FIG 3.

Alteration of BbHtrA cleavage site in BB0323 impairs processing of N-terminal domains but does not affect spirochete growth and biology in vitro. (A) Diagram of the vector construct (pXLF14301-bb0323^NL) for introducing mutants in B. burgdorferi. The construct was generated via the chromosomal insertion of bb0323^N236AL237A into the bb0323 deletion mutant, in order to produce bb0323^NL complemented B. burgdorferi isolates (designated Bb^bb0323NL). (B) Production of mature N-terminal BB0323 domains in B. burgdorferi isolates. The wild type (WT), bb0323 mutant (bb0323-), bb0323 complement (bb0323com), and cleavage site mutant (Bb^bb0323NL) were grown at different temperatures (25 to 37°C). The spirochetes were harvested and lysates were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and blotted with antiserum against BB0323-N (upper portion) and FlaB (lower portion). Mature N-terminal proteins with expected molecular weights of ∼27 kDa (larger arrows), as well as polypeptides of slightly lower molecular weights (small arrows), are indicated. (C) The Bb^bb0323NL cleavage site mutants display normal growth in culture compared to wild-type cells. B. burgdorferi cells were diluted to a density of 10⁵/mL and grown at 34°C and 37°C in BSK-H medium. Samples were counted under a dark-field microscope every 24 h using a Petroff-Hausser cell counter. The bb0323 deletion mutant (Bb^bb0323-) display significant growth defects compared to other isolates (*, P < 0.05). (D) N-terminal BB0323 proteins are associated with spirochete membrane fractions. Spirochete lysates were subjected to Triton X-114 phase partitioning of aqueous and detergent phases and immunoblotted using antibodies against BB0323, FlaB, and BB0238.