TRANSFORMATION AND GENE INACTIVATION IN BORRELIA BURGDORFERI

伯氏疏螺旋体是莱姆病的感染源，通过受感染的硬蜱叮咬传播给哺乳动物。我们的主要目标是用遗传学方法阐明伯氏杆菌适应和变异的分子机制及其在感染循环中的作用。已经为其他细菌开发的大多数遗传方法不能直接应用于伯氏杆菌。我们之前开发了一种通过等位基因交换来使基因失活的方法。我们最近开发了更多的遗传工具，这些工具极大地促进了我们灭活基因、引入外来DNA和改进伯氏杆菌转化方法的努力。其中包括两个抗生素耐药基因，它们的特性比最初的可选择标记提供了更大的效用。更多遗传标记的出现也使更详细的研究成为可能，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，以解决与大肠杆菌连接的可行性，作为一种更有效地将DNA导入伯氏杆菌的方法。它们还使我们能够展示培养的螺旋体之间的遗传物质交换，这些遗传物质可以通过其耐药性表型来区分，并研究最近描述的一种创建随机、标记突变的方法。也许最重要的是，改进的可选择标记直接促进了穿梭载体的发展，以便在大肠杆菌和伯氏杆菌中稳定地引入自主复制的DNA。这是一种标准和重要的遗传工具，以前在伯氏杆菌的研究中是缺乏的。除了作为一种遗传工具，穿梭载体目前还被用于研究伯氏假单胞菌的质粒结构和功能。这些研究的结果是定义了一个具有自主复制和维护功能的伯氏杆菌质粒区。这是首次在疏螺旋体中鉴定复制的质粒源。穿梭载体也被用来探索伯氏杆菌线性和环状质粒之间的结构关系。最近的一个同样显着的进展是证明了第一个通过等位基因交换在感染性螺旋体中失活的基因。以前，所有成功的基因失活都是在模式菌株B31的减毒、非传染性变体中进行的，B31菌株更容易通过电穿孔转化。我们已经能够通过使用最近开发的可选择标记仔细地优化电穿孔条件来克服这一障碍。基因失活、靶向插入和穿梭载体转化现在都已在感染性分离株中进行。然而，尽管有可能，对传染性伯氏杆菌的基因操作仍然是低效的，也不是常规的。目前正在努力改进现有技术，并研究转化致病伯氏杆菌的其他方法。所有这些最新进展使我们更接近分析特定螺旋体基因对传播、感染和疾病的贡献的目标。

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.