Borrelia burgdorferi, 3 × 107 cells mL−1, were harvested by centr

Borrelia burgdorferi, 3 × 107 cells mL−1, were harvested by centrifugation, and diluted in triplicate to a density of 5 × 105 cells mL−1 in PBS containing 0, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 4 and 5 mM H2O2 (Sigma Chemical Co.). After incubation for 1 h at 34 °C, cells were washed Dasatinib order with PBS, resuspended in complete BSK with appropriate antibiotics and cultured in capped 0.5-mL tubes or in 96-well plates in 3% CO2 at 34 °C for 12 days. End points were determined by the change of color of the medium, indicating bacterial growth (Terekhova et al., 2002). Results from two to four independent

experiments have been combined and are reported as minimal inhibitory concentrations (MIC). NaNO2 (10, 25, 50, 100, 150 mM), (Z)-1-[N-(3-ammoniopropyl)-N-[4-(3-aminopropylammonio) butyl]-amino]-diazen-1-ium-1,2-diolate (0.01, 0.1, 1 mM) (SPER/NO, Sigma Chemical Co.) and S-nitroso-N-acetylpenicillamine (0.05, 0.1, 0.5, 1 mM) (SNAP, Sigma Chemical Co.) were used as sources of NOS.

For treatment with NaNO2, 5 × 105 borrelia were inoculated into capped tubes containing 1 mL complete BSK-H and various concentrations of NaNO2 and cultured at 34 °C. For treatment with SPER/NO and SNAP, 5 × 105 cells were incubated in PBS with various concentrations of these reagents for 1 h at 37 °C, harvested by centrifugation, and resuspended and cultured at 34 °C in 1 mL complete BSK-H with appropriate antibiotics. Growth of B. burgdorferi was determined by counting under dark field microscopy every 2–3 days for 8 days. Results Seliciclib mw from two independent experiments have been combined. Acidity of complete BSK-H (pH 7.5) was adjusted to pH 5.5, 6.0, 6.5 and 6.8 by addition of HCl. Borrelia burgdorferi, 5 × 105 cells, were inoculated into 1 mL of pH unadjusted and adjusted medium, and cultured at 34 °C for 9 days. Bacterial growth was assessed

by counting under dark field microscopy. Results from two independent experiments have been combined. Data were analyzed by one-way anova with a post hoc Bonferroni PtdIns(3,4)P2 multiple comparisons test. The level of significance was set at P<0.05. To inactivate uvrABbu, a 2.3-kb DNA segment was constructed by long PCR (Shevchuk et al., 2004). This segment contained a small portion of the original uvrABbu gene lacking a domain necessary for function and an inserted kanamycin resistance gene (Fig. 1a). It was cloned into pGEM-T (a plasmid that cannot replicate in B. burgdorferi) to yield the suicide plasmid pBL12. After electroporation of pBL12 into low passage, infectious B. burgdorferi 297, multiple kanamycin-resistant clones were obtained; two were selected for genotyping. Genetic inactivation of uvrABbu in these clones was confirmed by PCR of genomic DNA using primers 12.1 and 12.4 (Supporting Information, Fig. S1a, compare lanes 1 and 2). Sequencing a 5.8-kb PCR fragment obtained with primers 12.5 (upstream gene BB0835) and 12.

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