Chemotactic Sensory Transduction in Bacillus Subtilis

枯草芽孢杆菌的趋化感觉转导

基本信息

批准号：
7931640
负责人：
George W. Ordal
金额：
$ 7.38万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7931640
关键词：
Affect Applications Grants Archaea Asparagine Bacillus subtilis Bacteria Behavior Binding Binding Sites Biochemical Burkholderia C-terminal Campylobacter jejuni Cations Charge Chemoreceptors Chemotaxis Complex Computer Simulation Coupling Cytoplasmic Tail Disabled Persons Disulfides Elements Enzymes Escherichia coli Evolution Excision Family Feedback Flagella Funding Genes Goals Gram-Positive Bacteria Group Meetings Helicobacter Homologous Gene Homologous Protein In Vitro Ions Joints Laboratories Measures Mediating Methylation Microbial Genome Sequencing Modeling Molecular Models Motion N-terminal Organism Pathway interactions Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Play Positioning Attribute Process Proline Protein Dephosphorylation Proteins Regulation Role Rotation Salmonella Sensory Site Structure System Tertiary Protein Structure Testing Thermotoga maritima Time Treponema pallidum United States National Institutes of Health Virulence Factors Work Writing Zinc alpha helix antibiotic design cell motility crosslink dimer insight interest member molecular modeling mutant novel pathogen progenitor receptor receptor binding research study response success thermophilic organism

项目摘要

Our goal during the past thirty years of NIH funding has been to understand the mechanism of chemotaxis in the Gram-positive bacterium Bacillus subtilis. During the past decade, the realization has emerged that the B. subtilis paradigm might be the ideal one for understanding these diverse mechanisms in the broad sweep of bacteria and archaea because of its inclusion of most known chemotaxis proteins and its similarity to the mechanism used in many archaea and the primitive thermophile Thermotoga maritima. This similarity suggests that the B. subtilis mechanism is an ancient mechanism and close to the progenitor mechanism that existed just before the divergence of bacteria and archaea. Thus, in studying this mechanism, we can understand how chemotaxis in bacteria and archaea might have evolved and gain insights into how these diverse pathways may function. During the last funding period, we have made tremendous progress in elucidating the biochemical function of the three proteins not found in Escherichia coli chemotaxis: CheC, CheD, and CheV. Collectively, this work has enabled us to formulate a working hypothesis for how the integrated pathway functions. The key element of this hypothesis is that there are three adaptation systems in B. subtilis (unlike just one in E. coli). In addition to the previously identified methylation and CheV systems, we have discovered a third adaptation system involving CheY, CheC, and CheD. The available evidence indicates that CheD is an allosteric activator of the receptor complex. Furthermore, we now have a molecular model for how the selective methylation of specific residues on the receptors differentially affects CheA kinase activity, a key element of which is a newly discovered zinc ion in the methylation region. These breakthroughs have been greatly facilitated by the recent structures for T. maritima CheC, CheD, and the cytoplasmic domain of TM1143 (a chemoreceptor). As a result, we are now in a position to propose detailed experiments to elucidate key mechanisms in B. subtilis chemotaxis. Specifically, we wish to develop a computational model for the integrated pathway and test various aspects experimentally. We will primarily focus on selective methylation and CheD binding, determining how specific methylation sites affect CheA kinase activation and CheD binding and how CheD binding affects CheA kinase activation and methylation. We will also focus on the relation between the CheC/D/Yp and CheV adaptation systems and the very slow receptor remethylation process. The proposed experiments include (among others): (i) measuring timing of methylation at each site in response to the addition and removal of attractants in McpB (weak CheD requirement) and McpC (strong CheD requirement) in strains having the CheV and CheC/D/Yp adaptation systems and lacking one or both; (ii) determining the role of each methylated site on CheD binding, CheA activation, and on receptor structure as reflected by disulfide crosslinking; and (iii) discovering where CheD binds and how CheD binding affects receptor methylation and CheA kinase activity.

在过去的三十年中，我们的目标是了解趋化性的机制革兰氏阳性细菌枯草芽孢杆菌。在过去的十年中，意识到 B.枯草式范式可能是理解这些不同机制的理想选择扫描细菌和古细菌，因为它包含了最著名的趋化蛋白及其与许多古细菌和原始的嗜热疗法马里匹马中使用的机制相似。这种相似性表明枯草芽孢杆菌的机制是一种古老的机制，靠近祖细胞在细菌和古细菌发散之前存在的机制。因此，在研究这种机制时，我们可以理解细菌和古细菌的趋化性如何发展并获得有关如何如何发展的这些不同的途径可能起作用。在最后的资金期间，我们在阐明大肠杆菌趋化性中未发现的三种蛋白质的生化功能：CHEC，切德和雪佛兰。总的来说，这项工作使我们能够为如何集成途径功能。该假设的关键要素是有三个适应系统在枯草芽孢杆菌中（与大肠杆菌中的一个不同）。除了先前鉴定的甲基化和CHEV 系统，我们发现了涉及Chey，Chec和Ched的第三个适应系统。可用证据表明CHED是受体复合物的变构激活剂。此外，我们现在有一个分子模型，用于如何在受体上特定残基的选择性甲基化差异影响 CHEA激酶活性，其关键要素是甲基化区域中新发现的锌离子。这些最近的Maritima Chec，Ched和The The The Maritima Chec的最新结构极大地促进了突破 TM1143（化学感受器）的细胞质结构域。结果，我们现在可以提出详细的信息实验阐明枯草芽孢杆菌趋化性中的关键机制。具体来说，我们希望开发一个集成途径和各个方面的计算模型实验。我们将主要专注于选择性甲基化和切成结合，确定了特定甲基化位点影响CHEA激酶激活和CHED结合，而Ched结合如何影响CHEA激酶激活和甲基化。我们还将重点关注CHEC/D/YP和CHEV适应之间的关系系统和非常缓慢的受体再甲基化过程。提出的实验包括其他）：（i）测量每个位点的甲基化时间，以响应吸引剂的添加和去除在MCPB（CHED弱需求）和MCPC（强大的CHED需求）中，具有CHEV和 CHEC/D/YP适应系统，缺乏一个或两个；（ii）确定每个甲基化位点在通过二硫键交联所反映的结合，CHEA激活和受体结构；（iii）发现Ched结合的位置以及Ched结合如何影响受体甲基化和CHEA激酶活性。