School of Biological Sciences, University of Utah, East Salt Lake City, Utah, USA.
School of Biological Sciences, University of Utah, East Salt Lake City, Utah, USA
J Bacteriol. 2019 Mar 26;201(8). doi: 10.1128/JB.00626-18. Print 2019 Apr 15.
While the protein complex responsible for controlling the direction (clockwise [CW] or counterclockwise [CCW]) of flagellar rotation has been fairly well studied in and , less is known about the switch complex in or other Gram-positive species. Two component proteins (FliG and FliM) are shared between and , but in place of the protein FliN found in , the complex contains the larger protein FliY. Notably, in the signaling protein CheY-phosphate induces a switch from CW to CCW rotation, opposite to its action in Here, we have examined the architecture and function of the switch complex in using targeted cross-linking, bacterial two-hybrid protein interaction experiments, and characterization of mutant phenotypes. In major respects, the switch complex appears to be organized similarly to that in The complex is organized around a ring built from the large middle domain of FliM; this ring supports an array of FliG subunits organized in a similar way to that of , with the FliG C-terminal domain functioning in the generation of torque via conserved charged residues. Key differences from involve the middle domain of FliY, which forms an additional, more outboard array, and the C-terminal domains of FliM and FliY, which are organized into both FliY homodimers and FliM heterodimers. Together, the results suggest that the CW and CCW conformational states are similar in the Gram-negative and Gram-positive switches but that CheY-phosphate drives oppositely directed movements in the two cases. Flagellar motility plays key roles in the survival of many bacteria and in the harmful action of many pathogens. Bacterial flagella rotate; the direction of flagellar rotation is controlled by a multisubunit protein complex termed the switch complex. This complex has been extensively studied in Gram-negative model species, but little is known about the complex in or other Gram-positive species. Notably, the switch complex in Gram-positive species responds to its effector CheY-phosphate (CheY-P) by switching to CCW rotation, whereas in or CheY-P acts in the opposite way, promoting CW rotation. In the work here, the architecture of the switch complex has been probed using cross-linking, protein interaction measurements, and mutational approaches. The results cast light on the organization of the complex and provide a framework for understanding the mechanism of flagellar direction control in and other Gram-positive species.
虽然负责控制鞭毛旋转方向（顺时针[CW]或逆时针[CCW]）的蛋白质复合物在和中已经得到了相当好的研究，但在或其他革兰氏阳性菌中，关于开关复合物的了解较少。两种组成蛋白（FliG 和 FliM）在和中共享，但在中发现的蛋白 FliN 的位置，复合物中含有较大的蛋白 FliY。值得注意的是，在中，信号蛋白 CheY-磷酸诱导从 CW 到 CCW 旋转的转换，与在中相反。在这里，我们使用靶向交联、细菌双杂交蛋白相互作用实验和突变表型特征研究了中开关复合物的结构和功能。在主要方面，开关复合物的组织似乎与的相似。该复合物围绕由 FliM 大亚域构建的环组织；该环支持以类似于的方式组织的 FliG 亚基阵列，其中 FliG C 端结构域通过保守带电残基在产生扭矩中起作用。与的关键区别涉及 FliY 的中间结构域，它形成了另一个更外侧的阵列，以及 FliM 和 FliY 的 C 端结构域，它们组织成 FliY 同源二聚体和 FliM 异源二聚体。总之，结果表明，革兰氏阴性和革兰氏阳性开关中的 CW 和 CCW 构象状态相似，但 CheY-磷酸在两种情况下驱动相反方向的运动。鞭毛运动在许多细菌的生存和许多病原体的有害作用中起着关键作用。细菌鞭毛旋转；鞭毛旋转的方向由一个称为开关复合物的多亚基蛋白复合物控制。该复合物在革兰氏阴性模型物种中得到了广泛研究，但对或其他革兰氏阳性物种中的复合物知之甚少。值得注意的是，革兰氏阳性物种中的开关复合物通过切换到 CCW 旋转对其效应物 CheY-磷酸（CheY-P）做出反应，而在或中，CheY-P 以相反的方式起作用，促进 CW 旋转。在目前的工作中，使用交联、蛋白相互作用测量和突变方法探测了的开关复合物的结构。结果揭示了复合物的组织，并为理解和其他革兰氏阳性物种中鞭毛方向控制的机制提供了框架。
