Structure and fonctions of bacterial’s cytoskeleton in stationary phase during the development of competence and sporulation. – CytoStat

In this research project, we propose a multi disciplinary approach
(transcriptional regulation, microscopy, biochemistry) in order to characterize the organization, the eventual restructuring and the functions of the actin cytoskeleton in stationary phase. For example, the candidate will be able to bring his expertise in the use of the luciferase of Photinus pyralys as a transcriptional reporter in order to study the expression profiles of the cytoskeleton genes in different media or genetic backgrounds. In addition, the host laboratory, which is a leader in the description and comprehension of the roles of the actin cytoskeleton in the prokaryotes, has mastered several advanced microscopy techniques (TIRFM, microfluidic) allowing the optimal visualization of the bacterial cytoskeleton. Finally, several techniques allowing the identification of protein-protein interactions will be possible, in situ, in the lab or the department, in order to establish eventual links between cytoskeleton and competence or sporulation.

We are making progress in the 3 main tasks composing this project:
- we have constructed the different transcriptional fusions and are now analysing them in different medium and genetic backgrounds. We already have interesting results. For example, we confirm the induction the mreB expression during competence.
- We also constructed several GFP-MreB fusions under the control of a native promoter (essential step for our project). We are starting to look at these fusions under the microscope et few localizations are already promising. For example, GFP-MreB is localized as patches during exponential growth but becomes diffuse in stationary phase in LB, while GFP-Mbl stays as patches all the time.
- finally, we started the pull-downs with MreB and Mbl during the competence development. Numerous potential partners have been identified. These partners are implicated in cell division, cell wall synthesis or, as we expected, competence.

We have now constructed almost all the tools needed for this project. New ones could be added to the list later if needed.
- about the transcriptional profils, we have to continue and test different medium and genetic backgrounds.
- about the microscopy, we have to characterize in detail the observed localizations and focus on the competence. To do so, we will need to co-localize the members of the AC with proteins produced during competence.
- finally, for the pull-downs, we have to go forward with our list of conditions and baits. We will also have to perform controls experiments in order to confirm that the eventual partners identified are really specific because they are in close proximity with our baits.

None yet

For decades, scientists believed that the rigid wall surrounding bacterial cells (prokaryotes) was the only shape-determining factor. In higher organisms (eukaryotes), cells have a meshwork of protein filaments, particularly actin filaments, which control cellular shape. This complex meshwork is known as the “cytoskeleton”. More recently, it has been shown that bacteria also have a dynamic cytoskeleton involved in the spatial organization of processes essentially associated with cellular growth.
In this fundamental research project, we propose to study the Actin cytoskeleton (encoded by the mre genes) during the development, in stationary phase, of the two main environmental adaptations that are competence and sporulation in the model organism Bacillus subtilis. The development of these two processes, which have been studied for more than 80 years now, requires a large reorganization of the genes expression, the proteins localization but also the cell shape in B. subtilis. Because they are energetically very costly, these physiological adaptations impose a growth arrest in the cells that initiated them. However, it’s been shown that the expression of the mre genes is activated during the development of competence or sporulation. But why inducing the actin cytoskeleton expression, one of the major cellular growth determinants by control of the peptidoglycan synthesis, while we know that cells developing competence or sporulation stop dividing?
Several results, published or preliminary, allow us to speculate about this observation. In the case of competence, numerous preliminary results suggest that both MreB and Mbl could play a role in the reorganization of the peptidoglycan (cell wall), in the localization of some competence proteins and in the processing of the transforming DNA during its entry into the cytoplasm until the recombination in the chromosome. In the case of sporulation, the maturation of the spore inside the mother cell almost inevitably implies a massive reorganization of the bacterial cytoskeleton. It has been shown in Streptomyces coelicolor, that a mreB mutant generates spores that double their volume and lose their cell wall consistency. This work also suggests that the sporulation-associated functions of MreB could be conserved, at least, in the Streptomyces.
In this research project, we propose a multi disciplinary approach (transcriptional regulation, microscopy, biochemistry) in order to characterize the organization, the eventual restructuration and the functions of the actin cytoskeleton in stationary phase. For example, the candidate will be able to bring its expertise in the use of the luciferase of Photinus pyralys as a transcriptional reporter in order to study the expression profiles of the cytoskeleton genes in different medium or genetic backgrounds. In addition, the host laboratory, which is leader in the description and comprehension of the roles of the actin cytoskeleton in the prokaryotes, masters several advanced microscopy techniques (TIRFM, microfluidic) allowing the optimal visualization of the bacterial cytoskeleton. Finally, several techniques allowing the identification of protein-protein interactions will be possible, in situ, in the lab or the department, in order to establish eventual links between cytoskeleton and competence or sporulation.