We have determined that Borrelia burgdorferi strain B31 MI carries 21 extrachromosomal DNA elements, the largest number known for any bacterium. Among these are 12 linear and nine circular plasmids, whose sequences total 610 694 bp. We report here the nucleotide sequence of three linear and seven circular plasmids (comprising 290 546 bp) in this infectious isolate. This completes the genome sequencing project for this organism; its genome size is 1 521 419 bp (plus about 2000 bp of undetermined telomeric sequences). Analysis of the sequence implies that there has been extensive and sometimes rather recent DNA rearrangement among a number of the linear plasmids. Many of these events appear to have been mediated by recombinational processes that formed duplications. These many regions of similarity are reflected in the fact that most plasmid genes are members of one of the genome's 161 paralogous gene families; 107 of these gene families, which vary in size from two to 41 members, contain at least one plasmid gene. These rearrangements appear to have contributed to a surprisingly large number of apparently non‐functional pseudogenes, a very unusual feature for a prokaryotic genome. The presence of these damaged genes suggests that some of the plasmids may be in a period of rapid evolution. The sequence predicts 535 plasmid genes ≥300 bp in length that may be intact and 167 apparently mutationally damaged and/or unexpressed genes (pseudogenes). The large majority, over 90%, of genes on these plasmids have no convincing similarity to genes outside Borrelia, suggesting that they perform specialized functions.

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...

This study demonstrates a strict temporal requirement for a virulence determinant of the Lyme disease spirochete Borrelia burgdorferi during a unique point in its natural infection cycle, which alternates between ticks and small mammals. OspC is a major surface protein produced by B. burgdorferi when infected ticks feed but whose synthesis decreases after transmission to a mammalian host. We have previously shown that spirochetes lacking OspC are competent to replicate in and migrate to the salivary glands of the tick vector but do not infect mice. Here we assessed the timing of the requirement for OspC by using an ospC mutant complemented with an unstable copy of the ospC gene and show that B. burgdorferi's requirement for OspC is specific to the mammal and limited to a critical early stage of mammalian infection. By using this unique system, we found that most bacterial reisolates from mice persistently infected with the initially complemented ospC mutant strain no longer carried the wild-type copy of ospC. Such spirochetes were acquired by feeding ticks and migrated to the tick salivary glands during subsequent feeding. Despite normal behavior in ticks, these ospC mutant spirochetes did not infect naive mice. ospC mutant spirochetes from persistently infected mice also failed to infect naive mice by tissue transplantation. We conclude that OspC is indispensable for establishing infection by B. burgdorferi in mammals but is not required at any other point of the mouse-tick infection cycle.

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Bacterial shape usually is dictated by the peptidoglycan layer of the cell wall. In this paper, we show that the morphology of the Lyme disease spirochete Borrelia burgdorferi is the result of a complex interaction between the cell cylinder and the internal periplasmic flagella. B. burgdorferi has a bundle of 7-11 helically shaped periplasmic flagella attached at each end of the cell cylinder and has a flat-wave cell morphology. Backward moving, propagating waves enable these bacteria to swim in both low viscosity media and highly viscous gel-like media. Using targeted mutagenesis, we inactivated the gene encoding the major periplasmic flagellar filament protein FlaB. The resulting flaB mutants not only were nonmotile, but were rod-shaped. Western blot analysis indicated that FlaB was no longer synthesized, and electron microscopy revealed that the mutants were completely deficient in periplasmic flagella. Wild-type cells poisoned with the protonophore carbonyl cyanide-m-chlorophenylhydrazone retained their flat-wave morphology, indicating that the periplasmic flagella do not need to be energized for the cell to maintain this shape. Our results indicate that the periplasmic flagella of B. burgdorferi have a skeletal function. These organelles dynamically interact with the rodshaped cell cylinder to enable the cell to swim, and to confer in part its flat-wave morphology.spirochete ͉ Lyme disease ͉ allelic exchange ͉ morphology S pirochetes have a unique position among the bacteria. These motile bacteria are one of the few bacterial phyla that can be identified by both 16S ribosomal RNA sequence analysis and morphology (1). Outermost is a membrane sheath, and within this sheath are the cell cylinder and periplasmic flagella. A given periplasmic flagellum is attached subterminally at only one end of the cell cylinder, and it resides within the periplasmic space (2). Depending on the species, the cell morphology is either a helix, a flat wave, or an irregularly shaped helix (2-4). The size of the spirochete, the number of periplasmic flagella attached at each end, and whether the filaments overlap at the center of the cell varies from species to species (2, 5). This phylum contains many medically important bacteria including Treponema pallidum (syphilis), several Borrelia species (relapsing fever), Borrelia burgdorferi (Lyme disease), Leptospira interrogans (leptospirosis), Brachyspira sp. (human diarrheal disease, swine dysentery), and oral treponemes associated with periodontal disease (5-8).The periplasmic flagella of spirochetes have been characterized in detail. Genetic evidence, including targeted mutagenesis studies in Treponema denticola and Brachyspira hyodysenteriae, have shown that these organelles are directly involved in motility (9, 10) (C. Li and N.W.C., unpublished observations). By analyzing protruding periplasmic flagella from certain motility mutants of T. phagedenis, and from stationary-phase cells of several spirochete species, these organelles have been shown to rotate in a manner similar to th...

We have characterized seven different 32-kb circular plasmids carried by Borrelia burgdorferi isolate B31. Restriction endonuclease recognition site mapping and partial sequencing of these plasmids indicated that all seven are probably closely related to each other throughout their lengths and have substantial relationships to cp8.3, an 8.3-kb circular plasmid of B. burgdorferi sensu lato isolate Ip21. With the addition of the seven 32-kb plasmids, this bacterial strain is known to carry at least 10 linear and 9 circular plasmids. Variant cultures of B. burgdorferi B31 lacking one or more of the 32-kb circular plasmids are viable and, at least in some cases, infectious. We have examined a number of different natural isolates of Lyme disease borreliae and found that all of the B. burgdorferi sensu stricto isolates and most of the B. burgdorferi sensu lato isolates tested appear to carry multiple 32-kb circular plasmids related to those of B. burgdorferi B31. The ubiquity of these plasmids suggests that they may be important in the natural life cycle of these organisms. They may be highly conjugative plasmids or prophage genomes, which could prove to be useful in genetically manipulating B. burgdorferi.Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic ailment of humans that is spread through the bite of certain species of Ixodes ticks (13, 61). As a spirochete, B. burgdorferi is a member of a morphologically and phylogenetically distinct order of eubacteria (11,28,(42)(43)(44)70). Although classically defined as gram-negative organisms (28), recent phylogenetic studies based on rRNA sequences indicated that the spirochetes are as distantly related to the gramnegative Escherichia coli as they are to gram-positive bacteria (42,70).One of the most striking differences between B. burgdorferi and other bacteria is its unusual genome, which includes a small (approximately 1 Mb) linear chromosome (12,16,19,21,23,41) and numerous linear and circular plasmids, sometimes approaching 20 different plasmids in one bacterium (7,8,10,22,27,30,35,57,65,71). A curious feature of these different plasmids is that they often appear to share regions of homologous DNA (58,60,65,71,73,74). Homologs of DNA sequences mapped to circular plasmids have even been found on linear plasmids (74), although in a related bacterium, Borrelia hermsii, at least one plasmid exists in both linear and circular forms (24), blurring the distinction between these two forms of DNA. Several of the plasmids that these bacteria harbor appear to be present in all natural isolates; therefore, the term minichromosome may be a more apt description of their nature. For example, a 49-to 54-kb linear plasmid and a 26-kb circular plasmid, which carry the outer surface protein genes ospAB and ospC, respectively, are ubiquitous (7,34,37,52,54,63,71). It is not known whether members of either of these two plasmid families have similar overall gene orders in different bacterial isolates, but their nearly invariant sizes support the idea that they...