Toward More Productive, Efficient, and Competitive Nitrogen-Fixing Symbiotic Bacteria

Abstract

The prospects of developing strains of legume nodule bacteria that provide higher productivity of leguminous plants are described. The generic, biochemical, physiological, regulatory, and economic constraints that govern the ability of private and public efforts to construct better inoculants for legume nodulation are discussed. Success in constructing better inoculants requires a two-pronged approach. First, strains need to be improved in order to compete successfully with indigenous strains for root nodulation of legumes. Several loci have been identified to date that affect competitiveness for strain nodule occupancy. Usually mutations in these loci affect the ability of a strain to form nodules rapidly and efficiently. Other loci, such as those that confer antibiotic production, can be added to strains to enhance nodulation competitiveness when co-inoculated with antibiotic-sensitive strains. Second, the inoculum strains must be improved with respect to symbiotic nitrogen fixation. Efforts to enhance the symbiotic productivity of legume nodule bacteria either by mutation or genetic engineering are also described. The best characterized example of these is the hydrogenase system. Due to nitrogenase-dependent catalysis of proton reduction, diazotrophs evolve large amounts of H,. An approach to maximize the efficiency of symbiotic N, fixation, and therefore of legume productivity, is to construct strains of Rhizobium with the ability to oxidize this otherwise wasted H,. The electrons produced by H, oxidation are funneled through energy-conserving electron transport chains. Our knowledge of the genetics and biochemistry of H, oxidation in Bradyrhizobium japonicum and Rhizobium leguminosarum has developed rapidly in recent years. At least 20 genes are needed for these bacteria to manufacture and efficiently express a nickel-containing H,-uptake hydrogenase. These genes include those encoding regulatory elements, posttranslational processing enzymes, nickel-sensing and nickel-metabolism proteins, and electron transport components for integrating the electrons from H, oxidation into the respiratory chain. Some of the components for oxidizing H, in the symbiotic N, fixing bacteria are distinct from the analogous components in (nonsymbiotic) H, oxidizing bacteria.

Hydrogenase genes from Rhizobium leguminosarum bv. viciae are controlled by the nitrogen fixation regulatory protein NifA

“…1 A). This is in contrast to the situation with other root-nodule bacteria, such as B. japonicum (5) and Azorhizobium caulinodans (40), where the hydrogenase system can be induced under appropriate culture conditions (low pO 2 , presence of H 2 and Ni 2ϩ ). Expression of R. leguminosarum hydrogenase structural genes (hupSL) in pea bacteroids is controlled from a promoter located upstream of hupS (hupS p ), which has the structure of a Ϫ24͞Ϫ12-type, RpoN-dependent promoter (31).…”

“…Since then, a cluster of genes containing, in addition to the structural genes (hupSL), an array of at least 16 genes coding for accessory proteins required for the synthesis of an active hydrogenase has been identified and sequenced in Bradyrhizobium japonicum and Rhizobium leguminosarum bv. viciae (4,5). Many of these genes are similar to those found in other H 2 -oxidizing bacteria such as Alcaligenes eutrophus, Rhodobacter capsulatus, Azotobacter vinelandii, and Escherichia coli (6,7).…”

Molecular and functional characterization of the ORS571 hydrogenase gene cluster

“…In the Rhizobium-legume symbiosis, hydrogen evolution is considered a major source of inefficiency [1]. Hydrogen oxidation by symbiotically expressed uptake hydrogenases has been postulated as a way to improve the efficiency of the nitrogen fixation process and enhance legume productivity [2].…”

Hydrogenase genes are uncommon and highly conserved in Rhizobium leguminosarum bv. viciae

“…Some rhizobia possess an uptake hydrogenase that allows them to recycle the hydrogen generated by nitrogenase. The potential of such a hydrogen uptake system to increase the energy efficiency of the symbiosis has been repeatedly emphasized [4][5][6] and clearly shown for the B. japonicum-soybean symbiosis [3,7].…”