Molecular cloning of the hydrogen oxidizing genes of Alcaligenes eutrophus strain H1

Hydrogen evolution by the enzyme nitrogenase during nitrogen fixation is considered to cause higher energy requirements for nitrogen fixation. This loss of energy may indirectly inhibit plant growth during symbiotic nitrogen fixation between leguminous plants and Rhizobium. The loss could conceivably be reversed by introduction of a hydrogenase enzyme into hydrogen-evolving Rhizobium to recycle the lost energy. The unique physiological and biochemical characteristics of the hydrogenase system of Alcaligenes eutrophus provides a potential source of hydrogen oxidizing genes for introduction into Rhizobium. The genes for hydrogen oxidation have been identified to be coded on a 200 Mdal plasmid in several A. eutrophus strains. The goal of this study was to map and isolate plasmid-encoded genes involved in hydrogen oxidation in A. eutrophus and also to determine if these hydrogen-oxidizing genes will function in other bacteria;The identification and isolation of hydrogen-oxidizing genes was achieved by Tn5-mutagenesis and genetic complementation by using a total genomic DNA library constructed in a broad-host-range vector. The cloned fragment of DNA carrying the hydrogenase genes was identified, but this fragment alone was not functional in Rhizobium;During the search for the hydrogenase gene clone, several unique phenomena were observed. These include (1) transposition of endogenous insertion element during Tn5 mutagenesis, (2) high frequency of plasmid curing during Tn5 mutagenesis, and (3) a plasmid region that seemed to be highly sensitive to mutagenesis treatment and consequently resulted in plasmid deletion. In addition, the 200 Mdal plasmid also demonstrated a high degree of homology with its chromosomal counterpart