Data_Sheet_1_Interdependent Polar Localization of FlhF and FlhG and Their Importance for Flagellum Formation of Vibrio parahaemolyticus.PDF

Failure of the cell to properly regulate the number and intracellular positioning of their flagella, has detrimental effects on the cells’ swimming ability. The flagellation pattern of numerous bacteria is regulated by the NTPases FlhF and FlhG. In general, FlhG controls the number of flagella produced, whereas FlhF coordinates the position of the flagella. In the human pathogen Vibrio parahaemolyticus, its single flagellum is positioned and formed at the old cell pole. Here, we describe the spatiotemporal localization of FlhF and FlhG in V. parahaemolyticus and their effect on swimming motility. Absence of either FlhF or FlhG caused a significant defect in swimming ability, resulting in absence of flagella in a ΔflhF mutant and an aberrant flagellated phenotype in ΔflhG. Both proteins localized to the cell pole in a cell cycle-dependent manner, but displayed different patterns of localization throughout the cell cycle. FlhF transitioned from a uni- to bi-polar localization, as observed in other polarly flagellated bacteria. Localization of FlhG was strictly dependent on the cell pole-determinant HubP, while polar localization of FlhF was HubP independent. Furthermore, localization of FlhF and FlhG was interdependent and required for each other’s proper intracellular localization and recruitment to the cell pole. In the absence of HubP or FlhF, FlhG forms non-polar foci in the cytoplasm of the cell, suggesting the possibility of a secondary localization site within the cell besides its recruitment to the cell poles.

细胞无法精准调控鞭毛的数量与胞内定位，会对细胞的游泳运动能力产生有害影响。多数细菌的鞭毛排布模式由FlhF与FlhG这两种核苷三磷酸酶（NTPase）调控。总体而言，FlhG负责调控鞭毛的合成数量，而FlhF则协调鞭毛的胞内定位。在人类致病菌副溶血性弧菌（Vibrio parahaemolyticus）中，其单根鞭毛会在细胞旧极处形成并定位。本研究阐明了副溶血性弧菌中FlhF与FlhG的时空定位特征，以及二者对细胞游泳运动能力的影响。单独缺失FlhF或FlhG均会显著损害细胞的游泳运动能力：flhF基因缺失突变株（ΔflhF）完全无法合成鞭毛，而flhG基因缺失突变株（ΔflhG）则呈现异常的鞭毛表型。两种蛋白均以细胞周期依赖的方式定位于细胞极区，但在整个细胞周期中呈现出不同的定位模式。与其他极性鞭毛细菌的情况一致，FlhF的定位会从单极转变为双极。FlhG的极区定位严格依赖于细胞极区决定因子HubP，而FlhF的极区定位则不依赖HubP。此外，FlhF与FlhG的定位相互依赖，二者均需要对方才能实现正常的胞内定位并被招募至细胞极区。在缺失HubP或FlhF的情况下，FlhG会在细胞胞质中形成非极区的聚集位点，这提示除了被招募至细胞极区之外，细胞内可能存在其他的FlhG定位位点。