Cosmids containing hydrogen uptake genes have previously been isolated in this laboratory. Four new cosmids that contain additional hup gene(s) have now been identified by conjugal transfer of a Rhizobium japonicum 122DES gene bank into a Tn5-generated Hup-mutant and screening for the acquisition ofHup activity. The newly isolated cosmids, pHU50-pHU53, contain part of the previously isolated pHU1 but extend as far as 20 kilobases beyond its border. pHU52 complements five of six Hup-mutants and confers activity on several Hup y wild-tpe R. japonicum strains in the free-living state and where tested in nodules. Transconjugants obtained from interspecies transfer of pHU52 to Rhizobium meliloti 102F28, 102F32, and 102F51 and Rhizobium leguminosarum 128C53 showed hydrogen-dependent methyleneblue reduction, performed the oxyhydrogen reaction, and showed hydrogen-dependent autotrophic growth by virtue of the introduced genes. The identity of the presumptive transconjugants was confirmed by antibiotic-resistance profiles and by plant modulation tests.Hydrogen evolution that accompanies dinitrogen reduction results in an inefficient use of the energy needed for nitrogen fixation. Most N2-fixing free-living microorganisms are able to oxidize the H2 produced by the nitrogenase reaction and recapture some of the energy lost through H2 evolution (1, 2). Recent experiments in which soybeans were inoculated with otherwise isogenic Hup+ and Hup-Rhizobium japonicum strains indicate a significant increase in plant nitrogen content and total weight when the plants were grown to maturity before harvesting.t However, only a minority of R. japonicum strains possess an active H2 uptake system (3). Highly active H2 uptake systems are lacking in Rhizobium meliloti and most other fast-growing rhizobia. A symbiotic plasmid possessing hydrogen uptake genes has been transferred from Rhizobium leguminosarum to R. meliloti (4, t).Cantrell et al. (5) constructed a gene bank of R. japonicum strain 122DES, from which overlapping cosmids containing hydrogen uptake genes were isolated by complementation of the Hup-point mutant PJ17 by using a colony assay (6). However, several observations indicated that more hupspecific DNA remained to be isolated (7).Since hydrogen recycling may be a beneficial trait, it is desirable to transfer hydrogen uptake (hup) genes from R, japonicum to other Hup-rhizobia. Here, we report that a cosmid (pHU52) apparently encoding all essential Hup determinants has been isolated. This cosmid confers Hup activity and autotrophic growth capability to Hup-R. japonicum strains, R. meliloti, and R. leguminosarum. MATERIALS AND METHODSBacterial Strains, Growth Media, and Growth Conditions. Strains of Escherichia coli and R. japonicum used in this study are listed elsewhere (5, 8). E. coli was grown in LB medium (9); rhizobial strains were routinely grown in yeast extract mannitol medium (8). Rhizobia were cultured chemolithotrophically (10, 11) on Repaske's liquid medium (12, 13) with 1.5% Noble agar added for plates. Pr...

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A method was devised that allows measurement in vivo of hydrogenase-catalysed H2 evolution from the cyanobacterium Anabaena cylindrica, independent of nitrogenase activity, which is also present. Addition of low concentrations of reduced Methyl Viologen (1-10mM) to intact heterocystous filaments of the organism resulted in H2 evolution, but produced conditions giving total inhibition of nitrogenase (acetylene-reducing and H2-evolving) activity. That the H2 formed under these conditions was not contributed to by nitrogenase was also supported by the observation that its rate of formation was similar in the dark or with Ar replaced by N2 in the gas phase, and also in view of the pattern of H2 evolution at very low Methyl Viologen concentrations. Conclusive evidence that the H2 formed in the presence of Methyl Viologen was solely hydrogenase-mediated was its evolution even from nitrogenase-free (non-heterocystous) cultures; by contrast 'uptake' hydrogenase activity in such cultures was greatly decreased. The hydrogenase activity was inhibited by CO and little affected by acetylene. Finally the hydrogenase activity was shown to be relatively constant at different stages during the batch growth of the organism, as opposed to nitrogenase activity, which varied.

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