Investigating E. coli cell envelope proteins and processes through colicin intoxication

革兰氏阴性菌已经进化到可以在不同的生态位中生存。许多物种是致病的，而另一些则不是，例如，在哺乳动物的肠道中起共生作用，帮助消化食物。与革兰氏阳性菌相比，革兰氏阴性菌的主要区别特征是存在额外的膜屏障，即外膜（OM），这也是在细菌学过程中没有革兰氏染料染色的原因。虽然为机体提供了重要的屏障功能，但OM并不是一个充满能量的系统。这对需要能量来源的过程提出了重大问题，例如引入太大而无法通过天然存在于OM中的蛋白质孔的必需营养素。这与细菌的内膜（IM）形成对比，内膜是生物体新陈代谢的能量系统。质子动力（pmf）是质子从内质和内质（外周质）之间的空间流回细胞质的一个基本要素。pmf是所有生物体中许多依赖能量的过程的动力源。在革兰氏阴性细菌中，它也负责生物体在基质中激活生化事件的方式，利用嵌入在基质中的长蛋白，这些长蛋白指向基质，在那里它们与伴侣蛋白相遇。执行这种类型的能量连接的两个最重要的蛋白质是TonB和TolA，它们都是通常被称为Ton和toll系统的较大蛋白质组合的一部分。Ton参与将必需的营养物质带入细胞，而Tol参与维持OM的屏障功能，尽管它是如何做到这一点尚不清楚。同样不清楚的是，尽管这已经被大量研究了多年，这些系统是如何以一种促进它们在OM中的特定功能的方式对pmf作出反应的。这个LOLA应用程序的目的是利用一种叫做粘菌素的蛋白质抗生素家族的行为来探测革兰氏阴性OM的能量依赖过程，重点是大肠杆菌。大肠杆菌产生的Colicins可以在竞争期间杀死邻近的细菌，是非常有效的抗菌素；进入细菌的单个分子就足以引起细胞死亡。粘菌素通过与大肠杆菌OM中的营养受体结合，开始进入大肠杆菌细胞的旅程。随后与Ton或Tol系统的相互作用催化它们进入细胞（一个称为易位的过程），该过程被认为依赖于跨IM的pmf，但这尚未得到证实。我们建议利用粘菌素作为OM过程的探针，使用生化，生物物理和结构方法。我们将测量粘附在细胞外表面的黏菌所受的力，并确定这些力是否完全依赖于黏菌。我们将确定这些有效的抗菌剂如何利用它们与外周质中的Tol蛋白的关联来穿透细胞的OM防御，这可能为开发新的抗生素指明方向。我们还将利用一系列引人注目的观察结果，首次将大肠杆菌细胞外表面与受体蛋白结合的单个大肠杆菌素分子可视化。这些观察结果突出了OM的一个特性，这与标准的生化和微生物教科书相矛盾，在这些教科书中，嵌入OM的蛋白质分子的运动被认为是自由和不受限制的，就像IM的情况一样。相反，我们发现运动并不是不受限制的，而是被划分成不同的区域。我们将研究这种区隔化的原因，并确定它是否在粘菌素易位中起作用。最终，该LOLA将为革兰氏阴性OM及其组织提供根本性的新见解。

Gram-negative bacteria have evolved to survive in diverse ecological niches. Many species are pathogenic while others are not, for example serving a symbiotic role in the mammalian gut helping to digest food. The major distinguishing feature of Gram-negative organisms compared to their Gram-positive counterparts is the existence of an additional membrane barrier, the outer membrane (OM), which is also responsible for the absence of staining with Gram dye in bacteriological procedures. Although serving an important barrier function for the organism, the OM is not an energised system. This presents significant problems for processes that require an energy source, such as the bringing in of essential nutrients that are too big to pass through the protein-pores that naturally exist in the OM. This is in contrast to the inner membrane (IM) of the bacterium which is an energised system by virtue of the organism's metabolism. An essential element of an energised IM is the flow of protons from the space between the OM and IM (the periplasm) back across the IM into the cell's cytoplasm, which is called the proton motive force (pmf). The pmf is a powersource for many energy-dependent processes in all organisms. In Gram-negative bacteria it is also responsible for the way in which the organism energises biochemical events at the OM, using long proteins that are embedded in the IM and which point towards the OM where they meet partner proteins. Two of the most important proteins that perform this type of energy linkage are TonB and TolA, each of which is part of larger protein assemblies usually referred to as the Ton and Tol systems. Ton is involved in bringing essential nutrients into the cell while Tol is involved in maintaining the barrier functions of the OM although how it does this is not clear. What is also not clear, even though this has been heavily studied for many years, is how these systems respond to pmf in a way that promotes their specific functions at the OM. This LOLA application aims to exploit the behaviour of a family of protein antibiotics called colicins to probe energy-dependent processes at the Gram-negative OM, focusing on Escherichia coli. Colicins are made by E. coli to kill neighbouring bacteria during times of competition and are very potent antibacterials; a single molecule entering the bacterium is sufficient to elicit cell death. Colicins begin their journey into an E. coli cell by binding to a nutrient receptor in the OM. Subsequent interactions with either the Ton or Tol systems catalyse their entry into the cell (a process called translocation) which is thought to be dependent on the pmf across the IM, but this has yet to be proven. We propose exploiting colicins as probes of OM processes using biochemical, biophysical and structural approaches. We will measure the forces that are exerted on colicins bound to the external surface of a cell and determine whether these forces are wholly pmf-dependent. We will establish how these potent antimicrobials use their associations with Tol proteins in the periplasm to penetrate the cells' OM defences, which may point the way toward new antibiotics. We will also capitalise on a remarkable series of observations that have for the first time visualised single colicin molecules bound to receptor proteins diffusing on the external surface of an E. coli cell. These observations highlight a property of the OM that contradicts standard biochemical and microbiological textbooks, where the motion of protein molecules embedded in the OM is assumed to be free and unrestricted, as is the case for the IM. In contrast, we find that movement is not unrestricted but rather demarcated into compartments. We will investigate the reason for such compartmentalisation and determine whether it plays a role in colicin translocation. Ultimately, this LOLA will provide fundamental new insight into the Gram-negative OM and its organisation.