Conservation of symbiotic nitrogen fixation gene sequences in Rhizobium japonicum and Bradyrhizobium japonicum.

Southern hybridization with nif (nitrogen fixation) and nod (nodulation) DNA probes from Rhizobium meliloti against intact plasmid DNA of Rhizobium japonicum and Bradyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid DNA in most R. japonicum strains. An exception is found with R. japonicum strain USDA194 and all B. japonicum strains where nif and nod sequences are on the chromosome. In R. japonicum strains, with the exception of strain USDA205, both nif and nod sequences are on the same plasmid. In strain USDA205, the nif genes are on a 112-megadalton plasmid, and nod genes are on a 195-megadalton plasmid. Hybridization to EcoRI digests of total DNA to nif and nod probes from R. meliloti show that the nif and nod sequences are conserved in both R. japonicum and B. japonicum strains regardless of the plasmid or chromosomal location of these genes. In addition, nif DNA hybridization patterns were identical among all R. japonicum strains and with most of the B. japonicum strains examined. Similarly, many of the bands that hybridize to the nodulation probe isolated from R. meliloti were found to be common among R. japonicum strains. Under reduced hybridization stringency conditions, strong conservation of nodulation sequences was observed in strains of B. japonicum. We have also found that the plasmid pRjaUSDA193, which possess nif and nod sequences, does not possess sequence homology with any plasmid of USDA194, but is homologous to parts of the chromosome of USDA194. Strain USDA194 is unique, since nif and nod sequences are present on the chromosome instead of on a plasmid as observed with all other strains examined.     

Genetic relatedness of Bradyrhizobium japonicum field isolates as revealed by repeated sequences and various other characteristics.

Forty-nine isolates of Bradyrhizobium japonicum indigenous to a field where soybeans were grown for 45 years without inoculation were characterized by using four DNA hybridization probes from B. japonicum. nifDK-specific hybridization clearly divided the isolates into two divergent groups. Diversity in repeated-sequence (RS)-specific hybridization was observed; 44 isolates derived from 41 nodules were divided into 33 different RS fingerprint groups. Cluster analysis showed that the RS fingerprints were correlated with the nif and hup genotypes. We found multiple bands of RS-specific hybridization for two isolates that differed from the patterns of the other isolates. These results suggest that RS fingerprinting is a valuable tool for evaluating the genetic structure of indigenous B. japonicum populations.     

Genetic relatedness of Bradyrhizobium japonicum field isolates as revealed by repeated sequences and various other characteristics.

Forty-nine isolates of Bradyrhizobium japonicum indigenous to a field where soybeans were grown for 45 years without inoculation were characterized by using four DNA hybridization probes from B. japonicum. nifDK-specific hybridization clearly divided the isolates into two divergent groups. Diversity in repeated-sequence (RS)-specific hybridization was observed; 44 isolates derived from 41 nodules were divided into 33 different RS fingerprint groups. Cluster analysis showed that the RS fingerprints were correlated with the nif and hup genotypes. We found multiple bands of RS-specific hybridization for two isolates that differed from the patterns of the other isolates. These results suggest that RS fingerprinting is a valuable tool for evaluating the genetic structure of indigenous B. japonicum populations.     

Variability in molybdenum uptake activity in Bradyrhizobium japonicum strains.

Twenty naturally occurring strains of Bradyrhizobium japonicum in 11 serogroups were screened for the ability to take up Mo as bacteroids from soybean root nodules. The strains varied greatly in their ability to take up Mo in a 1-min period. The best strain was USDA 136, which had an Mo uptake activity of almost 3.0 pmol/min per mg of bacteroid (dry weight). In contrast, the poorest strain, USDA 62, had an Mo uptake activity of 0.35 pmol of Mo per min per mg of bacteroid. There were similarities in Mo uptake ability among most of the same serogroup members. The variability in Mo uptake rates between the best (USDA 136 and USDA 122) and poorest (USDA 62 and USDA 140) strains was attributed to their differing affinities for Mo. Double-reciprocal plots of velocity versus substrate indicated a Km for USDA 136 and USDA 122 of 0.045 and 0.054 microM, respectively, whereas strains USDA 62 and USDA 140 both exhibited an apparent Km for MoO42- of about 0.36 microM. The two strains with the higher-affinity Mo binding also accumulated four to five times as much Mo over a 30-min period as the other strains. Soybeans were grown in Mo-deficient and Mo-supplemented conditions after inoculation with the three top-ranking Mo uptake strains and the three poorest Mo uptake strains. Two separate greenhouse studies indicated that Mo supplementation significantly increased the N2 fixation activity of USDA 140 nodules; up to a 35% increase in specific nitrogen fixation activity of nodules due to Mo supplementation was observed. Strain USDA 62 nodule N2 fixation responded positively to Mo supplementation in one of the two experiments. The results indicate that MoO42- transport and, specifically, affinity for Mo by the bacteroid may ultimately affect symbiotic N2 fixation activity. Attempts to reactivate nitrogenase by adding molybdate to bacteroids from plants grown in Mo-deficient conditions were unsuccessful.     

Efficiency of different formulations of Bradyrhizobium japonicum and effect of co-inoculation of Bacillus subtilis with two different strains of Bradyrhizobium japonicum

A key constraint in successfully obtaining an effective inoculant is overcoming difficulties in formulating a viable and user-friendly final product and maintaining the microbial cells in a competent state. Co-cultures of rhizobia and PGPR (Plant Growth Promoting Rhizobacteria) are a logical next subject for formulation researchers as they can influence the efficacy of rhizobia. A greenhouse experiment was set to assess the formulation effect of one strain i.e. Bradyrhizobium japonicum, 532c (granules, liquid and broth) and also to determine the efficiency of co-inoculation of Bacillus with two commercial strains of B. japonicum (532c and RCR 3407) on 2 soybean (Glycine max L.) varieties. PCR-RFLP analysis was used to determine the nodule occupancy in each treatment. Most of the inoculants showed increased nodulation and biomass yields (by approximately 2-5 and 4-10 g plant(-1) respectively) as compared to the uninoculated controls. TGx1740-2F showed no significant differences in nodule fresh weights for the formulation effect while the co-inoculants increased the nodule fresh weights by up to 4 g plant(-1). The liquid and granule-based inoculants induced higher biomass yields (4-8 g plant(-1)) suggesting a possible impact of formulation on the effectiveness of the inoculants. The co-inoculants also gave higher yields but showing no significant differences to the rhizobial inoculants. Nodule occupancy was 100 % for the rhizobial inoculants as well as the co-inoculants emphasizing the infectivity and high competitiveness of 532c and RCR 3407 strains despite the high population of indigenous rhizobia.     

IS1631 Occurrence in Bradyrhizobium japonicum Highly Reiterated Sequence-Possessing Strains with High Copy Numbers of Repeated Sequences RSα and RSβ

From Bradyrhizobium japonicum highly reiterated sequence-possessing (HRS) strains indigenous to Niigata and Tokachi in Japan with high copy numbers of the repeated sequences RSα and RSβ (K. Minamisawa, T. Isawa, Y. Nakatsuka, and N. Ichikawa, Appl. Environ. Microbiol. 64:1845–1851, 1998), several insertion sequence (IS)-like elements were isolated by using the formation of DNA duplexes by denaturation and renaturation of total DNA, followed by treatment with S1 nuclease. Most of these sequences showed structural features of bacterial IS elements, terminal inverted repeats, and homology with known IS elements and transposase genes. HRS and non-HRS strains of B. japonicum differed markedly in the profiles obtained after hybridization with all the elements tested. In particular, HRS strains of B. japonicum contained many copies of IS1631, whereas non-HRS strains completely lacked this element. This association remained true even when many field isolates of B. japonicum were examined. Consequently, IS1631 occurrence was well correlated with B. japonicum HRS strains possessing high copy numbers of the repeated sequence RSα or RSβ. DNA sequence analysis indicated that IS1631 is 2,712 bp long. In addition, IS1631 belongs to the IS21 family, as evidenced by its two open reading frames, which encode putative proteins homologous to IstA and IstB of IS21, and its terminal inverted repeat sequences with multiple short repeats.     

Differential symbiotic response of phage-typed strains of Bradyrhizobium japonicum with soybean cultivars

In this study, native Bradyrhizobium strains were isolated from the host plant, Glycine max, harvested from fields in Madhya Pradesh, India, and were typed by lytic rhizobiophages. Eight indigenous (Soy2, ASR011, ASR031, ASR032, MSR091, ISR050, ISR076 and ISR078) and two exotic strains (USDA123 and CB1809), all of which evidenced a distinct reaction with six phages, were employed in this study. The symbiotic interaction of these strains was studied initially using soybean cultivar JS335 in a sand culture in a controlled environment, and the efficiency was assessed based on the nodule number, nodule dry weight, plant dry weight, nitrogenase activity, and total accumulation of N per plant. Symbiotic effectiveness was found to be highest with the native phage-sensitive isolate ASR011, whereas it was at a minimum with the phage-resistant isolates, ISR050 and ISR078. Additionally, the effectiveness of these strains was evaluated using six soybean cultivars belonging to different maturity groups; namely, Bragg, Lee, Pusa20, PK416, JS335 and NRC37. Analysis of variance data evidenced significant differences due to both symbionts, for the majority of the tested parameters. The CB1809, USDA123, and ASR011 strains evidenced relatively superior symbiotic effectiveness with soybean cultivars Bragg, Lee and JS335. Strain ISR078 evidenced no significant responses with any of the cultivars. The ASR031 strain performed moderately well with all tested cultivars. The symbiotic response of all the strains was quite poor with cultivar PK416. Our studies showed that a significant relationship existed between the phage sensitivity and symbiotic efficiency of the bacterial strains with the host-cultivars.     

Short communication: Screening of native and foreign Bradyrhizobium japonicum strains for high N2 fixation in soybean

Four local rhizobia isolates selected after two screening experiments and five USDA Bradyrhizobium japonicum strains were estimated for N₂ fixation in soybean using the ¹⁵N isotope dilution technique. Strain USDA 110 was superior to the local isolates in nodulation and N₂ fixation when inoculated onto soybean cv TGX 1497-ID in a Nigerian soil and could therefore be used as an inoculant for enhanced N₂ fixation in soybean in Nigeria.     

Nickel accumulation and storage in Bradyrhizobium japonicum.

Hydrogenase-derepressed (chemolithotrophic growth conditions) and heterotrophically grown cultures of Bradyrhizobium japonicum accumulated nickel about equally over a 3-h period. Both types of cultures accumulated nickel primarily in a form that was not exchangeable with NiCl2, and they accumulated much more Ni than would be needed for the Ni-containing hydrogenase. The nickel accumulated by heterotrophically incubated cultures could later be mobilized to allow active hydrogenase synthesis during derepression in the absence of nickel, while cells both grown and derepressed without nickel had low hydrogenase activities. The level of activity in cells grown with Ni and then derepressed without nickel was about the same as that in cultures derepressed in the presence of nickel. The Ni accumulated by heterotrophically grown cultures was associated principally with soluble proteins rather than particulate material, and this Ni was not lost upon dialyzing an extract containing the soluble proteins against either Ni-containing or EDTA-containing buffer. However, this Ni was lost upon pronase or low pH treatments. The soluble Ni-binding proteins were partially purified by gel filtration and DEAE chromatography. They were not antigenically related to hydrogenase peptides. Much of the 63Ni eluted as a single peak of 48 kilodaltons. Experiments involving immunoprecipitation of 63Ni-containing hydrogenase suggested that the stored source of Ni in heterotrophic cultures that could later be mobilized into hydrogenase resided in the nonexchangeable Ni-containing fraction rather than in loosely bound or ionic forms.     

Variation among alfalfa somaclones in copy number of repeated DNA sequences.

Repeated DNA sequences of alfalfa (Medicago sativa L.) somaclonal variants were analyzed to determine if changes in copy number had occurred during tissue culture. DNA clones containing highly repeated nuclear sequences from the diploid line HG2 (2x = 16) were slot blotted and probed with labeled DNAs from HG2 and several somaclones of HG2. Two DNA clones that differed visually in hybridization intensity among the plant DNAs and one clone that had constant hybridization intensity were selected and used as probes on Southern blots and slot blots containing equal quantities of DNAs from HG2 and 15 régénérants. Statistically significant differences were detected in the copy number of two anonymous DNA sequences initially selected as variable and in the copy number of sequences homologous to pea ribosomal DNA. Based on Southern blot analysis, these sequences appeared to be arranged as tandem repeats. The cloned sequence initially selected as stable did not vary significantly in copy number and it appeared to be arranged as a dispersed repeat. Both increases and decreases in copy number of repeated sequences were observed in plants from successive regeneration cycles. Results from this study indicate that specific repeated nuclear DNA sequences have changed copy number in plants regenerated from tissue culture.     

Fine-tuning of nif and fix gene expression by upstream activator sequences in Bradyrhizobium japonicum

The significance of Bradyrhizobium japonicum upstream activator sequences (UASs) for differential NifA-mediated fix and nif gene expression was investigated by two means: (i) hybrid fixA- and fixB-lacZ fusions were constructed by transposing a nifH-UAS cartridge in front of their promoters; and (ii) B. japonicum mutants were generated carrying specific chromosomal deletions or UAS cartridge insertions within the fixA, fixB or nifH promoter-upstream regions. Expression of fixA was not affected, and expression of fixB decreased only to 42%, when the respective fixA and fixB promoter-upstream DNAs were deleted. This shows that in B. japonicum the NifA-dependent activation of at least the fixA promoter does not require the presence of a closely adjacent UAS. Deletion of the UASs in front of the nifH gene not only reduced the expression of nifH down to 2.5% but, surprisingly, also resulted in a reduction of the fixB mRNA level to less than 20%. This suggests that the nifH-UASs may exert a long-range effect on the expression of the 3-kb-distant fixBCX operon in nif cluster I or B. japonicum. Artificial transposition of the nifH-UASs in front of the fixA and fixB promoters strongly enhanced fixA and fixB expression.     

Protein phosphorylation in Bradyrhizobium japonicum bacteroids and cultures.

Protein phosphorylation was demonstrated in Bradyrhizobium japonicum bacteroids in vivo and in cultures in vivo and in vitro. Comparison of in vivo-labeled phosphoproteins of bacteroids and of cultured cells showed differences in both the pattern and intensity of labeling. In cultured cells, comparison of the labeling patterns and intensities of in vivo- and in vitro-labeled phosphoproteins showed a number of similarities; however, several phosphoproteins were found only after one of the two labeling conditions. The labeling intensity was time dependent in both in vivo and in vitro assays and was dependent on the presence of magnesium in in vitro assays. Differences in the rates of phosphorylation and dephosphorylation were noted for a number of proteins. The level of incorporation of 32P into protein was only 2% or less of the total phosphate accumulated during the in vivo labeling period. Several isolation and sample preparation procedures resulted in differences in labeling patterns. Phosphatase inhibitors and several potential metabolic effectors had negligible effects on the phosphorylation pattern. There were no significant changes in the phosphorylation patterns of cells cultured on mannitol, acetate, and succinate, although the intensity of the labeling did vary with the carbon source.