Genetic exchange and plasmid transfers in Borrelia burgdorferi sensu stricto revealed by three-way genome comparisons and multilocus sequence typing

Abstract

Comparative genomics of closely related bacterial isolates is a powerful method for uncovering virulence and other important genome elements. We determined draft sequences (8-fold coverage) of the genomes of strains JD1 and N40 of Borrelia burgdorferi sensu stricto, the causative agent of Lyme disease, and we compared the predicted genes from the two genomes with those from the previously sequenced B31 genome. The three genomes are closely related and are evolutionarily approximately equidistant ( approximately 0.5% pairwise nucleotide differences on the main chromosome). We used a Poisson model of nucleotide substitution to screen for genes with elevated levels of nucleotide polymorphisms. The three-way genome comparison allowed distinction between polymorphisms introduced by mutations and those introduced by recombination using the method of phylogenetic partitioning. Tests for recombination suggested that patches of high-density nucleotide polymorphisms on the chromosome and plasmids arise by DNA exchange. The role of recombination as the main mechanism driving B. burgdorferi diversification was confirmed by multilocus sequence typing of 18 clinical isolates at 18 polymorphic loci. A strong linkage between the multilocus sequence genotypes and the major alleles of outer-surface protein C (ospC) suggested that balancing selection at ospC is a dominant force maintaining B. burgdorferi diversity in local populations. We conclude that B. burgdorferi undergoes genome-wide genetic exchange, including plasmid transfers, and previous reports of its clonality are artifacts from the use of geographically and ecological isolated samples. Frequent recombination implies a potential for rapid adaptive evolution and a possible polygenic basis of B. burgdorferi pathogenicity.

Fig. 1.

Variability of HDNP loci. Pairwise nucleotide differences of B. burgdorferi HDNPs are shown. Data for some pairs are missing (no vertical bar). (A) HDNPs on the main chromosome. (B) Plasmid HDNPs. Note that most ORFs are polymorphic in two of the three pairwise genome comparisons and varied little in the third pairwise comparison (low vertical bar). BBB19 (ospC), BBA24, and BB0144 stand out as the only ORFs that are significantly (P < 0.001) variable in all three pairwise comparisons. Note the scale difference in the vertical axis between A and B. *, ORFs used in MLST.

Fig. 2.

Multilocus sequence types of the three genomes and 18 clinical isolates. For each gene (column), colors represent various major-group alleles. Blank spaces indicate that data were not obtained because of either a lack of amplification or multiple amplicons in PCR. Isolates of the same ospC types (labeled on the right) share alleles across all surveyed loci except at loci enclosed in the red boxes, to which divergent alleles were introduced by recent genetic exchange.

Fig. 3.

Rates of synonymous and nonsynonymous substitutions in HDNP ORFs. Ka vs. Ks of HDNPs between B31 and N40 are shown. (A) Chromosomal HDNPs. ORFs are mostly far below the Ka = Ks neutrality line (solid line) and there is no Ka ≈ Ks correction (regression line in dashes; R² not significant), indicating strong selective constraints on amino acid substitution. (B). Plasmid HDNPs. ORFs are closer to the Ka = Ks neutrality line and Ka is strongly correlated (R² = 0.83, P « 0.001) with Ks, likely resulting from balancing selection. Note the difference in scale between A and B.