Intra- and interspecies transfer and expression of Rhizobium japonicum hydrogen uptake genes and autotrophic growth capability

Abstract

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...

Roles of HoxX and HoxA in biosynthesis of hydrogenase in Bradyrhizobium japonicum

“…These genes are in an area of the hup gene cluster previously known to be necessary for hydrogenase activity under free-living conditions (18) and have been proposed, based on homology to regulatory elements, to encode a two-component regulatory system that affects the transcription of hydrogenase (35). Bacterial twocomponent regulatory systems form a superfamily, consisting of (i) a family of sensor proteins, all of which have homology in a carboxy-terminal transmitter domain, and (ii) a family of regulator proteins, all of which have homology in an amino-terminal receiver domain (10).…”

Plant gene expression in early stages of Rhizobium-legume symbiosis

“…Prior to derepression for hydrogenase activity cells were grown on plates containing H2 uptake medium (Maier ef a/., 1978, with modifications of Cantrell ef a/., 1983). The cultures were derepressed for 10 days in a gas atmosphere maintained at 89.5% N2, 5% H2, 5% CO2, and 0.5% O2 (Lambert et al, 1985).…”

Cloning and characterization of hydrogen uptake genes from Rhizobium leguminosarum

“…Cosmid pHU1 was shown by site-directed TnS insertion mutagenesis to contain at least 15 kilobase pairs (kb) of hup-specific DNA (17), but not all the B. japonicum Hup-mutants were complemented by this cosmid. Additional hup genes are present on cosmid pHU52 which apparently encodes all essential hup determinants to confer hydrogenase activity to wild-type Hup-strains of B. japonicum and R. leguminosarum (20). Both cosmids pHU1 * Corresponding author.…”

“…As can be seen, the relative order of the R. leguminosarum presumptive hup genes is the same as that of B. japonicum hup genes. When the 7.2-kb EcoRI DNA fragment from pAL704 was used as a probe to hybridize EcoRI-restricted DNA from pHU1 and pHU52, no hybridization signals were observed to the unique 5.5-kb EcoRI fragment of pHU52, reported to be essential for autotrophic growth in B. japonicum (20).…”