Acidification of the cytoplasm as a poorly understood stimulus for the activation of acid stress adaptation in Escherichia coli

在地球上，有许多栖息地具有低pH值，例如脊椎动物的胃肠道或酸性土壤的地区。虽然大多数细菌是嗜中性的，但它们能够在酸性环境中生存。酸胁迫传感和适应允许这些细菌在中等酸胁迫下保持恒定的细胞内pH。然而，在强酸胁迫下，嗜中性细菌的细胞内pH值降低约一个pH单位，这对所有生物分子的质子化状态具有相当大的影响，并且可以影响它们的电荷、结构和功能。在该项目的初步工作中，我们已经获得了证据表明，大肠杆菌细胞质的这种酸化被用作刺激物，以激活有助于抗酸胁迫的调节过程。该项目的目的是识别和表征调节剂，成为积极的响应细胞质的pH值下降。为了实现这一目标，我们将该项目分为两个部分。在第一部分中，我们将应用系统的方法来量化细胞对酸胁迫强度的反应。基于我们的初步工作，我们将进行核糖体分析，以测量所有蛋白质合成速率的变化，从而确定每个细胞的拷贝数。然后，我们将描述所有新鉴定的调节剂，并确定它们在酸胁迫下的细胞功能。在项目的第二部分中，我们计划对转录因子AdiY进行深入的表征，AdiY是大肠杆菌Adi系统的激活因子。大肠杆菌，其在细胞质酸化时被激活。调查的重点是pH依赖性的结构-功能的研究和识别的配体，结合AdiY。此外，我们将测试令人兴奋的假设，增加质子化，使AdiY找到它的结合位点更快地促进一维滑动沿着DNA。通过对细胞质酸化过程的分子特征的研究，我们将重点放在一个在E.大肠杆菌，作为革兰氏阴性菌的代表。

On Earth, there are many habitats that have a low pH, such as the gastrointestinal tract of vertebrates or areas with acidic soils. Although most bacteria are neutralophiles, they are able to survive in acidic environments. Acid stress sensing and adaptation allow these bacteria to maintain a constant intracellular pH under moderate acid stress. However, under strong acid stress, the intracellular pH of neutralophilic bacteria decreases by approximately one pH unit, which has a considerable impact on the protonation states of all biological molecules, and can affect their charge, structure and function. In the preliminary work for this project, we have gained evidence that this acidification of the cytoplasm of Escherichia coli is used as stimulus to activate regulatory processes that contribute to acid stress resistance. The aim of the project is to identify and characterize regulators that become active in response to a fall in the pH of the cytoplasm.To achieve this goal, we have divided the project into two parts. In the first part, we will apply a systemic approach to quantify cellular changes in response to the strength of acid stress. Based on our preliminary work, we will perform ribosome profiling to measure the changes in the rates of synthesis of all proteins and thus determine their copy numbers per cell. We will then characterize all newly identified regulators and ascertain their cellular functions under acid stress. In the second part of the project, we plan an in-depth characterization of the transcription factor AdiY, the activator of the Adi system in E. coli, which is activated upon acidification of the cytoplasm. The investigations focus on pH-dependent structure-function studies and the identification of the ligand that binds to AdiY. Furthermore, we will test the exciting hypothesis that increased protonation enables AdiY to find its binding sites faster by promoting 1D sliding along the DNA. With the investigations on the molecular characterisation of the cellular processes as a result of an acidification of the cytoplasm, we focus on a poorly understood stimulus in the acid stress adaptation of E. coli, as a representative of the Gram-negative bacteria.