Plasmid pXO2 Replication in Bacillus anthracis

描述(申请人提供)：炭疽杆菌是一种重要的人类病原体，也是一种潜在的生物武器。两个大的质粒pXO1和pXO2在该菌的毒力中起主要作用。关于这两个毒力质粒的复制和稳定性所涉及的分子机制，人们知之甚少。炭疽杆菌与蜡状芽孢杆菌、苏云金芽孢杆菌和真菌芽孢杆菌等亲缘关系密切的物种之间经常发生基因转移，这使得pXO1和pXO2质粒很可能从炭疽杆菌自然转移到对一种或多种抗生素具有抗药性的相关物种中。此外，生物恐怖分子可能会将pXO1和pXO2质粒引入多种抗药性菌株，以产生“超级生物恐怖剂”，这一可能性不容忽视。鉴于这些可能性，重要的是要找出针对pXO2(和pXO1)的特异性药物，这些药物可以干扰质粒复制，并可用于消除炭疽杆菌和相关生物中的质粒。本R21建议的目的是研究炭疽杆菌pXO2质粒的复制特性。将确定pXO2的最小复制子，并通过其在炭疽杆菌和其他革兰氏阳性细菌如蜡状芽孢杆菌、苏云金杆菌、枯草杆菌、金黄色葡萄球菌、产气荚膜梭菌和肺炎链球菌中的建立能力来研究其宿主范围。通过估计微型pXO2在不同宿主中的拷贝数和测量细菌生长过程中每代质粒损失的百分比，将研究pXO2的RepB蛋白在质粒拷贝数控制和稳定性中的作用。将通过电泳迁移率改变分析和DMS足迹来研究REPS启动子蛋白与pXO2复制来源之间的相互作用。与REPS蛋白相互作用的pXO2起源区域将发生突变，这些突变体支持复制的能力将得到测试。将建立REPS起源的相互作用和质粒pXO2复制之间的关联。我们还将制作炭疽杆菌的无细胞提取物，并用这些提取物在体外研究pXO2的复制。我们的研究可能会为治疗揭示新的分子靶点，影响感染期间的质粒复制和/或维持。

DESCRIPTION (provided by applicant): Bacillus anthracis is an important human pathogen and a potential biological weapon. Two large plasmids, pXO1 and pXO2 play a major role in the virulence of this organism. Very little is known about the molecular mechanisms involved in the replication and stability of the two virulence plasmids. Gene transfer can frequently occur between B. anthracis and closely related species such as Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides, making it likely that the pXO1 and pXO2 plasmids could naturally transfer from B. anthracis into related species that are resistant to one or more antibiotics. Also, the possibility that bioterrorists may introduce the pXO1 and pXO2 plasmids into multiple drug resistant strains to generate "super bioterror agents" cannot be discounted. Given these possibilities, it is important to identify plasmid pXO2 (and pXO1)-specific drugs that could interfere with plasmid replication and can be used for the elimination of plasmids from B. anthracis and related organisms. The goal of this R21 proposal is to study the replication properties of the pXO2 plasmid of B. anthracis. The minimal replicon of pXO2 will be identified and the host range of the mini pXO2 plasmid studied by its ability to be established in B. anthracis and other Gram-positive bacteria such as B. cereus, B. thuringiensis, Bacillus subtilis, Staphylococcus aureus, Clostridium perfringens and Streptococcus pneumoniae. The role of the RepB protein of pXO2 in plasmid copy number control and stability will be investigated by estimating the copy number of mini pXO2 in different hosts and by measuring percent plasmid loss per generation during bacterial growth. The interaction between the RepS initiator protein and the origin of replication of pXO2 will be studied by electrophoretic mobility-shift assays and by DMS footprinting. Regions of pXO2 origin that interact with the RepS protein will be mutated and the ability of these mutants to support replication will be tested. A correlation between RepS-origin interaction and plasmid pXO2 replication will be established. We will also make cell-free extracts from B. anthracis and use these to study pXO2 replication in vitro. Our studies may reveal new molecular targets for therapeutics that affect plasmid replication and/or maintenance during infection.