GENETIC ANALYSIS OF RICKETTSIA PROWAZEKII

描述（改编自申请人的摘要）： 立克次体是落基山热和其他斑点热的病原体 以及地方性、丛林性和流行性斑疹伤寒， 全球威胁。 普氏立克次体，流行病的病原体 斑疹伤寒是一种专性细胞内寄生菌， 在真核宿主细胞的细胞质中。 立克次体的能力， 在节肢动物和人类等多种动物中利用这种细胞内生态位 并随后导致严重的人类疾病， study. 该提案重点关注基因工程的发展和应用 技术来解决有关致病菌R. prowazekii及其专性胞质内存在。 它利用了 R的可用性。prowazekii基因组序列和开发 立克次氏体遗传学技术检验立克次氏体基因相关假说 功能、DNA复制和致病机制。 具体目标1 PI的目标是利用立克次体转化系统， 可表达的可选择抗生素抗性基因的鉴定 in R. prowazekii通过敲除来区分，在 单基因水平。特定靶基因包括那些编码 与其他细菌、基因的已知毒力基因具有同源性的产物 假设仅在节肢动物载体中表达，假设 是立克次体还原过程的一部分 进化，最后，与R. prowazekii基因组 此外，将使用基于转座子的方法来产生随机 插入突变体在具体目标2中，PI的目标是隔离功能 复制起点一种方法是尝试产生立克次体 通过将假定的起源片段与可选择的 红霉素耐药基因ereB。另一种方法将确定来源 通过结合立克次体DnaA。具体目标3将继续PI 立克次体的转录终止特征和鉴定 在一些实施方案中，转录改变是在宿主细胞裂解之前发生的。使用 核糖核酸酶保护研究，PI将确定这些变化是否 通过检查额外的 非固有终止位点和细胞数量对终止的影响 这些网站。Rho的调节及其与这些变化的相关性将被 处理。

DESCRIPTION (Adapted from the Applicant's Abstract): Members of the genus Rickettsia are the etiologic agents of rocky mountain and other spotted fevers and endemic, scrub and epidemic typhus, diseases that pose a pernicious health threat worldwide. Rickettsia prowazekii, the etiologic agent of epidemic typhus is an obligate intracellular parasitic bacterium that can grow only within the cytoplasm of a eucaryotic host cell. The ability of rickettsiae to exploit this intracellular niche in animals as diverse as arthropods and humans and to subsequently cause serious human disease provides the impetus for this study. This proposal focuses on the development and application of genetic techniques to address questions regarding the pathogenic bacterium R. prowazekii and its obligate intracytoplasmic existence. It exploits the availability of the R. prowazekii genome sequence and the development of rickettsial genetic technologies to test hypotheses related to rickettsial gene function, DNA replication, and pathogenic mechanisms. In Specific Aim 1 the PI's goal is to capitalize on a rickettsial transformation system and identification of a selectable antibiotic resistance gene that can be expressed in R. prowazekii to discriminate, via knockouts, essential function at the level of single genes. Specifically targeted genes include those that encode products with homology to known virulence genes of other bacteria, genes hypothesized to be expressed only in the arthropod vector, genes hypothesized to be non-functional and part of the process of rickettsial reductive evolution, and finally, genes with homologs within the R. prowazekii genome. In addition, a transposon-based approach will be used to generate random insertion mutants. In Specific Aim 2,the PI's goal is to isolate the functional origin of replication. One approach will attempt to generate a rickettsial mini-chromosome by linking putative origin fragments with the selectable erythromycin-resistant gene, ereB. An alternate method will identify the origin by binding of rickettsial DnaA. Specific Aim 3 will continue the PI's characterization of transcription termination and identification of rickettsial transcriptional changes that occur just prior to lysis of the host cell. Using ribonuclease protection studies, the PI will determine whether these changes reflect a general property of the rickettsiae by examining additional non-intrinsic termination sites and the effect of cell number on termination at these sites. Modulation of Rho and its correlation to these changes will be addressed.