Genetic studies in Borrelia burgdorferi

Borrelia burgdorferi, the agent of Lyme disease, has recently joined a grow ing number of microorganisms for which the entire genomic sequence is known . Despite this wealth of information, little is known about the contributio n of specific spirochetal components to the pathogenesis of Lyme disease or their function in the normal life cycle of the organism. This discrepancy is due in part to the lack of a well-developed genetic system in B. burgdor feri, which in turn can be attributed to its relatively recent isolation an d the dissimilarity of Borrelia from other genetically tractable bacteria. We are interested in several plasmid-encoded gene products in B. burgdorfer i that may play a role in sensing and adaptation to the different environme nts the spirochete encounters as it completes an infectious cycle between t he tick vector and the mammalian host. We are developing genetic tools with which to test the roles of specific B. burgdorferi gene products in the tr ansmission cycle in an animal model of Lyme disease. We have demonstrated targeted gene inactivation by allelic exchange, using the gyrB(r) gene encoding coumermycin-resistant topoisomerase as a selectab le marker. Spirochetes are transformed by electroporation and coumermycin-r esistant colonies are screened by PCR for allelic exchange at the targeted locus. We have successfully inactivated several genes of interest in the ty pe strain B31. We are investigating the utility of additional antibiotic re sistance genes as selectable markers in B. burgdorferi. Targeted gene inactivation is a powerful tool with which to investigate the role of particular proteins in the basic biology and virulence of a pathog enic microorganism. We have made significant advances in our ability to gen etically manipulate B. burgdorferi in order to address these issues. Howeve r, the available methods are incomplete and far from routine. We are curren tly improving existing methods as well as developing additional genetic too ls with which to augment genetic studies in B. burgdorferi.