Systematic Cys mutagenesis of FlgI, the flagellar P-ring component of Escherichia coli.
Systematic Cys mutagenesis of FlgI, the flagellar P-ring component of Escherichia coli.
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DOI：
10.1099/mic.0.2007/013854-0
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发表时间：
2008-03
期刊：
影响因子：
--
通讯作者：
Homma M
中科院分区：
文献类型：
--
作者：
Hizukuri Y;Kojima S;Yakushi T;Kawagishi I;Homma M
细菌鞭毛马达包埋在细胞质膜中，并穿透肽聚糖层和外膜。被称为P环的基体的环状结构位于肽聚糖层中，被认为是平滑旋转所需的，并起到衬套的作用。在这项工作中，我们表征了32种半胱氨酸取代的大肠杆菌P环蛋白FlgI变体，这些变体旨在取代FlgI 346个aa成熟形式中的每10个残基。 对FlgI蛋白的免疫印迹分析表明，5个FlgI变体的细胞量显着减少。Swarm分析表明，几乎所有的变体都具有接近野生型的功能，但有五种变体显著降低了细胞的运动性，其中一种特别是FlgI G21 C，完全破坏了FlgI功能。这五个残基损害的运动的细胞被定位在N末端的FlgI。为了证明FlgI的哪些残基暴露于蛋白质表面上的溶剂，我们通过使用巯基特异性试剂甲氧基聚乙二醇5000马来酰亚胺检查了半胱氨酸修饰，并将FlgI Cys变体分为三组：良好标记、中度标记和较少标记。有趣的是，FlgI的良好和适度标记的残基从未与已知对蛋白质量或运动性重要的残基重叠。从这些结果和多种FlgI蛋白的氨基酸序列的多重比对，推测FlgI的N末端的高度保守区域，残基1-120，在FlgI结构的稳定和通过与FlgI分子和/或其它鞭毛组分相互作用形成P环中起重要作用。
The bacterial flagellar motor is embedded in the cytoplasmic membrane, and penetrates the peptidoglycan layer and the outer membrane. A ring structure of the basal body called the P ring, which is located in the peptidoglycan layer, is thought to be required for smooth rotation and to function as a bushing. In this work, we characterized 32 cysteine-substituted Escherichia coli P-ring protein FlgI variants which were designed to substitute every 10th residue in the 346 aa mature form of FlgI. Immunoblot analysis against FlgI protein revealed that the cellular amounts of five FlgI variants were significantly decreased. Swarm assays showed that almost all of the variants had nearly wild-type function, but five variants significantly reduced the motility of the cells, and one of them in particular, FlgI G21C, completely disrupted FlgI function. The five residues that impaired motility of the cells were localized in the N terminus of FlgI. To demonstrate which residue(s) of FlgI is exposed to solvent on the surface of the protein, we examined cysteine modification by using the thiol-specific reagent methoxypolyethylene glycol 5000 maleimide, and classified the FlgI Cys variants into three groups: well-, moderately and less-labelled. Interestingly, the well- and moderately labelled residues of FlgI never overlapped with the residues known to be important for protein amount or motility. From these results and multiple alignments of amino acid sequences of various FlgI proteins, the highly conserved region in the N terminus, residues 1–120, of FlgI is speculated to play important roles in the stabilization of FlgI structure and the formation of the P ring by interacting with FlgI molecules and/or other flagellar components.
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DOI：
10.1073/pnas.0509932103
发表时间：
2006-05-23
影响因子：
11.1
作者：
Reid, Stuart W.;Leake, Mark C.;Berry, Richard M.
通讯作者：
Berry, Richard M.
影响因子：
11.4
作者：
Akiyama, Y;Kanehara, K;Ito, K
通讯作者：
Ito, K
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL
影响因子：
64.5
作者：
BLAIR, DF;BERG, HC
通讯作者：
BERG, HC
影响因子：
64.8
作者：
Leake, Mark C.;Chandler, Jennifer H.;Armitage, Judith P.
通讯作者：
Armitage, Judith P.