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.     

Distribution of twelve linear extrachromosomal DNAs in natural isolates of Lyme disease spirochetes

We have analyzed a panel of independent North American isolates of the Lyme disease agent spirochete, Borrelia burgdorferi (sensu stricto), for the presence of linear plasmids with sequence similarities to the 12 linear plasmids present in the B. burgdorferi type strain, isolate B31. The frequency of similarities to probes from each of the 12 B31 plasmids varied from 13 to 100% in the strain panel examined, and these similarities usually reside on plasmids similar in size to the cognate B31 plasmid. Sequences similar to 5 of the 12 B31 plasmids were found in all of the isolates examined, and >66% of the panel members hybridized to probes from 4 other plasmids. Sequences similar to most of the B. burgdorferi B31 plasmid-derived DNA probes used were also found on linear plasmids in the related Eurasian Lyme agents Borrelia garinii and Borrelia afzelii; however, some of these plasmids had uniform but substantially different sizes from their B. burgdorferi counterparts.     

High-throughput plasmid content analysis of Borrelia burgdorferi B31 by using Luminex multiplex technology

Borrelia burgdorferi, the causative agent of Lyme disease in North America, is an invasive pathogen that causes persistent multiorgan manifestations in humans and other mammals. Genetic studies of this bacterium are complicated by the presence of multiple plasmid replicons, many of which are readily lost during in vitro culture. The analysis of B. burgdorferi plasmid content by plasmid-specific PCR and agarose gel electrophoresis or other existing techniques is informative, but these techniques are cumbersome and challenging to perform in a high-throughput manner. In this study, a PCR-based Luminex assay was developed for determination of the plasmid content of the strain B. burgdorferi B31. This multiplex, high-throughput method allows simultaneous detection of the plasmid contents of many B. burgdorferi strains in a 96-well format. The procedure was used to evaluate the occurrence of plasmid loss in 44 low-passage B. burgdorferi B31 clones and in a library of over 4,000 signature-tagged mutagenesis (STM) transposon mutant clones. This analysis indicated that only 40% of the clones contained all plasmids, with (in order of decreasing frequency) lp5, lp56, lp28-1, lp25, cp9, lp28-4, lp28-2, and lp21 being the most commonly missing plasmids. These results further emphasize the need for careful plasmid analysis in Lyme disease Borrelia studies. Adaptations of this approach may also be useful in the evaluation of plasmid content and chromosomal gene variations in additional Lyme disease Borrelia strains and other organisms with variable genomes and in the correlation of these genetic differences with pathogenesis and other biological properties.     

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.     

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.     

Detection of a large linear plasmid in Borrelia spielmanii isolate

Borrelia spielmanii belongs to human pathogenic species within the Borrelia burgdorferi sensu lato complex in Europe, which is a causative agent of Lyme disease. So far, the human disease caused by B. spielmanii has been associated with skin manifestations. The aim of the study was to analyze 4 human B. spielmanii isolates by pulsed-field gel electrophoresis and to localize genes of 3 important Borrelia proteins: OspA, DbpA, and VlsE. The analysis revealed variation within linear plasmid profiles among the strains; isolate PSigII contained a large plasmid of 100 kb compared with a 50 kb plasmid present in the 3 other B. spielmanii isolates, all carried the genes ospA and dbpA. Differences in the size of linear plasmids among the Borrelia strains may be a result of host-pathogen interactions, as the PSigII strain was the only strain of the 4 tested strains to be isolated from a patient with a previous history of Lyme disease, whereas 3 other patients were diagnosed with this disease for the first time.     

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.     

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.     

Cloning and sequence analysis of linear plasmid telomeres of the bacterium Borrelia burgdorferi

Borrelia burgdorferi, the Lyme disease agent, has double-stranded linear plasmids with covalently closed ends. DNA at the ends, or telomeres, of two linear plasmids of B. burgdorferi strain B31 was examined. Telomeric sequences from both ends of a 16 kb linear plasmid and from one end of a 49 kb linear plasmid were cloned and sequenced. An 18 bp AT-rich inverted repeat was found at each end of the 16 kb linear plasmid. The sequences of the two ends of this plasmid were different beyond these short inverted terminal repeats. The cloned end of the 49 kb linear plasmid had sequence identity with one end of the 16 kb linear plasmid. The end sequence common to both plasmids contained a series of phased, short direct repeats and a 52 bp palindrome adjacent to a highly AT-rich region. These findings indicate that Borrelia linear plasmid telomeres have structural features different from those of other known replicons.     

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.     

Comparison of Borrelia isolated from UK foci of Lyme disease

Abstract Restriction endonuclease digestion of linear borrelial chromosomal DNA showed that three isolates of UK Lyme disease spirochaetes differed markedly from each other and from published data for other isolates from North America and continental Europe. Analysis of linear plasmid bands revealed that UK isolates differed from each other in the number and sizes of the plasmids in isolates from different foci of UK Lyme disease. Fatty acid analysis (of fatty acid methyl ester (FAME) profiles) showed the UK isolates clustering together with the relapsing fever spirochaetes, Borrelia turicatae and Borrelia parkeri . These data are discussed in respect of current knowledge of Lyme borreliosis in the UK.     

Linear plasmids of Borrelia burgdorferi have a telomeric structure and sequence similar to those of a eukaryotic virus.

Spirochetes of the genus Borrelia have double-stranded linear plasmids with covalently closed ends. The physical nature of the terminal connections was determined for the 16-kb linear plasmid of the B31 strain of the Lyme disease agent Borrelia burgdorferi. Native telomeric fragments representing the left and right ends of this plasmid were isolated and subjected to Maxam-Gilbert sequence analysis. At the plasmid ends the two DNA strands formed an uninterrupted, perfectly palindromic, AT-rich sequence. This Borrelia linear plasmid consisted of a continuous polynucleotide chain that is fully base paired except for short single-stranded hairpin loops at each end. The left and right telomeres of the 16-kb plasmid were identical for 16 of the first 19 nucleotide positions and constituted an inverted terminal repeat with respect to each other. The left telomere of the 49-kb plasmid of strain B31 was identical to the corresponding telomere of the 16-kb plasmid. Different-sized plasmids of other strains of B. burgdorferi also contained sequences homologous to the left end of the 16-kb plasmid. When the borrelia telomeres were compared with telomeric sequences of other linear double-stranded DNA replicons, sequence similarities were noted with poxviruses and particularly with the iridovirus agent of African swine fever. The latter virus and a Borrelia sp. share the same tick vector. These findings suggest that the novel linear plasmids of Borrelia originated through a horizontal genetic transfer across kingdoms.     

The essential nature of the ubiquitous 26-kilobase circular replicon of Borrelia burgdorferi

The genome of the type strain (B31) of Borrelia burgdorferi, the causative agent of Lyme disease, is composed of 12 linear and 9 circular plasmids and a linear chromosome. Plasmid content can vary among strains, but one 26-kb circular plasmid (cp26) is always present. The ubiquitous nature of cp26 suggests that it provides functions required for bacterial viability. We tested this hypothesis by attempting to selectively displace cp26 with an incompatible but replication-proficient vector, pBSV26. While pBSV26 transformants contained this incompatible vector, the vector 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 the BBB29 gene, which has sequence homology to a gene encoding a glucose-specific phosphotransferase system 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 merodiploid transformants, suggesting that resT is required for B. burgdorferi growth. To determine if resT is the only cp26 gene essential for growth, we introduced resT into B. burgdorferi on pBSV26. This did not result in displacement of cp26, suggesting that additional cp26 genes encode vital functions. We concluded that B. burgdorferi plasmid cp26 encodes functions critical for survival and thus shares some features with the chromosome.     

The nucleotide sequence of a linear plasmid of Borrelia burgdorferi reveals similarities to those of circular plasmids of other prokaryotes.

A linear plasmid of Borrelia burgdorferi had 16,927 bp, a G+C content of 23.1%, a relative deficiency of CpG dinucleotides, and open reading frames A to O. The OrfC and OrfE proteins were similar to hypothetical proteins encoded by circular plasmids of B. burgdorferi. The OrfM and OrfN proteins were similar to replication proteins of circular plasmids of other bacteria.     

Intra- and Interbacterial Genetic Exchange of Lyme Disease Spirochete <Emphasis Type="Italic">erp</Emphasis> Genes Generates Sequence Identity Amidst Diversity

All isolates of the spirochete Borrelia burgdorferi contain multiple, different plasmids of the cp32 family, each of which contains a locus encoding Erp surface proteins. Many of these proteins are known to bind host complement regulatory factor H, enabling the bacteria to avoid killing by the alternative complement pathway during vertebrate infection. In the present study, we characterized the erp loci and cp32 plasmids of strains N40, Sh-2-82, and 297 and compared them to the previously determined cp32 sequences of type strain B31. Bacteria of strain N40 contain 6 different cp32s, those of Sh-2-82 contain 10, and 297 bacteria contain 9 cp32s. Significant conservation between all strains was noted for the cp32 loci responsible for plasmid maintenance, indicating close relationships that appear to correspond with incompatibility groups. In contrast, considerable diversity was found between erp gene sequences, both within individual bacteria and between different strains. However, examples of identities among erp loci were found, with strains Sh-2-82, 297, and B31 each containing three identical loci that likely arose through intrabacterial genetic rearrangements. These studies also found the first evidence of large-scale genetic exchanges between Lyme disease spirochetes in nature, including the apparent transfer of an entire cp32 plasmid between two different bacteria.     

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.     

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.     

Borrelia genomes in the year 2000

All analyzed members of the spirochete genus Borrelia contain a linear chromosome about 910 kbp long. The complete sequence of the B. burgdorferi B31 genome predicts that its chromosome carries essentially all of this organism's housekeeping genes. In accordance with these bacterial species' obligatory parasitic lifestyle, its genes encode enzymes that are capable of only a minimal metabolism, in which all nucleotides, amino acids, fatty acids and enzyme cofactors must be scavenged from the host. In addition to the chromosome, all Borrelia isolates examined carry multiple linear and circular plasmids with lengths between 5 and 200 kbp. The plasmids, which account for over 600 kbp in isolate B31, carry very few genes with homology to genes outside of the Borrelia genus. But they do carry numerous predicted lipoprotein genes, many of which are have been shown to be or are expected to be outer surface proteins. Ten of the linear plasmids have strikingly low protein coding potential for bacterial DNA. These plasmids have enjoyed numerous past duplicative rearrangements, which have resulted in the presence of a substantial fraction of the DNA that appears to be currently undergoing mutational decay, presumably because it is no longer under selection for function.