ofBradyrhizobium Japonicum Research Articles

Revision of the taxonomic status of the species Rhizobium lupini and reclassification as Bradyrhizobium lupini comb. nov.

The species Rhizobium lupini was isolated from Lupinus nodules and included in the Approved Lists of Bacterial Names in 1980. Nevertheless, on the basis of the analysis of the type strain of this species available in DSMZ, DSM 30140(T), whose 16S rRNA gene was identical to that of the type strain of Bradyrhizobium japonicum , R. lupini was considered a later synonym of this species. In this study we confirmed that the strain DSM 30140(T) belongs to the species B. japonicum , but also that it cannot be the original strain of R. lupini because this species effectively nodulated Lupinus whereas strain DSM 30140(T) was able to nodulate soybean but not Lupinus. Since the original type strain of R. lupini was deposited into the USDA collection by L. W. Erdman under the accession number USDA 3051(T) we analysed the taxonomic status of this strain showing that although it belongs to the genus Bradyrhizobium instead of genus Rhizobium , it is phylogenetically distant from B. japonicum and closely related to Bradyrhizobium canariense . The type strains R. lupini USDA 3051(T) and B. canariense BTA-1(T) share 16S rRNA, recA and glnII gene sequences with similarities of 99.8%, 96.5% and 97.1%, respectively. They presented a DNA-DNA hybridization value of 36% and also differed in phenotypic characteristics and slightly in the proportions of some fatty acids. Therefore we propose the reclassification of the species Rhizobium lupini as Bradyrhizobium lupini comb. nov. The type strain is USDA 3051(T) ( = CECT 8630(T) = LMG 28514(T)).

Nitrate-Dependent N 2 O Emission from Intact Soybean Nodules via Denitrification by Bradyrhizobium japonicum Bacteroids

In the presence of nitrate, N₂O emission increased markedly from soybean roots inoculated with nosZ mutant of Bradyrhizobium japonicum, but not from soybean roots inoculated with a napA nosZ double mutant, indicating that B. japonicum bacteroids in soybean nodules are able to convert the exogenously supplied nitrate into N₂O via a denitrification pathway.

Bradyrhizobium huanghuaihaiense sp. nov., an effective symbiotic bacterium isolated from soybean (Glycine max L.) nodules

In a survey of the biodiversity and biogeography of rhizobia associated with soybean (Glycine max L.) in different sites of the Northern (Huang-Huai-Hai) Plain of China, ten strains were defined as representing a novel genomic species in the genus of Bradyrhizobium. They were distinguished from defined species in restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene and the 16S-23S rRNA gene intergenic spacer (IGS). In BOX-PCR, these strains presented two patterns that shared 94% similarity, demonstrating that they were a homogenous group with limited diversity. In phylogenetic analyses of the 16S rRNA gene, IGS and housekeeping gene sequences, four representative strains formed a distant lineage within the genus Bradyrhizobium, which was consistent with the results of DNA-DNA hybridization. The strains of this novel group formed effective nodules with G. max, Glycine soja and Vigna unguiculata in cross-nodulation tests and harboured symbiotic genes (nodC and nifH) identical to those of reference strains of Bradyrhizobium japonicum, Bradyrhizobium liaoningense and 'Bradyrhizobium daqingense' originating from soybean, implying that the novel group may have obtained these symbiotic genes by lateral gene transfer. In analyses of cellular fatty acids and phenotypic features, some differences were found between the novel group and related Bradyrhizobium species, demonstrating that the novel group is distinct phenotypically from other Bradyrhizobium species. Based upon the data obtained, these strains are proposed to represent a novel species, Bradyrhizobium huanghuaihaiense sp. nov., with CCBAU 23303(T) ( = LMG 26136(T) = CGMCC 1.10948(T) = HAMBI 3180(T)) as the type strain. The DNA G+C content of strain CCBAU 23303(T) is 61.5 mol% (T(m)).

Genetic diversity ofBradyrhizobium japonicum within soybean growing regions of the north-eastern Great Plains of North America as determined by REP-PCR and ERIC-PCR profiling

While soybean (Glycine max [L.] Merr.) has been grown for several decades in several northern states in the United States, the introduction of early maturing cultivars of soybean in western Canada in the late 1990’s has resulted in a exponential increase in soybean production in the region. Soybean grows in a symbiotic association withBradyrhizobium japonicum [Kirchner] Jordan, which carries out biological nitrogen fixation within the plant roots. Previous studies have shown that rhizobia introduced from commercial inoculants tend to evolve quickly in soil. In this study, we examined the genetic diversity of 105B. japonicum isolates from the soybean growing areas of the north-eastern Great Plains of North America by genomic fingerprinting techniques — REP-PCR and ERIC-PCR profiling. High genetic diversity was detected among theB. japonicum isolates sampled across various sites in North Dakota, South Dakota and Minnesota in the United States, and southern Manitoba in Canada. Analysis of the genetic diversity by the unweighted pair group method with an arithmetic mean algorithm(UPGMA) indicated an interesting segregation of isolates between US and Canadian sites. Results of this study also suggest a relatively rapid rate of genetic change within theB. japonicum populations and some evidence that soil texture may influence genetic diversity of the bacterium in the region.

Mitogen-activated protein kinase is involved in the symbiotic interaction betweenBradyrhizobium japonicum USDA110 and soybean

Mitogen-activated protein kinase (MAPK) plays an important role in mediating the intracellular transmission and amplification of extracellular stimuli. We examined whether MAPK is involved in the signaling process during the early step of nodule formation. A genistein induced culture filtrate (GCF) ofBradyrhizobium japonicum was prepared for inducing an early response by soybean root hairs via Nod factor. Upon treatment, several types of deformations were seen, demonstrating that GCF contains active Nod factor molecules. In-gel kinase assays showed that treating soybean roots with GCF induced the rapid activation of two protein kinases (molecular masses of 47 kD and 44 kD), which phosphorylate myelin basic protein (MBP). To identify the activated kinase, we prepared an antibody againstGMK1 (Glycine max MAP kinase 1), based on information from SIMK (an alfalfa MAP kinase) and a soybean EST database. An immunocomplex kinase assay with the GMK1-specific antibody revealed that the 47-kD kinase in GCF-treated seedlings is indeed GMK1. Consistent with many other MAP kinases, GMK1 is likely to be under post-translational regulation. Considering these results and previous reports from soybean, GMK1 seems to be a signaling mediator with a broad range of stimuli, including a fungal elicitor, wounding, and the symbiotic interaction between soybean and B.japonicum.

A Novel Deaminase Involved in Chloronitrobenzene and Nitrobenzene Degradation with Comamonas sp. Strain CNB-1

Comamonas sp. strain CNB-1 degrades nitrobenzene and chloronitrobenzene via the intermediates 2-aminomuconate and 2-amino-5-chloromuconate, respectively. Deamination of these two compounds results in the release of ammonia, which is used as a source of nitrogen for bacterial growth. In this study, a novel deaminase was purified from Comamonas strain CNB-1, and the gene (cnbZ) encoding this enzyme was cloned. The N-terminal sequence and peptide fingerprints of this deaminase were determined, and BLAST searches revealed no match with significant similarity to any functionally characterized proteins. The purified deaminase is a monomer (30 kDa), and its V(max) values for 2-aminomuconate and 2-amino-5-chloromuconate were 147 micromol x min(-1) x mg(-1) and 196 micromol x min(-1) x mg(-1), respectively. Its catalytic products from 2-aminomuconate and 2-amino-5-chloromuconate were 2-hydroxymuconate and 2-hydroxy-5-chloromuconate, respectively, which are different from those previously reported for the deaminases of Pseudomonas species. In the catalytic mechanism proposed, the alpha-carbon and nitrogen atoms (of both 2-aminomuconate and 2-amino-5-chloromuconate) were simultaneously attacked by a hydroxyl group and a proton, respectively. Homologs of cnbZ were identified in the genomes of Bradyrhizobium japonicum, Rhodopseudomonas palustris, and Roseiflexus sp. strain RS-1; these genes were previously annotated as encoding hypothetical proteins of unknown function. It is concluded that CnbZ represents a novel enzyme that deaminates xenobiotic compounds and/or alpha-amino acids.

Indole-3-acetic acid biosynthesis is deficient in Gluconacetobacter diazotrophicus strains with mutations in cytochrome c biogenesis genes.

Gluconacetobacter diazotrophicus is an endophyte of sugarcane frequently found in plants grown in agricultural areas where nitrogen fertilizer input is low. Recent results from this laboratory, using mutant strains of G. diazotrophicus unable to fix nitrogen, suggested that there are two beneficial effects of G. diazotrophicus on sugarcane growth: one dependent and one not dependent on nitrogen fixation. A plant growth-promoting substance, such as indole-3-acetic acid (IAA), known to be produced by G. diazotrophicus, could be a nitrogen fixation-independent factor. One strain, MAd10, isolated by screening a library of Tn5 mutants, released only approximately 6% of the amount of IAA excreted by the parent strain in liquid culture. The mutation causing the IAA(-) phenotype was not linked to Tn5. A pLAFR3 cosmid clone that complemented the IAA deficiency was isolated. Sequence analysis of a complementing subclone indicated the presence of genes involved in cytochrome c biogenesis (ccm, for cytochrome c maturation). The G. diazotrophicus ccm operon was sequenced; the individual ccm gene products were 37 to 52% identical to ccm gene products of Escherichia coli and equivalent cyc genes of Bradyrhizobium japonicum. Although several ccm mutant phenotypes have been described in the literature, there are no reports of ccm gene products being involved in IAA production. Spectral analysis, heme-associated peroxidase activities, and respiratory activities of the cell membranes revealed that the ccm genes of G. diazotrophicus are involved in cytochrome c biogenesis.

Specific detection of Bradyrhizobium and Rhizobium strains colonizing rice (Oryza sativa) roots by 16S-23S ribosomal DNA intergenic spacer-targeted PCR.

In addition to forming symbiotic nodules on legumes, rhizobial strains are members of soil or rhizosphere communities or occur as endophytes, e.g., in rice. Two rhizobial strains which have been isolated from root nodules of the aquatic legumes Aeschynomene fluminensis (IRBG271) and Sesbania aculeata (IRBG74) were previously found to promote rice growth. In addition to analyzing their phylogenetic positions, we assessed the suitability of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (IGS) sequences for the differentiation of closely related rhizobial taxa and for the development of PCR protocols allowing the specific detection of strains in the environment. 16S rDNA sequence analysis (sequence identity, 99%) and phylogenetic analysis of IGS sequences showed that strain IRBG271 was related to but distinct from Bradyrhizobium elkanii. Rhizobium sp. (Sesbania) strain IRBG74 was located in the Rhizobium-Agrobacterium cluster as a novel lineage according to phylogenetic 16S rDNA analysis (96.8 to 98.9% sequence identity with Agrobacterium tumefaciens; emended name, Rhizobium radiobacter). Strain IRBG74 harbored four copies of rRNA operons whose IGS sequences varied only slightly (2 to 9 nucleotides). The IGS sequence analyses allowed intraspecies differentiation, especially in the genus Bradyrhizobium, as illustrated here for strains of Bradyrhizobium japonicum, B. elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium sp. (Chamaecytisus) strain BTA-1. It also clearly differentiated fast-growing rhizobial species and strains, albeit with lower statistical significance. Moreover, the high sequence variability allowed the development of highly specific IGS-targeted nested-PCR assays. Strains IRBG74 and IRBG271 were specifically detected in complex DNA mixtures of numerous related bacteria and in the DNA of roots of gnotobiotically cultured or even of soil-grown rice plants after inoculation. Thus, IGS sequence analysis is an attractive technique for both microbial ecology and systematics.

Further studies of the role of cyclic beta-glucans in symbiosis. An NdvC mutant of Bradyrhizobium japonicum synthesizes cyclodecakis-(1-->3)-beta-glucosyl.

The cyclic beta-(1-->3),beta-(1-->6)-D-glucan synthesis locus of Bradyrhizobium japonicum is composed of at least two genes, ndvB and ndvC. Mutation in either gene affects glucan synthesis, as well as the ability of the bacterium to establish a successful symbiotic interaction with the legume host soybean (Glycine max). B. japonicum strain AB-14 (ndvB::Tn5) does not synthesize beta-glucans, and strain AB-1 (ndvC::Tn5) synthesizes a cyclic beta-glucan lacking beta-(1-->6)-glycosidic bonds. We determined that the structure of the glucan synthesized by strain AB-1 is cyclodecakis-(1-->3)-beta-D-glucosyl, a cyclic beta-(1-->3)-linked decasaccharide in which one of the residues is substituted in the 6 position with beta-laminaribiose. Cyclodecakis-(1-->3)-beta-D-glucosyl did not suppress the fungal beta-glucan-induced plant defense response in soybean cotyledons and had much lower affinity for the putative membrane receptor protein than cyclic beta-(1-->3),beta-(1-->6)-glucans produced by wild-type B. japonicum. This is consistent with the hypothesis presented previously that the wild-type cyclic beta-glucans may function as suppressors of a host defense response.

Identification ofRhizobium strains and evaluation of their competitiveness

Six strains ofRhizobium leguminosarum bv.viciœ, three strains ofBradyrhizobium japonicum and three strains ofRhizobium fredii were evaluated by the polymerase chain reaction (PCR). The possibility of identification of individual rhizobial strains and the way of product analysis were verified. The result of amplifications proved rich spectra along the whole length scale. Numerous identical bands could be found in related strains. Verification of the expected identity of some strains confirmed the applicability of this method for identification of individual bacterial strains of generaRhizobium andBradyrhizobium. Furthermore, competitiveness of two strains ofR. leguminosarum bv.viciœ against the native rhizobial population was evaluated in a pot experiment. When using PCR as the identification method, the presence of the strains in host plant's nodules was ascertained after inoculation by different rates of inoculum strains. With increasing the inoculum rate, the presence of inoculum strains in pea nodules also increased. On the basis of mathematical models by Amarger and Lobreu the competitiveness of the mentioned strains was estimated at certain inoculum rates. Both tested strains displayed a higher competitiveness than native rhizobia in the soil used. As they are also effective N2 fixators (one strain being HUP+), one may expect successful field inoculations with them.

Synthesis of lipooligosaccharide nodulation signals NodBj-V(RCO, MeFuc) and NodBj-IV(RCO, MeFuc) of Bradyrhizobium japonicum.

First total syntheses of Nod factors ofBradyrhizobium japonicum were described in a stereo- and regio-controlled manner.

Plant Defense Responses of Host Plants with Determinate Nodules Induced by EPS-DefectiveexoBMutants ofBradyrhizobium japonicum

Plant Defense Responses of Host Plants with Determinate Nodules Induced by EPS-Defective<i>exoB</i>Mutants of<i>Bradyrhizobium japonicum</i>

Cloning and Mutagenesis of a Cytochrome P-450 Locus from Bradyrhizobium japonicum That Is Expressed Anaerobically and Symbiotically

Cytochromes P-450, which in many organisms participate in the metabolism of a variety of endobiotic and xenobiotic substances, are synthesized by symbiotic bacteroids of Bradyrhizobium japonicum. Polyclonal antibodies were raised against two cytochromes P-450 (CYP112 and CYP114) purified from bacteroids. A lambda gt11 expression clone of B. japonicum USDA 110 DNA that reacted with the anti-CYP112 antibody was obtained and was used to screen a library of USDA 110 genomic DNA in pLAFR1 for a clone of the P-450 locus. Forced expression of subclones of the P-450 locus in Escherichia coli produced polypeptides that reacted with either the anti-CYP112 antibody or the anti-CYP114 antibody; no cross-reactivity was evident. A Western blot (immunoblot) analysis showed that neither protein was present in free-living aerobically grown B. japonicum cells, but that both proteins were present in cells grown anaerobically, as well as in bacteroids. A mutant strain disrupted in the CYP112 locus produced neither CYP112 nor CYP114, indicating that the mutation was polar for CYP114. The mutant produced effective nodules on soybeans, even though the bacteroids contained no detectable P-450. This suggests that the cytochromes P-450 which we examined are not involved in an essential symbiotic function.

Integration ofhup cosmid pHU52 into the chromosomal DNA ofCicer-Rhizobium using Tn5 as an homologous sequence

Cosmid pHU52, which carrieshup genes ofBradyrhizobium japonicum, has been integrated into theCicer-Rhizobium G36-84 genome via Tn5-mediated homologous recombination. Tn5 was inserted into both the cosmid pHU52 and the chromosome ofCicer-Rhizobium to provide a region of DNA homology, without affecting the expression of necessary genes. An incompatible plasmid, pPH1JI, was used to select those few cells that had undergone recombination. The integration of the cosmid was demonstrated by Southern blot analysis. Chromosomal integration of thehup genes maximized stability and minimized the potential for their horizontal transfer to other bacterial species. The integratedhup genes were found to expressex planta as well in nodules. The method described illustrates how a given gene can be stably integrated into the chromosome.

Bradyrhizobium strains and the nodulation, nodule efficiency and growth of soybean (Glycine max L.) in Egyptian soils

Six strains and a commercial inoculant ofBradyrhizobium japonicum were evaluated in association withGlycine max (L.) cultivar Clark. Inoculated and uninoculated plants were grown in pot and field experiments. Nodules were counted and weighed and roots and shoots were separated and analysed for total nitrogen. In pot experiments, two of six bacterial strains were superior to the other four, and to the commercial inoculant (Nitragin) in promoting greater root and top growth and plant nitrogen accumulation. In the field experiment, there were indications that environmental conditions may have affected nodulation by the bacteria. The strains could be divided into three groups according to nodule efficiencies, accumulation of plant dry matter, and total nitrogen content. The greater variations in nodule efficiencies of the tested strains could be attributed to the quantities of bacteroid, cytosol protein and leghaemoglobin in the nodules.

Accumulation of the phytoalexin, glyceollin, in root nodules of soybean formed by effective and ineffective strains ofBradyrhizobium japonicum

Nitrogen fixation in root nodules formed by strain 2143 ofBradyrhizobium japonicum andGlycine max (L.) Merr. cv Williams 82 reaches a maximum at 21 to 28 days postinoculation and then begins to decline. The phytoalexin, glyceollin, accumulates in nodules coincident with the decline in nitrogen fixation. Nodules formed by strain 3122, which are unable to fix nitrogen, accumulate even higher levels of glyceollin and do so beginning 21 days postinoculation even though these nodules contain no recoverable bacteria. The typical phytoalexin response occurs within days of infection. The mechanism by which this response in theBradyrhizobium japonicum-soybean combination is delayed 2 to 3 weeks after infection is presently unknown but phytoalexin accumulation could contribute to the inability of the soybean-Bradyrhizobium japonicum combination to maintain high levels of nitrogen fixation throughout the growing season.

Constitutive and nitrate-induced, membrane-bound nitrate reductase fromBradyrhizobium japonicum

Nitrate reductase (NR) activity was detected in membranes from cells ofBradyrhizobium japonicum cultured in defined medium either with glutamate or nitrate as the only nitrogen source. With gel filtration, the relative molecular mass (Mr) of the NR in cells growth with glutamate was estimated to be about 78 kDa. The enzyme from cells grown aerobically with nitrate had an Mr of 236 kDa, the same as that of the NR from microaerobically nitrate-grown cells. When cells that had been grown with glutamate were incubated microaerobically in both the absence and the presence of nitrate, the enzyme from each source resembled that of nitrate-grown cells in having an Mr of 236 kDa. In glutamate-grown cells that were further incubated, both microaerobiosis and nitrate were required for fully expression of the activity of the enzyme.

Real continuous biomass production ofBradyrhizobium japonicum in a veterinary vaccine production unit

In a real continuous culture,Bradyrhizobium japonicum strain 1755 was maintained in the 'Göttingen bioreactor unit' developed to produce veterinary bacterial vaccines for a period of 110 days without contamination or mutations. The average yield of viable bacteria over the whole period was more than 10(10)/ml.

Response of symbiotic interrelationship between soybean and the indigenous or inoculated microsymbiont,Bradyrhizobium japonicum, to soil application of manganese and molybdenum

Soybean nodulation was induced indigenously by three strains ofBradyrhizobium japonicum inoculated in each of the two previous seasons. The same strains were applied, as a peat-based inoculum in similar cell density as the indigenous ones, to a rhizobia-free soll from the same field. Inoculation was tested under soll application of 10 or 20 ppm Mn and/or 2 or 4 ppm Mo. Inoculation with the peat-based inoculum significantly increased nodule numbers and weight, dry weight, N content and mg N/g of the 50-day-old plants, number of pods and seeds per plant, seed yield, seed N content and percentage, and mg N/g of the empty pods. Increasing the application of Mn and Mo fed to further increases. There was a complex response of nodulation, vegetative growth and yield to the applications of Mn and Mo.

Rhizobiophage effects on nodulation, nitrogen fixation, and yield of field-grown soybeans (Glycine max L. Merr.)

Previous laboratory and greenhouse studies have shown that phages significantly reduce soil populations of homologous rhizobia. Reductions in nodulation and N2 fixation have also been observed. The purpose of the current study was to examine the effect of a phage specific ofBradyrhizobium japonicum USDA 117 on nodulation, nodule occupancy, N2 fixation and soybean growth and yield under field conditions. The phage was inoculated in combination withB. japonicum USDA 117 and/orB. japonicum USDA 110 (resistant strain) into a rhizobia-free sandy loam soil and planted toGlycine max (L.) Merr. “Williams”. When the phage was applied to soil inoculated withB. japonicum USDA 117 alone, significant reductions in nodule weight and number, shoot weight, foliar N, nitrogenase activity, and seed index were observed. When, however, the soil also contained the non-homologous strain,B. japonicum USDA 110, no significant effects on any of these parameters were found. Nodule occupancy by competing strains ofB. japonicum USDA 110 and USDA 117 was also affected by the phage. In soil which did not contain the phage, 46% and 44% of the identified nodules were occupied by USDA 110 and 117, respectively. When the phage was present in the soil, nodule occupancy byB. japonicum USDA 117 was reduced to 23%, while occupancy byB. japonicum USDA 110 was increased to 71%. These results suggest that nodulation by selected strains of rhizobia can be restricted and nodulation by more effective, inoculated strains can be increased through the introduction of a homologous phage to soils.