Cloning of the gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough) and determination of the NH 2 ‐terminal sequence

The gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough) has been cloned in Escherichia coli. D. vulgaris DNA was digested with the restriction endonucleases Eco RI and Sal I and ligated into the vector pUC9 [Vieira, J. & Messing, J. (1982) Gene 19 , 259–268], which had been cut with these same enzymes. Approximately 9000 recombinant clones were obtained by transformation of E. coli JM101 followed by growth on rich plates with ampicillin for selection and isopropyl‐β‐D‐thiogalactoside and 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside present for detection of recombinants. The recombinant clones were then screened for production of immunoreactive proteins with rabbit antisera against purified hydrogenase and 125 I‐labelled protein A. 28 positive clones were found in this initial screening. These were further tested in an immunocompetition experiment, which showed that the protein product from one clone behaved identically to purified hydrogenase. The plasmid pHV 15 isolated from this clone has a 4.7 × 10 3 ‐base‐pair Sal I/ Eco RI insert. Cells of E. coli JM 101 transformed with pHV 15 produce a hydrogenase polypeptide of molecular mass 46 kDa as detected by Western blotting. The mass, as well as the Cleveland mapping pattern of the polypeptide produced by E. coli , are identical with those of the hydrogenase isolated from D. vulgaris (Hildenborough). Southern blotting of restriction‐enzyme‐digested D. vulgaris DNA, using the nick‐translated 4.7 × 10 3 ‐base‐pair Sal I/ Eco RI fragment as a probe, indicates the presence of a single gene with an internal Pst I site. The NH 2 ‐terminal sequence of the hydrogenase was determined to be: Ser 1 ‐Arg‐Thr‐Val‐Met 5 ‐Glu‐Arg‐Ile‐Glu‐Tyr 10 ‐Glu‐ Met‐His‐Thr‐Pro 15 ‐Asp‐Pro‐Lys‐Ala‐Asp 20 . This information should allow an unambiguous identification of the hydrogenase gene.