Telomere exchange between linear replicons of Borrelia burgdorferi

Abstract

Spirochetes in the genus Borrelia carry a linear chromosome and numerous linear plasmids that have covalently closed hairpin telomeres. The overall organization of the large chromosome of Borrelia burgdorferi appears to have been quite stable over recent evolutionary time; however, a large fraction of natural isolates carry differing lengths of DNA that extend the right end of the chromosome between about 7 and 20 kbp relative to the shortest chromosomes. We present evidence here that a rather recent nonhomologous recombination event in the B. burgdorferi strain Sh-2-82 lineage has replaced its right chromosomal telomere with a large portion of the linear plasmid lp21, which is present in the strain B31 lineage. At least two successive rounds of addition of linear plasmid genetic material to the chromosomal right end appear to have occurred at the Sh-2-82 right telomere, suggesting that this is an evolutionary mechanism by which plasmid genetic material can become part of the chromosome. The unusual nonhomologous nature of this rearrangement suggests that, barring horizontal transfer, it can be used as a unique genetic marker for this lineage of B. burgdorferi chromosomes.

FIG. 1.

Sequence relationships among B. burgdorferi B31 MI linear plasmid lp21 and chromosomal right telomeric regions. Shading of the chromosomal and plasmid DNAs represented by rounded bars indicates the following: solid black, the constant portion of the chromosome (which extends from the left chromosomal end through gene BB0843); dark gray, the right-end extension on the B31 chromosome and very similar sequence on the Sh-2-82 chromosome; white, the unique (non-63-bp repeat) regions of B31 plasmid lp21 and the very similar sequence on the Sh-2-82 chromosome; hatched, the region of 63-bp tandem repeats on lp21 and similar sequence on the Sh-2-82 chromosome. Gray areas between DNAs highlight regions of similarity between adjacent DNAs; the darker gray (and percent values there) indicate similarities to sequence determined in this study. Black arrows indicate putative intact genes (pseudogenes are not shown). Numbered black bars show the locations of DNA probes used in this study (also Table 1). The Sh-2-82 nucleotide sequences determined here are indicated by open bars immediately above the kilobase pair scale, whose zero point is the right end of the constant portion of the chromosome.

FIG. 2.

The B. burgdorferi Sh-2-82 chromosome contains strain B31 linear plasmid lp21-like sequences. Whole-genomic Sh-2-82 DNA was prepared in agarose blocks and subjected to CHEF electrophoresis, and Southern analysis was performed with probe 2 (Table 1 and Fig. 1) as described in Materials and Methods. Culture passage number is indicated above the lanes; “std” is HindIII cut plus a ladder of whole bacteriophage λ DNA.

FIG. 3.

Determination of the length and location of the 63-bp repeat tract present in B. burgdorferi Sh-2-82 DNA. Whole-genomic Sh-2-82 DNA was prepared in agarose blocks, restricted, and subjected to CHEF electrophoresis. Southern analysis was performed with 63-bp repeat probe 4 (Table 1 and Fig. 1). Culture passage number and restriction enzymes used are indicated above the lanes; “std” is HindIII cut plus a ladder of whole bacteriophage λ DNA.

FIG. 4.

Linear plasmid lp21-like sequences lie near the right end of some B. burgdorferi chromosomes. Bacterial DNA was prepared, restricted, separated by CHEF electrophoresis, and subjected to Southern analysis with probe 5 (Table 1 and Fig. 1) DNA from the right unique region of plasmid lp21. Strain names and restriction enzyme cleavage are indicated above the lanes (the p7, p166, and p320 lanes contain Sh-2-82 DNA from cultures with those passage numbers); “std” is HindIII cut plus a ladder of whole bacteriophage λ DNA.

FIG. 5.

There is little sequence similarity between partners at deduced recombination points. The nucleotide sequence of one strand is shown with the 5′ end at the left; vertical lines indicate identical base pairs in adjacent sequences. The parts of the putative parental sequences that fused to give rise to the Sh-2-82 sequence are underlined. (A) Nucleotide sequences of the two putative parental participants (upper, B31-like chromosome; lower, B31 lp21-like plasmid) in the nonhomologous recombination event that gave rise to the present Sh-2-82 nucleotide sequence (center). (B) Nucleotide sequences of the two putative parental participants (upper, B31-like left location; lower, B31-like right location) in the nonhomologous recombination event that gave rise to the apparent 264-bp deletion in the present Sh-2-82 nucleotide sequence (center).

FIG. 6.

Conservation of telomeric sequences. The 23 terminal bp present at the right ends of the Sh-2-82 chromosome and the B31 lp21 plasmid are shown in the middle, with the covalently closed hairpin telomere on the left for ease of comparison with previous publications (3, 6, 12, 27). Gray boxes highlight the TATAAT and TAGTAYANA conserved regions (see text). Casjens (3) and Tourand et al. (32a) have pointed out that the previously characterized Borrelia telomeres appear to fall into two categories, type 1 and type 2, in which an apparently conserved TATAAT sequence is present 1 or 4 bp, respectively, from the terminus. Among the nine previously sequenced telomeres, five are type 1 and four are type 2; the consensus sequences of the two types are shown at the top and bottom of the figure (R = A or G; Y = C or T; W = A or T; K = G or T; M = A or C; N indicates that three different base pairs are present among the known members of that type).