Borrelia burgdorferi, the cauative agent of Lyme disease, is maintained in nature through an infectious cycle between wild mammals and ticks. Like many bacterial pathogens, B. burgdorferi must cope with a changing array of environmental conditions in order to successfully persist, proliferate and be transmitted between hosts. B. burgdorferi has an unusual genomic structure composed of a linear chromosome and a large number of linear and circular plasmids. Abundant evidence indicates that plasmid-encoded genes are critical for adaptation in the infectious cycle. In contrast to their essential role in vivo, plasmids are often lost during in vitro cultivation of B. burgdorferi. A major focus of our research is to determine the contributions of individual B. burgdorferi genes and plasmids at each stage of the infectious cycle, taking a molecular genetic approach. Environmentally responsive synthesis of surface proteins represents a hallmark of the infectious cycle of B. burgdorferi. Dorothee Grimm and colleagues in the lab created and analyzed a B. burgdorferi mutant lacking outer surface protein C (OspC), an abundant plasmid-encoded Osp that spirochetes normally synthesize in the tick vector during the blood meal and down-regulate after transmission to the mammal. These experiments demonstrated that B. burgdorferi strictly requires OspC to infect mice but not to localize or migrate appropriately in the tick. The induction of a spirochetal virulence factor preceding the time and host in which it is required demonstrates a developmental sequence for transmission of this arthropod-borne pathogen. Chitobiose is the dimer subunit of chitin, a component of tick cuticle and peritrophic matrix, which is not found in mammals. We previously determined that the plasmid-encoded chbC gene of B. burgdorferi is required for the use of chitobiose as a source of the essential nutrient N-acetyl glucosamine during growth in vitro. In order to investigate the role of chitobiose transport in the infectious cycle, Kit Tilly constructed isogenic chbC mutant and wild-type strains in an infectious B. burgdorferi background and confirmed that the mutants were defective in chitobiose utilization. The defect in the mutants was shown to be in chitobiose transport, consistent with the predicted function of the ChbC protein as the membrane component of a phosphotransferase transporter for chitobiose. The mutant was then tested to determine whether the chbC locus is also required for any stage of the experimental mouse-tick infectious cycle. Both wild-type and mutant bacteria were found to successfully infect both mice and ticks and were transmitted between the two hosts. These results demonstrate that B. burgdorferi growth in vivo is independent of chitobiose transport, even in an environmental niche in which the sugar is likely to be present. The genome of the type strain (B31) of B. burgdorferi has 12 linear and 9 circular plasmids, in addition to the linear chromosome. Plasmid content can vary among strains, but one 26-kb circular plasmid (cp26) is always present. The genes encoding OspC and ChbC are among the 29 open reading frames carried by cp26. The ubiquitous nature of cp26 suggests that it provides functions required for bacterial viability. Rebecca Byram tested this hypothesis by attempting to selectively displace cp26 with an incompatible but replication-proficient vector, pBSV26. While pBSV26 transformants contained this incompatible vector, it 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 a gene (BBB29) with homology to a glucose-specific phosphotransferase component, and the resT gene, 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 of resT resulted in merodiploids, suggesting that resT is required for B. burgdorferi growth. To determine if resT is the only cp26 gene essential for growth, the resT gene was introduced into B. burgdorferi on pBSV26. This did not result in displacement of cp26, suggesting that additional cp26 genes encode vital functions. We conclude that B. burgdorferi plasmid cp26 encodes functions critical for survival and thus shares some features with the chromosome. Experimental data suggest that two linear plasmids, lp25 and lp28-1, play essential roles for infectivity in mice, although they are dispensable for in vitro growth of B. burgdorferi. Dorothee Grimm and colleagues recently proved the essential nature of these plasmids by selectively displacing lp25 and lp28-1 from an infectious wild-type clone with incompatible vectors derived from the native plasmids, rendering the respective transformants noninfectious to mice. Conversely, restoration of plasmid lp25 or lp28-1 in noninfectious clones that naturally lack the corresponding plasmid reestablished infectivity in mice. This approach establishes the ability to manipulate the plasmid content of strains by eliminating or introducing entire plasmids in B. burgdorferi and will be valuable in assessing the roles of plasmids even in unsequenced B. burgdorferi strains. Lyme disease is the leading vector-borne illness in the United States. Many of the genetic factors affecting spirochete morphology and physiology are unknown due to the limited genetic tools available and the large number of open reading frames with unknown function. By adapting a mariner transposon system to work in B. burgdorferi, Philip Stewart has developed a random mutagenesis system that tags the mutated locus for rapid identification. Transposition occurs at saturating levels in B. burgdorferi and appears to be random, targeting both linear and circular replicons. Combining the transposon system with a screen for factors affecting growth rate, mutations were readily identified in genes putatively involved in cell division, chemotaxis, and a hypothetical open reading frame involved in outer membrane integrity. The successful adaptation of a mariner transposon to function in B. burgdorferi should aid in identifying virulence factors and novel gene products related to spirochete physiology.
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