Genetic studies in Borrelia burgdorferi

Borrelia burgdorferi, the agent of Lyme disease, has recently joined a growing number of microorganisms for which the entire genomic sequence is known. Despite this wealth of information, little is known about the contribution 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. burgdorferi, which in turn can be attributed to its relatively recent isolation and the dissimilarity of Borrelia from other genetically tractable bacteria. We are interested in several plasmid-encoded gene products in B. burgdorferi that may play a role in sensing and adaptation to the different environments the spirochete encounters as it completes an infectious cycle between the 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 transmission 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 selectable marker. Spirochetes are transformed by electroporation and coumermycin- resistant colonies are screened by PCR for allelic exchange at the targeted locus. We have successfully inactivated several genes of interest in the type strain B31. We are investigating the utility of additional antibiotic resistance 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 pathogenic microorganism. We have made significant advances in our ability to genetically manipulate B. burgdorferi in order to address these issues. However, the available methods are incomplete and far from routine. We are currently improving existing methods as well as developing additional genetic tools with which to augment genetic studies in B. burgdorferi.