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. 2008 Jan 21;205(1):133–141. doi: 10.1084/jem.20070962

Figure 4.

Figure 4.

Construction and analysis of the bmpA/B− B. burgdorferi. (A) Schematic drawings of the wild-type isolate (WT) and the bmpA/B− mutant at the bmpAB locus. Genes bb0381, bmpB, bmpA, bmpC, and bmpD (white box-arrows) and the kanamycin-resistance cassette driven by the B. burgdorferi flaB promoter (flaB-Kan, black box-arrow) are indicated. Nucleotide positions of primers P1-P4 in the B. burgdorferi genomic database (www.tigr.org) are indicated within parentheses. The 5′ and the 3′ arms for homologous recombination, flanking up- and downstream of the bmpAB locus were amplified using primers P1-P2 and P3-P4 and ligated to the flaB-Kan cassette, as detailed in the Materials and methods. (B) RT-PCR analysis of the bmpA and B transcripts. Total RNA were isolated from either the wild-type (WT) or mutant (bmpA/B−) B. burgdorferi, converted to cDNA, and subjected to PCR analysis with flaB, bmpA, bmpB, bmpC, or bmpD primers and analyzed on a 2% agarose gel. (C) Protein analysis of wild-type (WT) or bmpA/B− spirochetes. Equal amounts of proteins from wild-type or bmpA/B− B. burgdorferi were separated on a SDS-PAGE gel, and either stained with Coomassie blue (left) or transferred onto a nitrocellulose membrane and probed with the affinity-purified BmpA (top right) or affinity-purified BmpB (bottom right) antibody. Migration of protein standards is shown to the left in kilodaltons. (D) Comparable levels of wild-type and bmpA/B− B. burgdorferi in the dermis of infected mice. Total DNA was isolated from the skin of mice 1 wk after B. burgdorferi challenge. The B. burgdorferi burden was analyzed by qPCR measurement of flaB copies and expressed as flaB/mouse β-actin. Wild-type (WT) and bmpA/B− B. burgdorferi were evident in similar numbers. n = 3. P > 0.5. (E) The bmpA/B− B. burgdorferi were capable of dissemination from the dermis of infected mice. Blood was collected from mice 1 wk after B. burgdorferi challenge, and spirochete burden was analyzed by qPCR measurement of flaB copies and expressed as flaB/mouse β-actin. Similar levels of wild-type (WT) and bmpA/B− B. burgdorferi were detected. n = 3. P > 0.5.