Figure 4. Generation of isogenic B. burgdorferi p66 mutants and assessment of OM protein complexes.

(A) Schematic representation of wild type (WT) and p66 mutant (p66⁻) B. burgdorferi at the bb0603 (p66) locus. Genes bb0601- bb0605 (white box arrows) and the kanamycin-resistance cassette driven by the B. burgdorferi flaB promoter (flaBp-Kan, black box arrow) are indicated. The regions up- and down-stream of the p66 locus were amplified using primers P1-P4 (black arrow-heads) and cloned to BamHI-SacII and XhoI-KpnI sites flanking the flaBp-Kan cassette. (B) RT-PCR analysis for assessment of p66 transcripts and polar effects of mutagenesis. Total RNA was isolated from wild type (WT) and p66 mutant (p66⁻) B. burgdorferi, converted to cDNA for detection of p66, flaB, bb0602 and bb0604 transcripts. (C) Protein analysis of wild type (WT) and p66 mutant (p66⁻). Equal amounts of protein were separated on SDS-PAGE gels and either stained with Coomassie blue (upper panel) or transferred onto a nitrocellulose membrane and probed with P66 and FlaB antibodies (lower panels). Protein standards are shown to the left in kDa. Arrow indicates the missing P66 band in mutant lysates. (D) p66 mutant lacks detectable growth defects in vitro. Wild type (WT) and p66 mutant (p66⁻) spirochetes were diluted to a density of 10⁵cells/ml, grown at 33°C in BSK-H medium and counted under a dark-field microscope. (E) Analysis of OM protein complexes in p66 mutants. The OM fraction from wild type or p66 mutant B. burgdorferi was isolated, and protein complexes were separated using first dimensional BN/PAGE (left panel). A parallel first dimensional BN/PAGE gel containing protein complexes of the p66 mutants was transferred onto a nitrocellulose membrane and immunoblotted with anti-OM antibodies (right panel). Complex VI was absent in the p66 mutant (arrow), while monomeric protein group MGI was present (arrowhead).