Melanin production encoded by a cryptic plasmid in a Rhizobium leguminosarum strain

Rhizobium leguminosarum strain VF39, isolated from nodules of field-grown faba beans in the Federal Republic of Germany, was shown to contain six plasmids ranging in molecular weight from 90 to 400 Md. Hybridisation to nif gene probes, plasmid curing, and mobilisation to other strains of Rhizobium and to Agrobacterium showed that the third largest plasmid, pRleVF39d (220 Md), carried genes for nodulation and nitrogen fixation. This plasmid was incompatible with pRL10JI, the Sym plasmid of R. leguminosarum strain JB300. Of the other plasmids, the two smallest (pRleVF39a and pRleVF39b, 90 and 160 Md respectively) were shown to be self-transmissible at a low frequency. Although melanin production is as yet unreported in strains of R. leguminosarum biovar viceae, strain VF39 produced a dark pigment, which, since it was not produced on minimal media and its production was greatly enhanced by the presence of tyrosine in the media, is probably melanin-like. Derivatives of VF39 cured of pRleVF39a no longer produced this pigment, but regained the ability to produce it when this plasmid was transferred into them. Strains of Agrobacterium tumefaciens, R. meliloti, and some strains of R. leguminosarum carrying pRleVF39a did not produce this pigment, indicating perhaps that some genes elsewhere on the VF39 genome are also involved in pigment production. Plasmid pRleVF39a appeared to be incompatible with the cryptic Rhizobium plasmids pRle336b and pRL8JI (both ca. 100 Md), but was compatible with the R. leguminosarum biovar phaseoli Sym plasmids pRP1JI, pRP2JI and pRph51a, all of which also code for melanin production. The absence of pRleVF39a in cured derivatives of VF39 had no effect on the symbiotic performance or competitive ability of this strain.     

Negative effects of agrocin 84-encoding Agrobacterium plasmids on symbiotic properties of Rhizobium meliloti

Two plasmids, pAgK84::Tn5-Mob from Agrobacterium radiobacter carrying genes for the production of agrocin 84, and RP4-4 from E. coli were inserted either separately or together into a strain of Rhizobium meliloti. Each of these plasmid-containing R. meliloti transconjugants was less effective than the wild type strain in their ability to fix nitrogen in Medicago tornata. The pAgK84::Tn5-Mob-containing transconjugant was significantly less effective than that containing RP4-4. The transconjugant strains were inferior to the wild type strain in their ability to nodulate seedlings and to compete for nodulation.     

Decreased symbiotic effectiveness of Rhizobium leguminosarum strains carrying plasmid RP4

The presence of derivatives of the broad host range plasmid RP4 in strains of Rhizobium leguminosarum biovar viciae severely inhibited nitrogen fixation by these strains in nodules on cultivars of pea (Pisum sativum). The strains formed small white nodules. Yield and total nitrogen values were comparable with those obtained for plants inoculated with a non-nodulating mutant. Strains carrying the same derivatives gave rise to nitrogen fixing nodules when inoculated on cultivars of lentils (Lens culinaris). Similar results were observed with plasmid R702 but not with R751, suggesting that the effect is limited to plasmids of the IncPα classification. Histological examination of nodules induced by strains carrying RP4 indicated that there are fewer infected cells and starch granules are organised unusually in the infected cells. Tn5 mutagenesis of plasmid RP4-4 was undertaken and Tn5 inserts were screened for abolition of the effect on nitrogen fixation. Eight mutants, having no effect on nitrogen fixation, were isolated. Seven of these had lost the ability to transfer by conjugation and the eighth was greatly reduced in conjugation frequency. Physical analysis of the transposon inserts revealed that they were located in the Tra regions of RP4.     

Organisation of nodulation and nitrogen fixation genes on a Rhizobium trifolii symbiotic plasmid

A Rhizobium trifolii symbiotic plasmid specific gene library was constructed and the physical organisation of regions homologous to nifHDK, nifA and nod genes was determined. These symbiotic gene regions were localised to u 25 kb region on the sym-plasmid, pPN1. In addition four copies of a reiterated sequence were identified on this plasmid, with one copy adjacent to nifH. No rearrangement of these reiterated sequences was observed between R. trifolii bacterial and bacteroid DNA. Analysis of a deletion derivative of pPN1 showed that these sequences were spread over a 110 kb region to the left of nifA.     

Hydrogen production and uptake by pea nodules as affected by strains of Rhizobium leguminosarum

Hydrogen evolution from root nodules has been reported to make N₂ fixation by some legume-Rhizobium symbiotic systems inefficient. We have surveyed the extent of H₂ evolution and estimated relative efficiencies of nodules of Austrian winter peas formed by 15 strains of R. leguminosarum. Their rates of H₂ evolution in air were about 30% of the rates of H₂ evolution under an atmosphere in which N₂ was replaced by Ar. Relative efficiency values based on C₂H₂ reduction rates ranged from 0.55 to 0.80. With some of the strains, hydrogenase activities were demonstrated in intact nodules and in bacteroids, but the levels of activity were insufficient to recycle all the H₂ evolved by the nitrogenase system. In both intact nodules and bacteroids the hydrogenase is less sensitive to O₂ damage than the nitrogenase system, so H₂ uptake capacity was observed in intact nodules by suppressing the nitrogenase-dependent H₂ evolution with an atmosphere containing a high O₂ concentration, and in bacteroids by using aerobically prepared bacteroid suspensions. The hydrogenase activity of both was dependent on O₂ consumption. A K _mfor H₂ of near 4 M was determined in suspension of bacteroids from nodules formed by strains 128C53 and 128C56.     

Nitrate effects on the nodulation of legumes inoculated with nitrate-reductase-deficient mutants of Rhizobium

The effect of nitrate on the symbiotic properties of nitrate-reductase-deficient mutants of a strain of cowpea rhizobia (32H1), and of a strain of Rhizobium trifolii (TA1), were examined; the host species were Macroptilium atropurpureum (DC.) Urb. and Trifolium subterraneum L. Nitrate retarded initial nodulation by the mutant strains to an extent similar to that found with the parent strains. It is therefore unlikely that nitrite produced from nitrate by the rhizobia, plays a significant role in the inhibition of nodulation by nitrate. Nitrite is an inhibitor of nitrogenase, and its possible production in the nodule tissue by the action of nitrate reductase could be responsible for the observed inhibition of nitrogen fixation when nodulated plants are exposed to nitrate. However, the results of this investigation show that nitrogen fixation by the plants nodulated by parent or mutant strains was depressed by similar amounts in the presence of nitrate. No nitrite was detected in the nodules. Nodule growth, and to a lesser extent, the nitrogenase specific activity of the nodules (mol C₂H₄g^–1 nodule fr. wt. h^–1), were both affected by the added nitrate.     

Induced genetic variability in Rhizobium leguminosarum for nitrogen fixation parameters in Vicia faba L.

Sumary The objective of this work was to know the behaviour and variability of Rhizobium leguminosarum after irradiation. The induced variation was tested under greenhouse conditions on the variety JV 3 of broad beans (Vicia faba) in six replications. Induced genetic variabilty was observed for strain, parent and mutant versus parent. Out of 24 irradiated strains, strain 93-32 performed better with a greater number of nodules and higher dry weight of nodules per plant and biological yield. Environment played an important role in the expression of characters observed. High heritability and genetic advance of these traits indicated that the nitrogen fixation ability of Rhizobium can easily be improved by selection.     

Speed of nodulation and competitive ability among strains of Rhizobium leguminosarum bv phaseoli

This study examines the speed of nodulation of 20 strains of Rhizobium leguminosarum bv phaseoli, and relates this trait to the competitive performance of these strains with Phaseolus vulgaris L. At 25/20°C day/night temperature, and with 10⁷ cells applied per growth pouch, there was a strong positive correlation between the speed of nodulation and the competitiveness of strains with the nod ⁺ fix^– reference strain UMR 1116. Strains UMR 1084, 1125, 1165, 1173 and 1384 combined good competitive performance with extensive nodulation in the uppermost root regions. When inoculant levels in the RTM studies were reduced to 10³ cells per pouch no correlation between the apparent competitiveness of strains and their speed in nodulation was evident, presumably because cells had to undergo multiplication before infection. Nodulation was also delayed when growth temperatures were raised to 31/26°C, but a correlation was still evident between competitive performance and nodulation in the region 0.1 to 5.0 mm below the RTM at the time of inoculation. From these results speed of nodulation can be used to estimate the competitive potential of Rhizobium strains, but only under carefully regulated conditions. The effects of inoculation level and temperature on the relationship between speed of nodulation and strain competitiveness could explain the inconsistent results obtained in earlier studies on this topic.Journal paper No. 16962, Agricultural Experiment Station, University of Minnesota, St. Paul, MN 55108, USA     

The role of motility in the efficiency of nodulation by Rhizobium meliloti

Tn5 mutants of Rhizobium meliloti L5.30 defective in motility (Mot^-) were isolated and compared to the parent with respect to the nodulation activity. Each of the mutants was able to generate normal nodules on the alfalfa (Medicago sativa) but had slightly delayed nodule formation. Coinoculation of lucerne with wild type Mot⁺ and Mot^- cells in the wide range of ratios resulted in nodules occupied in the majority by a motile strain suggesting that motility is a factor involved in the competition for nodule formation.     

Interaction between alfalfa cultivars and Rhizobium strains for nitrogen Fixation

Summary Two experiments were conducted in the greenhouse to study the interaction between alfalfa cultivars (Medicago sativa L. and M. falcata L.) and strains of Rhizobium meliloti Dang. for acetylene reduction rate, plant height and dry weights of shoot, root and whole plant. Fifteen alfalfa cultivars were inoculated with 10 strains of Rhizobium in Experiment I. Variance component analysis revealed that more than 30% of the total variance was due to alfalfa cultivars for acetylene reduction rate and 26% was accounted for by Rhizobium strains. More than 36% of the total variation was attributed to the interaction between alfalfa cultivars and Rhizobium strains for this character. Twenty-five host cultivars and 11 Rhizobium strains were included in Experiment II. The results also showed that the interaction of alfalfa cultivars and Rhizobium strains contributed the largest portion of the total variation for dry weights of shoot, root and whole plant and acetylene reduction rate. The results clearly demonstrated that the non-additive effects were the major component of variation for these characters associated with nitrogen fixation in alfalfa. Therefore, an effective way of improving nitrogen fixation in alfalfa is to select for a favourable combination of specific Rhizobium strains and alfalfa cultivars.     

Root exuded nod-gene inducing signals limit the nodulation capacity of different alfalfa varieties with Rhizobium meliloti

Summary Different nodulation capacities were found among nine different varieties of alfalfa, cultivated in the Central region of Mexico, by Rhizobium meliloti 2011. A correlation between nodulation capacity and foliar dry weight was observed, which points to a genotype dependance on these parameters. A correlation between the nodulation capacity and the R. meliloti nod-gene inducing activity of the root exudates from the different varieties, as measured by -galactosidase induction in a test system consisting of a R. meliloti nodC-lacZ strain incubated with each root exudate, was established. When the root exudate from the best nodulating variety was added to the four poorest nodulating varieties, an increase in nodule formation was observed. We conclude that root exuded nod-gene inducing signals are a symbiotically-limiting component in natural populations of the poorest nodulating varieties of alfalfa.     

Rhizobium leguminosarum genes required for expression and transfer of host specific nodulation

The contributions of various nod genes from Rhizobium leguminosarum biovar viceae to host-specific nodulation have been assessed by transferring specific genes and groups of genes to R. leguminosarum bv. trifolii and testing the levels of nodulation on Pisum sativum (peas) and Vicia hirsuta. Many of the nod genes are important in determination of host-specificity; the nodE gene plays a key (but not essential) role and the efficiency of transfer of host specific nodulation increased with additional genes such that nodFE < nodFEL < nodFELMN. In addition the nodD gene was shown to play an important role in host-specific nodulation of peas and Vicia whilst other genes in the nodABCIJ gene region also appeared to be important. In a reciprocal series of experiments involving nod genes cloned from R. leguminosarum bv. trifolii it was found that the nodD gene enabled bv. viciae to nodulate Trifolium pratense (red clover) but the nodFEL gene region did not. The bv. trifolii nodD or nodFEL genes did significantly increase nodulation of Trifolium subterraneum (sub-clover) by R. leguminosarum bv. viciae. It is concluded that host specificity determinants are encoded by several different nod genes.     

Nitrate reductase activity of free-living and symbiotic uptake hydrogenase-positive and uptake hydrogenase-negative strains of Bradyrhizobium japonicum

The nitrate reductase activity (NR) of selected uptake hydrogenase-positive (hup ⁺) and uptake hydrogenase-negative (hup ^-) strains of Bradyrhizobium japonicum were examined both in free-living cells and in symbioses with Glycine max L. (Marr.) cv. Williams. Bacteria were cultured in a defined medium containing either 10 mM glutamate or nitrate as the sole nitrogen source. Nodules and bacteriods were isolated from plants that were only N₂-dependent or grown in the presence of 2 mM KNO₃. Rates of activity in nodules were determined by an in vivo assay, and those of cultured cells and bacteriods were assayed after permeabilization of the cells with alkyltrimethyl ammonium bromide. All seven strains examined expressed NR activity as free-living cells and as symbiotic forms, regardless of the hup genotype of the strain used for inoculation. Although the presence of nitrate increased nitrate reduction by cultures cells and nodules, no differences in NR activity were observed between bacteroids isolated from nodules of plants fed with nitrate or grown on N₂-fixation exclusively. Cultured cells, nodules and bacteriods of strains with hup ^- genotype (USDA 138, L-236, 3. 15B3 and PJ17) had higher rates of NR activity than those with hup ⁺ genotype (USDA 110, USDA 122 DES and CB1003). These results suggest that NR activity is reduced in the presence of a genetic determinant associated with the hup region of B. japonicum.Abbreviations EDTA ethylene-diamine tetraacetic acid - Hup hydrogen uptake - MOPS 3-(N-morpholino)-propane sulfonic acid - NR nitrate reductase - PVP polyvinyl-polypyrrolidone - Tris Tris(hydroxymethyl)-aminomethane     

Glucose uptake by free living and bacteroid forms of Rhizobium leguminosarum

Free living cells of Rhizobium leguminosarum contain a constitutive glucose uptake system, except when they are grown on succinate, which appears to prevent its formation. Bacteroids isolated from Pisum sativum L fail to accumulate glucose although they actively take up ¹⁴C-succinate. Glucose uptake in free living cells is an active process since uptake was inhibited by azide, cyanide, dinitrophenol and carbonyl-m-chlorophenyl hydrazone but not by fluoride or arsenate. The non-metabolizable analogue -methyl glucose was extracted unchanged from cells, showing that it was not phosphorylated during its transport. Galactose also appears to the transported via the glucose uptake system. Organic acids, amino acids and polyols had no effect on the actual uptake of glucose. The K _m for -methyl glucose uptake was 2.9×10^-4 M.     

Competition of strains of Rhizobium of the cowpea group in two soil types

Summary Competition of five strains of Rhizobium of the cowpea group, onVigna radiata (L) Wilcjeck variety ML 5, was tested in loamy clay and loamy sand soils. Strains RM 6 and RM 5 were effective nodulators in loamy clay soil, and strains MNH, M 20 and RM 6 were effective nodulators in loamy sand soil. Strains RM 6 and MNH predominated nodule formation in loamy clay and loamy sand soils respectively.     

Rhizobium strains expressing uptake hydrogenase in different host species of cowpea miscellany

Uptake hydrogenase activity in nodules of green gram (Vigna radiata (L.) (Wilczek)), black gram (Vigna mungo (L.) (Hepper)), cowpea (Vigna unguiculata (L.) and cluster bean (Cyamopsis tetragonoloba (L.) (Taub.)), formed with two Hup⁺ (S24 and CT2014) and one Hup⁻ (M11)Rhizobium strains, was determined at different levels of external H2 in air atmosphere. Nodules of all the 4 host species formed by inoculation with strains S24 and CT2014, showed H2 uptake but not those formed with strain M11. H₂ uptake rates were higher in 1 and 2% H₂ in air atmosphere (v/v) than at 5 or 10% levels in all the host species. Variations in the relative rates of H₂ uptake were observed both, due to host species as well as due toRhizobium strains. However, no host dependent complete repression of the expression of H₂ uptake activity was observed in nodules of any of the host species formed with Hup⁺ strains.     

Effect of glutamate transport and catabolism on symbiotic effectiveness in Rhizobium leguminosarum bv. phaseoli

Seven Tn5 induced mutants unable to use glutamate as sole carbon and nitrogen source were isolated from the effective Rhizobium leguminosarum bv. phaseoli strain P121-R. As indicated by restriction and hybridisation analysis, all the mutants arose from a single Tn5 insertion in the chromosome. The ¹⁴C-glutamate uptake rate of the mutants was 76 to 88% lower than that of strain P121-R. Inoculation of Phaseolus vulgaris cv. Labrador with these mutants significantly decreased shoot dry matter yield and the total nitrogen content respectively, as compared to inoculation with the parental strain P121-R. All the mutants formed nodules, however they were smaller, white to greenish and approximately 30% less numerous than those formed by strain P121-R. These observations suggest that glutamate transport and catabolism in R. leguminosarum bv. phaseoli P121-R may play an important role in the establishment of an effective symbiosis in field bean. None of the mutants isolated was an auxotroph. All mutants were unable to grow on aspartate suggesting that glutamate and aspartate, probably have the same transporter as indicated in Rhizobium meliloti and in Bacillus subtilis. All mutants readily used glutamine, proline, arginine as sole carbon and nitrogen source, but grew more slowly than the wild type strain. On the other hand, all the mutants were impaired in growth on histidine and -aminobutyrate as sole carbon and nitrogen source. As the catabolism of these amino acids occurs predominantly through glutamate, our results indicate that mutants are also impaired in their ability to use histidine and -aminobutyrate as a nitrogen source. Our results also suggest that other amino acids catabolized through the glutamate pathways may be an additional important carbon source for bacteroids in nodules.