The B. burgdorferi uvrA homologue (BB0837) encodes a protein of 950 amino acids (UvrABbu) whose deduced amino acid sequence has 23–54% homology to UvrA of Treponema pallidum, Leptospira interrogans, Bacillus subtilus and E. coli, and, like the others, contains two zinc finger motifs and two ATP-binding sites (Savery, 2007). The function of BB0837 has not been experimentally verified, and study of its function, expression and regulation in B. burgdorferi is therefore likely to shed
light on its role in DNA repair and bacterial survival. To this end, we inactivated uvrABbu and found that the resulting B. burgdorferi disruption mutant was more sensitive to UV radiation, mitomycin C (MMC) and ROS than the parental strain. This increased sensitivity was reversed by extrachromosomal complementation with a wild-type copy of uvrABbu. Low-passage Tanespimycin concentration infectious B. burgdorferi 297, clone BbAH130,
was obtained from Dr M. V. Norgard, University of Texas Southwestern Medical Center. PCR analysis using appropriate primers (Iyer ZD1839 research buy et al., 2003) indicated that this clone contained lp25, but lacked lp28-1. Cultures were routinely grown at 34 °C in Barbour–Stoenner–Kelly medium supplemented with 6% rabbit serum (BSK-H) (Sigma Chemical Co., St. Louis, MO). Escherichia coli DH5α (Gibco/Life Technologies, Grand Island, NY) was routinely used for cloning, and was grown and maintained in Luria–Bertani medium. Genomic DNA was isolated from pelletted B. burgdorferi grown at 34 °C to 3 × 108 cells mL−1 with High Pure PCR Template Preparation Kit (Roche Diagnostics Corporation, Indianapolis, IN) and total RNA was isolated using TRizol Reagent (Invitrogen Life Technology, Carlsbad, CA), both according to the manufacturer’s
instructions. Traces of genomic DNA were removed from isolated RNA by treatment with RNase-free DNase. RNA was dissolved in RNase-free water (Ambion, Austin, TX) and stored in aliquots at −80 °C. cDNA was generated by AMV reverse transcriptase with random primers using the Access RT-PCR system (Promega Corporation, Madison, WI). Controls with the omission of reverse transcriptase were always included in each experiment. PCR reactions were performed using Taq polymerase (Denville Scientific Inc., Metuchen, Thalidomide NJ) or Expend Long Template DNA polymerase mix (Roche Applied Science) using parameters according to Tm of primers. All constructs were confirmed by restriction enzyme analysis, PCR and DNA sequencing using standard procedures (Sambrook & Russell, 2001). The primers used in this study are listed in Table 1. The uvrABbu inactivation construct (Fig. 1a) was generated using overlap extension PCR fusion (Shevchuk et al., 2004). Flanking fragments of uvrA were amplified from B. burgdorferi 297 genomic DNA (Fraser et al., 1997) using target-specific primers. Briefly, the 544-bp upstream region of uvrABbu was amplified from B. burgdorferi genomic DNA using primers 12.4 and 12.3 (nt 889980–890523 in the B.