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

Abstract

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.

Figure 1. Length variation of B. burgdorferi chromosomes.

The relationships among the right end, plasmid-like, chromosomal extensions relative to known plasmids are indicated by gray shading; plasmid sizes are not drawn exactly to scale. There is a 1053 bp deletion and a 17 bp insertion in the 297 lp28-1 plasmid relative to the B31 extension. It is assumed that the 297 chromosome is essentially identical to that strain Sh-2-82 (see text and [51]).

Figure 2. Linear plasmid contents of B. burgdorferi strains B31, N40, 297 and JD1.

The linear plasmids and right end plasmid-like chromosomal extensions of are shown as horizontal bars with rounded ends. Identical colors indicate regions of nucleotide sequence that is ≥94% identical, and white denotes regions that are <94% identical to other sequences in the diagram. Each of the B31 plasmids was first defined with a different color and additional colors were added to the other plasmid sets as necessary. Arrows connect plasmids that have identical overall organization and high sequence similarity (ignoring small polymorphisms and indels <500 bp). Strain 297 plasmids lp28-3 and lp28-4 have not been sequenced to their termini (see table S1), so it is not known whether they are organizationally the same as their B31 or JD1 and N40 or JD1 cognates, respectively.

Figure 3. Organizational and open reading frame relationships among four lp17 plasmids.

The four lp17s are aligned vertically, and identical colored background rectangles indicate very similar sequence (the 297 plasmid is the same size as JD1 lp17 but is missing several Kbp of sequence from its termini; see text and table S1). Rectangles of the same color denote homologous sequences, and the percentage nucleotide sequence identity of parallel yellow sections are shown between the maps. The arrows denote annotated predicted genes, where red arrows have a predicted function, black have unknown function, orange are known antigens, and white is a pseudogene not annotated except in the B31 plasmid; alternate gene names or predicted function are noted in red text in figure. An “X” indicates that a gene is truncated or has a frame disruption relative to a known homolog. The blue “Δ” indicates a short deletion relative to orthologous sequence in another lp17 plasmid(s); blue numbers indicate the number of short tandem repeats present at that location; an asterisk (*) notes that the repeat sequence is not identical to that of the other lp17s; an (I) marks the locations of short inversions relative to the other lp17s.

Figure 4. Comparison of three lp25 plasmids.

Matrix plots with a 19 identities/23 bp window were created by DNA Strider . Percent identities of nucleotide sequences are indicated near the diagonal identity line for most orthologous regions. The predicted genes for B31 lp25 are shown between the two plots (open arrows with “X”s are putative pseudogenes), and regions of high similarity to other plasmids are noted on the right.

Figure 5. Comparison of lp28-1 plasmids and the vls cassette and vlsE loci.

Percent G+C plots for the plasmids were created by DNA Strider . Different background color indicates very different sequence in the different plasmids (note that the partition gene regions in the two lp28-1 plasmids are homologous, but moderately divergent from those of lp36; see text).

Figure 6. Organizational and open reading frame relationships among three lp28-5 plasmids.

Maps are labeled as in Figure 3. Yellow background between maps joins regions of homologous sequence in adjacent maps; paralogous family numbers (table S2) are indicated in black boxes above each putative gene; red boxes marked “new” indicate genes for which there is no homolog in the strain B31 genome; green bars mark the 133 bp repeat regions (see text); CdGMPBP, cyclic-di-GMP binding protein. Blue background marks regions of high similarity to regions in other B. burgdorferi genomes.

Figure 7. N40 DNA is not cut by restriction endonuclease SfoI.

DNAs were prepared in agarose blocks, cleaved with the indicated restriction endonuclease, and subjected to agarose gel pulsed-field agarose electrophoresis and stained with ethidium bromide as previously described , . Strain M is described in the text. Identical results to those with strain M were obtained with strains B31, JD1 and 297 (data not shown).

Figure 8. Organizational and open reading frame relationships among four lp36 plasmids.

Maps of the four lp36s are labeled as described in Figure 6; green genes encode predicted and proven surface lipoproteins; red, plasmid partitioning and other DNA and nucleotide metabolism proteins; magenta, and vls cassette region (see text). Yellow shading between maps marks regions of nucleotide sequence similarity (percent identity values in black text).

Figure 9. Organizational and open reading frame relationships among four lp38 plasmids.

Maps of the four lp38s are labeled as described in Figure 8. Yellow shading between maps marks regions of high nucleotide sequence similarity (percent identity values in black text). The pink horizontal bar indicates the region of 63 bp repeats in JD1 lp38; and blue arrows represent predicted transporter genes.

Figure 10. Comparison of JD1 and 297 lp28-6 plasmids.

The DNAs of JD1 plasmids lp28-6 and lp28-7 were aligned with DNA strider , and the percent identity was computed for sequential 200 bp windows across the region shown in the figure. These two plasmids have two regions of near identity from about 8 to 9 Kbp and 10.5 to 22.5 Kbp, which abut regions with lower similarity. The JD1 lp28-7 open reading frames and their paralogous family relationships are shown above.