TRANSFORMATION AND GENE INACTIVATION IN BORRELIA BURGDORFERI

伯氏疏螺旋体的转化和基因失活

• 批准号: 6431592
• 负责人: PATRICIA A ROSA
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6431592
• 关键词: Borrelia; bacterial genetics; borreliosis; drug resistance; gene expression; gene mutation; genetic markers; laboratory mouse; laboratory rabbit; life cycle; microorganism culture; molecular cloning; plasmids

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.

伯氏疏螺旋体是莱姆病的传染因子，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方法来阐明伯氏疏螺旋体的适应和变异的分子机制及其在感染周期中的作用。大多数为其他细菌开发的遗传方法不能直接应用于伯氏疏螺旋体。我们之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，极大地促进了我们在伯氏疏螺旋体中灭活基因、引入外源DNA和改进转化方法的努力。其中包括两种抗生素抗性基因，其特性比原来的选择性标记提供了更大的效用。其他遗传标记的可用性也允许进行更详细的研究，例如互补分析和产生具有双突变的菌株。这些改进的标记促进了正在进行的研究，以解决与大肠杆菌结合的可行性，作为一种更有效地将DNA引入伯氏疏螺旋体的方法。它们还使我们能够证明通过抗性表型区分的培养螺旋体之间的遗传物质交换，并研究最近描述的一种创建随机标记突变的方法。也许最重要的是，改进的选择性标记直接促进了穿梭载体的发展，用于在大肠杆菌和伯氏疏螺旋体中稳定地引入自主复制的DNA。这是一个标准的和重要的遗传工具，以前缺乏与伯氏疏螺旋体的研究。除了作为一种遗传工具外，穿梭载体目前还被用于研究伯氏疏螺旋体的质粒结构和功能。这些研究已经导致伯氏疏螺旋体质粒区域赋予自主复制和维持功能的定义。这是首次鉴定出伯氏疏螺旋体复制的质粒起源。穿梭载体也被用于探索伯氏疏螺旋体线性质粒和圆形质粒之间的结构关系。最近同样重要的进展是在感染性螺旋体中首次通过等位基因交换实现基因失活。在此之前，所有成功的基因失活都是在B31型菌株的减毒、非传染性变体中进行的，这种变体更容易通过电穿孔转化。我们已经能够克服这一障碍，通过精心优化电穿孔条件与最近开发的选择性标记。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完成。然而，尽管有可能，传染性伯氏疏螺旋体的遗传操作仍然效率低下，而且不常规。正在进行的工作旨在改进现有技术并研究转化致病性伯氏疏螺旋体的其他方法。所有这些最近的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。