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环境中生存。许多物种是致病的，而其他物种则不是，例如在哺乳动物肠道中发挥共生作用，帮助消化食物。与革兰氏阳性微生物相比，革兰氏阴性微生物的主要区别特征是存在额外的膜屏障，即外膜（OM），这也是细菌学程序中不存在革兰氏染料染色的原因。虽然OM对生物体起着重要的屏障作用，但它不是一个能量系统。这对于需要能量源的过程提出了重大问题，例如引入太大而不能通过OM中天然存在的蛋白质孔的必需营养素。这与细菌的内膜（IM）形成对比，细菌的内膜是一个通过生物体代谢的能量系统。能量化IM的一个基本要素是质子从OM和IM之间的空间（周质）穿过IM返回细胞的细胞质，这被称为质子动力（pmf）。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OM处激发生化事件的方式，使用嵌入IM中的长蛋白质，并指向OM，在那里它们遇到伴侣蛋白质。执行这种类型的能量连接的两个最重要的蛋白质是TonB和托拉，它们中的每一个都是通常被称为Ton和Tol系统的较大蛋白质组装体的一部分。Ton参与将必需营养素带入细胞，而Tol参与维持OM的屏障功能，尽管它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很多年，这些系统如何以促进它们在OM中的特定功能的方式对pmf做出反应。该LOLA应用旨在利用称为大肠杆菌素的蛋白质抗生素家族的行为来探测革兰氏阴性OM的能量依赖性过程，重点是大肠杆菌。大肠杆菌素是由大肠杆菌E.在竞争期间，大肠杆菌能够杀死邻近的细菌，并且是非常有效的抗菌剂;单个分子进入细菌就足以引起细胞死亡。大肠杆菌素开始它们进入E.大肠杆菌细胞中的营养受体结合的OM。随后与Ton或Tol系统的相互作用催化它们进入细胞（称为易位的过程），这被认为取决于穿过IM的pmf，但这尚未得到证实。我们建议利用大肠杆菌素作为探针的OM过程中使用的生物化学，生物物理和结构的方法。我们将测量施加在与细胞外表面结合的大肠杆菌素上的力，并确定这些力是否完全依赖于pmf。我们将确定这些有效的抗菌剂如何利用它们与周质中的Tol蛋白的结合来穿透细胞的OM防御，这可能为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，这些观察结果首次可视化了单个大肠杆菌素分子与在大肠杆菌外表面扩散的受体蛋白结合。coli细胞。这些观察结果突出了OM的一个属性，这与标准的生物化学和微生物学教科书相矛盾，其中嵌入OM中的蛋白质分子的运动被假定为是自由和不受限制的，就像IM的情况一样。与此相反，我们发现，运动不是不受限制的，而是划分成隔间。我们将调查这种区室化的原因，并确定它是否在大肠杆菌素易位中发挥作用。最终，该LOLA将为革兰氏阴性OM及其组织提供基本的新见解。

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.