Plasmid pXO2 Replication in Bacillus anthracis

炭疽杆菌中的质粒 pXO2 复制

• 批准号: 6825626
• 负责人: SALEEM A. KHAN
• 金额: $ 25.49万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6825626
• 关键词: Bacillus anthracis; DNA footprinting; DNA replication; bacterial genetics; bacterial proteins; bioterrorism /chemical warfare; chemical stability; frameshift mutation; gel mobility shift assay; intermolecular interaction; messenger RNA; microorganism growth; nucleic acid sequence; plasmids; replicon; site directed mutagenesis; virulence

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.

描述（由申请人提供）：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。两个大质粒pXO 1和pXO 2在该微生物的毒力中起主要作用。关于这两种毒力质粒的复制和稳定性的分子机制知之甚少。基因转移经常发生在B之间。炭疽杆菌和密切相关的物种，如蜡状芽孢杆菌、苏云金芽孢杆菌和蕈状芽孢杆菌，使得pXO 1和pXO 2质粒可能可以从B天然转移。炭疽菌转化为对一种或多种抗生素有抗性的相关物种。此外，生物恐怖分子可能将pXO 1和pXO 2质粒引入多种耐药菌株以产生“超级生物恐怖剂”的可能性也不能排除。鉴于这些可能性，重要的是要确定质粒pXO 2（和pXO 1）特异性药物，可以干扰质粒复制，并可用于消除质粒从B。炭疽菌和相关生物。该R21提案的目标是研究B的pXO 2质粒的复制特性。炭疽病将鉴定pXO 2的最小复制子，并通过其在B中建立的能力研究微型pXO 2质粒的宿主范围。炭疽菌和其它革兰氏阳性菌如B。蜡状芽孢杆菌（B. cereus）、B.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。将通过估计不同宿主中微型pXO 2的拷贝数和测量细菌生长期间每代质粒损失百分比，研究pXO 2的RepB蛋白在质粒拷贝数控制和稳定性中的作用。RepS起始蛋白和pXO 2复制起点之间的相互作用将通过电泳迁移率变动分析和DMS足迹法进行研究。将突变与RepS蛋白相互作用的pXO 2起源区域，并测试这些突变体支持复制的能力。将建立RepS-起点相互作用和质粒pXO 2复制之间的相关性。我们还将从B中提取无细胞提取物。炭疽菌，并使用这些研究pXO 2在体外复制。我们的研究可能会揭示新的分子靶点的治疗，影响质粒复制和/或感染期间的维护。