Lipopolysaccharide epitope expression of Rhizobium bacteroids as revealed by in situ immunolabelling of pea root nodule sections.

To investigate the in situ expression of lipopolysaccharide (LPS) epitopes on nodule bacteria of Rhizobium leguminosarum, monoclonal antibodies recognizing LPS macromolecules were used for immunocytochemical staining of pea nodule tissue. Many LPS epitopes were constitutively expressed, and the corresponding antibodies reacted in nodule sections with bacteria at all stages of tissue infection and cell invasion. Some antibodies, however, recognized epitopes that were only expressed in particular regions of the nodule. Two general patterns of regulated LPS epitope expression could be distinguished on longitudinal sections of nodules. A radial pattern probably reflected the local physiological conditions experienced by endosymbiotic bacteria as a result of oxygen diffusion into the nodule tissue. The other pattern of expression, which followed a linear axis of symmetry along a longitudinal section of the pea nodule, was apparently associated with the differentiation of nodule bacteria and the development of the nitrogen-fixing capacity in bacteroids. Basically similar patterns of LPS epitope expression were observed for pea nodules harboring either of two immunologically distinct strains of R. leguminosarum bv. viciae, although these epitopes were recognized by different sets of strain-specific monoclonal antibodies. Furthermore, LPS epitope expression of rhizobia in pea nodules was compared with that of equivalent strains in nodules of French bean (Phaseolus vulgaris). From these observations, it is suggested that structural modifications of Rhizobium LPS may play an important role in the adaptation of endosymbiotic rhizobia to the surrounding microenvironment.     

Mixed Inoculations with Effective and Ineffective Strains of Rhizobium leguminosarum

An ineffective Rhizobium leguminosarum strain capable of forming green nodules of similar size and number as normally effective strains was tested for its ability to compete with an effective strain in nodule formation on the pea. The ineffective strain was found to be more competitive and influenced the pattern of nodulation by the effective strain on the same root system. Nodules containing both strains were pink and able to reduce acetylene.     

Identification of Rhizobium leguminosarum and Rhizobium sp. (Cicer) strains using a custom Fatty Acid Methyl Ester (FAME) profile library

The potential of using fatty acid methyl ester (FAME) profiles of Rhizobium leguminosarum bv. viceae , phaseoli and trifolii , and Rhizobium sp. ( Cicer ) strains, for the identification of unknown isolates was assessed. This was achieved by developing a Rhizobium FAME library using 16 different Rhizobium strains of Rh. leguminosarum bv. viceae ( n  ₌ 5), Rh. leguminosarum bv. phaseoli ( n  ₌ 5), Rh. leguminosarum bv. trifolii ( n  ₌ 1) and Rhizobium sp. ( Cicer ) ( n  ₌ 5). Although there were considerable differences between Rh. leguminosarum biovars and strains and Rhizobium sp. ( Cicer ) strains, the variation within a particular biovar of Rh. leguminosarum was not high. Nevertheless, the feature FAME profiles of the various groups in the library allowed 75 putative rhizobia obtained from surface-sterilized nodules of field-grown lentil and pea plants to be identified.     

Screening method for the detection of uptake hydrogenase activity of Rhizobium leguminosarum bacteroids

Abstract A method has been developed for screening Rhizobium leguminosarum wild-type strains and mutants for uptake hydrogenase (Hup) activity, using H₂-dependent methylene blue reduction. For this purpose, a simple device has been constructed which allows the simultaneous screening of 6 strains and 6 controls. Bacteroids of R. leguminosarum isolated from pea root nodules were suspended in buffer containing methylene blue and inhibitors of dehydrogenases. The suspensions were first sparged with argon (to remove oxygen) and then with hydrogen.     

Nitrate reductase activity in nodules of pea inoculated with hydrogenase positive and hydrogenase negative strains of Rhizobium leguminosarum

The nitrate reductase (NR, EC 1.6.6.1) activity in root nodules formed by hydrogenase positive (Hup⁺) and hydrogenase negative (Hup⁻) Rhizobium leguminosarum strains was examined in symbioses with the pea cultivar Alaska ( Pisum sativum L.), Rates of activity were determined by the in vivo assay in nodules from plants that were only N₂-dependent or grown in the presence of 2 m M KNO₃. The rates varied widely among strains, regardless of the Hup phenotype of the R. leguminosarum strain used for inoculation, but the overall results indicated that nodules formed by Hup⁻ strains accumulated more nitrite in the incubation medium than did those with Hup⁻ phenotypes. Total plant dry weight and reduced nitrogen content of pea plants grown in the presence of 2 m M KNO₃ and inoculated with single Hup⁺ and Hup⁻ R. leguminosarum strains were statistically different among some strains. These observations suggest that the possible advantages derived from the presence of the Hup system on whole plant growth may be counteracted by the higher rates of NR activity in the Hup⁻ strains in the R. leguminosarum -pea symbiosis.     

Identification of a Rhizobium trifolii plasmid coding for nitrogen fixation and nodulation genes and its interaction with pJB5JI, a Rhizobium leguminosarum plasmid

Rhizobium trifolii T37 contains at least three plasmids with sizes of greater than 250 megadaltons. Southern blots of agarose gels of these plasmids probed with Rhizobium meliloti nif DNA indicated that the smallest plasmid, pRtT37a, contains the nif genes. Transfer of the Rhizobium leguminosarum plasmid pJB5JI, which codes for pea nodulation and the nif genes and is genetically marked with Tn5, into R. trifolii T37 generated transconjugants containing a variety of plasmid profiles. The plasmid profiles and symbiotic properties of all of the transconjugants were stably maintained even after reisolation from nodules. The transconjugant strains were placed into three groups based on their plasmid profiles and symbiotic properties. The first group harbored a plasmid similar in size to pJB5JI (130 megadaltons) and lacked a plasmid corresponding to pRtT37a. These strains formed effective nodules on peas but were unable to nodulate clover and lacked the R. trifolii nif genes. This suggests that genes essential for clover nodulation as well as the R. trifolii nif genes are located on pRtT37a and have been deleted. The second group harbored hybrid plasmids formed from pRtT37a and pJB5JI which ranged in size from 140 to ca. 250 megadaltons. These transconjugants had lost the R. leguminosarum nif genes but retained the R. trifolii nif genes. Strains in this group nodulated both peas and clover but formed effective nodules only on clover. The third group of transconjugants contained a hybrid plasmid similar in size to pRtT37b. These strains contained the R. trifolii and R. leguminosarum nif genes and formed N2-fixing nodules on both peas and clover.     

Gene centres,a source for genetic variants in symbiotic nitrogen fixation: The symbiotic response of the cultivated pea toRhizobium leguminosarum strains from Europe and the Middle East

Summary Soil samples from several European countries; Sweden, the Netherlands, Spain, Italy and Greece, contained rhizobial populations capable of forming an effective symbiosis with the cultivated pea cv. Rondo from the Netherlands. The range of variation among the European Rhizobium strains, as expressed on pea cv. Rondo, was not so large and almost the same variation could be found within the rhizobial population within each country. Superior Rhizobium strains for the Dutch pea were not restricted to soils from the Netherlands but were also found in those from Sweden and Italy.Soils from Turkey and Israel also contained Rhizobium strains capable of nodulating pea cv. Rondo. However, the genetic variation among these Middle East Rhizobium strains was much larger than that of the European strains. When tested on pea cv. Rondo the majority of the Middle East strains belonged to the medium or low effective classes and only a few strains were comparable with European Rhizobium strains.Dutch Rhizobium strains induced effective nodules on both the Dutch pea cv. Rondo and the Swedish cv. L 110. However, in association with a Turkish Rhizobium strain effective nodules were formed on pea cv. Rondo and ineffective nodules on cv. L 110.We suggest that the genetic uniformity of EuropeanR. leguminosarum strains is the result of selection and domestication of Rhizobium strains originally derived from the gene centres of the pea plant.     

Identification of a rhizosphere protein encoded by the symbiotic plasmid of Rhizobium leguminosarum.

A protein was identified which was made by wild-type strains of Rhizobium leguminosarum but not by nodulation-deficient derivatives which had deletions of their symbiotic plasmids. The protein, which had a subunit molecular weight of ca. 24,000 ( 24K ), was found to be present in large amounts within bacteria that had been reisolated from the surface of inoculated pea roots but was not detected in bacteroids isolated from nodules. The protein could also be induced during growth of R. leguminosarum on nutrient medium and was purified from the cytoplasmic fraction of broken cells. Antiserum raised against the purified protein was used to screen transposon-induced mutants of R. leguminosarum, and four independent mutants were isolated which lacked the protein. The sites of the Tn5 insertions were found to map between the nitrogenase and nodulation genes on symbiotic plasmid pRL1JI , ca. 5 kilobases from the nitrogenase genes and 13 kilobases from the nodulation genes. Genetic determinants for the 24K protein were found to be closely linked to plasmid-borne nodulation genes for all strains of R. leguminosarum tested. However, the mutants which lacked the 24K protein still formed normal nitrogen-fixing nodules on peas, and the function of the protein is unknown.     

Direct amplification of nodD from community DNA reveals the genetic diversity of Rhizobium leguminosarum in soil

Sequences of nodD , a gene found only in rhizobia, were amplified from total community DNA isolated from a pasture soil. The polymerase chain reaction (PCR) primers used, Y5 and Y6, match nodD from Rhizobium leguminosarum biovar trifolii , R. leguminosarum biovar viciae and Sinorhizobium meliloti . The PCR product was cloned and yielded 68 clones that were identified by restriction pattern as derived from biovar trifolii [11 restriction fragment length polymorphism (RFLP) types] and 15 clones identified as viciae (seven RFLP types). These identifications were confirmed by sequencing. There were no clones related to S. meliloti nodD . For comparison, 122 strains were isolated from nodules of white clover ( Trifolium repens ) growing at the field site, and 134 from nodules on trap plants of T. repens inoculated with the soil. The nodule isolates were of four nodD RFLP types, with 77% being of a single type. All four of these patterns were also found among the clones from soil DNA, and the same type was the most abundant, although it made up only 34% of the trifolii -like clones. We conclude that clover selects specific genotypes from the available soil population, and that R. leguminosarum biovar trifolii was approximately five times more abundant than biovar viciae in this pasture soil, whereas S. meliloti was rare.     

Strain Identification in Rhizobium Using Intrinsic Antibiotic Resistance

The variation in intrinsic resistance to low levels of eight antibiotics was used as an identifying characteristic for 26 Rhizobium leguminosarum strains. The pattern of antibiotic resistance of each strain was a stable property by which rhizobia isolated from root nodules of inoculated Pisum sativum could be recognized. The antibiotic tests for strain identification with R. leguminosarum were applied to R. phaseoli . It was necessary to include reference cultures in tests with this species, as the tests most suitable for the R. leguminosarum strains showed some variability with R. phaseoli .     

根瘤菌同工酶图谱分析和分类

利用聚丙烯酰胺凝胶电泳分析了９株根瘤菌酯酶及ＳＯＤ同工酶图谱,结果显示快生型根瘤菌与慢生型根瘤菌有明显区别,不仅根瘤菌种间有明显差别,而且菌株间也存在差异。Ｒｈｉｚｏｂｉｕｍｌｅｇｕｍｉｎｏｓａｒｕｍｂｉｏｖａｒｓｖｉｃｅａｅ和ｐｈａｓｅｏｌｉ具有相同的ＳＯＤ图谱和相似的酯酶图谱。快生型大豆根瘤菌的上述同工酶图谱不同于慢生型大豆根瘤菌和其它快生型根瘤菌。     

Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within HupRhizobium leguminosarum Strains

Thirteen Rhizobium leguminosarum strains previously reported as H(2)-uptake hydrogenase positive (Hup) or negative (Hup) were analyzed for the presence and conservation of DNA sequences homologous to cloned Bradyrhizobium japonicum hup-specific DNA from cosmid pHU1 (M. A. Cantrell, R. A. Haugland, and H. J. Evans, Proc. Natl. Acad. Sci. USA 80:181-185, 1983). The Hup phenotype of these strains was reexamined by determining hydrogenase activity induced in bacteroids from pea nodules. Five strains, including H(2) oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H(2)-uptake hydrogenase activity and contained DNA sequences homologous to three probe DNA fragments (5.9-kilobase [kb] HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) from pHU1. The pattern of genomic DNA HindIII and EcoRI fragments with significant homology to each of the three probes was identical in all five strains regardless of the H(2)-dependent ATP generation trait. The restriction fragments containing the homology totalled about 22 kb of DNA common to the five strains. In all instances the putative hup sequences were located on a plasmid that also contained nif genes. The molecular sizes of the identified hup-sym plasmids ranged between 184 and 212 megadaltons. No common DNA sequences homologous to B. japonicum hup DNA were found in genomic DNA from any of the eight remaining strains showing no significant hydrogenase activity in pea bacteroids. These results suggest that the identified DNA region contains genes essential for hydrogenase activity in R. leguminosarum and that its organization is highly conserved within Hup strains in this symbiotic species.     

Gene centres,a source for genetic variants in symbiotic nitrogen fixation: Host-induced ineffectivity inPisum sativum ecotype fulvum

Summary Pisum sativum ecotype fulvum forms ineffective nodules with Rhizobium strains, isolated from effective nodules of the cultivated pea in Europe. Rhizobium strains isolated from nodules of fulvum peas in Israel are fully effective on this host plant, but in association with the cultivated pea they induce nodules of poor N₂-fixing activity. The distribution of these fulvum-specific Rhizobium strains is restricted to regions where the fulvum pea occurs naturally. Rhizobium strains from other geographical regions induce mainly ineffective, or partially effective nodules on fulvum plants.A wide genetic variation, with regard to symbiotic response to a standard set of Rhizobium strains, was demonstrated in the fulvum plants collected in Israel. Based on variation in N₂-fixation three groups of plants can be distinguished. These plants offer the possibility for the study of host-genetic control on symbiotic nitrogen fixation.     

Comparison of characteristics of the nodX genes from various Rhizobium leguminosarum strains

We have analyzed the nucleotide sequences of the nodX genes from two strains of Rhizobium leguminosarum bv. viciae able to nodulate Afghan peas (strains A1 and Himalaya) and from two strains of R. leguminosarum bv. trifolii (ANU843 and CSF). The nodX genes of strains A1 and ANU843 were shown to be functional for the induction of nodules on Afghan peas. To analyze the cause of phenotypic differences of strain A1 and strain TOM we have studied the composition of the lipochitin-oligosaccharides (LCOs) produced by strain A1 after induction by the flavonoid naringenin or various pea root exudates. The structural analysis of the LCOs by mass spectrometry revealed that strain A1 synthesizes a family of at least 23 different LCOs. The use of exudates instead of naringenin resulted only in quantitative differences in the ratios of various LCOs produced.     

Genetic structure of a soil population of nonsymbiotic Rhizobium leguminosarum.

The genetic structure of a population of nonsymbiotic Rhizobium leguminosarum strains was determined by the electrophoretic mobilities of eight metabolic enzymes. Nonsymbiotic strains were isolated from the rhizosphere of bean plants and characterized by growth on differential media and at different temperatures, intrinsic antibiotic resistance, the lack of homology to a nifH probe, and their inability to form nodules on bean roots. All the isolates clustered with R. leguminosarum bv. phaseoli reference strains and did not encompass any other Rhizobium taxa. Their rRNA operon restriction fragment length polymorphisms and the nucleotide sequence of a fragment of the 16S rRNA gene were also found to be identical to those of R. leguminosarum bv. phaseoli reference strains. When complemented with an R. leguminosarum bv. phaseoli symbiotic plasmid (p42d), the nonsymbiotic isolates were able to fix nitrogen in symbiosis with bean roots at levels similar to those of the parental strain. The symbiotic isolates were found at a relative frequency of 1 in 40 nonsymbiotic R. leguminosarum strains.     

Genetic structure of a soil population of nonsymbiotic Rhizobium leguminosarum.

The genetic structure of a population of nonsymbiotic Rhizobium leguminosarum strains was determined by the electrophoretic mobilities of eight metabolic enzymes. Nonsymbiotic strains were isolated from the rhizosphere of bean plants and characterized by growth on differential media and at different temperatures, intrinsic antibiotic resistance, the lack of homology to a nifH probe, and their inability to form nodules on bean roots. All the isolates clustered with R. leguminosarum bv. phaseoli reference strains and did not encompass any other Rhizobium taxa. Their rRNA operon restriction fragment length polymorphisms and the nucleotide sequence of a fragment of the 16S rRNA gene were also found to be identical to those of R. leguminosarum bv. phaseoli reference strains. When complemented with an R. leguminosarum bv. phaseoli symbiotic plasmid (p42d), the nonsymbiotic isolates were able to fix nitrogen in symbiosis with bean roots at levels similar to those of the parental strain. The symbiotic isolates were found at a relative frequency of 1 in 40 nonsymbiotic R. leguminosarum strains.     

Distribution of Symbiotic Genotypes in Rhizobium leguminosarum biovar viciae Populations Isolated Directly from Soils

The distribution of symbiotic (Sym) plasmid types across background genotypes was investigated in two field populations of Rhizobium leguminosarum biovar viciae isolated directly from soils. PCR-based methods were used to characterize the background genotypes and the Sym gene types. Identical Sym gene types were associated with a variable range of background genotypes, while the same background genotype could harbor distinct Sym gene types. Random distributions of Sym gene types in the background genotypes were observed in the two soil populations. These results suggest that Sym plasmid transfer is less restricted than previously thought on the basis of the analysis of strains isolated from legume nodules.     

Specific flavonoids induced nod gene expression and pre-activated nod genes of Rhizobium leguminosarum increased pea (Pisum sativum L.) and lentil (Lens culinaris L.) nodulation in controlled growth chamber environments.

The gram-negative soil bacteria Rhizobium spp. infect and establish a nitrogen-fixing symbiosis with legume crops which involves the mutual exchange of diffusable signal molecules. In this study, Rhizobium leguminosarum containing a nod-lacZ gene fusion was used to screen the most effective plant-to-bacteria signal molecules for pea and lentil and the induction conditions. Out of a number of signal compounds including apigenin, daidzein, genistein, hesperetin, kaempferol, luteolin, naringenin, and rutin, hesperetin and naringenin were found to be the most effective plant-to-bacteria signal molecules. The induction of nod genes was temperature-dependent, where nod gene induction was decreased with dropping incubation temperature. The combination of hesperetin at 7 microM and naringenin at 3 microM resulted in better induction of nod gene activities compared to either hesperetin or naringenin alone. Nodulation and plant dry matter accumulation of pea and lentil plants receiving preinduced R. leguminosarum were higher than those of plants receiving uninduced R. leguminosarum cells in controlled environment growth chamber conditions. Preinduced Rhizobium with hesperetin at a concentration of 10 microM increased nodule number on average by 60.5% and dry matter accumulation by 14% in field pea at 17 degrees C, while it was 32% and 9% at 24 degrees C, respectively. Similarly, averaged over two rhizobial strains, a 59% and 6% increase in nodule number and biomass production at 17 degrees C, and a 39% and 27% at 24 degrees C, were obtained from lentil inoculated with hesperetin-induced R. leguminosarum, respectively.     

Genetic factors in Rhizobium affecting the symbiotic carbon costs of N2 fixation and host plant biomass production

The effect of genetic factors in Rhizobium on host plant biomass production and on the carbon costs of N₂ fixation in pea root nodules was studied. Nine strains of Rhizobium leguminosarum were constructed, each containing one of three symbiotic plasmids in combination with one of three different genomic backgrounds. The resulting strains were tested in symbiosis with plants of Pisum sativum using a flow-through apparatus in which nodule nitrogenase activity and respiration were measured simultaneously under steady state conditions. Nodules formed by strains containing the background of JI6015 had the lowest carbon costs of N₂ fixation (7.10–8.10 μmol C/μmol N₂), but shoot dry weight of those plants was also smaller than that of plants nodulated by strains with the background of B151 or JI8400. Nodules formed by these two strain types had carbon costs of N₂ fixation varying between 11.26 and 13.95 μmol C/μmol N₂. The effect of symbiotic plasmids on the carbon costs was relatively small. A time-course experiment demonstrated that nodules formed by a strain derived from JI6015 were delayed in the onset of nitrogenase activity and had a lower rate of activity compared to nodules induced by a strain with the background of B151. The relationship between nitrogenase activity, carbon costs of N₂ fixation and host plant biomass production is discussed.