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

Abstract

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.Lyme disease, a tick-borne zoonosis (5,7,36,37), is caused by Borrelia burgdorferi, Borrelia garinii, and Borrelia afzelii (1,6,19,20,38). Related species of uncertain pathogenic potential, i.e., Borrelia japonica (18) and Borrelia andersonii (21), have also been identified. These five species are collectively referred to as B. burgdorferi sensu lato (BBsl). The BBsl genome is composed of variable linear and circular plasmids (15, 30) and a linear chromosome (11). The ospAB operon is carried on the largest of the linear plasmids, which ranges from 48 to 60 kb (31). We refer to this variably sized plasmid as the 50-kb ospAB plasmid or pAB50. The importance of the BBsl plasmids is underscored by the fact that they are present in all isolates and that they carry genes encoding metabolic enzymes (24). In view of this, the extent of plasmid variability among isolates is surprising. The significance of and the mechanisms involved in generating plasmid variability are not completely understood. Plasmid loss (32), recombination (17,22,28), and lateral transfer of plasmids among BBsl species (22) have been demonstrated to be contributing processes. Here we demonstrate that linear plasmid dimer formation also contributes to plasmid variability and discuss how these findings may provide information concerning the mechanisms of linear plasmid replication.Bacterial isolates analyzed in this study were cultivated at 34ЊC in BSK-H (Sigma) supplemented with 6% rabbit serum. Pulsed-field gel electrophoresis (PFGE) was performed to analyze plasmid content as previously described (21). Several isolates carrying 92-to 105-kb plasmids, 40 to 50 kb larger than those typically observed (2-4, 8, 16, 22, 31-35, 39), were identified (Table 1). Only B. burgdorferi R100 had been previously shown to carry a plasmid in this size range (26). Southern blot analyses with an ospA probe (ospA-5Ј-GGCTGCTAACATTT-TGCTTACATGC) revealed that the ospAB operon was carried by the large plasmids in IKA2, HO14 (Fig. 1), BO23, and R100 and by a 50-kb plasmid in VS116 (data not shown). In IKA2, a second plasmid of 47 kb also bound the probe (Fig. 1); however, after continued in vitro cultivation, this plasmid was lost and an ospAB-hybridizing ...

Distribution of Twelve Linear Extrachromosomal DNAs in Natural Isolates of Lyme Disease Spirochetes

“…For example, B31 plasmid lp54 clone CM64 hybridizes to 53-to 56-kbp plasmids in all three species. This agrees with the observations that the ospAB and P27 genes have been found on a plasmid of this size in members of these species that have been analyzed (5,18,23,24). The hybridization target of lp17 probe CL47 is universally present on 21-to 28-kbp plasmids in B. garinii and B. afzelii, but is present in less than half of the B. burgdorferi isolates we examined.…”

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

“…When directing insertion mutations to the oppAIV and guaB genes, we isolated transformants with both wild-type and mutant copies of the targeted gene (K. Tilly and P. Rosa, unpublished results). These bacteria had double-length cp26, as was previously found for lp49 (Marconi et al, 1996), although the lp49 dimer structure was a tail-tail linear molecule, whereas the cp26 dimer has a directly repeated structure. Homozygous guaB and oppAIV mutant cp26 transformants are also viable (K. Tilly and P. Rosa, unpublished results; J. L. Bono et al, manuscript in preparation), so the stability of the heterozygous dimers was not conferred by selection for both a wild-type copy of the targeted gene and the coumermycin-resistant gyrase derived from the mutated gene.…”

Comparative Genome Hybridization Reveals Substantial Variation among Clinical Isolates ofBorrelia burgdorferiSensu Stricto with Different Pathogenic Properties

“…Linear plasmid alterations may include deletions and/or rearrangement of fragments between plasmids, as initially suggested by Casjens et al (9), and were observed for lp17, lp25, lp28-1, lp28-2, lp28-3, and lp36 by Palmer et al (42). Plasmid dimerization is also possible, as was described for lp54-like plasmids in B. burgdorferi sensu lato (36). Thus, in this latter plasmid class, ORFs located on a specific plasmid in B31MI may either be linked to the same partition locus, be present on a different replicon, or be completely absent from other isolates.…”