A second allele of eppA in Borrelia burgdorferi strain B31 is located on the previously undetected circular plasmid cp9-2

Abstract

Although sequence analysis of Borrelia burgdorferi isolate B31 was recently declared "complete," we found that cultures of this strain can contain a novel 9-kb circular plasmid, cp9-2. The newly described plasmid contains both sequence similarities with and differences from the previously identified B31 plasmid cp9-1 (formerly cp9). cp9-1 and cp9-2 each encode a unique allele of EppA, a putative membrane protein synthesized by B. burgdorferi during mammalian infection.

FIG. 1

Comparisons of eppA1 and eppA2 and their predicted proteins. (A) Nucleotides common to both eppA alleles are boxed and shaded. Predicted start and stop codons of each gene are indicated by boldface and asterisks. Additional DNA flanking eppA1 that corresponds with oligonucleotides EA-A and EA-B is also shown. (B) Amino acids predicted to be contained in both EppA1 and EppA2 are boxed and shaded.

FIG. 2

(A) Two-dimensional Southern blotting of B31-RML plasmids hybridized with an eppA2-derived probe. Three forms of cp9-2 are evident and marked as CCC (covalently closed circular), OC (open circular), and Linear. (B) Diagram of cp9-2 indicating the locations of sequences complementary to oligonucleotides EA-G and EA-H, with 5′-to-3′ directions indicated by arrows. (C) Results of long-range PCR of B31-RML and B31-4a. The approximately 9-kb PCR product of cp9-2 is indicated by an arrow. A smaller PCR product was obtained from both cultures and is marked with an asterisk. Positions of DNA size standards (in kilobases) are indicated to the left of panels A and C.

FIG. 3

Southern blot analysis of B. burgdorferi isolated from human Lyme disease patients (22) using a B31 eppA2-derived probe. Positions of DNA size standards (in kilobases) are indicated to the left.