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

描述（由申请人提供）：炭疽芽孢杆菌是一种重要的人类病原体，是人类炭疽的病原，也是潜在的生物武器。pXO1和pXO2两个大的质粒在这种生物的毒力中起主要作用。炭疽芽孢杆菌与蜡样芽孢杆菌和苏云金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这些对抗生素有耐药性的物种中。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。虽然pXO1在炭疽杆菌的生长中是不可缺少的，但它是高度稳定的，很少在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。本研究旨在分析炭疽杆菌编码炭疽毒素的pXO1质粒的复制特性。我们计划表征pXO1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pXO1的复制子，并证明它编码一种新的复制蛋白，RepX。RepX类似于FtsZ/微管蛋白，我们已经证明纯化的RepX蛋白是一种gtp酶，可以在体外进行gtp依赖的动态聚合。我们最近也证明了RepX在炭疽杆菌体内聚合。我们假设RepX可能参与了高度稳定的pXO1质粒的复制和分裂。利用荧光标记蛋白和/或pXO1 DNA的细胞生物学方法，我们将在无性生长和产孢的炭疽杆菌细胞中进行实验，研究RepX在体内的聚合和定位，以及它与pXO1质粒的共定位和细胞分裂机制。通过突变分析，我们将确定RepX中对其复制和分裂活动很重要的区域。最后，我们将利用“下拉”实验来鉴定宿主和pXO1编码的蛋白，这些蛋白与RepX相互作用，可能在pXO1的复制和稳定性中发挥重要作用。我们的研究有助于更好地了解pXO1质粒稳定复制和维持的相关因素，并可能有助于未来开发质粒特异性联合治疗药物，以降低炭疽芽胞杆菌和相关生物的毒力。公共卫生相关性：本提案的目的是研究RepX蛋白在炭疽芽孢杆菌编码炭疽毒素的pXO1质粒的复制和分离中的作用。我们还计划在体内研究RepX和pXO1 DNA的共定位。我们还将研究pXO1的稳定性和拷贝数控制。我们计划鉴定RepX蛋白的功能域，并鉴定与RepX相互作用的质粒和染色体编码蛋白。

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.