The role of pseudopilins during transformation in Bacillus subtilis

假菌毛蛋白在枯草芽孢杆菌转化过程中的作用

• 批准号: 7754713
• 负责人: Jessica Marie Calzola
• 金额: $ 3.02万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7754713
• 关键词: Antibiotic Resistance; Bacillus subtilis; Bacteria; Binding; Biochemical; Biological Assay; Cell Wall; Cells; Characteristics; Competence; Complex; Cytosol; DNA; DNA Binding; DNA delivery; DNA-Binding Proteins; Dimensions; Electron Microscopy; Escherichia coli; Evolution; Fluorescence Microscopy; Genomics; Gold Colloid; Horizontal Gene Transfer; Human; Immunoblotting; In Vitro; Ion Channel; Light; Mediating; Membrane; Microbe; Minor; Modeling; Molecular Structure; Movement; Pattern; Polyacrylamide Gel Electrophoresis; Proteins; Research; Role; Structure; System; Testing; Time; Type IV Secretion System Pathway; disulfide bond; in vivo; monomer; pathogen; pill; protein function; protein protein interaction; uptake

DESCRIPTION (provided by applicant): The study of natural transformation, along with conjugation and transduction, has gained importance since genomic sequencing revealed horizontal gene transfer to be a significant factor in the evolution of bacteria and antibiotic resistance. The machinery that mediates transformation in Bacillus subtilis is highly conserved among competent microbes, both gram-positive and negative. Also, B. subtilis pseudopilins and assembly proteins, which are essential components of the transformation machinery, share homology to proteins of type 2 secretion systems (T2SS) and type IV pill (T4P), found in many gram-negative human pathogens. Ultimately, studying the competence proteins in B. subtilis will shed light on general characteristics of transformation proteins and the functions of T2SS and T4P proteins. The focus of this proposal is to decipher the functions of ComGD, ComGE, and ComGG, collectively called the minor pseudopilins, and ComGC, the major pseudopilin which comprises the competence pseudopilus, of Bacillus subtilis. These proteins are essential for both DNA-binding and transport to the cytosol during transformation, but their specific roles are unknown. We hypothesize that the minor pseudopilins interact to form a heterotrimeric complex similar to the one formed by the minor pseudopilins of Escherichia coli T2SS. The E. coli minor pseudopilin complex is believed to bind to the tip of the T2SS pseudopilus and mediate protein-protein interactions. Accordingly, we believe the B. subtilis complex localizes at the tip of the competence pseudopilus, comprised of ComGC, and binds to ComEA, the integral membrane DNA-binding protein. As a result, ComEA will be connected to the competence pseudopilus such that retraction of the pseudopilus will cause movement in ComEA, which will allow for delivery of the DNA to the membrane channel composed of ComEC. Analysis of our model will require in vitro and in vivo biochemical protein-protein interaction studies like pull-downs and immunoprecipitiation. Fluorescence microscopy will serve to evaluate the localization and possible co-localization of ComEA and the pseudopilins with other competence proteins. Overall, we seek to decipher the function and interactions of the pseudopilins during transformation. Bacillus subtilis shares DNA using the same mechanism and machinery that some human pathogens use to become antibiotic resistant. The focus of this research is on studying this conserved machinery in B. subtilis because it can be easily manipulated.

描述（由申请人提供）：自然转化的研究，沿着接合和转导，已经变得重要，因为基因组测序显示水平基因转移是细菌进化和抗生素耐药性的重要因素。枯草芽孢杆菌中介导转化的机制在革兰氏阳性和阴性的感受态微生物中是高度保守的。还有B枯草杆菌假青霉素和装配蛋白是转化机制的基本组分，与在许多革兰氏阴性人类病原体中发现的2型分泌系统（T2 SS）和IV型pill（T4 P）的蛋白具有同源性。最后，对B.本研究为进一步研究转化蛋白的一般特性以及T2 SS和T4 P蛋白的功能奠定了基础。 该建议的重点是破译ComGD、ComGE和ComGG（统称为次要假青霉素）和ComGC（包含枯草芽孢杆菌的感受态假青霉素的主要假青霉素）的功能。这些蛋白质在转化过程中对DNA结合和转运到胞质溶胶都是必不可少的，但它们的具体作用尚不清楚。我们假设，次要的假磷脂相互作用，形成一个异源三聚体复合物类似于大肠杆菌T2 SS的次要的假磷脂形成的。急诊大肠杆菌次要假纤毛蛋白复合物被认为与T2 SS假纤毛的尖端结合并介导蛋白质-蛋白质相互作用。因此，我们认为B。枯草杆菌复合物定位于感受态假菌毛的顶端，由ComGC组成，并结合到ComEA，整合膜DNA结合蛋白。因此，ComEA将连接到感受态假纤毛，使得假纤毛的缩回将引起ComEA的移动，这将允许将DNA递送到由ComEC组成的膜通道。 我们的模型的分析将需要在体外和体内生化蛋白质-蛋白质相互作用的研究，如下拉和免疫沉淀。荧光显微镜将用于评估ComEA和假磷脂与其他感受态蛋白的定位和可能的共定位。总的来说，我们试图破译的功能和相互作用的pseudopilins在转化过程中。 枯草芽孢杆菌使用与一些人类病原体用于产生抗生素抗性相同的机制和机器来共享DNA。本研究的重点是研究这种保守的机制在B。枯草杆菌，因为它可以很容易地操纵。