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

在地球上，有许多栖息地的pH值很低，比如脊椎动物的胃肠道或酸性土壤。虽然大多数细菌是嗜中性菌，但它们能够在酸性环境中生存。酸胁迫感知和适应使这些细菌在中等酸胁迫下保持恒定的细胞内pH值。然而，在强酸胁迫下，嗜中性细菌的细胞内pH降低了大约一个pH单位，这对所有生物分子的质子化状态产生了相当大的影响，并能影响它们的电荷、结构和功能。在该项目的前期工作中，我们已经获得证据表明，大肠杆菌细胞质的酸化被用作刺激，激活有助于抗酸胁迫的调节过程。该项目的目的是确定和表征在响应细胞质pH值下降时变得活跃的调节因子。为了实现这一目标，我们将项目分为两部分。在第一部分中，我们将采用系统的方法来量化细胞变化，以响应酸胁迫的强度。基于我们的初步工作，我们将执行核糖体分析来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，我们将描述所有新发现的调节和确定其在酸性胁迫下的细胞功能。在项目的第二部分，我们计划深入表征转录因子AdiY，这是大肠杆菌中Adi系统的激活剂，它在细胞质酸化时被激活。研究重点是ph依赖性结构-功能研究和与AdiY结合的配体的鉴定。此外，我们将验证一个令人兴奋的假设，即增加质子化使AdiY通过促进沿DNA的1D滑动来更快地找到其结合位点。随着细胞质酸化对细胞过程分子特征的研究，我们将重点放在大肠杆菌（革兰氏阴性菌的代表）酸性胁迫适应中的一种鲜为人知的刺激上。

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.