Proton-Coupling Strategies in Alkaliphilic Bacillus

描述(由申请人提供)：这个项目将测试关于呼吸系统细菌生物能工作的两个主要假说，即由跨膜的质子的电化学梯度质子动力(PMF)提供能量的与膜相关的过程。大部分工作的实验系统是一种嗜碱芽孢杆菌，它表现出结构性准备，以迎接高生物能量工作负荷的挑战。嗜碱假坚固芽孢杆菌4也具有特殊的适应性，有利于其在低PMF条件下的生物能量工作。第一个主要假设是，特殊的结构特征增强了亲碱性ATP合成酶及其唯一复杂的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动态平衡。自适应特征被假设为使这些PMF用户复合体能够利用从呼吸泵转移质子的隔离路径，并在质子贫乏的环境中收集质子。生物能机械的其他功能，包括ATP合成酶，可以防止氧化磷酸化(OXPHOS)期间的质子泄漏。前两个具体目标集中在亲碱机械的结构-功能特征上。特定目标#1测试了F1F0-ATP合成酶c-转子的独特P51XXEXXP基序防止质子泄漏的假设。这将通过对野生型和cP51a突变体的稳定c-转子环的比较结构研究来评估。将分析2D投影图，并从3D晶体中寻找高分辨率结构信息。特定目标#2测试了这样的假设，即7蛋白MRP异源低聚物是相互依赖和协同的反转运体和其他转运体的联盟，例如共同呈现一个为质子聚集而设计的大外表面。拟议的实验包括使用一种新的MRP突变体来筛选可能的阴离子转运功能，该突变体具有高Na+抗性，但缺乏反转运活性。第二个最重要的假设是，主要的PMF消耗复合体，如ATP合成酶和MRP反向转运体，利用细胞成分和调节器的网络来应对生物能量挑战。在非亲碱的人中，对于繁重的生物能量工作负荷来说，通过钠碱挑战激活主要的PMF消耗者会引起系统响应，从而促进挑战的管理。第三和第四个具体目标侧重于生物能量工作的生理背景。具体目的#3探讨低PMF下OXPHOS中亲碱呼吸链超复合体、心磷脂和ATP合成酶之间的相互作用。具体目标#4将模拟和测试一个假想的系统对金黄色葡萄球菌MNH(一种MRP同源物)活性的反应，这会导致跨膜电位的净增加，影响膜脂流动性以及对抗菌肽的敏感性。该模型将通过一系列遗传、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全细胞响应的理解方面的空白。 公共卫生相关性：氧化磷酸化是一个中心生理过程，在这个过程中，功能障碍与先天遗传疾病和年龄相关疾病有关，其机制将在已建立的模型系统中进行探索。MRP型逆向转运蛋白是拟议工作的另一个重点，其非常不寻常的性质和生理影响尚不完全清楚，但具有重要意义，因为这些膜转运蛋白广泛存在于革兰氏阴性和革兰氏阳性病原菌中，它们对毒力和抗菌素耐药性有影响。

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.