Genetic basis for retention of a critical virulence plasmid of Borrelia burgdorferi

The genome of Borrelia burgdorferi is composed of one linear chromosome and approximately 20 linear and circular plasmids. Although some plasmids are required by B. burgdorferi in vivo, most plasmids are dispensable for growth in vitro. However, circular plasmid (cp) 26 is present in all natural isolates and has never been lost during in vitro growth. This plasmid carries ospC, which is critical for mammalian infection. We previously showed that cp26 encodes essential functions, including the telomere resolvase, ResT, and hence cannot be displaced. Here we identify two additional essential genes on cp26, bbb26 and bbb27, through a systematic attempt to inactivate each open reading frame (ORF). Furthermore, an incompatible plasmid carrying resT, bbb26 and bbb27 could displace cp26. Computational and experimental analyses suggested that both BBB26 and BBB27 are membrane-associated, periplasmic proteins. These data indicate that bbb26 and bbb27 encode essential but possibly redundant functions and that one or the other of these cp26 genes, in addition to resT, is required for bacterial viability. We conclude that the genetic linkage of critical physiological and virulence functions on cp26 is pertinent to its stable maintenance throughout the evolution of B. burgdorferi.     

Isolation of a circular plasmid region sufficient for autonomous replication and transformation of infectious Borrelia burgdorferi

Borrelia 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 non-infectious 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.     

Identification of loci critical for replication and compatibility of a Borrelia burgdorferi cp32 plasmid and use of a cp32-based shuttle vector for the expression of fluorescent reporters in the lyme disease spirochaete

The 32kb circular plasmid (cp32) family of Borrelia burgdorferi has been the subject of intensive investigation because its members encode numerous differentially expressed lipoproteins. As many as nine different cp32s appear to be capable of stable replication within a single spirochaete. Here, we show that a construct (pCE310) containing a 4 kb fragment from the putative maintenance region of a B. burgdorferi CA-11.2A cp32 was capable of autonomous replication in both high-passage B. burgdorferi B31 and virulent B. burgdorferi 297. Deletion analysis revealed that only the member of paralogous family 57 and the adjacent non-coding segment were essential for replication. The PF32 ParA orthologue encoded by the pCE310 insert was almost identical to the PF32 orthologues encoded on the B31 and 297 cp32-3 plasmids. The finding that cp32-3 was selectively deleted in both B31 and 297 transformants carrying pCE310 demonstrated the importance of the PF32 protein for cp32 compatibility and confirmed the prediction that cp32 plasmids expressing identical PF32 paralogues are incompatible. A shuttle vector containing the CA-11.2A cp32 plasmid maintenance region was used to introduce green, yellow and cyan fluorescent protein reporters into B. burgdorferi. Flow cytometry revealed that the green fluorescent protein was well expressed by almost 90% of both avirulent and infectious transformants. In addition to enhancing our understanding of B. burgdorferi plasmid biology, our results further the development of genetic systems for dissecting pathogenic mechanisms in Lyme disease.     

Requirements for Borrelia burgdorferi plasmid maintenance

Borrelia burgdorferi has multiple linear and circular plasmids that are faithfully replicated and partitioned as the bacterium grows and divides. The low copy number of these replicons implies that active partitioning contributes to plasmid stability. Analyzing the requirements for plasmid replication and partition in B. burgdorferi is complicated by the complexity of the genome and the possibility that products may act in trans. Consequently, we have studied the replication-partition region (bbb10-13) of the B. burgdorferi 26kb circular plasmid (cp26) in Escherichia coli, by fusion with a partition-defective miniF plasmid. Our analysis demonstrated that bbb10, bbb11, and bbb13 are required for stable miniF maintenance, whereas bbb12 is dispensable. To validate these results, we attempted to inactivate two of these genes in B. burgdorferi. bbb12 mutants were obtained at a typical frequency, suggesting that the bbb12 product is dispensable for cp26 maintenance as well. We could not directly measure cp26 stability in the bbb12 mutant, because cp26 carries essential genes, and bacteria that have lost cp26 are inviable. Conversely, we were unable to inactivate bbb10 on cp26 of B. burgdorferi. Our results suggest that bbb12 is dispensable for cp26 maintenance, whereas bbb10, bbb11, and bbb13 play crucial roles in that process.     

Conservation of plasmid maintenance functions between linear and circular plasmids in Borrelia burgdorferi

The Lyme disease agent Borrelia burgdorferi maintains both linear and circular plasmids that appear to be essential for mammalian infection. Recent studies have characterized the circular plasmid regions that confer autonomous replication, but the genetic elements necessary for linear plasmid maintenance have not been experimentally identified. Two vectors derived from linear plasmids lp25 and lp28-1 were constructed and shown to replicate autonomously in B. burgdorferi. These vectors identify internal regions of linear plasmids necessary for autonomous replication in B. burgdorferi. Although derived from linear plasmids, the vectors are maintained in circular form in B. burgdorferi, indicating that plasmid maintenance functions are conserved, regardless of DNA form. Finally, derivatives of these vectors indicate that paralogous gene family 49 is apparently not required for either circular or linear plasmid replication.     

Characterization of cp18, a naturally truncated member of the cp32 family of Borrelia burgdorferi plasmids.

We have mapped the genes encoding the antigenic lipoproteins OspE and OspF to an approximately 18-kb circular plasmid in Borrelia burgdorferi N40. Sequencing and restriction mapping have revealed that this plasmid, cp18, is homologous to an 18-kb region of the cp32 circular plasmids found in the Lyme disease spirochetes. Our data show that cp18 may have arisen from an ancestral cp32 plasmid by deletion of a 14-kb region of DNA, indicating that a significant portion of the cp32 plasmid is not essential in cis for plasmid maintenance. These findings suggest that a relatively small recombinant plasmid capable of being stably maintained in B. burgdorferi could be constructed from a cp32 plasmid.     

aadA confers streptomycin resistance in Borrelia burgdorferi

To enhance genetic manipulation of the Lyme disease spirochete Borrelia burgdorferi, we assayed the aadA gene for the ability to confer resistance to the antibiotics spectinomycin and streptomycin. Using the previously described pBSV2 as a backbone, a shuttle vector, termed pKFSS1, which carries the aadA open reading frame fused to the B. burgdorferi flgB promoter was constructed. The hybrid flgB promoter-aadA cassette confers resistance to spectinomycin and streptomycin in both B. burgdorferi and Escherichia coli. pKFSS1 has a replication origin derived from the 9-kb circular plasmid and can be comaintained in B. burgdorferi with extant shuttle vector pCE320, which has a replication origin derived from a 32-kb circular plasmid, or pBSV2, despite the fact that pKFSS1 and pBSV2 have the same replication origin. Our results demonstrate the availability of a new selectable marker and shuttle vector for genetically dissecting B. burgdorferi at the molecular level.     

Mapping of essential replication functions of the linear plasmid lp17 of B. burgdorferi by targeted deletion walking

The genome of the Lyme disease pathogen Borrelia burgdorferi strain B31 MI includes one linear chromosome, 10 circular and 12 linear plasmids. Members of four paralogous gene families, revealed by genome sequencing, have been suggested as replication/partition functions for both the linear and circular plasmids. Some of these genes have been experimentally shown to be essential for the replication of the B. burgdorferi replicons that encode them. In this study, we located the region essential for replication of lp17, the second smallest linear plasmid in B. burgdorferi. We used a novel in vivo method, targeted deletion walking, to systematically delete DNA from either the left or right end of lp17. We report that the region essential for replication of lp17 is 1.8 kb (bp 7946-9766) and contains only one intact open reading frame (BBD14). Expression of BBD14 is required for the replication, suggesting that it is the replication initiator for lp17. The BBD14 protein is a member of paralogous family (PF) 62 and we present the first experimental evidence for the role of a PF 62 member. Adjacent non-coding sequences are also required, suggesting that the origin lies at least partially outside the coding region. Surprisingly, deletion of BBD21, the ParA orthologue (PF 32), had little effect upon plasmid stability or incompatibility. Finally, data are presented suggesting that lp17 replication occurs preferentially on a linear rather than a circular DNA molecule.     

The Borrelia burgdorferi circular plasmid cp26: conservation of plasmid structure and targeted inactivation of the ospC gene

The 26 to 28 kb circular plasmid of B. burgdorferi sensu lato (cp26) is ubiquitous among bacteria of this group and contains loci implicated in the mouse–tick transmission cycle. Restriction mapping and Southern hybridization indicated that the structure of cp26 is conserved among isolates from different origins and culture passage histories. The cp26 ospC gene encodes an outer surface protein whose synthesis within infected ticks increases when the ticks feed, and whose synthesis in culture increases after a temperature upshift. Previous studies of ospC coding sequences showed them to have stretches of sequence apparently derived from the ospC genes of distantly related isolates by homologous recombination after DNA transfer. We found conservation of the promoter regions of the ospC and guaA genes, which are divergently transcribed. We also demonstrated that the increase in OspC protein after a temperature upshift parallels increases in mRNA levels, as expected if regulatory regions adjoin the conserved sequences in the promoter regions. Finally, we used directed insertion to inactivate the ospC gene of a non-infectious isolate. This first example of directed gene inactivation in B. burgdorferi shows that the OspC protein is not required for stable maintenance of cp26 or growth in culture.     

Comparative genome hybridization reveals substantial variation among clinical isolates of Borrelia burgdorferi sensu stricto with different pathogenic properties

Clinical and murine studies suggest that there is a differential pathogenicity of different genotypes of Borrelia burgdorferi, the spirochetal agent of Lyme disease. Comparative genome hybridization was used to explore the relationship between different genotypes. The chromosomes of all studied isolates were highly conserved (>93%) with respect to both sequence and gene order. Plasmid sequences were substantially more diverse. Plasmids lp54, cp26, and cp32 were present in all tested isolates, and their sequences and gene order were conserved. The majority of linear plasmids showed variation both in terms of presence among different isolates and in terms of sequence and gene order. The data strongly imply that all B. burgdorferi clinical isolates contain linear plasmids related to each other, but the structure of these replicons may vary substantially from isolate to isolate. These alterations include deletions and presumed rearrangements that are likely to result in unique plasmid elements in many isolates. There is a strong correlation between complete genome hybridization profiles and other typing methods, which, in turn, also correlate to differences in pathogenicity. Because there is substantially less variation in the chromosomal and circular plasmid portions of the genome, the major differences in open reading frame content and genomic diversity among isolates are linear plasmid driven.     

Linear- and circular-plasmid copy numbers in Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease agent, and other members of the spirochetal genus Borrelia have double-stranded linear plasmids in addition to supercoiled circular plasmids. The copy number relative to the chromosome was determined for 49- and 16-kb linear plasmids and a 27-kb circular plasmid of the type strain, B31, of B. burgdorferi. All three plasmids were present in low copy number, about one per chromosome equivalent, as determined by relative hybridizations of replicon-specific DNA probes. The low copy number of Borrelia plasmids suggests that initiation of DNA replication and partitioning are carefully controlled during the cell division cycle. The copy numbers of these three plasmids of strain B31 were unchanged after approximately 7,000 generations in continuous in vitro culture. A clone of B. burgdorferi B31 that did not contain the 16-kb linear plasmid was obtained after exposure of a culture to novobiocin, a DNA gyrase inhibitor. The plasmid-cured strain contains only one linear plasmid, the 49-kb plasmid, and thus has the smallest genome reported to date for B. burgdorferi.     

Homology throughout the multiple 32-kilobase circular plasmids present in Lyme disease spirochetes.

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.     

Genome stability of Lyme disease spirochetes: comparative genomics of Borrelia burgdorferi plasmids

Lyme disease is the most common tick-borne human illness in North America. In order to understand the molecular pathogenesis, natural diversity, population structure and epizootic spread of the North American Lyme agent, Borrelia burgdorferi sensu stricto, a much better understanding of the natural diversity of its genome will be required. Towards this end we present a comparative analysis of the nucleotide sequences of the numerous plasmids of B. burgdorferi isolates B31, N40, JD1 and 297. These strains were chosen because they include the three most commonly studied laboratory strains, and because they represent different major genetic lineages and so are informative regarding the genetic diversity and evolution of this organism. A unique feature of Borrelia genomes is that they carry a large number of linear and circular plasmids, and this work shows that strains N40, JD1, 297 and B31 carry related but non-identical sets of 16, 20, 19 and 21 plasmids, respectively, that comprise 33-40% of their genomes. We deduce that there are at least 28 plasmid compatibility types among the four strains. The B. burgdorferi ～900 Kbp linear chromosomes are evolutionarily exceptionally stable, except for a short ≤20 Kbp plasmid-like section at the right end. A few of the plasmids, including the linear lp54 and circular cp26, are also very stable. We show here that the other plasmids, especially the linear ones, are considerably more variable. Nearly all of the linear plasmids have undergone one or more substantial inter-plasmid rearrangements since their last common ancestor. In spite of these rearrangements and differences in plasmid contents, the overall gene complement of the different isolates has remained relatively constant.     

Plasmid location of Borrelia purine biosynthesis gene homologs.

The Lyme disease spirochete Borrelia burgdorferi must survive in both its tick vector and its mammalian host to be maintained in nature. We have identified the B. burgdorferi guaA gene encoding GMP synthetase, an enzyme involved in de novo purine biosynthesis that is important for the survival of bacteria in mammalian blood. This gene encodes a functional product that will complement an Escherichia coli GMP synthetase mutant. The gene is located on a 26-kb circular plasmid, adjacent to and divergent from the gene encoding the outer surface protein C (OspC). The guaB gene homolog encoding IMP dehydrogenase, another enzyme in the purine biosynthetic pathway, is adjacent to guaA. In Borrelia hermsii, a tick-borne relapsing fever spirochete, the guaA and guaB genes are located on a linear plasmid. These are the first genes encoding proteins of known function to be mapped to a borrelial plasmid and the only example of genes encoding enzymes involved in the de novo purine biosynthesis pathway to be mapped to a plasmid in any organism. The unique plasmid location of these and perhaps other housekeeping genes may be a consequence of the segmented genomes in borreliae and reflect the need to adapt to both the arthropod and mammalian environments.     

Directed insertion of a selectable marker into a circular plasmid of Borrelia burgdorferi.

Studies of the biology of Borrelia burgdorferi and the pathogenesis of Lyme disease are severely limited by the current lack of genetic tools. As an initial step toward facile genetic manipulation of this pathogenic spirochete, we have investigated gene inactivation by allelic exchange using a mutated borrelial gyrB gene that confers resistance to the antibiotic coumermycin A1 as a selectable marker. We have transformed B. burgdorferi by electroporation with a linear fragment of DNA in which this selectable marker was flanked by sequences from a native borrelial 26-kb circular plasmid. We have identified coumermycin A1-resistant transformants in which gyrB had interrupted the targeted site on the 26-kb plasmid via homologous recombination with the flanking sequences. Antibiotic resistance conferred by the mutated gyrB gene on the plasmid is dominant, and transformed spirochetes carrying this plasmid do not contain any unaltered copies of the plasmid. Coumermycin A1 resistance can be transferred to naive B. burgdorferi by transformation with borrelial plasmid DNA from the initial transformants. This work represents the first example of a directed mutation in B. burgdorferi whereby a large segment of heterologous DNA (gyrB) has been inserted via homologous recombination with flanking sequences, thus demonstrating the feasibility of specific gene inactivation by allelic exchange.     

Repetition, conservation, and variation: the multiple cp32 plasmids of Borrelia species

Members of the spirochete genus Borrelia contain large numbers of extrachromosomal DNAs. Sequence analysis of the B. burgdorferi strain B31 genome indicated that its many plasmids contain large quantities of repeated sequences, the most obvious of which are the cp32 plasmid family. Individual spirochetes may carry nine or more different, but homologous, cp32 plasmids. Every other species of Borrelia examined thus far also contains multiple plasmids related to the B. burgdorferi cp32s. These plasmids are arguably the best characterized of all the borrelial plasmids, and epitomize the apparent redundancy evident in the many plasmids carried by these bacteria. Despite their extensive similarities, cp32 plasmids contain some open reading frames whose sequences often vary between plasmids, and which encode proteins synthesized by the bacteria during vertebrate infection. In this review, we analyze the hypervariable and conserved regions of the cp32 plasmid family, and discuss possible reasons why borreliae harbor multiple gene paralogs.     

Analysis of linear plasmid dimers in Borrelia burgdorferi sensu lato isolates: implications concerning the potential mechanism of linear plasmid replication.

The Borrelia genome is composed of a linear chromosome and a number of variable circular and linear plasmids. Atypically large linear plasmids of 92 to 105 kb have been identified in several Borrelia burgdorferi sensu lato isolates and characterized. These plasmids carry the p27 and ospAB genes, which in other isolates reside on a 50-kb plasmid. Here we demonstrate that these plasmids are dimers of the 50-kb ospAB plasmid (pAB50). The 94-kb plasmid from isolate VS116, pVS94, was an exception and did not hybridize with any plasmid gene probes. When this plasmid was used as a probe, homologous sequences in other isolates were not detected, suggesting that it is unique to isolate VS116. These analyses provide insight into the mechanism of linear plasmid replication and the mechanisms by which plasmid variability can arise.     

Characterization of Circular Plasmid Dimers in Borrelia burgdorferi

We have inactivated the ospC, oppAIV, and guaB genes on the 26-kb circular plasmid of Borrelia burgdorferi (cp26) by allelic exchange. On several occasions following such transformations, the cp26 of transformants had an aberrant mobility through agarose gels. Characterization of these cp26 molecules showed that the plasmid had dimerized. These dimers were quite stable during either selective or nonselective passage. Subsequent transformations with dimer DNA supported the hypothesis that in B. burgdorferi, transforming cp26 DNA most likely does not displace the resident homologous plasmid but rather must recombine in order to donate sequences that it carries. These serendipitous findings provide a mechanism for obtaining heterozygous complemented control strains when mutant phenotypes are characterized.