GENETIC ANALYSIS OF RICKETTSIA PROWAZEKII

但前提是。 立克次体属的成员是落基山和其他斑点热病和地方性疾病的病原体， 灌木和流行性斑疹伤寒，这些疾病在世界范围内构成了有害的健康威胁。太赫兹立克次体病因学 流行性斑疹伤寒杆菌是一种专性的细胞内寄生细菌，只能在斑疹伤寒杆菌的细胞质内生长。 真核宿主细胞。立克次体利用细胞内生态位的能力，动物包括节肢动物和 并由此引发严重的人类疾病，为本研究提供了动力。 这项提案侧重于基因技术的开发和应用，以解决有关 病原菌R.prowazekii及其专性胞质内的存在。它利用了R的可用性。 Prowazekii基因组序列和立克次体基因技术的发展以检验与立克次体相关的假说 基因功能、DNA复制和致病机制。在具体目标1中，我们的目标是利用立克次体 普氏根瘤菌转化系统及一个可选择表达的抗生素抗性基因的鉴定 通过基因敲除，在单基因水平上区分必要的功能。特定靶向的基因包括 编码与其他细菌已知毒力基因同源的产物，假设基因仅在 节肢动物载体，假设为无功能的基因和立克次体还原进化过程的一部分，以及 最后，在普罗瓦泽克乳杆菌基因组中有同源基因。此外，还将使用基于转座子的方法来 产生随机插入突变体。在特定的目标2中，我们的目标是分离复制的功能起源。一 该方法将试图通过将假定的起源片段与可选择的 红霉素抗性基因Ereb。另一种方法是通过与立克次体DNAA的结合来识别来源。特定的 目标3将继续我们对转录终止的描述和我们对立克次体转录的鉴定 在宿主细胞裂解前发生的变化。利用核糖核酸酶保护研究，我们将确定 这些变化反映了立克次体的一般性质，通过检查其他非固有终止位点和 细胞数量对这些部位终止的影响。RHO的调制及其与这些变化的相关性将被评估。

PROVIDED. Members of the genus Rickettsia are the etiologic agents of rocky mountainand other spotted fevers and endemic, scrub, and epidemic typhus, diseases that pose a pernicious health threat worldwide. Rickettsiaprowazekii, thz 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 intracellularniche in animalsas 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 about 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 torickettsial gene function, DNA replication, and pathogenic mechanisms. In Specific Aim 1 our goal is to capitalize on a rickettsial transformation system and the identification of a selectable antibiotic resistance gene that can beexpressed inR.prowazekii to discriminate, via knockouts, essential function at the level of single genes. Specifically targeted genes include those that encode products withhomology to known virulencegenes 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 reductiveevolution,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 our goal is to isolate the functional origin of replication. One approach will attempt to generate a rickettsial minichromosome by linkingputative origin fragments with the selectable erythromycin-resistant gene, ereB. An alternate method will identify the originby the bindingof rickettsial DnaA. Specific Aim 3 will continue our characterization of transcription termination and our identificationof rickettsialtranscriptional changes that occur just prior to lysis of the host cell. Using ribonuclease protection studies we 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 terminationat these sites. Modulation of Rho and its correlation to these changes will be: assessed.