the septum, while Pbp3 becomes more dynamic and Pbp4 more static.
- PBPs lose their localization patterns when exposed to vancomycin or penicillin G, indicating dependency on substrate availability.

## Abstract

We have used single molecule tracking to investigate dynamics of four penicillin-binding proteins (PBPs) in Bacillus subtilis to shed light on their possible modes of action. We show that Pbp2a, Pbp3, Pbp4, and Pbp4a, when expressed at very low levels, show at least two distinct states of mobility: a state of slow motion, likely representing molecules involved in cell wall synthesis, and a mode of fast motion, likely representing freely diffusing molecules. Except for Pbp4, all other PBPs showed about 50% molecules in the slow mobility state, suggesting that roughly half of all molecules are engaged in a substrate-bound mode. We observed similar coefficients for the slow mobility state for Pbp4 and Pbp4a on the one hand, and for Pbp2a and Pbp3 on the other hand, indicating possible joint activities, respectively. Upon induction of osmotic stress, Pbp2a and Pbp4a changed from a pattern of localization mostly at the lateral cell membrane to also include localization at the septum, revealing that sites of preferred positioning for these two PBPs can be modified during stress conditions. While Pbp3 became more dynamic after induction of osmotic stress, Pbp4 became more static, showing that PBPs reacted markedly differently to envelope stress conditions. The data suggest that PBPs could take over functions in cell wall synthesis during different stress conditions, increasing the resilience of cell wall homeostasis in different environmental conditions. All PBPs lost their respective localization pattern after the addition of vancomycin or penicillin G, indicating that patterns largely depend on substrate availability. Our findings show that PBPs rapidly alter between non-targeted motion through the cell membrane and capture at sites of active cell wall synthesis, most likely guided by complex formation with other cell wall synthesis enzymes.