GENETIC APPROACHES TO VIRULENCE IN B. BURGDORFERI

描述：（改编自申请人的摘要）：伯氏疏螺旋体是 一种能在体外培养的细菌，是莱姆病的病因。其小 基因组含有约1,000个染色体基因和400个质粒基因。 尽管已经知道了B的完整DNA序列。burgdorferi基因组， 鉴定和表征独特体内表达的B。burgdorferi 毒力决定因素已被推迟，由于缺乏迅速和 有效的遗传系统。我们开发了一个基因系统， B。burgdorfed，其由染色体外克隆载体pGKI 2， 增强型绿色荧光蛋白作为潜在的报告基因， 红霉素、卡那霉素和其他抗生素作为选择标记。我们有 也能够表明bmp基因簇在B之间是高度保守的。 burgdorferi sensu lato菌株，并且该簇的基因经历 环境调节的差异表达表明了潜在的作用 这些基因的毒性。我们提出的实验方案是基于 我们的初步工作和框架由两个假设：1）有效的分子 可以为B开发遗传系统。burgdorferi和2）BMP的作用 可以使用以下方法确定伯氏B螺旋体生物学和毒力中的基因簇 这些系统。长期目标是鉴定伯氏B菌的毒力 决定因素，提高我们对它们在体内表达的理解， 监管，我们提出以下具体目标：1）持续发展， B染色体外克隆系统改进burgdorferi，2） 分离和补充B。burgdorferi bmpD、bmpC和bmpA无效突变体， 确定这些基因在B中的可能作用。莱姆病毒力 感染的体外和体内模型系统;和3）表征启动子 和调控DNA序列的bmpC使用转录融合与 增强型绿色荧光蛋白和荧光激活细胞 分选（FACS）。我们希望这些实验将允许扩展 分子科赫的假设，以表征独特的或具体的 B的分子毒力决定因子。burgdorferi。

DESCRIPTION: (adapted from the applicant's abstract): Borrelia burgdorferi is an in vitro culturable bacterium that is the cause of Lyme disease. Its small genome contain approxnnately 1,000 chromosomal genes and 400 plasmid genes. Despite knowledge of the complete DNA sequence of the B. burgdorferi genome, identification and characterization of unique in vivo expressed B. burgdorferi virulence determinants has been delayed by the lack of expeditious and efficacious genetic systems in Borrelia. We have developed a genetic system in B. burgdorfed that consists of the extrachromosomal cloning vector, pGKI2, enhanced green fluorescent protein as a potential reporter gene, and resistance to erythromycin, kanamycin, and other antibiotics as selective markers. We have also been able to show that the bmp gene cluster is highly conserved among B. burgdorferi sensu lato strains, and that the genes of this cluster undergo environmentally modulated differential expression suggesting a potential role in virulence for these genes. The experimental protocol we propose is based on our preliminary work and framed by two hypotheses: 1) efficient molecular genetic systems can be developed for B. burgdorferi, and 2) the role of the bmp gene cluster in B.burgdorferi biology and virulence can be ascertained using these systems. With the long-term aim of identifying B.burgdorferi virulence determinants and improving our understanding of their in vivo expression and regulation, we propose the following Specific Aims: 1) Continue development and improvement of an extrachromosomal cloning system for B. burgdorferi, 2) isolate and complement B. burgdorferi bmpD, bmpC and bmpA null mutants to determine the possible role of these genes in B. burgdorferi virulence in in vitro and in vivo model systems of infection; and 3) characterize promoters and regulatory DNA sequences of bmpC using transcriptional fusions with enhanced green fluorescent protein (EGFP) and fluorescence-activated cell sorting (FACS). We expect these experiments will permit the extension of the molecular Koch's postulates to the characterization of unique aud specific molecular virulence determinants of B. burgdorferi.