TRANSFORMATION AND GENE INACTIVATION IN BORRELIA BURGDORFERI

伯氏疏螺旋体生物学及其致病机制的研究 莱姆病的发病率受到目前基因缺乏的严重限制 工具。作为通向BB基因操作的第一步，我们有 开发了一种通过等位基因交换来使基因失活的方法，使用了 对抗生素产生抗药性的突变疏螺旋体gyrB基因 香豆素A1作为一个可选择的标记。我们想要调查一下 猪瘟病毒26kb环状质粒(Cp26)上基因的潜在作用 Bb在环境感知和适应扁虱媒介方面以及 哺乳动物宿主。我们已经灭活了几个cp26基因 非传染性、实验室适应的BB菌株，目的是 随后将这些突变转移到一个有感染性的分离株中。到目前为止， 我们一直未能成功地使用 标准协议等一直无法直接测试效果 在感染循环中的这些突变。我们正在修改 程序和尝试其他转换方法，以便 克服这一技术障碍。我们已经测试了额外的基因 标记，目的是开发更多或更好的选择工具 以及转化子的筛选。执行常规遗传操作的能力 BB中的操作将极大地促进我们对适应的研究 和螺旋体的变异，以及多样化的研究 该领域其他调查人员的目标。

Studies of the biology of Borrelia burgdorferi (Bb) and the pathogenesis of Lyme disease are severely limited by the current lack of genetic tools. As an initial step toward genetic manipulation of Bb we have developed a method of gene inactivation by allelic exchange, using a mutated borrelial gyrB gene that confers resistance to the antibiotic coumermycin A1 as a selectable marker. We would like to investigate the potential roles of genes located on a 26-kb circular plasmid (cp26) of Bb in environmental sensing and adaptation to the tick vector and mammalian host. We have inactivated several cp26 genes in a non-infectious, laboratory-adapted Bb strain, with the intention of subsequently moving these mutations into an infectious isolate. To date, we have been unsuccessful in transforming infectious Bb using the standard protocol and so have been unable to directly test the effects of these mutations in the infectious cycle. We are modifying the procedure and trying other methods of transformation in order to overcome this technical hurdle. We have tested additional genetic markers with the aim of developing more or better tools for selection and screening of transformants. The ability to perform routine genetic manipulations in Bb will greatly facilitate our studies on adaptation and variation of the spirochete, as well as the diverse research objectives of other investigators in the field.