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道细菌中生物能工作的总体假设，即，与膜相关的过程，其通过质子穿过膜的电化学梯度（质子动力（PMF））来供能。大部分工作的实验系统是一个嗜碱芽孢杆菌，表现出组成准备，以满足高生物能量工作负荷的挑战。嗜碱假坚强芽孢杆菌OF 4也具有特殊的适应性，这有助于其在低pmf条件下的生物能工作。第一个总体假设是，特定的结构特征增强了嗜碱ATP合酶及其独特的复杂MRP型Na？在低pmf下，反向转运蛋白分别支持呼吸依赖的ATP合成和细胞质pH稳态。假设适应性特征使这些pmf用户复合体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。生物能机械的其他特征，包括ATP合酶，防止氧化磷酸化（OXPHOS）过程中的质子泄漏。前两个具体目标侧重于嗜碱机器的结构功能特征。具体目标#1测试了F1 F0-ATP合酶c-转子的独特P51 XXEXXP基序防止质子泄漏的假设。这将通过野生型和cP 51 A突变体的稳定c-转子环的比较结构研究进行评估。将分析二维投影图，并从三维晶体中寻找高分辨率的结构信息。具体目标#2测试了以下假设：7-蛋白质Mrp异源寡聚体是相互依赖和协同的反向转运蛋白和其他转运蛋白的联合体，例如，共同呈现为质子聚集而设计的大的外表面。建议的实验包括一个假定的阴离子转运功能，使用一个新的Mrp突变体，赋予高Na+抗性，但缺乏反向转运活性的屏幕。第二个总体假设是，主要的pmf消耗复合物，如ATP合成酶和Mrp反向转运蛋白利用细胞组分和调节剂的网络，以满足生物能量的挑战。在非嗜碱菌中，它们对于重的生物能量工作负荷不是“硬连线”的，通过钠-碱挑战激活主要的pmf消耗者可以激发系统响应，促进挑战的管理。第三和第四个具体目标侧重于生物能量工作的生理背景。具体目标#3探测在低pmf下OXPHOS中嗜碱呼吸链超复合物、心磷脂和ATP合酶之间的相互作用。具体目标#4将模拟和测试金黄色葡萄球菌中对Mnh（Mrp同系物）活性的假设系统响应，其导致跨膜电位净增加并影响膜脂质流动性以及对抗菌肽的敏感性。该模型将通过一组遗传、生物化学和分子测定进行测试和建立。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内的反应的理解中的空白。 公共卫生相关性：氧化磷酸化，其机制将在一个建立的模型系统中进行探讨，是一个中央的生理过程，其中功能障碍与先天性遗传疾病和年龄相关的疾病。非常不寻常的性质和生理影响的MRP型反向转运蛋白，其他重点的拟议工作，是不完全理解，但重要的是，因为这些膜转运蛋白是广泛存在于革兰氏阴性和革兰氏阳性病原体，它们的毒力和耐药性的抗菌剂的影响。

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. PUBLIC HEALTH RELEVANCE: Oxidative phosphorylation, whose mechanism will be probed in an established model system, is a central physiological process in which malfunctions are associated with inborn genetic disorders and age-associated diseases. The very unusual properties and physiological impacts of the Mrp-type antiporters, the other focus of the proposed work, are incompletely understood but are of importance because these membrane transporters are widespread in Gram-negative and Gram-positive pathogens where they have impacts on virulence and resistance to antimicrobial agents.