Symbiotic Expression of Cosmid-Borne Bradyrhizobium japonicum Hydrogenase Genes

Abstract

The expression of cosmid-borne Bradyrhizobium japonicum hydrogenase genes in alfalfa, clover, and soybean nodules harboring Rhizobium transconjugants was studied. Cosmid pHU52 conferred hydrogen uptake (Hup) activity in both free-living bacteria and in nodules on the different plant hosts, although in nodules the instability of the cosmid resulted in low levels of Hup activity. In contrast, cosmid pHU1, which does not confer Hup activity on free-living bacteria, gave a Hup + phenotype in nodules on alfalfa and soybean. Nodules formed by B. japonicum USDA 123Spc(pHU1) recycled about 90% of nitrogenase-mediated hydrogen evolution. Both subunits of hydrogenase (30- and 60-kilodalton polypeptides) were detected in enzyme-linked immunosorbent assays of bacteroid preparations from nodules harboring B. japonicum strains with pHU1 or pHU52. Neither pHU53 nor pLAFR1 conferred detectable Hup activity in either nodules or free-living bacteria. Based on the physical maps of pHU1 and pHU52, it is suggested that a 5.5-kilobase Eco RI fragment unique to pHU52 contains a gene or part of a gene required for Hup activity in free-living bacteria but not in nodules. This conclusion is supported by the observation that two Tn 5 insertions in the chromosome of B. japonicum USDA 122DES obtained by marker exchange with Tn 5 -mutagenized pHU1 abolished Hup activity in free-living bacteria but not in nodules.

Construction of a Symbiotically Effective Strain of Rhizobium leguminosarum bv. trifolii with Increased Nodulation Competitiveness

“…Isolation of nodulation competitiveness genes from a Rhizobium strain by cosmid cloning and their subsequent conjugal transfer to superior Rhizobium strains are insufficient to provide a useful inoculum for legume crops. Recombinant plasmids based on pLAFR3, such as pTFX1, are very unstable in the absence of selection pressure (11,13) (Fig. 4).…”

“…Thus, the tfx genes are probably chromosomal in the TA1::tfx exconjugants. In any case, data are presented here which illustrate the stability of trifolitoxin production in TA1:: [10][11][12][13][14][15] (Fig. 3).…”

“…However, recombinant plasmids based on pLAFR1 or pLAFR3, such as pTFX1, are commonly unstable in the absence of selection pressure (13,15). Thus, Rhizobium transconjugants with pTFX1 are not likely to be able to limit nodulation by trifolitoxin-sensitive indigenous strains of Rhizobium under agricultural conditions where tetracycline application is impractical.…”

Sequences and characterization of hupU and hupV genes of Bradyrhizobium japonicum encoding a possible nickel-sensing complex involved in hydrogenase expression

“…Bradyrhizobium japonicum, the nitrogen-fixing symbiont of soybean, contains a [Ni-Fe] hydrogenase composed of two subunits, a 33-kDa subunit and a 65-kDa subunit (1,26), encoded by the hupSL genes (24). In addition to the structural genes, some other loci on the hydrogenase gene cluster of B. japonicum have been found to be involved in hydrogenase processing (9), nickel metabolism (7), and regulation (16,17,19). B. japonicum SR139, a Hup-(hydrogen-uptake-deficient) mutant, has been characterized and localized to a region between less than 0.5 kb and 2.5 kb upstream from the transcriptional start site of the small subunit of hydrogenase (22).…”

Greenhouse and Field Evaluations of an Autoselective System Based on an Essential Thymidylate Synthase Gene for Improved Maintenance of Plasmid Vectors in Modified Rhizobium meliloti

“…Protocols to facilitate the development of plasmid-cured derivatives of rhizobia have been proposed (1,15), but curing of plasmids remains a challenging task (15,21). On the other hand, maintenance and stability of exogenous natural plasmids or recombinant plasmids introduced into Rhizobium meliloti or other bacteria are frequently poor in the absence of selective pressure (8,10,17,18,27,32). It is thought that plasmids that do not contribute to the fitness of the host can become lost from a population because the metabolic cost of their maintenance may decrease the specific growth rate of cells (14).…”