TRANSFORMATION AND GENE INACTIVATION IN BORRELIA BURGDORFERI

莱姆病的传染源伯氏疏螺旋体通过受感染的硬蜱叮咬传播给哺乳动物。我们的主要目标是利用遗传学方法阐明B适应和变异的分子机制。Burgdorferi及其在传染循环中的作用。大多数已经为其他细菌开发的遗传方法不能直接应用于B。burgdorferi。我们以前开发了一种通过等位基因交换使基因失活的方法。我们最近开发了额外的遗传工具，这些工具极大地促进了我们在B中复制基因、引入外源DNA和改进转化方法的努力。burgdorferi。这些包括两个抗生素抗性基因，其特性提供了比原始选择标记更大的实用性。另外的遗传标记的可用性也允许更详细的研究，例如互补分析和产生具有双重突变的菌株。这些改进的标记促进了正在进行的研究，以解决与大肠杆菌缀合作为更有效地将DNA引入B的方法的可行性。burgdorferi。它们还使我们能够证明通过耐药表型区分的培养螺旋体之间的遗传物质交换，并研究最近描述的创建随机标记突变的方法。也许最重要的是，改进的选择标记直接促进了穿梭载体的发展，用于在两个大肠杆菌中稳定引入自主复制的DNA。coli和B. burgdorferi。这是一个标准和重要的遗传工具，以前缺乏与B的研究。burgdorferi。除了作为遗传工具的实用性外，穿梭载体目前还被用于研究B burgdorferi中的质粒结构和功能。这些研究导致了B的定义。赋予自主复制和维持功能的burgdorferi质粒区域。这代表了第一次确定的质粒复制起点在疏螺旋体。穿梭载体也被用于探索B中线性和环状质粒之间的结构关系。最近一个同样重要的进展是证明了感染性螺旋体中等位基因交换导致的第一个基因失活。以前，所有成功的基因失活都是在B31型菌株的减毒的非感染性变体中进行的，该变体更容易通过电穿孔转化。我们已经能够克服这一障碍，仔细优化电穿孔条件与最近开发的选择标记。基因失活，靶向插入和转化的穿梭载体，现在都进行了感染性分离。然而，尽管可能，传染性B。burgdorferi仍然效率低下，不按常规办事。正在进行的努力是针对改进现有的技术和调查其他方法，用它来转化致病性B。burgdorferi。 所有这些最新的进展使我们更接近我们的目标，分析特定的螺旋体基因的传播，感染和疾病的贡献。

Borrelia burgdorferi, the infectious agent of Lyme disease, is transmitted to mammals through the bite of infected Ixodes ticks. Our broad objective is to use a genetic approach to elucidate the molecular mechanisms of adaptation and variation in B. burgdorferi and their roles in the infectious cycle. Most genetic methods that have been developed for other bacteria can not be directly applied to B. burgdorferi. We previously developed a method of gene inactivation by allelic exchange. We recently developed additional genetic tools that have greatly facilitated our efforts to inactivate genes, introduce foreign DNA and improve methods of transformation in B. burgdorferi. These include two antibiotic resistance genes, whose properties provide much greater utility than the original selectable marker. The availability of additional genetic markers has also permitted more detailed studies, such as complementation analyses and the generation of strains with double mutations. These improved markers have facilitated ongoing studies to address the feasibility of conjugation with Escherichia coli as a method to more efficiently introduce DNA into B. burgdorferi. They have also allowed us to demonstrate exchange of genetic material between cultured spirochetes that are distinguishable by their resistance phenotypes, and to investigate a recently described method to create random, tagged mutations. Perhaps most significantly, the improved selectable markers have directly contributed to the development of a shuttle vector for stable introduction of autonomously replicating DNA in both E. coli and B. burgdorferi. This is a standard and important genetic tool that was previously lacking for studies with B. burgdorferi. In addition to its utility as a genetic tool, the shuttle vector is currently being used to investigate plasmid structure and function in B burgdorferi. These studies have resulted in the definition of a B. burgdorferi plasmid region conferring autonomous replication and maintenance functions. This represents the first identification of a plasmid origin of replication in borrelia. The shuttle vector is also being used to explore structural relationships between linear and circular plasmids in B. burgdorferi An equally significant recent advance was the demonstration of the first gene inactivation by allelic exchange in infectious spirochetes. Previously, all successful gene inactivations were made in an attenuated, non-infectious variant of the type strain B31, which is more readily transformed by electroporation., We have been able to overcome this impediment by careful optimization of electroporation conditions with the recently developed selectable markers. Gene inactivation, targeted insertion and transformation with the shuttle vector have now all been performed in infectious isolates. However, while possible, genetic manipulation of infectious B. burgdorferi is still inefficient and not routine. Ongoing efforts are directed towards improving existing techniques and investigating additional methods with which to transform pathogenic B. burgdorferi. All of these recent advances have brought us closer to our goal of analyzing the contributions of specific spirochetal genes to transmission, infection and disease.