Symbiotic legume nodules employ both rhizobial exo- and endo-hydrogenases to recycle hydrogen produced by nitrogen fixation.

BACKGROUND: In symbiotic legume nodules, endosymbiotic rhizobia (bacteroids) fix atmospheric N(2), an ATP-dependent catalytic process yielding stoichiometric ammonium and hydrogen gas (H(2)). While in most legume nodules this H(2) is quantitatively evolved, which loss drains metabolic energy, certain bacteroid strains employ uptake hydrogenase activity and thus evolve little or no H(2). Rather, endogenous H(2) is efficiently respired at the expense of O(2), driving oxidative phosphorylation, recouping ATP used for H(2) production, and increasing the efficiency of symbiotic nodule N(2) fixation. In many ensuing investigations since its discovery as a physiological process, bacteroid uptake hydrogenase activity has been presumed a single entity. METHODOLOGY/PRINCIPAL FINDINGS: Azorhizobium caulinodans, the nodule endosymbiont of Sesbania rostrata stems and roots, possesses both orthodox respiratory (exo-)hydrogenase and novel (endo-)hydrogenase activities. These two respiratory hydrogenases are structurally quite distinct and encoded by disparate, unlinked gene-sets. As shown here, in S. rostrata symbiotic nodules, haploid A. caulinodans bacteroids carrying single knockout alleles in either exo- or-endo-hydrogenase structural genes, like the wild-type parent, evolve no detectable H(2) and thus are fully competent for endogenous H(2) recycling. Whereas, nodules formed with A. caulinodans exo-, endo-hydrogenase double-mutants evolve endogenous H(2) quantitatively and thus suffer complete loss of H(2) recycling capability. More generally, from bioinformatic analyses, diazotrophic microaerophiles, including rhizobia, which respire H(2) may carry both exo- and endo-hydrogenase gene-sets. CONCLUSIONS/SIGNIFICANCE: In symbiotic S. rostrata nodules, A. caulinodans bacteroids can use either respiratory hydrogenase to recycle endogenous H(2) produced by N(2) fixation. Thus, H(2) recycling by symbiotic legume nodules may involve multiple respiratory hydrogenases.

Rhizobial factors required for stem nodule maturation and maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 symbiosis.

The molecular and physiological mechanisms behind the maturation and maintenance of N(2)-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N(2)-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N(2) fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C(4) metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.

Azorhizobium doebereinerae sp. Nov. Microsymbiont of Sesbania virgata (Caz.) Pers.

Thirty-four rhizobium strains were isolated from root nodules of the fast-growing woody native species Sesbania virgata in different regions of southeast Brazil (Minas Gerais and Rio de Janeiro States). These isolates had cultural characteristics on YMA quite similar to Azorhizobium caulinodans (alkalinization, scant extracellular polysaccharide production, fast or intermediate growth rate). They exhibited a high similarity of phenotypic and genotypic characteristics among themselves and to a lesser extent with A. caulinodans. DNA:DNA hybridization and 16SrRNA sequences support their inclusion in the genus Azorhizobium, but not in the species A. caulinodans. The name A. doebereinerae is proposed, with isolate UFLA1-100 (=BR5401, =LMG9993=SEMIA 6401) as the type strain.

Evaluation of nutrient limitation in aquatic ecosystems with nitrogen fixing bacteria.

There has always been a great need for simple and accurate bioassays for evaluating nutrient limitation in aquatic ecosystems. Whereas organic carbon is usually considered to be the limiting nutrient for microbial growth in many aquatic ecosystems, there are, however, many water sources that are limited by phosphorus or nitrogen. A method named "nitrogen fixing bacterial growth potential" (NFBGP) test, which is based on pre-culturing of autochthonous (target) microorganisms was described. The method was applied to evaluate phosphorus or nitrogen nutrient limitation in lake and sewage water samples using an isolate of the nitrogen fixing bacterium, Azorhizobium sp. WS6. The results corresponded well to those from the traditional algal growth potential (AGP) test and the bacterial regrowth potential (BRP) test, suggesting that the NFBGP test is a useful supplementary method for evaluating the limiting nutrient, especially phosphorus, in an aquatic environment.