Elucidating the biological mechanisms underlying the motility of flagellated bacteria by understanding torque generation

有必要了解鞭毛细菌的运动生理学，如空肠弯曲杆菌（C.jejuni）、肠道沙门氏菌（S.enterica），其在世界范围内引起住院和死亡，以及细菌如新月柄杆菌（Caulobacter crescentus），其在其生命周期中经历显著的形状变化，现在被广泛研究。特别是，了解细菌运动的分子机制是重要的，因为运动在宿主感染中起着重要作用。细菌的运动是由嵌入细菌膜中的马达的活动决定的，钩将马达附着在被称为鞭毛的尾巴状结构上。在生物学上，运动性被认为是在鞭毛内通过两个结构（定子和C环）之间的相互作用而启动的。定子由两种蛋白质组成，五聚体运动蛋白A（5 motA）和二聚体运动蛋白B（2 motB）被认为是顺时针运动的。然后，C形环跟随定子启动的运动，但像齿轮一样沿逆时针方向运动。从机制上讲，已知motA与C环内的鞭毛马达开关蛋白FliG蛋白相互作用。在肠道沙门氏菌和大肠杆菌等细菌中，影响运动的定子的确切数量取决于细菌的环境。例如，在高粘性（即，高阻力）环境下，认为比在低粘性（即，低阻力）环境下更多的定子是活动的。其次，推动细菌前进的鞭毛的运动方向被认为受到当地环境的影响。鞭毛的默认运动方向是逆时针方向，然而，当遇到局部刺激（“趋化性”）时，运动方向可以改变为顺时针方向。目前关于细菌运动性的生物学基础的文献具有重要的局限性。特别是，以下内容仍然不完全理解。首先，没有研究报道蛋白质5 MotA-2 MotB如何通过C中的FliG C-末端与C-环相互作用。空肠，尽管已经提出了可能的模型。其次，在C语言中，定子的可用性是静态的（不变的）还是动态的（变化的）过程仍然没有完全理解。空肠。后者与了解细菌的进化有关，因为定子可以在C中可视化。空肠（一种更复杂的马达），但不是在肠道链球菌或大肠杆菌（一种简单的马达）。第三，它仍然是未知的定子是如何物理组装周围的C-环在肠道链球菌。第四，仍不清楚5 MotA-2 MotB如何通过C中的FliG C末端与C环相互作用。当空肠弯曲菌顺时针旋转而不是逆时针旋转时，鉴于此，本论文主要研究了以下几个方面。除此之外，我还承担了两个合作实验室项目，重点是马达中的其他蛋白质。首先，蛋白FlaY被认为是一种关键蛋白，决定运动是否可能。然而，FlaY在鞭毛内的确切定位仍然不完全未知。第二，介导空肠弯曲菌马达旋转的所谓抗断裂蛋白（0996）的定位。

There is an imperative for the understanding of the physiology of motility of flagellated bacteria such as Campylobacter jejuni (C.jejuni), Salmonella enterica (S.enterica) which cause hospitalisations and death worldwide, as well as bacteria such as Caulobacter crescentus (C.crescentus) which undergo significant shape shifting during their life cycle are now widely studied. In particular, it is important to understand the molecular mechanisms of bacterial motility as motility plays an important role in the infection of hosts. Bacterial movement is determined by activity of the motor embed in the membrane of the bacteria, the hook attaches the motor to the tail like structure known as a flagellum. Biologically, motility is known to be initiated within the flagellum by the interaction between two structures, the stator and the C-ring. The stator consists of two proteins, a pentameric motility protein A (5motA) and a dimeric motility protein B (2motB) is thought to move clockwise. Then, the C-ring follows movement initiated by the stator but in a counter clockwise direction like a cogwheel. Mechanistically, motA is known to interact with the flagellar motor switch protein FliG protein within the C-ring. In bacteria such as S.enterica and E.coli the exact number of stators that effects movement differs depending on the environment of the bacteria. For example, under high-viscous (i.e. high resistance) environment more stators are thought to be active than under low-viscous (i.e. low resistance) environment. Next, the direction of movement of the flagellum, which propels the bacteria forward, is thought to be affected by the local milieu. The default direction of movement of the flagellum is counter clockwise, however, when local stimuli are encountered ('chemotaxis') the direction of movement can change to clockwise. Current literature on the biology underlying bacterial motility has important limitations. In particular, the following remains incompletely understood. First, no studies have reported how the proteins 5MotA-2MotB interact with the C-ring via the FliG C-terminal in C. jejuni, although possible models have been presented. Second, it remains incompletely understood whether the availability of stators is static (non-changing) or a dynamic (changing) process in C. jejuni. The latter is relevant to gain insight in the evolution of bacteria, since stators can be visualised in C. jejuni (a more complex motor) but not in S.enterica or E.coli (a simple motor). Third, it remains unknown how the stators are physically assembled around the C-ring in S.enterica. Fourth, it remains unclear how 5MotA-2MotB interact with the C-ring via the FliG C-terminal in C. jejuni when C.jejuni is rotating clockwise as oppose to counterclockwise rotation. In view of the above, the present thesis investigated the following aims. In addition to this, I have taken on two collaboratory projects which focus on other proteins in the motor. First, the protein FlaY is thought to be a critical protein, determining whether motility is possible. However, the exact localisation of FlaY within the flagellum remains incompletely unknown. Second, the localisation of so-called anti-break protein (0996) which mediates the rotation of C.jejuni motor.