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.

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...

SummaryBorrelia burgdorferi contains abundant circular and linear plasmids, but the mechanism of replication of these extrachromosomal elements is unknown. A B. burgdorferi 9 kb circular plasmid (cp9) was amplified in its entirety by the polymerase chain reaction and used to construct a shuttle vector that replicates in Escherichia coli and B. burgdorferi. A 3.3 kb region of cp9 containing three open reading frames was used to construct a smaller shuttle vector, designated pBSV2. This vector was stably maintained in B. burgdorferi, indicating that all elements necessary for autonomous replication are probably located on this 3.3 kb fragment. A noninfectious B. burgdorferi strain was efficiently transformed by pBSV2. Additionally, infectious B. burgdorferi was also successfully transformed by pBSV2, indicating that infectious strains of this important human pathogen can now be genetically manipulated.

Genetic studies in Borrelia burgdorferi have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is gyrB r , a mutated form of the chromosomal gyrB gene that encodes the B subunit of DNA gyrase and confers resistance to the antibiotic coumermycin A 1 . The utility of the coumermycin-resistant gyrB r gene for targeted gene disruption is limited by a high frequency of recombination with the endogenous gyrB gene. A kanamycin resistance gene (kan) was introduced into B. burgdorferi, and its use as a selectable marker was explored in an effort to improve the genetic manipulation of this pathogen. B. burgdorferi transformants with the kan gene expressed from its native promoter were susceptible to kanamycin. In striking contrast, transformants with the kan gene expressed from either the B. burgdorferi flaB or flgB promoter were resistant to high levels of kanamycin. The kanamycin resistance marker allows efficient direct selection of mutants in B. burgdorferi and hence is a significant improvement in the ability to construct isogenic mutant strains in this pathogen.Borrelia burgdorferi, the spirochetal agent of Lyme disease (4), is maintained in nature by an infectious cycle involving tick vectors and small rodent hosts (13). The genome sequence of B. burgdorferi has provided a wealth of data about the genetic composition of this bacterium (7). However, relatively little is known about the function of most of the deduced proteins encoded by the genome. For example, 41% of the chromosomal open reading frames and 84% of plasmid open reading frames are either homologs to hypothetical proteins in other bacteria or have no match in databases (7). Although many B. burgdorferi gene products have been identified and characterized on the basis of antigenicity, abundance, membrane location, or pattern of synthesis, the functions of most of these proteins are unknown (5,12,20,31,32,36). As a consequence, relatively little is known about the molecular mechanisms mediating B. burgdorferi variation and adaptation and the roles of these processes in the infectious cycle.Identification of genes that encode virulence factors or participate in the transmission cycle of B. burgdorferi is hindered because this pathogen differs considerably from bacteria with well-developed genetic systems. These differences include an atypical outer membrane composition, unique genome structure with an undefined mechanism of replication, and complex, stringent in vitro growth requirements. One major factor that has impeded genetic studies in B. burgdorferi is the lack of an exogenous selectable marker. The only available selectable marker was derived by mutating the B. burgdorferi gene for the B subunit of DNA gyrase (gyrB), yielding a derivative gene (gyrB r ) whose product confers resistance to the antibiotic coumermycin A 1 (26). When the gyrB r gene is used for gene inactivation by allelic exchange or integration into the genome, the most common outcome (Ͼ99.5%) is recombination with the endogenous chr...

Borrelia burgdorferi spends a significant proportion of its life cycle within an ixodid tick, which has a cuticle containing chitin, a polymer of N-acetylglucosamine (GlcNAc). The B. burgdorferi celA, celB, and celC genes encode products homologous to transporters for cellobiose and chitobiose (the dimer subunit of chitin) in other bacteria, which could be useful for bacterial nutrient acquisition during growth within ticks. We found that chitobiose efficiently substituted for GlcNAc during bacterial growth in culture medium. We inactivated the celB gene, which encodes the putative membrane-spanning component of the transporter, and compared growth of the mutant in various media to that of its isogenic parent. The mutant was no longer able to utilize chitobiose, while neither the mutant nor the wild type can utilize cellobiose. We propose renaming the three genes chbA, chbB, and chbC, since they probably encode a chitobiose transporter. We also found that the chbC gene was regulated in response to growth temperature and during growth in medium lacking GlcNAc.

The genus Borrelia includes the causative agents of Lyme disease and relapsing fever. An unusual feature of these bacteria is a genome that includes linear DNA molecules with covalently closed hairpin ends referred to as telomeres. We have investigated the mechanism by which the hairpin telomeres are processed during replication. A synthetic 140 bp sequence having the predicted structure of a replicated telomere was shown to function as a viable substrate for telomere resolution in vivo, and was suf®cient to convert a circular replicon to a linear form. Our results suggest that the ®nal step in the replication of linear Borrelia replicons is a site-speci®c DNA breakage and reunion event to regenerate covalently closed hairpin ends. The telomere substrate described here will be valuable both for in vivo manipulation of linear DNA in Borrelia and for in vitro studies to identify and characterize the telomere resolvase.

We previously demonstrated that Borrelia burgdorferi requires OspC to colonize a mammalian host. To delineate this requirement, we analyzed the clearance of ospC mutant spirochetes and found that they were eliminated within 48 h. We conclude that B. burgdorferi uses OspC to resist innate host defenses immediately after transmission.

We previously described a bacteriophage of the Lyme disease agent Borrelia burgdorferi designated BB-1. This phage packages the host complement of the 32-kb circular plasmids (cp32s), a group of homologous molecules found throughout the genus Borrelia. To demonstrate the ability of BB-1 to package and transduce DNA, a kanamycin resistance cassette was inserted into a cloned fragment of phage DNA, and the resulting construct was transformed into B. burgdorferi CA-11.2A cells. The kan cassette recombined into a resident cp32 and was stably maintained. The cp32 containing the kan cassette was packaged by BB-1 released from this B. burgdorferi strain. BB-1 has been used to transduce this antibiotic resistance marker into naive CA-11.2A cells, as well as two other strains of B. burgdorferi. This is the first direct evidence of a mechanism for lateral gene transfer in B. burgdorferi.Borrelia burgdorferi, the causative agent of Lyme disease (5, 32), has a complex genome that contains both linear and circular DNA (7,13). A growing number of tools have been developed for studying the molecular biology of this organism, although elaborating a convenient manipulatable genetic system will require further effort. Despite indirect evidence for lateral gene transfer in these bacteria (19,33,38), natural mechanisms for the transfer of genetic material (including conjugative plasmids and transducing bacteriophages) remain elusive for B. burgdorferi and related spirochetes.In a number of other bacteria, bacteriophages are well characterized, naturally occurring mechanisms for lateral gene transfer (22). Bacteriophages have been observed in association with many spirochetes (reviewed in references 10 and 11), including Borrelia spp. (3,12,14,23), Leptospira biflexa (29), and Brachyspira hyodysenteriae (15,27). VSH-1, a generalized transducing phage of B. hyodysenteriae, has been used to demonstrate the transfer of antibiotic resistance markers between two different strains of this spirochete (16). Recently, a prophage from L. biflexa was developed into a shuttle vector (28). To our knowledge, no transduction or any other naturally occurring mechanism for the introduction of exogenous DNA has been demonstrated in B. burgdorferi or related species.We previously reported the preliminary characterization of a temperate bacteriophage (Fig. 1) (10-12), designated BB-1, which packages the 32-kb circular plasmids (cp32s) of the B. burgdorferi genome. The cp32s are a family of extrachromosomal elements, several of which can be maintained in a single cell (7,8,35). There are three regions of variability on these homologous plasmids: two that encode various lipoproteins, and one that may encompass a possible partitioning region (1, 6-8, 17, 24, 25, 33-36, 40, 42). Sequence analysis of these molecules suggests that they have evolutionarily undergone recombination, possibly from both endogenous and exogenous sources (6-8, 20, 33, 35, 38). Many features of the cp32 family are consistent with the hypothesis that these plasmids are temperate pro...