Moreover, by substituting BV/TTC ACY-1215 in vitro with nitroblue tetrazolium as an electron acceptor we could demonstrate that only the oxygen-tolerant Hyd-1 enzyme could catalyse hydrogen-dependent dye reduction, suggesting that this facile assay could be used to identify oxygen-tolerant hydrogenases in other microorganisms. However, the ability of Hyd-1 to reduce NBT was not dependent on the oxygen-tolerance of the enzyme because an oxygen-sensitive Hyd-1 variant in which the supernumerary Cys-19 was substituted by Gly retained the ability to reduce the redox dye. Methods Strains and growth conditions All strains used in this study are listed in Table 1. E. coli strains were

routinely grown at 37°C on LB-agar plates or with shaking in LB-broth [48]. Plates were solidified by adding 1.5% (w/v) agar to the media. Anaerobic growths were performed

at 37°C as standing liquid cultures. Cultures for determination of enzyme activity were grown in TGYEP media [49] containing 1% (w/v) peptone, 0.5% (w/v) yeast extract, 0.1 M potassium buffer pH 6.5 and the cultures were supplemented with 0.8% (w/v) of glucose. When required, the antibiotics kanamycin and chloramphenicol were added to the culture media to the final concentration of 50 μg and 12 μg per ml, respectively. The strains CPD17, CPD23 and CPD24 were constructed using P1kc phage transduction to move the respective defined deletion mutation from the appropriate strains obtained from the Keio collection [48, find more 50]. When required the plasmid pCP20 was used to remove the antibiotic resistance cassette as described [51]. Polyacrylamide gel electrophoresis Non-denaturing

Lumacaftor PAGE was performed using a discontinuous system with 7.5% (w/v) polyacrylamide separating gels in 250 mM Tris/HCl buffer, pH 8.5 including 0.1% (w/v) Triton X-100 [18]. As running buffer 0.1 M Tris/0.1 M glycine buffer was used. After reaching mid-exponential phase of growth cells were harvested from cultures by centrifugation at 10,000 x g for 15 min at 4 °C and after washing once in the same volume of 50 mM MOPS buffer pH 7.0, cells were resuspended in a tenth of their volume of 50 mM MOPS buffer pH 7.0, broken by sonification and cell debris and unbroken cells removed as described [20]. Samples of crude extract were resuspended at a protein concentration of 10 mg ml-1 in 50 mM MOPS buffer pH 7.0 and incubated with a final concentration of 5% (w/v) Triton X-100 prior to application of the solubilized sample (usually 25 μg of protein) to the gels. Alternatively, for neutral pH analyses the barbitone gel system was used. This system uses final concentrations of 34 mM Tris-phosphate buffered stacking gel, pH 5.5 and 62.5 mM Tris-HCl resolving gel pH 7.5. The running buffer consists of 82.5 mM Tris and 26.