Secondary antibody conjugated to horseradish

peroxidase was obtained from Bio-Rad. Visualisation was done by the enhanced chemiluminescent reaction (Stratagene). Non-denaturating PAGE was performed using 7.5% (w/v) polyacrylamide gels pH 8.5 and included 0.1% (w/v) Triton-X100 in the gels [14]. Samples (25 μg of protein) were incubated with 5% (v/v) Triton X-100 prior to application to the gels. Where indicated, the relative intensity Acalabrutinib of hydrogenase staining and protein amount from immunoblots was quantified using ImageJ from the National Institutes of Health [36]. Hydrogenase activity-staining was done as described in [14] except that the buffer used was 50 mM MOPS pH 7.0. Acknowledgements We are grateful to Nadine Taudte and Gregor Grass for supplying strains and the plasmid pFEO and to Frank Sargent for supplying anti-hydrogenase antisera. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG SA494/3-1). Electronic supplementary

material Additional file 1: Plasmid-encoded FeoB synthesis in MC4100 and PM06 ( feoB ::Tn 5 ). Extracts (25 μg protein in membrane sample buffer) from Selleck GDC-973 MC4100 and PM06, transformed with pECD1079 bearing feoB and pFEO bearing the whole feo operon, both cloned behind a tetracycline promotor and encoding an N-terminal StrepII-tag on FeoB encoded on pECD1079 were separated by SDS-PAGE (10% w/v polyacrylamide) and after transfer to nitrocellulose detected by incubation with Strep-tactin conjugated to horseradish peroxidase. Strains were grown either with or without aeration in TGYEP, pH 6.5 and

gene expression was induced with 0.2 μg ml-1 AHT (anhydrotetracycline) as indicated. Biotin carboxyl carrier protein (BCCP) served as a loading control. The sizes of the protein standards are shown on the right side of the gel. The angled arrow indicates the position of the Strep-FeoB polypeptide. Extracts Amino acid derived from MC4100 and PM06 transformed with pFEO did not synthesize Strep-tagged FeoB and therefore acted as a negative control. (TIFF 371 KB) References 1. Vignais P, Billoud B: Occurrence, classification, and biological function of hydrogenases: an overview. Chem Rev 2007, 4206–4272. 2. Forzi L, Sawers RG: Maturation of [NiFe]-hydrogenases in Escherichia coli . Biometals 2007, 20:565–578.PubMedCrossRef 3. Pinske C, Krüger S, Soboh B, Ihling C, Kuhns M, Braussemann M, Jaroschinsky M, Sauer C, Sargent F, Sinz A, Sawers RG: Efficient electron transfer from hydrogen to benzyl viologen by the [NiFe]-hydrogenases of Escherichia coli is dependent on the coexpression of the iron-sulfur cluster-containing small subunit. Arch Microbiol 2011, in press. 4. Lukey MJ, Parkin A, Roessler MM, Murphy BJ, Harmer J, Palmer T, Sargent F, Armstrong FA: How Escherichia coli is equipped to oxidize hydrogen under different redox conditions. J Biol Chem 2010, 285:3928–3938.PubMedCrossRef 5. Böck A, King P, Blokesch M, Posewitz M: Maturation of hydrogenases. Adv Microb Physiol 2006, 51:1–71.