Ono Hiroki, Takashima Akari, Hirata Hikaru, Homma Michio, Kojima Seiji
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya, 464-8602, Japan.
Mol Microbiol. 2015 Oct;98(1):130-41. doi: 10.1111/mmi.13109. Epub 2015 Jul 17.
FlhG, a MinD homolog and an ATPase, is known to mediate the formation of the single polar flagellum of Vibrio alginolyticus together with FlhF. FlhG and FlhF work antagonistically, with FlhF promoting flagellar assembly and FlhG inhibiting it. Here, we demonstrate that purified FlhG exhibits a low basal ATPase activity. As with MinD, the basal ATPase activity of FlhG can be activated and the D171A residue substitution enhances its ATPase activity sevenfold. FlhG-D171A localizes strongly at the cell pole and severely inhibits motility and flagellation, whereas the FlhG K31A and K36Q mutants, which are defective in ATP binding, do not localize to the poles, cannot complement a flhG mutant and lead to hyperflagellation. A strong polar localization of FlhF is observed with the K36Q mutant FlhG but not with the wild-type or D171A mutant FlhG. Unexpectedly, an Ala substitution at the catalytic residue (D60A), which abolishes ATPase activity but still allows ATP binding, only slightly affects FlhG functions. These results suggest that the ATP-dependent polar localization of FlhG is crucial for its ability to downregulate the number of polar flagella. We speculate that ATP hydrolysis by FlhG is required for the fine tuning of the regulation.
FlhG是一种与MinD同源的ATP酶，已知它与FlhF共同介导溶藻弧菌单极鞭毛的形成。FlhG和FlhF起拮抗作用，FlhF促进鞭毛组装，而FlhG抑制鞭毛组装。在此，我们证明纯化的FlhG表现出较低的基础ATP酶活性。与MinD一样，FlhG的基础ATP酶活性可以被激活，D171A残基取代使其ATP酶活性提高了7倍。FlhG-D171A强烈定位于细胞极，并严重抑制运动性和鞭毛形成，而在ATP结合方面存在缺陷的FlhG K31A和K36Q突变体则不定位于细胞极，不能互补flhG突变体，并导致超鞭毛形成。在K36Q突变体FlhG中观察到FlhF强烈定位于细胞极，而在野生型或D171A突变体FlhG中则未观察到。出乎意料的是，催化残基处的丙氨酸取代（D60A）消除了ATP酶活性，但仍允许ATP结合，仅对FlhG的功能有轻微影响。这些结果表明，FlhG依赖ATP的极性定位对其下调极鞭毛数量的能力至关重要。我们推测，FlhG的ATP水解对于调节的微调是必需的。
