Signals were detected with a 489-bp PCR product of the gls24 gene, obtained with primers fm20 (5′-GCAACTGCAGAGCCCCAGCAAAAGATCC) and fm21 (5′-GAGCTCTCGAGTGCTCAATTGCTGATTTGGC) and a 323-bp PCR product of orf1 obtained with primers sm45 (5′-GTCATCGATCCAGGTCAAAC) and sm46 (5′- ATCGACGGCGATTCATTTCC). PCR fragments were labeled and detected using the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche, Basel, Switzerland) according to the instructions of the manufacturer. Enterococcus hirae ATCC9790 was grown semi-anaerobically in capped, but not deoxygenated,
tubes at 37 °C in M17 medium (Terzaghi & Sandine, 1975). Mid-log cultures were RO4929097 cost induced as indicated under Results and discussion for 1 h at 37 °C. From 1 mL of culture, RNA was isolated with the Qiagen RNeasy miniprep column kit (Qiagen, Germantown, MD). Quantitative CB-839 clinical trial PCR was performed with the QuantiTect SYBR Green I PCR and RT-PCR kits (Qiagen), using 100 ng of RNA per reaction in a total volume of 20 μL in a LightCycler (Roche)
and primers js7 (5′-GGTGATGTGACATATGAAGATAAGG) and js8 (5′-CAACATCGACATTGACTTCAATGAC). Cycle conditions were as follows: 45 cycles each of 55 °C for 30 s, 72 °C for 30 s, and 95 °C for 1 s. Expression levels were normalized to 16S rRNA levels. Enterococcus hirae 2-mL cultures in M17 media were grown to an OD546 nm of 0.3–0.5 and induced as described under Results and discussion. Pellets were incubated with 50 μL of 10 mg mL−1
lysozyme in 1 mM EDTA, 10 mM Tris-Cl, pH 8, for 30 min at 25 °C, followed by a freeze–thaw cycle. Ten microliters of 1 mg mL−1 DNaseI in 100 mM MgCl2 were added and incubation was continued for 10 min at 25 °C. Cell debris was removed by centrifugation for 5 min at 12 000 g. Protein concentrations in the supernatants were determined using the BioRad protein assay (BioRad, Richmond) and 40 μg of protein/lane was used for Western blotting as described (Towbin et al., 1979). Gls24 antiserum was kindly provided Clomifene by Barbara E. Murray, University of Texas (Teng et al., 2005). The IAsys instrument (Affinity Sensors, Cambridge) was used to measure the binding of CopZ to Gls24. Purified Gls24 was desalted by dialysis against 50 mM Na-HEPES, pH 7.5. A dual-well carboxymethyl dextran cuvette was equilibrated with phosphate-buffered saline, pH 7.4, 0.05% Tween-20, 2% acetonitrile, and 20 μg of Gls24 cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. CopZ was added at 1–10 μM and the interactions were measured at 25 °C, with the vibro-stirrer set to 85. Coupling, washing, and calibration steps were performed according to the manufacturer’s instructions. The results were evaluated using the grafit software version 5. CD spectra were recorded on a JASCO J-715 instrument using a quartz cuvette with a light path of 1 mm. The temperature was controlled with a JASCO PTC-348WI Peltier cell.