Transfer of nitrogen fixation genes from a bacterium with the characteristics of both Rhizobium and Agrobacterium.

Strain T1K, reported to be Rhizobium trifolii strain T1 carrying the drug resistance plasmid RU-1drd, was able to transfer a cluster of nif+ genes to Escherichia coli K-12. Additional genetic material, resembling the gal-chlA region of E. coli, was also transferred from strain T1K. The segregation pattern of these transferred genes suggested that they were on a plasmid. Although strain TIK was able to nodulate red and white clover, it also formed very slow-growing galls on tomato stems and shared many physiological properties with Agrobacterium tumefaciens, to which it seemed more closely related than to R. trifolii. The R. trifolii hybrid T1 (R1-19drd), constructed by conjugation, did not share any of these properties of both A. tumefaciens. Thus, strain T1K appears to be a bacterium with properties of both A. tumefaciens and R. trifolii and with the capacity to transfer nif+ genes and other functions which it may have "cloned" from another bacterium such as Klebsiella.     

Comparative properties of glutamine synthetases I and II in Rhizobium and Agrobacterium spp

Some properties of glutamine synthetase I (GSI) and GSII are described for a fast-growing Rhizobium sp. (Rhizobium trifolii T1), a slow-growing Rhizobium sp. (Rhizobium japonicum USDA 83), and Agrobacterium tumefaciens C58. GSII of the fast-growing Rhizobium sp. and GSII of the Agrobacterium sp. were considerably more heat labile than GSII of the slow-growing Rhizobium sp. As previously shown in R. japonicum 61A76, GSI became adenylylated rapidly in all species tested in response to ammonium. GSII activity disappeared within one generation of growth in two of the strains, but the disappearance of GSII activity required two generations in another. Isoactivity points for transferase assay, which were derived from the pH curves of adenylylated GSI and deadenylylated GSI, were approximately pH 7.8 for both R. trifolii and A. tumefaciens. No isoactivity point was found for R. japonicum under the standard assay conditions used. When the feedback inhibitor glycine was used to inhibit differentially the adenylylated GSI and deadenylylated GSI of R. japonicum, an isoactivity point was observed at pH 7.3. Thus, the transferase activity of GSI could be determined independent of the state of adenylation. A survey of 23 strains of bacteria representing 11 genera indicated that only Rhizobium spp. and Agrobacterium spp. contained GSII. Thus, this enzyme appears to be unique for the Rhizobiaceae.     

Glycerol metabolism in Rhizobium.

Four strains of Rhizobium japonicum and one strain of R. trifolii were grown on glycerol and found to contain a soluble ATP-glycerol kinase and a particulate glycerolphosphate dehydrogenase. Both enzymes are induced by glycerol. The presence of NAD+-or NADP+-glycerol dehydrogenase was not detected in any of the strains. No significant differences were found in the glycerol metabolic pathway between fast-and slow-growing rhizobia.     

Chemotaxis to aromatic and hydroaromatic acids: comparison of Bradyrhizobium japonicum and Rhizobium trifolii

Rhizobia are bacteria well known for their ability to fix nitrogen in symbiosis with leguminous plants. Members of diverse rhizobial species grow at the expense of hydroaromatic and aromatic compounds commonly found in plant cells and plant litter. Using a quantitative capillary assay to measure chemotaxis, we tested the ability of hydroaromatic acids, selected aromatic acids, and their metabolites to serve as chemoattractants for two distantly related rhizobial species, Bradyrhizobium japonicum and Rhizobium trifolii. Slow-growing B. japonicum I-110 demonstrated positive chemotaxis to shikimate, quinate, protocatechuate, and vanillate; threshold concentrations for the compounds were as low as 10(-6) M. The dicarboxylic acids succinate and beta-ketoadipate, metabolites in the catabolism of many aromatic compounds, were positive chemoattractants with low threshold concentrations as well. Taxis to beta-ketoadipate occurred constitutively and, of the tested compounds, beta-ketoadipate gave the strongest peak response. Taxis to shikimate or quinate was induced by growth on either substrate but not by growth on protocatechuate or succinate. In contrast, fast-growing R. trifolii 2066 was only weakly attracted to quinate and other aromatic and dicarboxylic acids that were strong attractants for B. japonicum. The R. trifolii strain exhibited positive chemotaxis to shikimate, but the threshold concentration of shikimate required to elicit a response (10(-4) M) was 2 orders of magnitude higher than that for the B. japonicum strain.     

Exopolysaccharide depolymerases induced by Rhizobium bacteriophages.

Enzymes induced by two Rhizobium trifolii bacteriophages caused depolymerization of exopolysaccharides from most R. trifolii and R. leguminosarum strains tested, but did not, in general, attack the exopolysaccharides of R. meliloti, the slow-growing rhizobia, or Agrobacterium. Ca2+ and (or) Mg2+ were required for enzyme activity. In all strains tested, depolymerization of exopolysaccharide occurred when there was successful phage infection, but depolymerization also occurred with exopolysaccharides from nonsusceptible strains.     

Comparison of the host range of fast-growingR. japonicum strains with a fast-growing isolate from Lablab

Summary Fast-growingRhizobium japnicum strains derived from the People's Republic of China were compared with a fast-growingRhizobium isolate from Lablab for their ability to nodulate tropical legumes grown in Leonard-jars and test tube culture. Fast-growingR. japonicum strains were all effective to varying degrees in their symbiosis withVigna unguiculata. Two strains USDA 192 and USDA 201, effectively nodulatedGlycine whightii and one strain, USDA 193, effectively nodulatedMacroptilium atropurpureum. Other nodulation responses in tropical legumes were ineffective. The fast-growing isolate from Lablab was more promiscuous, effectively nodulating with a larger host range. The fast-growing Lablab strain was considered more akin, on a symbiotic basis, to the slow-growing cowpea type rhizobia than the fast-growing China strains ofR. japonicum whilst maintaining physiological characteristics of other fast-growing rhizobia.     

Metabolic properties, maximum growth temperature and phage sensitivity of Rhizobium sp. (Galega) compared with other fast-growing rhizobia

Abstract Rhizobium strains nodulating Galega species were characterized by metabolic tests, maximum growth temperature determinations in a temperature gradient incubator and phage typing, and compared with other fast-growing rhizobia. By numerical taxonomy it was shown that the Galega Rhizobium strains are closely related to each other and unrelated to the recognized species of Rhizobium . The maximum growth temperature of rhizobia nodulating G. orientalis was 33.0–34.0°C and of rhizobia nodulating G. officinalis 35.0–37.0°C. The Galega rhizobia were only lysed by their own phages, and not by typing phages for other Rhizobium species. The G + C% of Rhizobium sp. ( Galega ) strainswas 63%.44 previously unclassified fast-growing rhizobia from tropical plants, which were included in the experiments, were shown to form a heterogenous group with diverse properties. The results confirm and extend previous findings, and suggest that Rhizobum sp. ( Galega ) should be considered a new species of Rhizobium .     

Predominance of Fast-Growing Rhizobium japonicum in a Soybean Field in the People's Republic of China

Soybean rhizobia were isolated from two soils with different cropping histories from Hubei province in central China. The first, from Honghu county, has been under soybean cultivation for decades. All of the isolates obtained from nodules on soybeans growing in this soil were fast-growing, acid-producing rhizobia. However, slow-growing, alkali-producing isolates were obtained at higher dilutions of the same soil. The second soil, from Wuchang county, has been under rice cultivation with no record of previous soybean cultivation. All of the soybean rhizobia recovered from this soil, and at higher dilutions of the soil, were typical slow-growing, alkali-producing isolates. The isolates from both soils were grouped by using intrinsic antibiotic resistance, gel immunodiffusion, and fluorescent-antibody procedures. Representative isolates were tested for symbiotic effectiveness with four soybean cultivars (Peking, Davis, Williams, and Ai Jiao Zao) in a pot experiment. There were significant cultivar-rhizobial interactions. Moreover, on each cultivar, there was at least one fast-growing isolate among these new rhizobia that was as effective as the highly effective slow-growing reference strain USDA 110.     

Predominance of Fast-Growing Rhizobium japonicum in a Soybean Field in the People's Republic of China

Soybean rhizobia were isolated from two soils with different cropping histories from Hubei province in central China. The first, from Honghu county, has been under soybean cultivation for decades. All of the isolates obtained from nodules on soybeans growing in this soil were fast-growing, acid-producing rhizobia. However, slow-growing, alkali-producing isolates were obtained at higher dilutions of the same soil. The second soil, from Wuchang county, has been under rice cultivation with no record of previous soybean cultivation. All of the soybean rhizobia recovered from this soil, and at higher dilutions of the soil, were typical slow-growing, alkali-producing isolates. The isolates from both soils were grouped by using intrinsic antibiotic resistance, gel immunodiffusion, and fluorescent-antibody procedures. Representative isolates were tested for symbiotic effectiveness with four soybean cultivars (Peking, Davis, Williams, and Ai Jiao Zao) in a pot experiment. There were significant cultivar-rhizobial interactions. Moreover, on each cultivar, there was at least one fast-growing isolate among these new rhizobia that was as effective as the highly effective slow-growing reference strain USDA 110.     

Enzymatic Basis for Differentiation of Rhizobium into Fast- and Slow-Growing Groups

Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and other enzymes related to carbohydrate metabolism were studied in rhizobia. A nicotinamide adenine dinucleotide phosphate-6-phosphogluconate dehydrogenase was detected in strains of the fast-growing group of Rhizobium but not in strains of the slow-growing group. An enzymatic differentiation of rhizobia was established.     

Genetic locus in Rhizobium japonicum (fredii) affecting soybean root nodule differentiation.

A genetic locus in fast-growing Rhizobium japonicum (fredii) USDA 191 (Fix+ on several contemporary soybean cultivars) was identified by random Tn5 mutagenesis as affecting the development and differentiation of root nodules. This mutant (MU042) is prototrophic and shows no apparent alterations in its surface properties. It induces aberrant nodules, arrested at the same early level of differentiation, on all its host plants. An 8.1-kilobase EcoRI fragment containing Tn5 was cloned from MU042. In USDA 191 as well as another fast-growing strain, USDA 201, the affected locus was found to be unlinked to the large symbiotic plasmid and appears to be chromosomal. An analogous sequence has been shown to be present in Bradyrhizobium japonicum (J. Stanley, G.G. Brown, and D.P.S. Verma, J. Bacteriol. 163:148-154, 1985) as well as in R. trifolii and R. meliloti. MU042 was complemented for effective nodulation of soybean by a cosmid clone from USDA 201, and the complementing locus was delimited to a 6-kilobase EcoRI subfragment. An R. trifolii strain (MU225), whose indigenous symbiotic plasmid was replaced by that of strain USDA 191, induced more highly differentiated nodules on soybean than did MU042. This suggests that the mutation in MU042 can be functionally substituted by similar loci of other fast-growing rhizobia. Leghemoglobin and nodulin-35 (uricase II) were present in the differentiated Fix- nodules induced by MU225, whereas both were absent in MU042-induced pseudonodule structures.     

Nitrogenase activity in pure cultures of spectinomycin-resistant fast and slow-growing Rhizobium.

Several strains of Rhizobium resistant to spectinomycin also had nitrogenase activity (C₂H₂ reduction and H₂ production) in static culture under 95% Ar/1%O₂/4%C₂H₂. This relationship between nitrogenase activity and spectinomycin resistance was observed in both fast-growing (R. trifolii and R. leguminosarum) and slow-growing (R. japonicum) rhizobia. The effect of different media and various carbon sources on nitrogenase activity was investigated in more detail in R. trifolii strain TlSp. This communication demonstrates that fast-growing rhizobia can have nitrogenase activity in the absence of any plant component.     

Phylogeny of fast-growing soybean-nodulating rhizobia support synonymy of Sinorhizobium and Rhizobium and assignment to Rhizobium fredii.

We determined the sequences for a 260-base segment amplified by the polymerase chain reaction (corresponding to positions 44 to 337 in the Escherichia coli 16S rRNA sequence) from seven strains of fast-growing soybean-nodulating rhizobia (including the type strains of Rhizobium fredii chemovar fredii, Rhizobium fredii chemovar siensis, Sinorhizobium fredii, and Sinorhizobium xinjiangensis) and broad-host-range Rhizobium sp. strain NGR 234. These sequences were compared with the corresponding previously published sequences of Rhizobium leguminosarum, Rhizobium meliloti, Agrobacterium tumefaciens, Azorhizobium caulinodans, and Bradyrhizobium japonicum. All of the sequences of the fast-growing soybean rhizobia, including strain NGR 234, were identical to the sequence of R. meliloti and similar to the sequence of R. leguminosarum. These results are discussed in relation to previous findings; we concluded that the fast-growing soybean-nodulating rhizobia belong in the genus Rhizobium and should be called Rhizobium fredii.     

Growth of Fast- and Slow-Growing Rhizobia on Ethanol

Free-living soybean rhizobia and Bradyrhizobium spp. (lupine) have the ability to catabolize ethanol. Of the 30 strains of rhizobia examined, only the fast- and slow-growing soybean rhizobia and the slow-growing Bradyrhizobium sp. (lupine) were capable of using ethanol as a sole source of carbon and energy for growth. Two strains from each of the other Rhizobium species examined (R. meliloti, R. loti, and R. leguminosarum biovars phaseoli, trifolii, and viceae) failed to grow on ethanol. One Rhizobium fredii (fast-growing) strain, USDA 191, and one (slow-growing) Bradyrhizobium japonicum strain, USDA 110, grew in ethanol up to concentrations of 3.0 and 1.0%, respectively. While three of the R. fredii strains examined (USDA 192, USDA 194, and USDA 205) utilized 0.2% acetate, only USDA 192 utilized 0.1% n-propanol. None of the three strains utilized 0.1% methanol, formate, or n-butanol as the sole carbon source.     

Some antigenic properties of cultured cell and bacteroid forms of fast- and slow-growing strains of Lotus rhizobia.

Immunodiffusion cross-reactions of 62 fast- and 76 slow-growing of Lotus rhizobia with antisera to four of the fast-growing and five of the slow-growing strains were studied. No sharing of antigens by both fast- and slow-growing strains was found. Somatic antigens were very strain specific with only eight of the fast-growing and five of the slow-growing strains tested having somatic antigens identical to those of one or more of the strains of the same group used for antisera production. In contrast, internal antigens were shared by all fast-growing strains and with seven exceptions by all slow-growing strains. Antigens of cultured rhizobia, and bacteroids from nodules formed on different legumes by the same strain of Rhizobium, were similar. However, incontrast to cultured cells, bacteroids generally required no pretreatment (heat or ultrasonic disruption) to give a strong somatic antigen reaction in immunodiffusions.     

Studies of symbiotic plasmids in Rhizobium trifolii and fast-growing bacteria that nodulate soybeans

The Rhizobium trifolii symbiotic plasmid pRt5a was transferred to the fast-growing soybean strain USDA 194. Transconjugants carrying pRt5a were not able to nodulate clovers and one of the transconjugants had lost its smallest resident plasmid and did not fix nitrogen in soybean. Transconjugants of USDA 194 carrying pRt5a were able to transfer pRt5a back to a non-nodulating R. trifolii which inherited the symbiotic properties of the R. trifolii strain from which the plasmid was derived.     

Host-range related structural features of the acidic extracellular polysaccharides of Rhizobium trifolii and Rhizobium leguminosarum

Proton nuclear magnetic resonance (1H NMR) and fast atom bombardment mass spectrometric analyses were performed on enzymatically derived oligosaccharides from the acidic excreted polysaccharides (EPS) from representative bacterial strains of the pea-nodulating symbiont, Rhizobium leguminosarum (128C53, 128C63, and 300) and the clover-nodulating symbiont, Rhizobium trifolii (NA-30, ANU843, 0403, TA-1, LPR5035, USDA20.102, and 4S). The results revealed structural similarities and differences between EPS of these two species. Octasaccharide units containing galactose, glucuronic acid, alpha-L-threo-hex-4-enopyranosyluronic acid, and glucose in a molar ratio of 1:1:1:5 were obtained from the EPS of the three R. leguminosarum strains and had the same primary glycosyl sequence and location of pyruvate, acetate, and 3-hydroxybutyrate substituents. About 80% of the galactose residues were acylated with 3-hydroxybutyrate, and there were two acetyl groups per repeating unit distributed between the 2 glucose residues of the main chain-derived sequence of the octasaccharides. In contrast, the R. trifolii strains had varied EPS structures, each of which differed from the common R. leguminosarum EPS structure. The EPS from one group of R. trifolii strains (0403 and LPR5035) most closely resembled the R. leguminosarum EPS but differed in that a lower number of galactose and glucose residues were substituted by 3-hydroxybutyryl and acetyl groups, respectively. The EPS from a second group of R. trifolii strains (ANU843, TA-1, and NA-30) was even more different than the R. leguminosarum EPS. These R. trifolii octasaccharides bore a single acetyl group on O-3 of the glucuronic acid residue. In addition, the level of acylation by 3-hydroxybutyryl groups was 50% of that present in the R. leguminosarum EPS. The remaining two strains of R. trifolii (USDA20.102 and 4S) had very different patterns of acylation to each other and to all of the other strains. The EPS from strain USDA20.102 practically lacked 3-hydroxybutyryl groups and had a unique degree and pattern of acetylation. The oligomers from the EPS of R. trifolii strain 4S completely lacked 3-hydroxybutyryl groups and galactose. The latter EPS contained only one O-1-carboxyethylidene group and had a different degree and pattern of acetylation. Interestingly, these two latter strains differ from the other R. trifolii strains in nodulation rates on rare clover species in the Trifolium cross-inoculation group. Thus, we define several groups of R. trifolii based upon their EPS structures and establish their similarities and distinct differences with the EPS of R. leguminosarum.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preservation of Rhizobium viability and symbiotic infectivity by suspension in water

Three Rhizobium japonicum strains and two slow-growing cowpea-type Rhizobium strains were found to remain viable and able to rapidly modulate their respective hosts after being stored in purified water at ambient temperatures for periods of 1 year and longer. Three fast-growing Rhizobium species did not remain viable under the same water storage conditions. After dilution of slow-growing Rhizobium strains with water to 10(3) to 10(5) cells ml-1, the bacteria multiplied until the viable cell count reached levels of between 10(6) and 10(7) cells ml-1. The viable cell count subsequently remained fairly constant. When the rhizobia were diluted to 10(7) cells ml-1, they did not multiply, but full viability was maintained. If the rhizobia were washed and suspended at 10(9) cells ml-1, viability slowly declined to 10(7) cells ml-1 during 9 months of storage. Scanning electron microscopy showed that no major morphological changes took place during storage. Preservation of slow-growing rhizobia in water suspensions could provide a simple and inexpensive alternative to current methods for the preservation of rhizobia for legume inoculation.     

Nodulation and Nitrogen Fixation Efficacy of Rhizobium fredii with Phaseolus vulgaris Genotypes

Phaseolus plant introduction (PI) genotypes (consisting of 684 P. vulgaris, 26 P. acutifolius, 39 P. lunatus, and 5 P. coccineus accessions) were evaluated for their ability to form effective symbioses with strains of six slow-growing (Bradyrhizobium) and four fast-growing (Rhizobium fredii) soybean rhizobia. Of the 684 P. vulgaris genotypes examined, three PIs were found to form effective nitrogen-fixing symbioses with the R. fredii strains. While none of the Bradyrhizobium strains nodulated any of the genotypes tested, some produced large numbers of undifferentiated root proliferations (hypertrophies). A symbiotic plasmid-cured R. fredii strain failed to nodulate the P. vulgaris PIs and cultivars, suggesting that P. vulgaris host range genes are Sym plasmid borne in the fast-growing soybean rhizobia.     

Preservation of Rhizobium viability and symbiotic infectivity by suspension in water.

Three Rhizobium japonicum strains and two slow-growing cowpea-type Rhizobium strains were found to remain viable and able to rapidly modulate their respective hosts after being stored in purified water at ambient temperatures for periods of 1 year and longer. Three fast-growing Rhizobium species did not remain viable under the same water storage conditions. After dilution of slow-growing Rhizobium strains with water to 10(3) to 10(5) cells ml-1, the bacteria multiplied until the viable cell count reached levels of between 10(6) and 10(7) cells ml-1. The viable cell count subsequently remained fairly constant. When the rhizobia were diluted to 10(7) cells ml-1, they did not multiply, but full viability was maintained. If the rhizobia were washed and suspended at 10(9) cells ml-1, viability slowly declined to 10(7) cells ml-1 during 9 months of storage. Scanning electron microscopy showed that no major morphological changes took place during storage. Preservation of slow-growing rhizobia in water suspensions could provide a simple and inexpensive alternative to current methods for the preservation of rhizobia for legume inoculation.