All recombinant DNA experiments and the use of antibiotic resistance markers in strain 297 were reviewed and approved by the University of Texas Southwestern Medical Center Biological and Chemical Safety Advisory Committee. A 4-kb DNA fragment containing ospAB (genes BBA15 and BBA16) with flanking sequence was obtained by PCR amplification from 297 genomic DNA. PCR was performed by using the Expand High Fidelity PCR System (Roche Diagnostics). The primer design was based on published gene sequences for Bb strain B31 (25 (link)) (primers 1 and 2; Table S1, available at http://www.jem.org/cgi/content/full/jem.20031960/DC1). The PCR fragment was then cloned into the pCR-XL-TOPO cloning vector (Invitrogen). The resulting plasmid was designated as pXT-OspAB. Then, an aadA cassette (which confers streptomycin [Strep] resistance to Bb [26 (link)]) was inserted at the AccI site within the ospA gene, resulting in plasmid pXT-OspAB-Strep (Fig. 1 A). To generate the ospAB mutant, 20 μg of pXT-OspA-Strep plasmid DNA was electroporated into BbAH130. The transformation mixture was then diluted into 100 ml of BSK-H medium. After overnight recovery, Strep was added to the transformation mixture at a final concentration of 50 μg/ml, and the mixtures were aliquotted into multiple 96-well tissue culture plates (200 μl/well). 2–3 wk after plating, the wells that contained positive cultures were identified by color change of the medium; the presence of viable spirochetes subsequently was verified by dark field microscopy. In general, <10% of microtiter plate wells contained positive cultures, suggesting that each well culture was clonal in origin. PCR amplification with various primer pairs (Table S1) was performed on whole cell lysates of these transformants to verify that the streptomycin-resistance (Strepr) transformants contained the appropriate aadA insertion (Fig. 1). Although highly unlikely, the possibility that the suicide plasmid inserted into more than one site would not have influenced our studies because the mutant remained infectious and complementation was successful in restoring OspA/B function (see Results and Discussion). Also, plasmid contents of the OspA/B− mutants were examined by PCR as described previously (19 (link), 27 (link), 28 (link)).