Characterization of the actin-like MreB cytoskeleton dynamics and its role in cell wall synthesis in Bacillus subtilis

The peptidoglycan cell wall (CW) and the actin-like MreB
cytoskeleton are the majordeterminants of cell morphology in
non-spherical bacteria. Bacillus subtilis is a rod-shaped
Grampositive bacterium that has three MreB isoforms: MreB, Mbl
(MreB–like) and MreBH (MreBHomologue). Over the last decade, all
three proteins were reported to localize in dynamic filamentous
helical structures running the length of the cells underneath the
membrane. This helical pattern led to a model where the extended
MreB structures act as scaffolds to position CW-synthesizing
machineries along sidewalls. However, the dynamic relationship
between the MreB cytoskeleton and CW elongation complexes remained
to be elucidated. Here we describe the characterization of the
dynamics of the three MreB isoforms, CW synthesis and elongation
complexes in live Bacillus subtilis cells at high spatial and
temporal resolution. Using total internal reflection fluorescence
microscopy (TIRFM) we found that MreB, Mbl and MreBH actually do
not assemble into an extended helical structure but instead into
discrete patches that move processively along peripheral tracks
perpendicular to the long axis of the cell. We found similar patch
localization and dynamics for several morphogenetic factors and
CW-synthesizing enzymes including MreD, MreC, RodA, PbpH and PBP2a.
Furthermore, using fluorescent recovery after photobleaching
(FRAP), we showed that treadmilling of MreB filaments does not
drive patch motility, as expected from the structural homology to
actin. Blocking CW synthesis with antibiotics that target different
steps of the peptidoglycan biosynthetic pathway stopped MreB
patches motion, suggesting that CW synthesis is the driving force
of patch motility. On the basis of these findings, we proposed a
new model for MreB fuction in which MreB polymers restrict and
orient patch motility to ensure controlled lateral CW expansion,
thereby maintaining cell shape. To further investigate the
molecular mechanism underlying MreB action, we next performed a
site-directed mutagenesis analysis. Alanine substitutions of three
charged amino 2 acids of MreB generated a B. subtilis strain with
cell shape and growth defects. TIRFM analysis revealed that the
mutated MreB protein displayed wild-type localization and dynamics,
suggesting that it is still associated to the CW elongation
machinery but might be defective in an interaction important for
MreB morphogenetic function. Thus, this mutant appears as as a good
candidate to start characterizing the interactions between the
three MreB isoforms and components involved in CW elongation. It
might also help to understand the function of components of
theCWsynthetic complexes, and how they are coordinated to achieve
efficient CW synthesis. Finally, to investigate how the integrity
of the CW is maintained, we studied the localization and dynamics
of the LiaIH-system, which i s t he t arget o f L iaRS, a t
wo-component system involved in cell envelope stress response. We
found that under stress conditions, when liaI and LiaH genes are
expressed, the proteins form static complexes that coat the cell
membrane. LiaI is required for the even distribution of the LiaH in
the membrane. Taken together, these data suggest that LiaIH
complexes may protect the cell from CW damage. Taken together, the
findings described in this thesis provide valuable insights into
the understanding of CW synthesis in B. subtilis, which may open
new perspectives for the design of novel antimicrobial agents.