Stalk formation in C. crescentus is cell cycle dependent. The dimorphic life cycle of C. crescentus produces one motile (swarmer) cell and one adherent (stalked) cell at each cell cycle (8). The swarmer cell has a polar flagellum and pili and swims for roughly 30% of the cell cycle. The swarmer then sheds its flagellum and, at the same pole, produces a holdfast - the strongest measured biological adhesive (9). After the holdfast is secreted, the stalk is formed and elongated from the holdfast pole, thereby causing the holdfast to be pushed away from the cell body and localized to the tip of the stalk. During cell division, a new flagellum is synthesized at the opposite pole. As a result, following cytokinesis, the stalked cell remains unchanged while the daughter cell is flagellated and begins a new proliferative cycle. Swarmer and stalked cells can be separated by density-gradient centrifugation (10). Therefore, it is possible to obtain a pure population of cells that have not yet synthesized stalks enabling the study of stalk formation as well as mechanisms of stalk-protein localization. .
The experimental paradigm proposed has broad long-term applications. The wealth of data in our localisome will allow us to expand this project to identify localization mechanism for other subcellular regions such as the pole and midcell. It is likely that these mechanisms will be broadly applicable in various bacterial species due to the conservation of basic localization mechanisms. Finally, mechanistic insight into protein localization will be of great clinical value. Since proper protein localization is required for bacterial proliferation and virulence, drugs that target localization may be a new source of antimicrobials to fight the growing problem of drug resistance.
Research Design and Methods.
Specific Aim 1: What trans-acting mechanisms are required for stalk localization?.
Despite the lack of subcellular compartments or organelles in bacteria, it is now clear that proteins localize to distinct subcellular domains, often connected to the cell envelope (11).