Symbiotic Expression ofCosmid-Borne Bradyrhizobium japonicum Hydrogenase Genest

hydrogenase genesinalfalfa, clover, andsoybean nodules harboring Rhizobium transconjugants was studied. CosmidpHU52conferred hydrogen uptake (Hup) activity inbothfree-living bacteria andinnodules on thedifferent plant hosts, although innodulesthe instability ofthecosmid resulted inlowlevels ofHupactivity. Incontrast, cosmidpHUI,whichdoesnotconfer Hupactivity on free-living bacteria, gaveaHup+phenotype innodules on alfalfa andsoybean. Nodules formed byB.japonicum USDA123Spc(pHU1) recycled about90%ofnitrogenase-mediated hydrogen evolution. Both subunits ofhydrogenase (30- and60-kilodalton polypeptides) were detected inenzyme-linked immunosorbent assaysofbacteroid preparations fromnodules harboring B.japonicum strains withpHU1or pHU52.Neither pHU53nor pLAFRIconferred detectable Hupactivity ineither nodules orfree-living bacteria. Basedon the physical maps ofpHU1andpHU52,itissuggested that a 5.5-kilobase EcoRIfragment unique topHU52 contains a gene or partofa generequired forHupactivity infree-living bacteria butnotinnodules. This conclusion issupported bytheobservation thattwoTn5insertions inthechromosome ofB.japonicum USDA 122DESobtained bymarkerexchange withTnS-mutagenized pHUlabolished Hupactivity infree-living bacteria butnotinnodules. Thehydrogen recycling capability ofsome Rhizobium strains hasattracted attention because ofitsrelationship to theefficiency ofnitrogen fixation, whichhasbeendiscussed theoretically (9,30)andforwhichexperimental evidence, obtained by usingotherwise isogenic hydrogen-uptakepositive (Hup+)andHup- Bradyrhizobium japonicum strains inplant tests,hasbeenpresented (10). Genetic studies havefocused on thetransfer ofhydrogen uptake (hup) genestothemajority ofRhizobium species whichlack Hup activity. Inthefast-growing species Rhizobium leguminosarum, hydrogenase genes havebeentransferred along withnodulation ability on a large plasmid (4,7).With theslow-growing species B.japonicum, however, several Hup+strains, including USDA 122DES,lacked detectable plasmid DNA (6). Consequently, overlapping Hupcosmids were isolated froma B.japonicumUSDA 122DESgene bank(5)bycomplementation ofHup-mutants(23). H2 uptake cosmids havealsobeenisolated fromaB.japonicum USDA 110gene bank(17). Several observations suggested thatpHUl,one ofthe cosmids isolated byCantrell etal.(5), contains some butnot allofthehupgenes necessaryforfree-living expression of Hupactivity (14). Accordingly, several more overlapping cosmidswere recently isolated by transferring theB. japonicum genebankinto aTn5-generated Hup-mutantand screening fortheacquisition ofhydrogen-dependent methy