Plasmid pXO2 Replication in Bacillus anthracis

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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.