Chemical composition of extracellular polysaccharides of cowpea rhizobia

The extracellular polysaccharides (EPS) of six strains of cowpea rhizobia were examined. The strains (MI50A, M6-7B, IRC253) produced polysaccharides containing glucose, galactose and mannose in a molar ratio of 2:1.1:1, 1:1.3:3.1 and 1:1.3:3.5 respectively. Two strains (513-B and Ez-Aesch) produced polysaccharides containing galactose and mannose in a molar ratio of 2:3. Mannose was the only sugar detected in the EPS of strain IRC291. Pyruvate, acetate, glucuronic acid and galacturonic acid were not detected in any strain.Abbreviations EPS Extracellular polysaccharide - YEMA yeast-extract mannitol agar - YEMB yeast extract mannitol broth     

Competitive interaction between non-nodulating and nodulating strains for nodulation of cowpea (Vigna unguiculata)

Abstract: We have examined the effect of a non-nodulating mutant (JRW3-Sm^D) on the nodulation ability of cowpea rhizobia ( Bradyrhizobium sp.) strains JRW3 and IRC256. Nodulation of cowpea ( Vigna unguiculata ) by a nodulating Rhizobium strain is suppressed by the presence of a non-nodulating mutant. The msgnitude of suppression for nodule formation by nodulating strains varied between 40% and 80% depending on the strain and the time of inoculation.     

Intrinsic antibiotic resistance and streptomycin uptake in cowpea rhizobia

Abstract Uptake of [³H]dihydrostreptomycin in sensitive and resistant strains of cowpea rhizobia was examined to explain one of the possible mechanisms of intrinsic antibiotic resistance. The rate of accumulation of labelled streptomycin in resistant strains was drastically reduced when compared to that of sensitive strains. Our results indicated that high levels of intrinsic antibiotic resistance was due to decreased permeability causing failure to accumulate the drug.     

Bradyrhizobium elkanii, Bradyrhizobium yuanmingense and Bradyrhizobium japonicum are the main rhizobia associated with Vigna unguiculata and Vigna radiata in the subtropical region of China

Cowpea (Vigna unguiculata) and mung bean (Vigna radiata) are important legume crops yet their rhizobia have not been well characterized. In the present study, 62 rhizobial strains isolated from the root nodules of these plants grown in the subtropical region of China were analyzed via a polyphasic approach. The results showed that 90% of the analyzed strains belonged to or were related to Bradyrhizobium japonicum, Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense and Bradyrhizobium elkanii, while the remaining represented Rhizobium leguminosarum, Rhizobium etli and Sinorhizobium fredii. Diverse nifH and nodC genes were found in these strains and their symbiotic genes were mainly coevolved with the housekeeping genes, indicating that the symbiotic genes were mainly maintained by vertical transfer in the studied rhizobial populations.     

Growth and nutritional characteristics of cowpea rhizobia

Summary Twenty-five slow-growing strains of cowpea rhizobia were examined for growth and nutritional characteristics. Growth and nutritional data of these isolates were surprisingly homogeneous given their proposed genetic diversity. Most strains tested were capable of anaerobic growth in the presence of nitrate and all were found capable of autotrophic growth in a defined atmosphere of CO₂ and H₂ with oxygen or nitrate as terminal electron acceptors. These isolates grew heterotrophically with various carbohydrates and organic acids. Nitrogen utilization was consistent with that of other slow-growing rhizobia. Medium composition strongly affected the final pH of the culture. Cowpea rhizobia generally did not require vitamins; those requiring vitamins exhibited good growth when biotin was supplemented to the medium.     

Diversity in symbiotic specificity of cowpea rhizobia indigenous to Zimbabwean soils

Tropical cowpea rhizobia are often presumed to be generally promiscuous but poor N fixers. This study was conducted to evaluate symbiotic interactions of 59 indigenous rhizobia isolates (49 of them from cowpea (Vigna unguiculata)), with up to 13 other (mostly tropical) legume species. Host ranges averaged 2.4 and 2.3 legume species each for fast- and slow-growing isolates respectively compared to 4.3 for slow-growing reference cowpea strains. An average of 22% and 19% of fast- and slow-growing cowpea isolates respectively were effective on each of 12 legume species tested. We conclude that the indigenous cowpea rhizobia studied have relatively narrow host ranges. The ready nodulation of different legumes in tropical soils appears due to the diversity of indigenous symbiotic genotypes, each consisting of subgroups compatible with a limited number of legume species.     

Extracellular polysaccharides of cowpea rhizobia: compositional and functional studies

Polysaccharides excreted by cowpea Rhizobium strains JLn(c) and RA-1 were mixtures of complex acidic exopolysaccharides and low molecular weight neutral glucans. These polymers were fractionated using gel filtration chromatography. Purified fractions of the acidic heteropolymer reacted with peanut agglutinin to give precipitin bands when subjected to Ouchterlony gel diffusion. The acidic exopolysaccharide was found to contain mainly glucose, galactose, glucuronic acid, mannose and fucose. The non-carbohydrate substituents of the acidic heteropolymer were pyruvate, acetate and uronate which were identified by infrared and proton nuclear magnetic resonance spectroscopy as well as by chemical analysis.Abbreviations EPS Extracellular polysaccharide - YEM yeast extract mannitol - PNA peanut agglutination - ¹H-NMR proton nuclear magnetic resonance     

Development ofBradyrhizobium infections in supernodulating and non-nodulating mutants of soybean (Glycine max [L.] Merrill)

Summary The early events in the development of nodules induced byBradyrhizobium japonicum were studied in serial sections of a wild type (cv. Bragg), a supernodulating mutant (nts 382) and four non-nodulating mutants (nod49, nod139, nod772, andrj ₁) of soybean (Glycine max [L.] Merrill). Cultivar Bragg responded to inoculation in a similar manner to that described previously for cv. Williams; centres of sub-epidermal cell divisions were observed both with and without associated infection threads and most infection events were blocked before the formation of a nodule meristem. The non-nodulating mutants (nod49, nod772, andrj ₁) had, at most, a few centres of sub-epidermal cell divisions. In general, these were devoid of infection threads and did not develop beyond the very early stages of nodule ontogeny. Sub-epidermal cell divisions or infection threads were never observed on mutant nodl39. This mutant is not allelic to the other non-nodulating mutants and represents a defect in a separate complementation group or gene that is required for nodulation. The supernodulating mutant nts382, which is defective in autoregulation of nodulation, had a similar number of sub-epidermal cell divisions as the wild-type Bragg, but a much greater proportion of these developed to an advanced stage of nodule ontogeny. Mutant nts382, like Bragg, possessed other infection events that were arrested at an early stage of development. The results are discussed in the context of the progression of events in nodule formation and autoregulation of nodulation in soybean.Abbreviations nts nitrate tolerant symbiosis - RT root tip (i.e., position of the tap root tip at the time of inoculation) - SERH shortest emerging root hair (i.e., position of the shortest emerging root hair on the tap root at the time of inoculation) - SCD subepidermal cell divisions     

Symbiotic properties of 5-methyltryptophan-resistant mutants of Bradyrhizobium japonicum

The effect that resistance to 5-methyltryptophan (MT) has on the symbiotic properties of B. japonicum was examined in a survey of fourteen clones. Resistance to MT often involves a mutational alteration in the regulation of tryptophan biosynthesis.Resistant clones (MT^R) were isolated from agar plates containing MT. In the selection process care was taken to avoid pigmented clones that are likely to accumulate large amounts of indole compounds or show increased tryptophan catabolism. Wild-type control clones (WT_c) were isolated from plates containing no selective agent. In greenhouse studies. Tracy-M soybean plants were inoculated with the two types of clones. After six weeks, plants which were inoculated with the MT resistant clones showed a much greater range of symbiotic effectiveness than did plants that received the control clones.While most MT-resistant clones were poor symbionts or unchanged in their symbiotic performance, one clone was obtained that had significantly improved symbiotic properties. The procedure may offer a way of selecting for clones with improved symbiotic performance. These results also indicate a link between tryptophan biosynthesis and symbiotic effectiveness.     

Characterization of the Bradyrhizobium japonicum galE gene: its impact on lipopolysaccharide profile and nodulation of soybean

The galE gene from Bradyrhizobium japonicum 61A101C, a soybean endosymbiont, was cloned and characterized. Its deduced amino-acid sequence showed a high similarity with that of other rhizobia. Functional identification of the galE gene was achieved by complementation of a galE mutant strain, PL2, with a series of pKM subclones. Disruption of the B. japonicum galE gene affects the lipopolysaccharide profile compared with that of the wild type, suggesting that galE is responsible for alteration of lipopolysaccharide structure. Examination of nodule formation by the wild-type and galE mutant revealed that the former displayed normal nodule development on soybean roots, whereas the latter showed no nodule formation at all time points examined except for 20 days after inoculation when <10% of soybean formed pseudo-nodules.     

Isolation and characterization of rhizobitoxine mutants of Bradyrhizobium japonicum.

To explore the role of rhizobitoxine in Bradyrhizobium-legume symbiosis, 11 rhizobitoxine mutants of B. japonicum USDA61 were isolated on the basis of their inability to synthesize the toxin in culture. Each mutant is prototrophic and symbiotically effective on soybean, cowpea, siratro, and Glycine soja. The rhizobitoxine mutants differ in their chlorosis phenotypes and rhizobitoxine production in planta. As expected, one group of mutant fail to make toxin in planta, resulting in the absence of chlorosis. Another group of mutants causes severe chlorosis on all cultivars of soybean tested. Surprisingly, this group of mutants makes more rhizobitoxine in soybean nodules than the wild-type strain does. This phenotype is only observed on soybean and not on other hosts such as cowpea, siratro, or G. soja. The remaining mutants all produce rhizobitoxine in planta but vary in the amount of toxin they produce and the severity of chlorosis they induce in soybean plants. Biochemical analysis of mutants demonstrates that one mutant is unable to synthesize serinol, a molecule hypothesized to be an intermediate in rhizobitoxine biosynthesis. By using these mutants, it was found that rhizobitoxine plays no apparent role in the nodulation of rj1 soybeans. Recently, it was found that inhibition of ethylene biosynthesis allows Rhizobium meliloti to overcome nitrate inhibition of nodule formation on alfalfa. Because rhizobitoxine also inhibits ethylene biosynthesis, we tested the ability of mutants which accumulate high levels of toxin in planta to overcome nitrate inhibition of nodule formation on soybean plants and found that the nodule formation induced by the wild type and that induced by mutant strains were equally suppressed in the presence of nitrate.     

Characterization of Tn5-induced symbiotically defective mutants of cowpea rhizobia

Symbiotically defective mutants of cowpea rhizobia strain IRC256 were isolated by random Tn5 mutagenesis and characterized. One auxotroph (MS1) requiring adenine and thiamine was a non-nodulating mutant (Nod⁻) and three prototrophic mutants were Nod⁺ Fix⁻ which formed small and ineffective nodules on cowpeas (Vigna unguiculata). Acetylene reduction activity of the Nod⁺ Fix⁻ mutants was reduced to 80–94% of that of the wild-type strain. The non-nodulating mutant (MS1) induced root-hair curling but did not show any nodule initiation or nodule development. Ultrastructural examination of nodules formed by Fix⁻ mutants showed that these contained few bacteroids, indicating either early senescence or a reduction in bacterial release into the cytoplasm of the host cell. DNA hybridization of total DNAs from a representative number of Tn5 mutants showed that each of them had one copy of the transposon Tn5 which was randomly inserted into the genome of cowpea rhizobia.     

Utilization of carbon and nitrogen sources and acid/alkali production by cowpea rhizobia

Summary Sixteen slow-growing strains of rhizobia (15 cowpea rhizobia and oneR. japonicum) were examined to determine the effects of carbon and nitrogen sources on acid/alkali production in culture media. We found that the pH changes of the medium were more influenced by nitrogen sources than carbon sources (with the exception of ribose). When ammonium sulphate was used as a nitrogen source, all the cowpea rhizobia strains produced acid. When yeast-extract was used as a nitrogen source, however, a heterogenous pattern for acid/alkali production was found. The majority of the strains produced alkali from nitrate, glutamate and urea irrespective of carbon sources and acid from ribose irrespective of nitrogen sources.     

Bradyrhizobium japonicum mutants defective in root-nodule bacteroid development and nitrogen fixation

The genome of the slow-growing Bradyrhizobium japonicum (strain 110) was mutagenized with transposon Tn5. A total of 1623 kanamycin/streptomycin resistant derivatives were screened in soybean infection tests for nodulation (Nod) and symbiotic nitrogen fixation (Fix). In this report we describe 14 strains possessing a stable, reproducible Nod⁺Fix^- phenotype. These strains were also grown under microaerobic culture conditions to test them for free-living nitrogen fixation activity (Nif). In addition to strains having reduced Fix and Nif activities, there were also strains that had reduced symbiotic Fix activity but were Nif⁺ ex planta.Analysis of the genomic structure revealed that the majority of the strains had a single Tn5 insertion without any further apparent physical alteration. A few strains had additional insertions (by Tn5 or IS50), or a deletion, or had cointegrated part of the vector used for Tn5 mutagenesis. One of the insertions was found in a known nif gene (nifD) whereas all other mutations seem to affect different, hitherto unknown genes or operons.Several mutant strains had an altered nodulation phenotype, inducing numerous, small, widely distributed nodules. Light and electron microscopy revealed that most of these mutants were defective in different stages of bacteroid development and/or bacteroid persistence. The protein patterns of the mutants were inspected by two-dimensional gel electrophoresis after labelling microaerobic cultures with l-(³⁵S)methionine. Of particular interest were mutants lacking a group of proteins the synthesis of which was known to be under oxygen control. Such strains can be regarded as potential regulatory mutants.     

Competition between inoculum and native rhizobia for nodulation of cowpea (Vigna unguiculata (L) Walp): Use of a dark-nodule strain

Summary Cowpea rhizobia strains were examined with indigenous populations in nodulating cowpea (Vigna unguiculata (L) Walp) cv. Laura B. strain IRC256 formed dark nodules on cowpea, and were used as the standard against orthodox pink-nodule strains in evaluating nodulating competitiveness. The dark nodule phenotype and intrinsic antibiotic resistance pattern were used to identify the strains in the nodules. Our results showed the usefulness of the dark-nodule strain in evaluating nodulating competitiveness of cowpea rhizobia in soils where dark-nodule strains were not indigenous.     

Transfer of plasmids RP4 and R68.45 and chromosomal mobilization in cowpea rhizobia

R-plasmids RP4 and its derivatives R68.45 were transferred from Escherichia coli to two cowpea rhizobia strains. The frequency of RP4 transfer in cowpea rhizobia strains JRC23-SM20 and IRC256-HA409 was 1,000-fold higher than transfer frequency of R68.45. The transconjugants were further used to transfer R-plasmids within (isogenic) and between (non-isogenic) cowpea rhizobia strains. The plasmid transfer frequency was higher in isogenic than non-isogenic strains. The ability of R-plasmids to mobilize chromosomal genes in cowpea rhizobia was also examined. R-plasmids mediated the chromosomal transfer; however, mobilization of chromosomal markers Sm^R and Met⁺ by RP4 in isogenic strains was more efficient than by R68.45. Chromosomal mobilization has not previously been reported in cowpea rhizobia.Abbreviations Ap ampicillin - Km kanamycin - Tc tetracycline - Rif rifampicin - TYS tryptone yeast-extract sodium chloride - YEMA yeast-extract mannitol agar - YEMB yeast-extract mannitol broth Part of the work was presented in 6th International Symposium on Nitrogen Fixation at Oregon State University, Corvallis, August 4–10, 1985     

Ecology and genetics of tropical rhizobia species

Biological nitrogen fixation (BNF) technology with special reference to Rhizobium-legume symbiosis is growing very rapidly with the hope of combatting world hunger by producing cheaper protein for animal and human consumption in the Third World. One can see rapid progress made in the biochemistry and molecular biology of symbiotic nitrogen fixation in general; however, less progress has been made on the ecological aspects despite the fact that an enormous amount of literature is available on inoculation problems and on agronomic aspects of symbiotic nitrogen fixation. So far most information on Rhizobium concerns fast-growing rhizobia and their host legume. Although it is essential that food production using BNF technology should be maximized in the Third World, the least work has been done on slow-growing rhizobia, which are generally found in tropical and sub-tropical soils. The majority of the developing countries are in tropical and sub-tropical regions. Except for R. japonicum, a microsymbiont partner of soybean (Glycine max), the majority of the slow-growing rhizobia belong to the cowpea group, and we refer to cowpea rhizobia as tropical rhizobia species. In this review we have tried to consolidate the recent progress made on ecology and genetics of tropical rhizobia. By using recombinant DNA technology techniques it is expected that super strains of rhizobia with desirable characteristics can be produced. One must evaluate the efficiency and effectiveness of these genetically manipulated laboratory strains under field conditions. In conclusion, if one aims at combatting hunger in the Third World using BNF technology, an intensive research programme on fundamental and applied aspects of tropical rhizobia species is suggested. This involves close cooperation between molecular biologists and microbial ecologists.     

Rapid and efficient selection of recombinant site-directed mutants of Bradyrhizobium japonicum by colony hybridization

Abstract Due to the high incidence of spontaneous antibiotic resistance and slow growth of Bradyrhizobium japonicum strains, screening for site-directed mutants is cumbersome and time-consuming. A rapid method for selection of recombinant site-directed mutants of B. japonicum was developed. A kanamycin (Km) and a spectinomycin (Sp) cassette were each used to replace DNA fragments in the chromosome by homologous recombination. The primary new features of this method involve a simple plate selection for the antibiotic (Km or Sp) resistant mutants, then colony streaking, and lysis for DNA hybridization on a nitrocellulose filter enabling direct identification of the recombinant site-directed mutants. This method has permitted us to quickly and easily identify a large number of positive recombinant mutants from a large number of indicidual colonies. The procedure eliminates the need to first isolate genomic DNA from each mutant for Southern hybridization. All of the tested site-directed mutants from this method were confirmed to exhibit the expected mutant phenotype.     

Isolation and characterization of functional insertion sequences of rhizobia

Rhizobia are a group of bacteria that form nodules on the roots of legume host plants. The sequenced genomes of the rhizobia are characterized by the presence of many putative insertion sequences (IS) elements. However, it is unknown whether these IS elements are functional and it is therefore relevant to assess their transposition activity. In this work, several functional insertion sequences belonging to the IS1256, IS3, IS5, IS166, and IS21 families were captured from Rhizobium tropici, Rhizobium sp. NGR234 and Sinorhizobium meliloti, using pGBG1 as a trapping system. In silico analysis shows that homologs of rhizobia mobile elements are present in distantly related genomes, suggesting that Rhizobium IS elements are prone to genetic transfer.