Two plasmids GSK1120212 molecular weight were constructed to complement this mutant. Because promoters of B. burgdorferi often overlap the preceding ORF (Cabello et al., 2006), one of these, pAB63 (Fig. 1b), contained both the
uvrABbu ORF and the 504 bp upstream of the translational start of uvrA. The other, pMS9 (Fig. 1b), contained the uvrABbu ORF under the control of the borrelial flaB promoter. Electroporation of these plasmids and the pKFSS1 vector control into B. burgdorferiΔuvrABbu, followed by selection and passaging yielded clones containing both full-length and disrupted uvrABbu (Fig. S1a) that expressed uvrA mRNA transcripts (Fig. S1a). Reactions performed without reverse transcriptase showed no amplicons and confirmed the lack of DNA contamination in total RNA samples (data not shown). UV irradiation damages
DNA by generating intrachain thymine dimers (Black et al., 1998; Aertsen et al., 2004; Fry et al., 2005; Maul & Sutton, 2005). Exposure of the parental strain to 800 and 1000 μJ cm−2 of UV radiation had little effect on its survival, while exposure of ΔuvrABbu or its derivative containing only the pKFSS1 cloning vector to these doses resulted in complete loss of viability (Fig. 2a and b). Significant complementation of the phenotypic defect of the inactivation mutant was obtained with both pAB63 and pMS9 (P<0.001). The inability of the inactivation mutant to survive UV radiation was partially corrected by pAB63 (uvrABbu and 504 bp 5′ up to the uvrABbu start codon, Fig. 2a) and fully corrected by pMS9 (Fig. 2b). This indicates that the uvrABbu gene product is involved in the ability of B. burgdorferi find more to repair Baricitinib intrachain DNA damage. MMC, a nucleotide-akylating agent, cross-links DNA (Iyer & Szybalski, 1963). Bacterial mutants with various defects in DNA repair have been found to be more susceptible to growth inhibition by this agent than are wild type (Bijlsma et al., 2000; Liveris et al., 2004). In the absence of MMC, wild type, the ΔuvrABbu inactivation mutant and its pAB63 (not shown), pMS9 or pKFSS1 derivatives (Fig. 3a) grew equally well in complete BSK-H. All strains reached log-phase
density (about 108 cells mL−1) by day 4 of culture. In the presence of MMC, the growth of ΔuvrABbu was significantly (P<0.001) inhibited [concentrations examined: 0.1 μg mL−1 (data not shown), 1 μg mL−1 (data not shown), 5 μg mL−1 (Fig. 3b), 10 μg mL−1 (Fig. 3c)]. This growth inhibition was reversed by extrachromosomal complementation of ΔuvrABbu with pMS9 (uvrABbu under the control of flaBp) but not with the cloning vector pKFSS1 (Fig. 3b and c). Similar results were obtained using pAB63 (uvrABbu under the control of 504 upstream nucleotides) to complement ΔuvrABbu (data not shown). This indicates that the uvrABbu gene product is involved in repair of interchain repair of DNA damage in B. burgdorferi, in striking difference to the situation in E.