FIG. 4.

In vitro expression of BBA65, BBA66, BBA71, and BBA73 proteins in association with infectious B. burgdorferi isolates and with the borrelial outer surface. All proteins were separated by SDS-PAGE and either stained with silver (upper panels) or immunoblotted with antibodies specific for BBA65, BBA66, BBA71, BBA73, FlaB, or OppAI (lower panels). OspA and OspC are indicated to the right and molecular mass markers, in kilodaltons, to the left of each silver-stained gel. Arrows point to the BBA65 protein band, the lower of two bands recognized by our anti-BBA65 polyclonal serum (see text). (A) Association of protein expression with infectious isolates. Total membrane fractions were prepared from infectious isolates (B31 and A3) and noninfectious isolates (A and A-34), and 7.5 μg of protein was loaded per lane. (B) Membrane association. Intact B31 cells were treated with TX-114, and an equivalent of 1 × 10⁸ cells of the no-treatment (NT) control was loaded to each lane. CP, insoluble cold pellet; WP, insoluble warm pellet; AQ, aqueous phase; DT, detergent phase. FlaB and OppAI served as controls and appropriately partitioned to the expected fractions. (C) Surface localization. Intact B31 cells were treated with 400 μg/ml of the indicated protease for 1 h, and 1 × 10⁸ cells were loaded to each lane. FlaB and OppA1 are controls for cell integrity and loading. Pn, pronase SC.