The identification and partial characterization of a plasmid containing the gene for the membrane-associated...

Escherichia coli DNA was digested with restriction endonuclease PstI and ligated into the PstI site of plasmid pBR322. Recombinant plasmids that were constructed in this manner were used to transform E. coli H61, a mutant with a decreased level of hydrogenase activity. Complementation of this hydrogenase mutation identified a bacterial clone carrying the gene for the membrane-associated E. coli hydrogenase in plasmid pBL101. In E. coli minicells, the pBL101 DNA directed the synthesis of a protein of a size corresponding to that of the precursor of the E. coli membrane-associated hydrogenase, which appears to contain an uncleaved leader peptide. A restriction map of the cloned DNA was determined for 14 endonucleases.