Spatiotemporal analysis of cell wall synthesis related enzymes and the influence of cell wall stress factors

Bacteria have a highly varied appearance and shape, which is primarily affected by the cell wall and other components that give shape to the cells. Despite decades of research in the field of cell wall synthesis there are still many open questions regarding the organisation of the bacterial peptidoglycan sacculus, but also regarding the spatiotemporal distribution of proteins that are involved in peptidoglycan synthesis. An essential class of these, the so-called Penicillin Binding Proteins, or PBPs for short, are all performing the last steps and some additional steps of the peptidoglycan synthesis. The Bacillus subtilis genome codes for a large number of different PBPs, which have different enzymatic properties, and it is not yet clear for all of these proteins, when – or under which conditions – they become active. Some of the PBPs are known to interact with cell wall synthesis machineries, the so called elongasome or divisome, both being partly organised by cytoskeletal elements like MreB or FtsZ. To better understand their localisation and function, different PBPs and MreB were used in this thesis as indicators for the location of cell wall synthesis. They were investigated under different conditions, where the main focus was the spatiotemporal distribution of these proteins. Five different PBPs from different classes were selected, and n-terminally tagged with an mVenus fluorophore. These were then investigated with the help of a high-resolution microscopy method called single-molecule tracking, and the diffusion coefficients of the molecules were determined. Two different dynamic molecular populations could be discovered, one a slow-diffusing, likely enzymatically active population, and the other a fast-moving inactive population. Most PBPs demonstrate a 50% slow diffusing population, which means that roughly half of the molecules are bound to their substrate. Additionally, the reactions involving the PBPs were tested in the presence of different environmental conditions – to achieve this, the cells were treated with osmotic stressors (NaCl and sorbitol), as well as antibiotics (vancomycin, penicillin G, nisin, fosfomycin, bacitracin). Through these experiments, changes in the diffusion coefficients of the proteins were observed. Particularly the availability of peptidoglycan building blocks was altered by the presence of antibiotics. MreB also reacts to the available amounts of peptidoglycan building blocks through a change in its dynamic populations, but not as strongly as most studied PBPs. Additionally, this thesis builds on the results of a previous doctoral thesis from the same research group (written by Dr. Simon Dersch). Therefore, this thesis (written by Lisa Stuckenschneider) also investigated the elongasome components PbpH, Pbp2a and MreB through corresponding deletion (∆pbpA and ∆pbpH) and depletion genetic backgrounds (MreB levels were strongly reduced in the cell by a Cas9 system), with the same antibiotics. The dynamics of both of the redundant transpeptidases PbpH and Pbp2a change strongly in the corresponding deletion strains and under the influence of antibiotics. Although the decreased MreB levels in the cell had only a minor effect on the diffusion constants of PbpH or Pbp2a. The absence of MreB made the diffusion of PbpH more susceptible to antibiotic treatment, and to a similar degree as in the ∆pbpA background