Plasmid pXO2 Replication in Bacillus anthracis

描述（申请人提供）：炭疽芽孢杆菌是一种重要的人类病原体和潜在的生物武器。px1和pXO2两个大的质粒在这种生物体的毒力中起主要作用。关于这两种毒性质粒的复制和稳定性所涉及的分子机制知之甚少。炭疽芽孢杆菌与蜡样芽孢杆菌、苏云金芽孢杆菌、霉菌芽孢杆菌等密切相关的物种之间经常发生基因转移，这使得px1和pXO2质粒很可能从炭疽芽孢杆菌自然转移到对一种或多种抗生素具有抗性的相关物种中。此外，生物恐怖分子可能会将pXO1和pXO2质粒引入多种耐药菌株中，以产生“超级生物恐怖剂”，这种可能性也不容忽视。考虑到这些可能性，鉴定能够干扰质粒复制的质粒pXO2（和pXO2）特异性药物并用于消除炭疽芽胞杆菌和相关生物的质粒是很重要的。本R21提案的目的是研究炭疽芽孢杆菌pXO2质粒的复制特性。通过在炭疽芽孢杆菌和其他革兰氏阳性菌（如蜡样芽孢杆菌、苏云金芽孢杆菌、枯草芽孢杆菌、金黄色葡萄球菌、产气荚膜梭菌和肺炎链球菌）中建立pXO2质粒的能力，鉴定pXO2的最小复制子，并研究其宿主范围。通过估算不同寄主中pXO2的拷贝数和测量细菌生长过程中每代质粒损失百分比，研究pXO2的RepB蛋白在质粒拷贝数控制和稳定性中的作用。RepS引发蛋白与pXO2复制起源之间的相互作用将通过电泳迁移转移试验和DMS足迹法进行研究。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被测试。将建立rep -origin相互作用与质粒pXO2复制之间的相关性。我们还将从炭疽杆菌中提取无细胞提取物，并利用这些提取物在体外研究pXO2的复制。我们的研究可能会揭示影响感染期间质粒复制和/或维持的治疗的新分子靶点。

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.