Suppressor analysis of the MotB(D33E) mutation to probe bacterial flagellar motor dynamics coupled with proton translocation
Suppressor analysis of the MotB(D33E) mutation to probe bacterial flagellar motor dynamics coupled with proton translocation
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DOI：
10.1128/jb.00503-08
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发表时间：
2008-10-01
影响因子：
3.2
通讯作者：
Minamino, Tohru
中科院分区：
文献类型：
--
作者：
Che, Yong-Suk;Nakamura, Shuichi;Minamino, Tohru
MOTA和MOTB形成了质子驱动的细菌鞭毛马达的定子，该马达传导质子并将质子流与马达旋转耦合。鼠伤寒沙门氏菌的ASP-33是一个可能的质子结合位点，对扭矩的产生是至关重要的。然而，能量耦合的机制仍不清楚。在这里，我们对一个运动缓慢的MOTB(D33E)突变体及其假恢复株进行了遗传和运动学分析。我们首先证实MOTB(D33E)突变体的运动能力较差既不是由于蛋白质不稳定、定位错误，也不是由于与MOTA的相互作用减弱。我们分离了17个假逆转株，并鉴定了MOTA的跨膜螺旋TM2和TM3以及MOTB的TM和周质结构域中的抑制突变。MOTB(D33E)突变马达产生的失速扭矩约为野生型的一半，而假回复马达产生的失速扭矩几乎恢复到野生型水平。然而，发动机在低负载条件下的高速旋转仍然显著受损，这表明质子转移速率在高速下仍然受到严重限制。这些结果表明，第二位点突变恢复了一个扭矩产生步骤，包括定子-转子相互作用和Glu-33的质子化/去质子化，但不是最大的质子传导性。
MotA and MotB form the stator of the proton-driven bacterial flagellar motor, which conducts protons and couples proton flow with motor rotation. Asp-33 of Salmonella enterica serovar Typhimurium MotB, which is a putative proton-binding site, is critical for torque generation. However, the mechanism of energy coupling remains unknown. Here, we carried out genetic and motility analysis of a slowly motile motB(D33E) mutant and its pseudorevertants. We first confirmed that the poor motility of the motB(D33E) mutant is due to neither protein instability, mislocalization, nor impaired interaction with MotA. We isolated 17 pseudorevertants and identified the suppressor mutations in the transmembrane helices TM2 and TM3 of MotA and in TM and the periplasmic domain of MotB. The stall torque produced by the motB(D33E) mutant motor was about half of the wild-type level, while those for the pseudorevertants were recovered nearly to the wild-type levels. However, the high-speed rotations of the motors under low-load conditions were still significantly impaired, suggesting that the rate of proton translocation is still severely limited at high speed. These results suggest that the second-site mutations recover a torque generation step involving stator-rotor interactions coupled with protonation/ deprotonation of Glu-33 but not maximum proton conductivity.