Role of RepX Protein in Replication/Partitioning of Anthrax Toxin Plasmid pXO1

描述（由申请人提供）：炭疽杆菌是一种重要的人类病原体，是人类炭疽病的病原体，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。基因转移经常发生在B之间。炭疽菌和密切相关的物种，如B。cereus和B. pXO 1和pXO 2质粒可以从B中自然转移。炭疽菌转化为对抗生素有抗药性的物种。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。虽然pXO 1对B的生长有抑制作用。在炭疽杆菌中，它非常稳定，很少从生长的细菌细胞中丢失。关于这种质粒的复制和稳定性所涉及的分子机制知之甚少。本研究旨在分析B炭疽毒素编码质粒pXO 1的复制特性。炭疽病我们计划表征pXO 1的最小复制子，包括涉及质粒稳定性和拷贝数控制的区域。我们已经分离出pXO 1的复制子，并表明它编码一种新的复制蛋白RepX。RepX类似于FtsZ/微管蛋白蛋白，我们已经证明，纯化的RepX蛋白是一个GTP酶，可以在体外进行GTP依赖的动态聚合。我们最近还表明RepX在体内B中聚合。炭疽病我们推测RepX可能参与了高度稳定的pXO 1质粒的复制和分配。利用荧光标记蛋白和/或pXO 1 DNA的细胞生物学方法，我们将进行实验来研究RepX在体内的聚合和定位，以及它与pXO 1质粒的共定位和植物生长和孢子形成B中的细胞分裂机制。炭疽菌细胞通过突变分析，我们将确定区域的RepX是重要的复制和分区活动。最后，我们将利用“下拉”试验，以确定主机和pXO 1编码的蛋白质与RepX相互作用，并可能发挥重要作用，pXO 1的复制和稳定性。我们的研究可能会导致更好地了解pXO 1质粒的稳定复制和维持所涉及的元素，并可能有助于未来开发质粒特异性的共同治疗药物，可以降低B的毒力。炭疽菌和相关生物。公共卫生相关性：该提案的目的是研究RepX蛋白在炭疽杆菌炭疽毒素编码pXO 1质粒的复制和分离中的作用。我们还计划在体内研究RepX和pXO 1 DNA的共定位。我们还将研究pXO 1的稳定性和拷贝数控制。我们计划确定RepX蛋白的功能结构域，并确定质粒和染色体编码的蛋白质与RepX相互作用。

DESCRIPTION (provided by applicant): Bacillus anthracis is an important human pathogen that is the etiological agent of anthrax in humans and a potential biological weapon. Two large plasmids, pXO1 and pXO2, play a major role in the virulence of this organism. Gene transfer can frequently occur between B. anthracis and closely related species such as B. cereus and B. thuringiensis, making it likely that the pXO1 and pXO2 plasmids could naturally transfer from B. anthracis into such species that are resistant to 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. Although pXO1 is dispensable for the growth of B. anthracis, it is highly stable and rarely lost from growing bacterial cells. Very little is known about the molecular mechanisms involved in the replication and stability of this plasmid. The aims of this proposal are to analyze the replication properties of the anthrax toxin-encoding pXO1 plasmid of B. anthracis. We plan to characterize the minimal replicon of pXO1, including regions involved in plasmid stability and copy number control. We have isolated the replicon of pXO1 and shown that it encodes a novel replication protein, RepX. RepX resembles the FtsZ/tubulin proteins and we have shown that the purified RepX protein is a GTPase that can undergo GTP-dependent dynamic polymerization in vitro. We have also recently shown that RepX polymerizes in vivo in B. anthracis. We hypothesize that RepX may be involved in both the replication and partitioning of the highly stable pXO1 plasmid. Using cell biological approaches with fluorescently labeled proteins and/or pXO1 DNA, we will carry out experiments to study polymerization and localization of RepX in vivo, as well as its co-localization with the pXO1 plasmid and the cell division machinery in vegetatively growing and sporulating B. anthracis cells. By mutational analysis, we will identify regions of RepX that are important for its replication and partitioning activities. Finally, we will utilize "pull-down" assays to identify host- and pXO1-encoded proteins that interact with RepX and may play important roles in pXO1 replication and stability. Our studies could lead to a better understanding of the elements involved in the stable replication and maintenance of the pXO1 plasmid and may contribute to the future development of plasmid-specific co-therapeutic drugs that can reduce the virulence of B. anthracis and related organisms. PUBLIC HEALTH RELEVANCE: The aims of this proposal are to study the role of the RepX protein in the replication and segregation of the anthrax toxin-encoding pXO1 plasmid of Bacillus anthracis. We also plan to study the colocalization of RepX and pXO1 DNA in vivo. We will also investigate the stability and copy number control of pXO1. We plan to identify the functional domains of the RepX protein and identify plasmid- and chromosome-encoded proteins that interact with RepX.