The role of pseudopilins during transformation in Bacillus subtilis

描述(由申请人提供)：自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一个重要因素以来，自然转化以及接合和转导的研究变得越来越重要。介导枯草芽孢杆菌转化的机制在革兰氏阳性和阴性的合格微生物中高度保守。此外，枯草杆菌假纤毛杆菌和组装蛋白是转化机制的重要组成部分，与许多革兰氏阴性人类病原体中发现的2型分泌系统(T2SS)和IV型药丸(T4P)的蛋白质具有同源性。最终，研究枯草杆菌中的竞争蛋白将有助于了解转化蛋白的一般特征以及T2SS和T4P蛋白的功能。 这项建议的重点是破译枯草芽孢杆菌的ComGD、ComGE和ComGG，统称为次要伪杆菌，以及ComGC，即组成能力伪杆菌的主要伪杆菌的功能。这些蛋白质在转化过程中对DNA结合和运输到细胞质都是必不可少的，但它们的具体作用尚不清楚。我们推测，次要伪宾相互作用形成一个异三聚体复合体，类似于大肠杆菌T2SS中次要伪宾所形成的复合体。据信，大肠杆菌微小的伪细菌素复合体与T2SS伪乳头末端结合，并介导蛋白质-蛋白质相互作用。因此，我们认为枯草杆菌复合体定位于ComGC组成的感受态假乳头的顶端，并与完整的膜DNA结合蛋白COMEA结合。因此，COMEA将与能力假纤毛相连，从而使伪纤毛的回缩将导致COMEA的运动，这将允许将DNA输送到由COMEC组成的膜通道。 对我们的模型的分析将需要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和伪毛蛋白与其他活性蛋白的定位和可能的共定位。总体而言，我们试图破译假性粘连蛋白在转化过程中的功能和相互作用。 枯草芽孢杆菌使用一些人类病原体产生抗生素抗药性的相同机制和机制分享DNA。本研究的重点是研究枯草芽孢杆菌的这一保守机制，因为它易于操作。

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.