Ikeda T, Oosawa K, Hotani H
Department of Microbiology, School of Dentistry, Aichi-Gakuin University, Nagoya, Japan.
J Mol Biol. 1996 Jun 21;259(4):679-86. doi: 10.1006/jmbi.1996.0349.
A bacterial flagellum has a cap structure at the tip of the external filament. The cap is composed of the FliD protein (Mr, 49 x 10(3)), and plays an essential role in the polymerization of the filament protein, flagellin, which is believed to be transported through a central channel in the flagellum. A fliD-deficient mutant becomes non-motile because it lacks flagellar filaments and leaks flagellin monomer out into the medium. We have constructed a FliD-overproducing plasmid and purified the protein. The purified FliD at high concentration formed a large complex (Mr, ca. 600 x 10(3)) under physiological conditions. The complex was found by electron microscopy to be ring shaped. Image analysis revealed that the complex consisted of five substructures arranged in a pentagonal shape. Its outer diameter, approximately 10 nm, was about the same as that of the cap at the tip of the wild-type flagella. When the annular structure was added to the culture medium of a Salmonella fliD mutant, almost all of the cells became able to swim. Overall, about ten molecules of FliD self-assemble into an annular structure in vitro, forming the functional capping structure by incorporating flagellin at the tip of the flagellar filament in vivo.
细菌鞭毛在外部鞭毛丝的顶端有一个帽状结构。该帽由FliD蛋白（分子量为49×10³）组成，在鞭毛蛋白（据信通过鞭毛中的中央通道运输）的丝聚合过程中起关键作用。一个fliD缺陷型突变体变得不能运动，因为它缺乏鞭毛丝且将鞭毛蛋白单体泄漏到培养基中。我们构建了一个过量表达FliD的质粒并纯化了该蛋白。纯化后的高浓度FliD在生理条件下形成了一个大的复合物（分子量约为600×10³）。通过电子显微镜发现该复合物呈环状。图像分析表明该复合物由五个呈五边形排列的亚结构组成。其外径约为10纳米，与野生型鞭毛顶端的帽的外径大致相同。当将该环状结构添加到沙门氏菌fliD突变体的培养基中时，几乎所有细胞都能够游动。总体而言，大约十个FliD分子在体外自组装成一个环状结构，通过在体内将鞭毛蛋白纳入鞭毛丝的顶端形成功能性帽状结构。
