Molecular and evolutionary characterization of the cp32/18 family of supercoiled plasmids in Borrelia burgdorferi 297

Abstract

In this study, we characterized seven members of the cp32/18 family of supercoiled plasmids in Borrelia burgdorferi 297. Complete sequence analysis of a 21-kb plasmid (cp18-2) confirmed that the strain 297 plasmids are similar in overall content and organization to their B31 counterparts. Of the 31 open reading frames (ORFs) in cp18-2, only three showed sequence relatedness to proteins with known functions, and only one, a ParA/SopA ortholog, was related to nonborrelial polypeptides. Besides the lipoproteins, none of the ORFs appeared likely to encode a surface-exposed protein. Comparison with the B31 genomic sequence indicated that paralogs for most of the ORFs in cp18-2 can be identified on other genetic elements. cp18-2 was found to lack a 9- to 10-kb fragment present in the 32-kb homologs which, by extrapolation from the B31 cp32 sequences, contains at least 15 genes presumed to be unnecessary for plasmid maintenance. Sequence analysis of the lipoprotein-encoding variable loci provided evidence that recombinatorial processes within these regions may result in the acquisition of exogenous DNA. Pairwise analysis with random shuffling revealed that the multiple lipoproteins (Mlp; formerly designated 2.9 LPs) fall into two distinct homology groups which appear to have arisen by gene fusion events similar to those recently proposed to have generated the three OspE, OspF, and Elp lipoprotein families (D. R. Akins, M. J. Caimano, X. Yang, F. Cerna, M. V. Norgard, and J. D. Radolf, Infect. Immun. 67:1526-1532, 1999). Comparative analysis of the variable regions also indicated that recombination within the loci of each plasmid may occur independently. Last, comparison of variable loci revealed that the cp32/18 plasmid complements of the B31 and 297 isolates differ substantially, indicating that the two strains have been subject to divergent adaptive pressures. In addition to providing evidence for two different types of recombinatorial events involving cp32/18 plasmids, these findings underscore the need for genetic analysis of diverse borrelial isolates in order to elucidate the Lyme disease spirochete's complex parasitic strategies.

FIG. 1

Schematic linear representation of cp18-2. ORFs are represented as open arrows. Striped arrows represent ORFs belonging to paralogous families in the B31 strain; the boxed numbers above each arrow indicate the Borrelia paralogous family to which the ORF belongs (BBGD). Black arrows designate the previously characterized, differentially expressed p21 and Mlp9 lipoproteins. Predicted leader peptides (LP) are represented by curved lines; IRs are indicated as solid arrowheads. The names of previously characterized proteins are given above the corresponding ORFs. Asterisks indicate possible pseudogenes. The approximate distances in base pairs are indicated below.

FIG. 2

Distribution of cp18-2 paralogous families within the B. burgdorferi B31 genome.

FIG. 3

Organization of IRs and/or related flanking ORFs in selected B. burgdorferi plasmids. ORFs are depicted as shaded boxes, with similarly shaded boxes indicating paralogous ORFs; the arrow below each ORF indicates the direction of transcription. Percent similarities between cp18-2 and corresponding paralogs (using amino acid sequences) and IRs are indicated.

FIG. 4

The cp18-2 deletion. (A) cp32 ORFs predicted to be deleted from cp18-2. Small arrows designate the nested primer pairs used to confirm the similarity between the cp18-2 deletion in a full-length 297 cp32 (represented by cp32-3) and the corresponding segments from the B31 homologs (represented by cp32-7). (B) The deletions in cp18-2 and cp18 of N40 are shown alongside a representative B31 plasmid (cp32-7).

FIG. 5

Schematic representation of the variable loci in B. burgdorferi 297 (A) and B31 (B). Similarly shaded boxes are used to indicate paralogous ORFs; the arrow below each ORF indicates the direction of transcription. In panel A, paralogs of cp18-2 ORF30 and ORF31 are designated 30 and 31; nonparalogous ORFs are indicated by asterisks.

FIG. 6

Unrooted neighbor-joining phylograms of OspE, OspF, and Elp (A) and Mlp (B) homologs in B. burgdorferi 297 and B31 cp32/18 plasmids. Scale bars represent the base pair substitutions per site.

FIG. 7

Sequence relatedness among the Mlp lipoproteins. The sequences of class I (A), class II (B), and the entire complement of proteins (C) were aligned using the ClustalW algorithm. Shaded residues represent identical or conserved matches.