MOLECULAR MECHANISMS OF VARIATION AND ADAPTATION IN BORRELIA BURGDORFERI

我们的主要目标是了解疏螺旋体 burgdorfer（Bb）建立感染：蜱虫之间的传播周期 载体和哺乳动物储库宿主，这两者都需要维持 自然界中的螺旋体 1.外表面蛋白的功能和表达。 的外膜 Bb含有几种丰富的脂蛋白（Osps），它们的大小不同， 表情 不同的OSP可能赋予不同的 螺旋体上的特性， 它必须生存的环境。 至少有三个OSP显示 温度相关差异表达，可能伴随蜱 喂养或传播给温血动物。 我们已经开发出一种 模式系统研究Osp基因在E.大肠杆菌， 使用与指示基因lacZ融合的osp启动子， 用于改变启动子表达的反式作用Bb基因产物。我们有 克隆了一个osp基因家族，该家族至少包含6个相关成员， 并正在研究这些基因的表达过程中， 感染 Bb生物学特性及莱姆病致病机理的研究 由于目前缺乏遗传工具，对疾病的研究受到严重限制。 我们 利用对抗生素的抗药性进行等位基因交换 香豆霉素A1作为选择标记。 这是第一个 通过同源重组证明靶基因失活 在BB。 我们目前正在使用这种方法来克隆基因， 研究它们在蜱间Bb感染循环中的作用 和哺乳动物。 2.质粒结构和复制。 我们已经确认了至少五个 Bb中分离的32 kb环状质粒，其中所有质粒都可以保留 在一个单一的细菌。 每个质粒含有不同的等位基因， 温度调节的Osp，并且所有质粒都含有同源的 整个长度的序列。 这些质粒可能提供了一个模型 用于研究Bb中质粒的复制和分离。 我们 研究它们作为质粒载体的潜在效用。 我们有 鉴定的基因的肽结合组分的同源物， 环状和线性Bb质粒上的寡肽通透酶（OppA）以及 染色体 在其他细菌中，OppA可以是肽的受体， 对环境条件做出适应性反应的信息素。 我们正在研究质粒和染色体oppA的表达 基因. 我们已经靶向oppA和另外的质粒基因， 通过我们最近开发的Bb等位基因交换系统的失活。

Our broad objective is to understand the means by which Borrelia burgdorfer (Bb) establishes infection:transmission cycle between the tick vector and mammalian reservoir host, both of which are needed to maintain the spirochete in nature. 1. Outer surface protein function and expression. The outer membrane of Bb contains several abundant lipoproteins (Osps) that vary in size and expression. It is likely that the different Osps confer distinct properties on the spirochete that are pertinent to the different environments in which it must survive. At least three Osps exhibit temperature-related differential expression, as might accompany tick feeding or transmission to a warm-blooded animal. We have developed a mode system for studying regulation of Osp gene expression in E. coli, using osp promoter fusions to the indicator gene lacZ and are screening for trans-acting Bb gene products that alter promoter expression. We have cloned an osp gene family, which contains at least six related members, and are investigating the expression of these genes during the course of an infection. Studies on the biology of Bb and the pathogenesis of Lyme disease are severely limited by the current lack of genetic tools. We have developed allelic exchange using resistance to the antibiotic coumermycin A1 as the selectable marker. This represents the first demonstration of targeted gene inactivation by homologous recombination in Bb. We are currently using this method to inactivate osp genes in order to study their roles in the infectious cycle of Bb between ticks and mammals. 2. Plasmid structure and replication. We have identified at least five separate 32 kb circular plasmids in Bb, al of which can be maintained within a single bacterium. Each plasmid contains a different allele of a temperature-regulated Osp, and all plasmids contain homologous sequences throughout their lengths. These plasmids may provide a model for the study of plasmid replication and segregation in Bb. We are investigating their potential utility as plasmid vectors. We have identified genes for homologs of the peptide binding component of oligopeptide permease (OppA) on circular and linear Bb plasmids and on the chromosome. In other bacteria, OppA can be a receptor for peptide pheromones that signal adaptive responses to environmental conditions. We are investigating the expression of plasmid and chromosomal oppA genes. We have targeted oppA and additional plasmid genes for gene inactivation by our recently developed system of allelic exchange in Bb.