Lyme disease spirochetes, Borrelia burgdorferi sensu lato, are maintained in zoonotic cycles involving ticks and small mammals. In unfed ticks, the spirochetes produce one outer surface protein, OspA, but not OspC. During infection in mammals, immunological data suggest that the spirochetes have changed their surface, now expressing OspC but little or no OspA. We find by in vitro growth experiments that this change is regulated in part by temperature; OspC is produced by spirochetes at 32-370C but not at 24°C. Furthermore, spirochetes in the midgut of ticks that have fully engorged on mice now have OspC on their surface. Thus two environmental cues, an increase in temperature and tick feeding, trigger a major alteration of the spirochetal outer membrane. This rapid synthesis of OspC by spirochetes during tick feeding may play an essential role in the capacity of these bacteria to successfully infect mammalian hosts, including humans, when transmitted by ticks.Many infectious agents pathogenic in humans are maintained in natural zoonotic cycles involving wild vertebrates and obligate blood-feeding arthropods (1, 2). Although much is known about the clinical description and diagnosis of these human diseases, the physiological and morphological adaptations of these agents, especially bacteria, while in their arthropod vectors are not well understood. Several non-vector-borne bacterial pathogens, including species of Salmonella, Shigella, and Bordetella, display an impressive repertoire of adaptive molecular responses to environmental signals on entry into mammalian hosts (3-5). Specific changes by bacterial pathogens in arthropod vectors during feeding on blood have not been described. Identifying such events would broaden our knowledge of how these agents are perpetuated and transmitted in nature and assist in the development of effective vaccines and diagnostic tests.Borrelia burgdorferi is one of at least three closely related species of spirochetes that cause a spectrum of clinical syndromes in humans, collectively called Lyme disease or Lyme borreliosis (6-8). These spirochetes are maintained in zoonotic cycles involving a diversity of wild mammals and ticks primarily in the genus Ixodes (9). These microbes' adaptation to tick and mammalian environments likely involves very different surface components so as to ensure their transmission and survival in two very different hosts. Several lipoproteins have been described on the surface of B. burgdorferi (10)(11)(12)(13)(14)(15)(16), some of which are variably expressed during serial passage in culture (17)(18)(19). The apparent flexibility in the spirochete's synthesis of some outer surface proteins (Osps) in vitro may have relevance to its alternation of hosts. Previous studies demonstrate that B. burgdorferi produces one surface protein, OspA, and likely OspB, in the midgut of Ixodes ticks that have not yet engorged on blood (10). However, it is unclear how long spirochetes continue to produce this protein after entering mammalian hosts because few anim...

Environmentally responsive synthesis of surface proteins represents a hallmark of the infectious cycle of the Lyme disease agent, Borrelia burgdorferi. Here we created and analyzed a B. burgdorferi mutant lacking outer-surface protein C (OspC), an abundant Osp that spirochetes normally synthesize in the tick vector during the blood meal and down-regulate after transmission to the mammal. We demonstrate 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.

A major obstacle to studying the functions of particular gene products in the mouse-tick infectious cycle of Borrelia burgdorferi has been an inability to knock out genes in pathogenic strains. Here, we investigated conditions for site-directed mutagenesis in B31 MI, the low-passage-number, infectious B. burgdorferi strain whose genome was sequenced. We inactivated several plasmid and chromosomal genes in B31 MI and determined that clones carrying these mutations were not infectious for mice. However, we found extensive heterogeneity among clones and mutants derived from B31 MI based on colony phenotype, growth rate, plasmid content, protein profile, and transformability. Significantly, several B31 MI clones that were not subjected to mutagenesis but that lacked particular plasmids also exhibited defects at various stages in the infectious cycle. Therefore, the high degree of clonal polymorphism within B31 MI complicates the assessment of the contributions of individual genes to the observed phenotypes of the mutants. Our results indicate that B31 MI is not an appropriate strain background for genetic studies in infectious B. burgdorferi, and a well-defined isogenic clone is a prerequisite for targeted mutagenesis. To this end, we derived several wild-type clones from B31 MI that were infectious for mice, and gene inactivation was successful in one of these clones. Due to the instability of the genome with in vitro propagation, careful monitoring of plasmid content of derived mutants and complementation of inactivated genes will be crucial components of genetic studies with this pathogen.Lyme disease is caused by Borrelia burgdorferi, a spirochete transmitted by ticks of the genus Ixodes and maintained within an enzootic cycle between the tick vector and mammalian hosts, most importantly small rodents (7,11,19). The clinical manifestations of this zoonosis can include a multisystem disorder affecting skin and joints and the nervous, lymphoreticular, and cardiovascular systems (39,40).The organization of the B. burgdorferi genome is unique among bacteria in that the genome is composed of a linear chromosome and a large number of linear and circular plasmids (8, 14). The complete genome sequence of an infectious B. burgdorferi isolate, the type strain B31, identified 21 linear and circular plasmids (8). In vitro propagation of B. burgdorferi can lead to plasmid loss and concurrent loss of infectivity for mice (3,21,22,30,33). Although increasing evidence suggests that certain Borrelia plasmids are important for infection in mice (18,25), this hypothesis has not been experimentally verified, and the roles of most plasmid-encoded genes in the infectious cycle are unknown. A number of plasmid-and chromosomally encoded genes have been inactivated in the highpassage-number, noninfectious clone B31-A (4,5,12,17,20,43,45,46), but gene inactivation in a low-passage-number, infectious strain background has not been reported.Here, we investigate conditions for site-directed mutagenesis in B31 MI, the low-passage-number, inf...