Proton-Coupling Strategies in Alkaliphilic Bacillus

描述（由申请人提供）：该项目将测试两个关于呼吸细菌生物能量工作的总体假设，即通过膜上质子的电化学梯度，质子动势（pmf）提供能量的膜相关过程。大部分工作的实验系统是一种亲碱芽孢杆菌，它表现出本构准备迎接高生物能量工作负荷的挑战。嗜碱性假硬杆菌OF4也具有特殊的适应性，有助于其在低pmf条件下的生物能工作。第一个主要假设是，特定的结构特征增强了亲碱ATP合成酶及其独特的复杂mrp型Na？在低pmf下分别支持呼吸依赖性ATP合成和细胞质pH稳态的反向转运蛋白。假设自适应特征使这些pmf使用者复合物能够利用来自呼吸泵的质子转移的隔离路径，并在质子贫乏的环境中收集质子。生物能量机制的其他特征，包括ATP合成酶，在氧化磷酸化（OXPHOS）过程中防止质子泄漏。前两个特定目标侧重于亲碱机械的结构功能特征。Specific Aim #1验证了F1F0-ATP合成酶c-转子独特的P51XXEXXP基序防止质子泄漏的假设。这将通过比较野生型和cP51A突变体的稳定c转子环的结构研究来评估。将分析二维投影图，并从三维晶体中寻求高分辨率的结构信息。特异性目标#2测试了7蛋白Mrp异聚寡聚物是反转运蛋白和其他转运蛋白相互依赖和协同作用的联合体的假设，例如，共同呈现一个用于质子聚集的大外表面。拟议的实验包括使用新的Mrp突变体筛选假定的阴离子传输功能，该突变体具有高Na+抗性，但缺乏反港活性。第二个总体假设是，主要的pmf消耗复合物，如ATP合成酶和Mrp反转运蛋白，利用细胞成分和调节因子的网络来应对生物能量挑战。在非嗜碱菌中，对于重的生物能量工作负荷，非嗜碱菌不是“硬连接”的，通过钠碱挑战激活主要的pmf消费者会引起系统反应，从而促进对挑战的管理。第三和第四个具体目标侧重于生物能量工作的生理背景。特异性目的#3探讨低pmf下OXPHOS中亲碱呼吸链超复合物、心磷脂和ATP合成酶之间的相互作用。特异性目标#4将模拟和测试金黄色葡萄球菌对Mnh （Mrp同源物）活性的假设系统反应，该反应导致跨膜电位的净增加，并影响膜脂流动性以及对抗菌肽的敏感性。该模型将通过一组基因、生化和分子分析进行测试和构建。总体目标是填补我们对生物能量工作的机制和可塑性以及管理生物能量工作负荷变化的细胞范围反应的理解空白。

DESCRIPTION (provided by applicant): This project will test two overarching hypotheses about bioenergetic work in respiratory bacteria, i.e., membrane-associated processes that are energized by an electrochemical gradient of protons across the membrane, the protonmotive force (pmf). The experimental system for much of the work is an alkaliphilic Bacillus that exhibits constitutive readiness to meet the challenge of a high bioenergetic work-load. Alkaliphilic Bacillus pseudofirmus OF4 also has special adaptations that facilitate its bioenergetic work under conditions of low pmf. The first overarching hypothesis is that specific structural features enhance the ability of the alkaliphile ATP synthase and its uniquely complex Mrp-type Na? antiporter to respectively support respiration- dependent ATP synthesis and cytoplasmic pH homeostasis at low pmf. Adaptive features are hypothesized to enable these pmf-user complexes to take advantage of sequestered paths of proton transfer from respiratory pumps and to gather protons in proton-poor environments. Other features of bioenergetic machinery, including ATP synthase, prevent proton leaks during oxidative phosphorylation (OXPHOS). The first two Specific Aims focus on structural-functional features of alkaliphile machinery. Specific Aim #1 tests the hypothesis that the unique P51XXEXXP motif of the F1F0-ATP synthase c-rotor prevents proton leakiness. This will be assessed through comparative structural studies of the stable c-rotor rings from wild-type and a cP51A mutant. 2-D projection maps will be analyzed and high resolution structural information from 3D crystals will be sought. Specific Aim #2 tests the hypothesis that the 7-protein Mrp hetero-oligomer is a consortium of antiporters and other transporters that are interdependent and synergistic, e.g. jointly presenting a large external surface engineered for proton-gathering. Proposed experiments include a screen for a putative anion transport function using a new Mrp mutant that confers high Na+resistance but lacks antiport activity. The second overarching hypothesis is that major pmf-consuming complexes such as ATP synthase and Mrp antiporter draw upon a network of cell components and regulators to meet bioenergetic challenges. In non-alkaliphiles that are not "hard-wired" for heavy bioenergetic work-loads, activation of a major pmf-consumer by sodium-alkali challenge elicits a systems response that facilitates management of challenge. The third and fourth Specific Aims focus on physiological contexts of bioenergetic work. Specific Aim #3 probes the interplay between alkaliphile respiratory chain supercomplexes, cardiolipin and ATP synthase in OXPHOS at low pmf. Specific Aim #4 will model and test a hypothesized systems response to Mnh (a Mrp homologue) activity in Staphylococcus aureus that results in a net increase in the transmembrane potential and impacts membrane lipid fluidity as well as sensitivity to antimicrobial peptides. The model will be tested and built via a panel of genetic, biochemical and molecular assays. The overall goal is to fill in gaps in our understanding of the mechanisms and plasticity of bioenergetic work and of the cell-wide responses that manage changes in bioenergetic work-load.