The 700-bp downstream region of uvrABbu was amplified using primers 12.2 and 12.1 (nt 891827–892526). The kanamycin www.selleckchem.com/screening/protease-inhibitor-library.html resistance gene aph(3′)-IIIa from Enterococcus faecalis was amplified with its own promoter and stop codon from pBLS500 using primers III and IV (Shevchuk et al., 2004). Parameters for PCR reactions were denaturation at 94 °C for 2 min, 32 cycles of 94 °C for 15 s, 56 °C for 20 s, 68 °C for 2 min, and a final extension at 68 °C for 5 min. PCR fragments were fused by long PCR (Shevchuk et al., 2004), and the final 2279-kb PCR product containing
the uvrABbu gene with a kanamycin resistance gene insertion was cloned into pGEM-T (Promega), a vector that cannot replicate in B. burgdorferi, to yield pBL12. Selection and maintenance of E. coli DH5α transformants with pBL12 was performed using solid and liquid Luria–Bertani medium containing 100 μg mL−1 of ampicillin. To obtain pAB63 (Fig. 1b), a 3.4-kb PCR fragment containing uvrABbu and 504 bp 5′ to
its translational start site (possible promoter INCB024360 region) were amplified from B. burgdorferi 297 genomic DNA using primers AVB3 (containing a SacI restriction site) (Table 1) and AVB4 (containing a PstI restriction site) (Table 1), and ligated into the multiple cloning site of pKFSS1 (Frank et al., 2003) digested with SacI and PstI. To obtain pMS9 (Fig. 1b), the flaBBbu promoter and uvrABbu were amplified from B. burgdorferi 297 genomic DNA using primers FflaB/RflaB (containing SacI and KpnI restriction sites) (Table 1) and FuvrA/RurvA (containing KpnI and PstI restriction sites) (Table 1), respectively, and cloned into pKFSS1, first the flaBBbu promoter, then the ORF for uvrABbu, using the appropriate aminophylline restriction enzymes. Spirochetes grown to mid-logarithmic phase were electroporated with 5–20 μg of plasmid DNA (Samuels, 1995). Individual clones were obtained by serial dilution of aliquots taken from antibiotic-resistant cultures in complete BSK-H containing antibiotics.
Borrelia burgdorferi cells (1 × 105) (midlog phase) were inoculated into 0.5 mL of complete BSK-H containing 0.01, 0.1, 1, 5 or 10 μg of MMC (Sigma Chemical Co.) and cultured at 34 °C for 12–13 days, and spirochetes were counted in duplicate every 1–4 days by dark-field microscopy (Sicklinger et al., 2003). Bacteria were always kept in the dark during these experiments. Two independent experiments with each complementing plasmid were performed. Cells grown to a density of 3 × 107 cells mL−1 in complete BSK-H were harvested by centrifugation, resuspended in phosphate-buffered saline (PBS), pH 7.4, to 1 × 105 cells mL−1 and exposed to 800 or 1000 μJ cm−2 280-nm UV radiation (Spectrolinker XL-1000 UV crosslinker, Spectronics Corporation, Westbury, NY). Survival of cells after culture at 34 °C on semisolid BSK-H was determined at 14–18 days (Liveris et al., 2004). Borrelia burgdorferi not exposed to UV irradiation served as a control. Bacteria were always kept in the dark during these experiments.
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