Construction of dbpBA -deletion mutant BbKH500 and Prom- dbpA complementation vector and PCR confirmation. (A) Strategy for the replacement of the dbpBA operon with the PflgB-kan and complementation with pKH2000. pKHdbpBAko was the pGEM-T easy-based suicide plasmid used to transform Bb297 for the homologous recombination of the kanamycin-resistance gene into the dbpBA operon. "A" denotes Asc I sites. PflgB-kan denotes the kanamycin-resistance marker expressed from the flgB promoter. The borrelial shuttle vector containing the dbpBA Prom fused to the dbpA ORF (pKH2000) was transformed into BbKH500 to restore DbpA expression. Oligonucleotide primers used for PCR are indicated with short arrows. (B) PCR using primers ko5 and ko4 (shown in panel A). The first two lanes are undigested PCR products from Bb297 and BbKH500, whereas the second two lanes are the corresponding PCR products digested with Asc I. (C) Lanes 1, 3 and 5 are PCR products derived from BbKH500 template DNA and lanes 2, 4 and 6 are PCR products derived from Bb297 template DNA. Primer pairs used in PCR are indicated above the lanes. FlaB5' and FlaB3' primers amplify flaB of B. burgdorferi. DNA size standards (M) are shown in base pairs on the left.