Division of Microbiology, Graduate School of Life Science, Northwest A&F University, Yanglin, Japan.
J Bacteriol. 2011 Aug;193(15):3773-84. doi: 10.1128/JB.00113-11. Epub 2011 May 20.
The stator proteins PomA and PomB form a complex that couples Na(+) influx to torque generation in the polar flagellar motor of Vibrio alginolyticus. This stator complex is anchored to an appropriate place around the rotor through a putative peptidoglycan-binding (PGB) domain in the periplasmic region of PomB (PomB(C)). To investigate the function of PomB(C), a series of N-terminally-truncated and in-frame mutants with deletions between the transmembrane (TM) segment and the PGB domain of PomB was constructed. A PomB(C) fragment consisting of residues 135 to 315 (PomB(C₅) formed a stable homodimer and significantly inhibited the motility of wild-type cells when overexpressed in the periplasm. A fragment with an in-frame deletion (PomB(ΔL)) of up to 80 residues retained function, and its overexpression with PomA impaired cell growth. This inhibitory effect was suppressed by a mutation at the functionally critical Asp (D24N) in the TM segment of PomB, suggesting that a high level of Na(+) influx through the mutant stator causes the growth impairment. The overproduction of functional PomA/PomB(ΔL) stators also reduced the motile fractions of the cells. That effect could be slightly relieved by a mutation (L168P) in the putative N-terminal α-helix that connects to the PGB domain without affecting the growth inhibition, suggesting that a conformational change of the region including the PGB domain affects stator assembly. Our results reveal common features of the periplasmic region of PomB/MotB and demonstrate that a flexible linker that contains a "plug" segment is important for the control of Na(+) influx through the stator complex as well as for stator assembly.
定子蛋白 PomA 和 PomB 形成一个复合物，该复合物将 Na(+) 内流与 Vibrio alginolyticus 极性鞭毛马达的扭矩产生偶联。这个定子复合物通过 PomB 周质区中的一个假定肽聚糖结合 (PGB) 结构域锚定在转子的适当位置（PomB(C)）。为了研究 PomB(C) 的功能，构建了一系列 N 端截断和框内缺失突变体，缺失位于 PomB 的跨膜 (TM) 片段和 PGB 结构域之间。由残基 135 到 315 组成的 PomB(C₅)片段形成稳定的同源二聚体，当在周质中过表达时，显著抑制野生型细胞的运动性。一个框内缺失高达 80 个残基的片段保留了功能，其与 PomA 的共表达会损害细胞生长。这种抑制作用被 PomB 的 TM 片段中功能关键的天冬氨酸 (D24N) 突变所抑制，表明通过突变定子的 Na(+) 大量内流会导致生长受损。功能性 PomA/PomB(ΔL)定子的过表达也降低了细胞的运动分数。该效应可通过突变位于连接 PGB 结构域的假定 N 端 α-螺旋的 L168P 略微缓解，而不影响生长抑制，这表明包括 PGB 结构域在内的区域的构象变化会影响定子组装。我们的结果揭示了 PomB/MotB 周质区的共同特征，并证明包含“塞子”结构域的柔性连接对于控制定子复合物的 Na(+) 内流以及定子组装很重要。
