ABSTRACTThe genome of the type strain (B31) ofBorrelia burgdorferi, the causative agent of Lyme disease, is composed of 12 linear and 9 circular plasmids and a linear chromosome. Plasmid content can vary among strains, but one 26-kb circular plasmid (cp26) is always present. The ubiquitous nature of cp26 suggests that it provides functions required for bacterial viability. We tested this hypothesis by attempting to selectively displace cp26 with an incompatible but replication-proficient vector, pBSV26. While pBSV26 transformants contained this incompatible vector, the vector coexisted with cp26, which is consistent with the hypothesis that cp26 carries essential genes. Several cp26 genes with ascribed or predicted functions may be essential. These include the BBB29 gene, which has sequence homology to a gene encoding a glucose-specific phosphotransferase system component, and theresTgene, which encodes a telomere resolvase involved in resolution of the replicated telomeres of the linear chromosome and plasmids. The BBB29 gene was successfully inactivated by allelic exchange, but attempted inactivation ofresTresulted in merodiploid transformants, suggesting thatresTis required forB. burgdorferigrowth. To determine ifresTis the only cp26 gene essential for growth, we introducedresTintoB. burgdorferion pBSV26. This did not result in displacement of cp26, suggesting that additional cp26 genes encode vital functions. We concluded thatB. burgdorferiplasmid cp26 encodes functions critical for survival and thus shares some features with the chromosome.