Molecular and cell biology of bacterial motility – 0

The subcellular architecture of the bacterial cell has become a central theme in prokaryotic biology. Indeed, since the bacterial cytoskeleton and regulated polarity were discovered, bacterial cells are no longer considered simple compartments where proteins achieve their function by simple diffusion. As a post-doc, I showed that the social bacterium M. xanthus is a promising model system to study cellular processes involved in bacterial motility. Here, we describe three distinct aims to extend these researches. We have suggested that M. xanthus cells move by assembling periodic focal adhesion sites (A-Motor) that connect the cell surface and the cytoskeleton on which they exert traction. We propose to characterize the cell envelope adhesion components and the intracellular molecular motor that powers movement. Also, to study individual focal adhesions and dynamics we plan to measure the traction forces specifically exerted at each individual site with imaging methods inspired from eukaryotic cell systems. M. xanthus also uses a second motility system that pulls the cell body forward by extrusion of retractile adhesive fibers (Type-IV pili, S-motor). Components involved in separate motility systems S (FrzS) and A (AglZ) oscillate coordinately from pole-to-pole to trigger periodic cellular movement reversals. In this process, the identity of each cell pole is suddenly switched so that the initial leading pole rapidly becomes the lagging pole. We propose to elucidate how proteins involved in each motility systems are targeted to the cell poles and how such targeting can be switched. We will first study the MglA protein that we have shown to be essential for the oscillations of both FrzS and AglZ. Moreover, we will search polar targets of FrzS with a genetic screen that takes advantage of a FrzS mutant that can no longer reside at the leading cell pole. Second site mutations that restore proper localization to this mutant FrzS may reveal the polar target of FrzS. Finally, we will search FrzS and AglZ binding partners directly by affinity chromatography. Our model proposes that the A-engine pulls on the internal cytoskeleton to propel the cell body forward. Furthermore, the kinetics of FrzS oscillations suggest that they do not result from diffusion but rather from active transport along an intracellular track. Thus, we will analyze the role of the cytoskeleton in those processes. First, we will determine the localization of the bacterial actin MreB in moving M. xanthus cells and the effect of its depletion. If the MreB filament is not involved in movement and motors localization, we will attempt to identify the involved filament by direct affinity chromatography using an FrzS mutant that is unable to localize to the cell poles. Such a mutant is organized as a spiral that connects both cell poles, suggesting that it remains bound to the filament it tracks on. All together the proposed experiments aim to define both a novel molecular machine that powers surface movement in bacteria and the mechanisms responsible of its spatial regulation, such as coordination with the S-motility system. A general goal of the project is to extend our knowledge of cytoskeleton-dependant processes and protein localization in bacteria.