アイテム詳細

要旨

Understanding of the inner working of living cells requires the study of the organization of their components, and how such organization arises. The bacterial flagellum is an example of such organization, since its position in the cell body is carefully regulated. Many patterns of flagellation exist in nature, indicative of the diversity of mechanisms that bacteria use to regulate it. The GTPase FlhF and the MinD-like ATPase FlhG have emerged to regulate the position and number of flagella in many species with different flagellation patterns. In γ-proteobacteria that use this system, flagella are found at the cell pole, and its production is tied to the cell cycle to ensure that daughter cells are flagellated at only one pole. Here, we introduce Vibrio parahaemolyticus as a model to study the spatiotemporal regulation of flagellum synthesis. We show that FlhF and FlhG work in a similar manner to other γ-proteobacteria, although FlhF is strictly required for flagellum assembly. We also show that the main polar landmark protein HubP plays a role in regulating the localization of FlhG but not of FlhF. Furthermore, we describe a new protein, named FipA, as an interacting partner of FlhF. We show that the phenotype of deleting FipA is very similar to that of a deletion of FlhF: they are both required to start assembly of the flagellum. Furthermore, we could demonstrate that FipA and FlhF interact directly, that this interaction is possible due to key residues in the domain of unknown function of FipA, and that this interaction is responsible for recruiting FlhF to the cell pole. In fact, we show that FipA and HubP act cooperatively to recruit FlhF to the cell pole. FipA is conserved among the γ-proteobacteria that use FlhFG, and we could show that its function is also conserved in Pseudomonas putida. Finally, we used a heterologous system to show that FipA is responsible for anchoring FlhF to the membrane. We could also identify residues in FlhF that are necessary for the interaction, which suggest that FipA binds primarily to the monomeric, GDP-bound form of FlhF. This hypothesis also receives support from in vitro interaction studies, that suggest that GTP inhibits the interaction between FlhF and FipA. Altogether, this work identifies a new piece in the puzzle of spatiotemporal regulation of flagella, and we propose a model incorporating our new findings to what is already known about this system in γ-proteobacteria.