GENETIC ANALYSIS OF RICKETTSIA PROWAZEKII

描述(改编自申请人的摘要)：该属的成员 立克次体是落基山和其他斑点热病的病原体。 地方性斑疹伤寒、丛林斑疹伤寒和流行性斑疹伤寒 全球范围的威胁。流行的病原体普氏立克次体 斑疹伤寒是一种专性的细胞内寄生细菌，只能生长 在真核宿主细胞的细胞质内。立克次体的致病能力 在节肢动物和人类等各种动物身上开发这种细胞内生态位 而随后导致严重的人类疾病则为此提供了推动力 学习。本提案重点介绍了基因工程的开发和应用。 解决有关致病细菌R. Prowazekii及其专性胞质内的存在。它利用了 普罗瓦泽克乳杆菌基因组序列的可获得性及其发展 立克次体基因技术用于检验与立克次体基因相关的假说 功能、DNA复制和致病机制。在具体目标1中 Pi的目标是利用立克次体转化系统和 一个可选择表达的抗生素耐药基因的鉴定 在R.prowazekii中，通过基因敲除来区分在 单基因水平。特定靶向的基因包括编码 与其他细菌的已知毒力基因同源的产物，基因 假设只在节肢动物载体中表达，假设基因 是无功能的，是立克次体还原过程的一部分 进化，最后，基因与普罗瓦泽克杆菌基因组中的同源基因。 此外，还将使用基于转座子的方法来生成随机 插入突变体。在特定目标2中，PI的目标是隔离功能 复制的来源。一种方法将尝试产生立克次体 通过将假定的起源片段与可选择的片段相关联来进行微染色体 红霉素抗性基因Ereb。另一种方法将识别原点 通过与立克次体DNAA结合。《特殊目标3》将延续《少年派》 立克次体转录终止特性及鉴定 在宿主细胞裂解前发生的转录变化。vbl.使用 核糖核酸酶保护研究中，等电点是否会决定这些变化 通过检查其他因素反映立克次体的一般特性 非本征终止位点及细胞数对终止的影响 这些网站。Rho的调制及其与这些变化的相关性将是 地址。

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.