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.     

Plant gene expression during effective and ineffective nodule development of the tropical stem-nodulated legume Sesbania rostrata

The expression of plant genes during symbiosis of Sesbania rostrata with Rhizobium sp. and Azorhizobium caulinodans was studied by comparing two-dimensional PAGE patterns of in vitro translation products of poly(A)⁺ RNA from uninfected roots and stems with that of root and stem nodules. Both types of nodules are essentially similar, particularly when stem nodules are formed in the dark. We detected the specific expression of at least 16 genes in stem and root nodules and observed the stimulated expression of about 10 other genes in both nodules. Six of the nodule-specific translation products (apparent molecular masses around 16 kDa) cross-react with an antiserum raised against leghemoglobin purified from Sesbania rostrata stem nodules. During stem nodule development, most of the nodule-stimulated genes are expressed concomitantly with leghemoglobin at day 12 after inoculation. However, some genes are already stimulated at days 6–7, some others later in development (day 18), and some are transiently activated. Patterns of root nodules induced by either Azorhizobium caulinodans strain ORS571, capable of effective root and stem nodulation, or Rhizobium sp. strain ORS51, capable of effective root nodulation only, are very similar except for a specific 37.5 kDa polypeptide. Several types of ineffective stem and root nodules were studied; in every case the amount of leghemoglobin components appeared reduced together with most of the nodule-stimulated polypeptides.     

Identification and cloning of nodulation genes from the stem-nodulating bacterium ORS571

Summary After random Tn5 mutagenesis of the stem-nodulating Sesbania rostrata symbiont strain ORS571, Nif^-, Fix^- and Nod^- mutants were isolated. The Nif^- mutants had lost both free-living and symbiotic N₂ fixation capacity. The Fix^- mutants normally fixed N₂ in the free-living state but induced ineffective nodules on S. rostrata. They were defective in functions exclusively required for symbiotic N₂ fixation. A further analysis of the Nod^- mutants allowed the identification of two nod loci. A Tn5 insertion in nod locus 1 completely abolished both root and stem nodulation capacity. Root hair curling, which is an initial event in S. rostrata root nodulation, was no longer observed. A 400 bp region showing weak homology to the nodC gene of Rhizobium meliloti was located 1.5 kb away from this nod Tn5 insertion. A Tn5 insertion in nod locus 2 caused the loss of stem and root nodulation capacity but root hair curling still occurred. The physical maps of a 20.5 kb DNA region of nod locus 1 and of a 40 kb DNA region of nod locus 2 showed no overlaps. The two nod loci are not closely linked to nif locus 1, containing the structural genes for the nitrogenase complex (Elmerich et al. 1982).     

Characterization of the fixABC region of Azorhizobium caulinodans ORS571 and identification of a new nitrogen fixation gene

Summary The fast growing strain, Azorhizobium caulinodans ORS571, isolated from stem nodules of the tropical legume Sesbania rostrata, can grow in the free-living state at the expense of molecular nitrogen. Five point mutants impaired in nitrogen fixation in the free-living state have been complemented by a plasmid containing the cloned fix-ABC region of strain ORS571. Genetic analysis of the mutants showed that one was impaired in fixC, one in fixA and the three others in a new gene, located upstream from fixA and designated nifO. Site-directed Tn5 mutagenesis was performed to obtain Tn5 insertions in fixB and fixC. The four genes are required for nitrogen fixation both in the free-living state and under symbiotic conditions. The nucleotide sequence of nifO was established. The gene is transcribed independently of fixA and does not correspond to fixX, recently identified in Rhizobium meliloti and R. leguminosarum. Biochemical analysis of the five point mutants showed that they synthesized normal amounts of nitrogenase components. It is unlikely that fixA, fixC and nifO are involved in electron transport to nitrogenase. FixC could be required for the formation of a functional nitrogenase component 2.     

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 N₂, an ATP-dependent catalytic process yielding stoichiometric ammonium and hydrogen gas (H₂). While in most legume nodules this H₂ is quantitatively evolved, which loss drains metabolic energy, certain bacteroid strains employ uptake hydrogenase activity and thus evolve little or no H₂. Rather, endogenous H₂ is efficiently respired at the expense of O₂, driving oxidative phosphorylation, recouping ATP used for H₂ production, and increasing the efficiency of symbiotic nodule N₂ 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 H₂ and thus are fully competent for endogenous H₂ recycling. Whereas, nodules formed with A. caulinodans exo-, endo-hydrogenase double-mutants evolve endogenous H₂ quantitatively and thus suffer complete loss of H₂ recycling capability. More generally, from bioinformatic analyses, diazotrophic microaerophiles, including rhizobia, which respire H₂ 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 H₂ produced by N₂ fixation. Thus, H₂ recycling by symbiotic legume nodules may involve multiple respiratory hydrogenases.     

Physiological comparisons of root and stem nodules of Aeschynomene scabra and Sesbania rostrata

A few legume species possess the ability to form N₂-fixing nodules on stems as well as on roots. Little is known of the functional characteristics of stem nodules, or to what extent they differ from root nodules. Stem and root nodules of greenhouse-grown plants of Aeschynomene scabra (inoculated with the photosynthetic rhizobial strain BTAi 1) and Sesbania rostrata (inoculated with Azorhizobium caulinodans strain BTSr 3) were examined for assimilation of ¹⁴CO₂ in the light and dark, soluble carbohydrate and starch contents, acetylene reduction activity, relative efficiency of nitrogenase in terms of uptake-hydrogenase activity, glutamine synthetase and glutamate synthase, and reduced N and ureide contents. In general, stem nodules possessed higher enzyme activities and metabolite contents than did root nodules, suggesting that they fix N₂ with greater energy efficiency. This greater efficiency correlated with photosynthesis in the cortex of stem nodules. Differences in enzyme activities and metabolite contents between the stem nodules on A. scabra and those on S. rostrata probably result either from legume-species characteristics or from the photosynthetic capability of strain BTAi 1.     

Free-Living Rhizobium Strain Able To Grow on N2 as the Sole Nitrogen Source

A Rhizobium strain isolated from stem nodules of the legume Sesbania rostrata was shown to grow on atmospheric nitrogen (N₂) as the sole nitrogen source. Non-N₂-fixing mutants isolated directly on agar plates formed nodules that did not fix N₂ when inoculated into the host plant.     

Variations in Outer-membrane Characteristics of Two Stem-nodulating Bacteria of <Emphasis Type="Italic">Sesbania rostrata</Emphasis> and its Role in Tolerance Towards Diverse Stress

Outer-membrane characteristics may determine the survivability of rhizobia under diverse abiotic and biotic stresses. Therefore, the role of lipopolysaccharides (LPS) and membrane proteins of two stem-nodulating bacteria of Sesbania rostrata (Azorhizobium caulinodans ORS571 and Rhizobium sp. WE7) in determining tolerance towards abiotic and biotic stresses (hydrophobics and phages) was investigated. Outer-membrane characteristics (LPS and membrane–protein profiles) of ORS571, WE7 and thirteen standard strains were distinct. ORS571 and WE7 also showed susceptibility towards morphologically distinct phages, i.e., ACSR16 (short-tailed) and WESR29 (long-tailed), respectively. ORS571 and WE7 were tolerant to hydrophobic compounds (triton X-100, rifampicin, crystal violet and deoxycholate). To ascertain the role of outer membrane characteristics in stress tolerance, phage-resistant transconjugant mutants of ORS571 (ORS571-M8 and ORS571-M20) and WE7 (WE7-M9) were developed. LPS- and membrane–protein profiles of mutants differed from that of respective wild types (ORS571 and WE7). In in vitro assay, phages got adsorbed onto purified LPS-membrane protein fractions of wild types. Phages did not adsorb onto membrane fraction of mutants and standard strains. Mutant with reduced expression of LPS (ORS571-M20 and WE7-M9) showed reduced tolerance towards hydrophobics. However, the tolerance was unaffected in mutant (ORS571-M8) where expression of LPS was not reduced but pattern was different. The tolerance level of mutants towards hydrophobics varied with the expression of LPS, whereas the specificity towards phages is correlated with the specific LPS pattern.     

Cloning and characterization of nifA and ntrC genes of the stem nodulating bacterium ORS571, the nitrogen fixing symbiont of Sesbania rostrata: Regulation of nitrogen fixation (nif) genes in the free living versus symbiotic state

Summary A cosmid bank of ORS571, a diazotrophic bacterium capable of inducing aerial stem and root nodules on Sesbania rostrata, was constructed in the vector pLAFR1. A DNA probe carrying the Klebsiella pneumoniae nifA gene was used to identify nifA-and ntrC-like regions of ORS571 in the cosmid bank by colony hybridization. Cosmids carrying these regions were mapped by restriction endonuclease analysis, Southern blotting and transposon Tn5 mutagenesis. Selected Tn5 insertion mutations in the nifA/ntrC homologous regions were used for gene-replacement experiments and the resulting ORS571 mutants were examined for Nif, Fix and Ntr phenotypes. Two clearly distinct regulatory loci were thus identified and named nifA and ntrC. Plasmids carrying gene fusions of the ORS571 nifH and nifD genes to lacZ were constructed and the regulation of the ORS571 nifHDK promoter, and of the Rhizobium meliloti nifHDK promoter, was studied under varying physiological conditions in ORS571, ORS571 nifA::Tn5 and ORS571 nitrC::Tn5 strains. A model for the role of nifA and ntrC in the regulation of ORS571 nif and other nitrogen assimilation genes is proposed.     

Growth of Sesbania rostrata (Brem) with stem nodules under controlled conditions

A series of experiments was carried out in an attempt to produce nodulated plants of Sesbania rostrata with qualities more closely resembling those in the wild than has been achieved to date. When groups of five plants were grown in a controlled climate chamber in pipes containing ～12dm³ modified Jensen's medium with 6mol m^?3 nitrate, the daily growth in height reached 5 cm and at 30 d the plants were ～40cm high. At this time, the stems were inoculated with Azorhizobium caulinodans ORS 571 and the medium replaced with Jensen's medium without nitrate. In the subsequent 19-d period ～300 nodules (representing >50% of the potential infection sites) developed on each stem. The nodules increased linearly in size over this time to ～15mg. Specific acetylene reduction activity, ARA ((μmol C₂H₄ mg^?1 h^?1) rose to 45 between days 5 and 10 after inoculation and plateaued; total ARA rose to ～200 μmol C₂H₄ plant^?1 h^?1. Under the conditions described the plants grew vigorously, and reproducibly uniform yields of nodules with high ARA activities were obtained. As outlined, the procedure offers a standard system in which, within a 2-week period after inoculation, individual strains of bacteria can be quantitatively compared in their ability to induce nodulation and N₂-fixation. Physiological and biochemical aspects of the nodulated system can be much more readily approached than with plants producing only root nodules. The inhibitory effects of stem nodules induced by wild type and two mutant strains of Azorhizobium on the development and activity of root nodules are described.     

Survival of Azorhizobium caulinodans in the Soil and Rhizosphere of Wetland Rice under Sesbania rostrata-Rice Rotation

The survival of indigenous and introduced strains of Azorhizobium caulinodans in flooded soil and in the rice rhizosphere, where in situ Sesbania rostrata was incorporated before the rice crop, is reported. The azorhizobia studied were both root and stem nodulating. In a pot experiment, two crop cycles each of inoculated and noninoculated Sesbania-rice were compared with two crop cycles of flooded fallow-rice. In a field experiment, the effect of repeated incorporation of in situ S. rostrata in the Sesbania-rice sequence was studied. Soils in which inoculated S. rostrata was incorporated contained about 3,000 times more azorhizobia than did soils in the flooded fallow treatment and about 50 times more azorhizobia than did soils in the noninoculated Sesbania treatment. Azorhizobial numbers in the inoculated Sesbania treatment declined toward rice harvest but remained much higher than in the flooded fallow-rice treatment. Repeated incorporation of S. rostrata increased the population density of indigenous soil azorhizobia, whereas the population of inoculated strain ORS571 (Str^r Spc^r) declined to an undetectable level; this finding suggested low competitiveness by the introduced strain. In the incorporated Sesbania treatment, the rice rhizosphere harbored significantly more A. caulinodans and supported higher nitrogenase activity per plant than did the rhizosphere of the flooded fallow-rice treatment. Sterile rice seedlings inoculated with A. caulinodans showed nitrogenase activity comparable to that of seedlings inoculated with Azospirillum lipoferum 34H, a rice root isolate. Rhizobia from Sesbania aculeata, Sesbania sesban, a Trifolium sp., and Vigna unguiculata did not support appreciable nitrogenase activity.     

Common nodABC genes in Nod locus 1 of Azorhizobium caulinodans: Nucleotide sequence and plant-inducible expression

Summary Azorhizobium caulinodans strain ORS571 induces nitrogen-fixing nodules on roots and stem-located root primordia of Sesbania rostrata. Two essential Nod loci have been previously identified in the bacterial genome, one of which (Nod locus 1) shows weak homology with the common nodC gene of Rhizobium mehloti. Here we present the nucleotide sequence of this region and show that it contains three contiguous open reading frames (ORFA, ORFB and ORFC) that are related to the nodABC genes of Rhizobium and Bradyrhizobium species. ORFC is followed by a fourth (ORF4) and probably a fifth (ORF5) open reading frame. ORF4 may be analogous to the nod[ gene of R. leguminosarum, whereas ORF5 could be similar to the rhizobial nodF genes. Coordinated expression of this set of five genes seems likely from the sequence organization. There is no typical nod promoter consensus sequence (nod box) in the region upstream of the first gene (ORFA) and there is no nodD-like gene. LacZ fusions constructed with ORFA, ORFB, ORFC, and ORF4 showed inducible -galactosidase expression in the presence of S. rostrata seedlings as well as around stem-located root primordia. Among a series of phenolic compounds tested, the flavanone naringenin was the most efficient inducer of the expression of this ORS571 nod gene cluster.     

Isolation and Characterization of a Competition-Defective Bradyrhizobium japonicum Mutant

Tn5 mutagenesis was coupled with a competition assay to isolate mutants of Bradyrhizobium japonicum defective in competitive nodulation. A double selection procedure was used, screening first for altered extracellular polysaccharide production (nonmucoid colony morphology) and then for decreased competitive ability. One mutant, which was examined in detail, was deficient in acidic polysaccharide and lipopolysaccharide production. The wild-type DNA region corresponding to the Tn5 insertion was isolated, mapped, and cloned. A 3.6-kb region, not identified previously as functioning in symbiosis, contained the gene(s) necessary for complementation of the mutation. The mutant was motile, grew normally on minimal medium, and formed nodules on soybean plants which fixed almost as much nitrogen as the wild type during symbiosis.     

Genetic analysis of nitrogen fixation in a tropical fast-growing Rhizobium

The Rhizobium strain ORS571, which is associated with the tropical legume Sesbania rostrata, has the property of growing in the free-living state at the expense of ammonia or N₂ as sole nitrogen source. Five mutants, isolated as unable to form colonies on plates under conditions of nitrogen fixation, were studied. All of them, which appear as Fix^- in planta, are nif mutants. With mutant 5740, nitrogenase activity of the crude extract was restored by addition of pure Mo-Fe protein of Klebsiella pneumoniae. A 13-kb BamHI DNA fragment from the wild-type strain, which hybridized with a probe carrying the nifHDK genes of K. pneumoniae, was cloned in vector pRK290 to yield plasmid pRS1. The extent of homology between the probe and the BamHI fragment was estimated at 4 kb and hybridization with K. pneumoniae nifH, nifK, and possibly nifD was detected. The pRS1 plasmid was introduced into the sesbania rhizobium nif mutants. Genetic complementation was observed with strain 5740(pRS1) both in the free-living state and in planta. It thus appears that biochemistry and genetics of nitrogen fixation in this particular Rhizobium strain can be performed with bacteria grown under non-symbiotic conditions.     

The Sinorhizobium meliloti ntrX Gene Is Involved in Succinoglycan Production,Motility, and Symbiotic Nodulation on Alfalfa

Rhizobia establish a symbiotic relationship with their host legumes to induce the formation of nitrogen-fixing nodules. This process is regulated by many rhizobium regulators, including some two-component regulatory systems (TCSs). NtrY/NtrX, a TCS that was first identified in Azorhizobium caulinodans, is required for free-living nitrogen metabolism and symbiotic nodulation on Sesbania rostrata. However, its functions in a typical rhizobium such as Sinorhizobium meliloti remain unclear. Here we found that the S. meliloti response regulator NtrX but not the histidine kinase NtrY is involved in the regulation of exopolysaccharide production, motility, and symbiosis with alfalfa. A plasmid insertion mutant of ntrX formed mucous colonies, which overproduced succinoglycan, an exopolysaccharide, by upregulating its biosynthesis genes. This mutant also exhibited motility defects due to reduced flagella and decreased expression of flagellins and regulatory genes. The regulation is independent of the known regulatory systems of ExoR/ExoS/ChvI, EmmABC, and ExpR. Alfalfa plants inoculated with the ntrX mutant were small and displayed symptoms of nitrogen starvation. Interestingly, the deletion mutant of ntrY showed a phenotype similar to that of the parent strain. These findings demonstrate that the S. meliloti NtrX is a new regulator of succinoglycan production and motility that is not genetically coupled with NtrY.     

Isolation and characterization of Tn5-induced NADPH-glutamate synthase (GOGAT-) mutants of Azorhizobium sesbaniae ORS571 and cloning of the corresponding glt locus

Summary Random Tn5 mutagenesis was used to isolate two independent Azorhizobium sesbaniae ORS571 mutants disturbed in ammonium assimilation (Asm^-). Both Asm^- mutant strains were shown to lack NADPH-glutamate synthase (NADPH-GOGAT) activity and to carry Tn5 insertions ca. 1.5 kb apart in the ORS571 chromosome. The Tn5-containing region of one of the GOGAT^- mutant strains was cloned in pACYC184 and used to identify the wild-type glt (GOGAT) locus in a phage clone bank of ORS571. The cloned region was shown to have DNA homology with the Escherichia coli glt locus and to complement the Asm^- phenotype of E. coli and ORS571 GOGAT^- strains. The ORS571 GOGAT^- mutations were found to interfere with free-living as well as symbiotic nitrogen fixation. Expression of ORS571 NADPH-GOGAT activity was shown to be independent of the nitrogen regulation (ntr) system.