Peanut agglutinin induced alterations in capsular and extracellular polysaccharide synthesis andex -planta nitrogenase activity of cowpea rhizobia

The root exudate ofArachis hypogea (groundnut) and its seed lectin peanut agglutinin were found to stimulate the synthesis of exopolysaccharide and capsular polysaccharide of the microsymbiont cowpeaRhizobium strain JLn (c). The synthesis of capsular polysaccharide was enhanced 1.5-fold and 2-fold in the presence of peanut agglutinin and root exudate, respectively. The synthesis of capsular polysaccharide was suppressed in the presence of different forms of combined nitrogen. Quantitative differences were also detected between the exopolysaccharide of cells grown in the presence and absence of root exudate. Electron microscopic examination of negatively stained lectin-treated JLn (c) cells showed an increased deposition of capsular polysaccharide surrounding the cells. Hurthermore,ex planta nitrogenase activity of JLn(c) cells in the presence of lectin was found to be enhanced by 63% in correlation with the increased synthesis of polysaccharides. Part of this work was presented at the colloquium session of the 4th Hederation of Asian and Oceanian Biochemists Congress, held at Singapore, in November 1986.     

Chemotaxonomic characterization of some fast-growing rhizobia nodulating leguminous trees

A total of about 50 strains of rhizobia from two leguminous trees (Acacia andProsopis) were described and compared with 20 reference strains of rhizobia, from other tree and herb legumes on the basis of protein, fatty acid and plasmid profiles, and DNA-DNA hybridization. The rhizobia formed thirteen clusters based on protein profile analysis. These clusters were not in complete agreement with a previously published cluster analysis based on numerical taxonomy of phenotypic characteristics and lipopolysaccharide (LPS) profile analysis (Zhanget al., Int. J. Syst. Bacteriol. 41, 104, 1991; Lindström and Zahran,FEMS Microbiol. Lett. 107, 327, 1993). The fatty acid methyl esters (FAME) of representative strains of rhizobia were analyzed. The rhizobia formed fourteen different clusters based on FAME analysis but the results also conflicted with the phenotypically based methods of analysis. Strains of rhizobia classified in one cluster by any of the above methods of analysis may have shown very different fatty acid profiles. The plasmid profile analysis of the tree rhizobia., on the other hand, was more consistent with the phenotype- and LPS-based numerical analysis. Some strains of the tree rhizobia showed medium or high levels of DNA homology withRhizobium meliloti. The DNA-DNA hybridization correlated well with protein and fatty acid profiles. The described methods provide a significant taxonomic tool for discrimination between rhizobia of leguminous trees. However, further DNA-DNA hybridization studies with other recognized species of rhizobia are needed for proper identification and classification of the diverse rhizobia from leguminous trees.     

Adsorption of slow- and fast-growing rhizobia to soybean and cowpea roots

Roots of soybean (Glycine max [L.] Merr. cv Hardee) and cowpea (Vigna unguiculata [L.] Walp. cv Pink Eye Purple Hull) were immersed in suspensions containing 10⁴Rhizobium cells per milliliter of a nitrogen-free solution. After 30 to 120 minutes the roots were rinsed, and the distal 2-centimeter segments excised and homogenized. Portions of the homogenates then were plated on a yeast-extract mannitol medium for bacterial cell counts. The adsorption capacities of four slow-growing rhizobia and a fast-growing R. meliloti strain varied considerably. Adsorption was independent of plant species and of the abilities of the Rhizobium strains to infect and nodulate. R. lupini 96B9 had the greatest adsorption capacity, and Rhizobium sp. 3G4b16 the least. Rhizobium sp. 229, R. japonicum 138, and R. meliloti 102F51 were intermediate, except on cowpea, where the adsorption of strain 102F51 was similar to that of strain 3G4b16. The initial adsorption rates of bacteria cultured in synthetic media and in the presence of soybean roots were about the same. Addition of soybean lectin to the bacterial inoculum failed to influence initial adsorption rates. Both treatments, however, reduced the numbers of bacteria that bound after incubation with roots for 120 minutes. The relationship between the logarithm of the number of strain 138 cells bound per soybean root segment and the logarithm of the density of bacteria in the inoculum was linear over five orders of magnitude. Binding of strain 138 to soybean roots was greatest at room temperature (27°C) and substantially attenuated at both 4 and 37°C. Although R. lupini 96B9 strongly rejected a model hydrophobic plastic surface, there were no simple correlations between bacterial binding to model hydrophobic and hydrophilic plastic surfaces and bacterial adsorption to roots.     

Synthesis and rheological properties of responsive thickeners based on polysaccharide architectures

New thermothickening copolymers were synthesized by grafting responsive poly(ethylene oxide-co-propylene oxide) [PEPO] onto three different polysaccharide backbones: carboxymethylcellulose [CMC], alginate [ALG], and carboxylated dextran [DEX]. The coupling reaction between carboxylic groups of biopolymers and the terminal amine of PEPO was activated at low temperature ( T < 10 degrees C) in water by using carbodiimide and N-hydroxysuccinimide. In these conditions it was shown that the formation of amide bonds strongly depends on the concentration of reactive groups, which is limited by the viscosity of the polymer sample. While a full conversion was obtained for the low molecular weight dextran, the efficiency of grafting remains low (between 30 to 40%) for CMC and alginate, which give a solution of high viscosity even at low concentration. When studied in the semidilute regime, all the copolymer solutions clearly exhibit thermothickening behavior with a large and reversible increase of viscosity upon heating. The association temperature and the gelation threshold were shown to depend on polymer concentration as it is expected from the phase diagram of PEPO precursor. Similarly, the influence of added salt on PEPO solubility in water has been used to control the self-assembling behavior of copolymer formulations. The relative comparison between the three copolymers reveals that the amplitude of the viscosity jump induced by heating mainly depends on the proportion of responsive material inside the macromolecular architecture rather than the dimensions of the main chain. The high increase of viscosity, which can reach several orders of magnitude between 20 degrees C and body temperature, clearly demonstrates the potentiality of these copolymers in biomedical applications like injectable gels for tissue engineering.     

Genetic diversity of indigenous tropical fast-growing rhizobia isolated from soybean nodules

This study characterized genetically 30 fast-growing rhizobial strains isolated from nodules of Asian and modern soybean genotypes that had been inoculated with soils from disparate regions of Brazil. Analyses by rep-PCR (ERIC and REP) and RAPD indicated a high level of genetic diversity among the strains. The RFLP-PCR and sequencing analysis of the 16S rRNA genes indicated that none of the strains was related to Sinorhizobium (Ensifer) fredii, whereas most were related to Rhizobium tropici (although they were unable to nodulate Phaseolus vulgaris) and to Rhizobium genomic species Q. One strain was related to Rhizobium sp. OR 191, while two others were closely related to Agrobacterium (Rhizobium) spp.; furthermore, symbiotic effectiveness with soybean was maintained in those strains. Five strains were related to Bradyrhizobium japonicum and B. elkanii, with four of them being similar to strains carried in Brazilian inoculants, therefore modifications in physiological properties, as a shorter doubling time might have resulted from adaptation to local conditions. Phospholipid fatty acid analysis (PFLA) was less precise in delineating taxonomic relationships. The strains fit into eight Nod-factor profiles that were related to rhizobial species, but not to N₂-fixation capacity or competitiveness. The data obtained highlight the diversity and promiscuity of rhizobia in the tropics, being capable of nodulating exotic legumes and might reflect ecological strategies to survive in N-poor soils; in addition, the diversity could also represent an important source of efficient and competitive rhizobial strains for the tropics. Putative new rhizobial species were detected only in undisturbed soils. Three species (R. tropici, B. japonicum and B. elkanii) were found under the more sustainable management system known as no-till, while the only species isolated from soils under conventional till was R. tropici. Those results emphasize that from the moment that agriculture was introduced into undisturbed soils rhizobial diversity has changed, being drastically reduced when a less sustainable soil management system was adopted.     

Unusual rheological properties of a new associative polysaccharide in salt media

New amphiphilic polysaccharides based on alginate-grafted-Poly (ε-caprolactone) or alg-g-PCL bearing two kinds of PCL chains with different molar masses (1250 and 530 gmol⁻¹) with various amounts from 3% to 15% were prepared. Rheological properties in aqueous solutions of such systems have been investigated as a function of polymer concentration, added salt and temperature in semi-dilute regime. Strong hydrophobic intermolecular associations were clearly demonstrated in pure water whatever the PCL chain length and extend of modification. Increasing polymer concentration, grafting rate and/or PCL chains length can lead to a structured liquid behaviour. Rheological properties of the most organized system have been found independent to the temperature (until 60 °C). In salt media, a strong dependence of hydrophobic interactions to the length of PCL chains was observed. For M_PCL = 1250 g.mol⁻¹ the screening of charges promotes the establishment of intermolecular interactions and leads to a strong physical gel for the highest grafting rates. For M_PCL = 530 g mol⁻¹, ionic strength leads to a decrease of rheological properties when increasing grafting rate. This result may indicate an increase of hydrophobic clusters even in the entangled regime. This unusual behaviour opens the ways for the preparation of suitable hydrogels for drug release.     

Purification, characterization and immunological properties of the capsular polysaccharide of Pasteurella haemolytica serotype T15: its identity with the K62 (K2ab) capsular polysaccharide of Escherichia coli and the capsular polysaccharide of Neisseria meningitidis serogroup H

Capsular polysaccharide from two strains of Pasteurella haemolytica serotype T15 was purified and characterized by chemical analysis and NMR spectroscopy. The polymer, a teichoic acid, proved to be very similar in structure to the capsular polysaccharide of P. haemolytica serotype T4 and identical to the previously described K62 (K2ab) capsular polysaccharide of Escherichia coli, and the capsular polysaccharide of Neisseria meningitidis serotype H, i.e. ----(2-glycerol-3)----(phosphate)----(4-alpha-D-galactopyranose -1)---- with partial O-acetylation on the galactose residues. Electron microscopy with Protein A-gold labelled antisera showed that the polysaccharide was peripherally located on the surface of all three organisms. Chemical removal of O-acetyl groups from the polysaccharide yielded a structure identical to that previously described for E. coli K2 (K2a). Both O-acetylated and de-O-acetylated P. haemolytica T15 polymers, when absorbed on to sheep erythrocytes in passive haemagglutination assays, yielded identical antibody titres with sera raised against P. haemolytica T15, E. coli K2 or N. meningitidis H whole cells. De-O-acetylation of the Pasteurella polysaccharide influenced its precipitability with immune sera, but this could not be related to the absence of O-acetyl groups because the non-acetylated E. coli K2 polymer readily precipitated with a line of 'identity' with the acetylated P. haemolytica T15 polymer.     

Genetic characterization of fast-growing rhizobia able to nodulate Prosopis alba in North Spain

Prosopis is a Mimosaceae legume tree indigenous to South America and not naturalized in Europe. In this work 18 rhizobial strains nodulating Prosopis alba roots were isolated from a soil in North Spain that belong to eight different randomly amplified polymorphic DNA groups phylogenetically related to Sinorhizobium medicae, Sinorhizobium meliloti and Rhizobium giardinii according to their intergenic spacer and 16S rRNA gene sequences. The nodC genes of isolates close to S. medicae and S. meliloti were identical to those of S. medicae USDA 1,037(T) and S. meliloti LMG 6,133(T) and accordingly all these strains were able to nodulate both alfalfa and Prosopis. These nodC genes were phylogenetically divergent from those of the isolates close to R. giardinii that were identical to that of R. giardinii H152(T) and therefore all these strains formed nodules in common beans and Prosopis. The nodC genes of the strains isolated in Spain were phylogenetically divergent from that carried by Mesorhizobium chacoense Pr-5(T) and Sinorhizobium arboris LMG 1,4919(T) nodulating Prosopis in America and Africa, respectively. Therefore, Prosopis is a promiscuous host which can establish symbiosis with strains carrying very divergent nodC genes and this promiscuity may be an important advantage for this legume tree to be used in reforestation.     

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 .     

Large plasmids of fast-growing rhizobia: homology studies and location of structural nitrogen fixation (nif) genes.

A single large plasmid was isolated from multiplasmid-harboring strains Rhizobium leguminosarum 1001 and R. trifolii 5. These single plasmids, as well as the largest plasmid detectable in R. phaseoli 3622, hybridized with part of the nif structural genes of Klebsiella pneumoniae. In contrast, the plasmids of R. meliloti strains V7 and L5-30 did not show hybridization with the nif genes of K. pneumoniae, indicating that these genes might be located either on the chromosome or on a much larger plasmid which as yet has not been isolated. Studies of the homology between plasmids of fast-growing Rhizobium species showed that a specific deoxyribonucleic acid sequence, which carries the structural genes for nitrogenase, is highly conserved on a plasmid in R. leguminosarum, R. trifolii, and R. phaseoli. Furthermore, it was found that this type of plasmid in the different species shares extensive deoxyribonucleic acid homology, suggesting that strains in the R. leguminosarum cluster have preserved a nif plasmid.     

Some properties of arctic rhizobia

Forty-eight strains of Rhizobium isolated from the root nodules of three species of legumes indigenous to the high tundra (Astragalus alpinus, Oxytropis maydelliana andOxytropis arctobia) are phenotypically heterogenous with respect to intrinsic antibiotic resistance, expression of nitrogenase activityex planta and plasmid content. All of the strains possess a 250–300 kb plasmid and are homologous to each other on the genomic DNA level but have little DNA homology with selected reference strains of well characterized species of rhizobia. The arctic rhizobia have an optimum growth temperature of 23°C and can grow slowly at 5°C. The DNA from four of the isolates, which were selected for detailed investigation, have sequences homologous tonif andnod genes fromRhizobium trifolii.     

Recognition of individual strains of fast-growing rhizobia by using profiles of membrane proteins and lipopolysaccharides.

Membrane protein and lipopolysaccharide profiles of Rhizobium leguminosarum (biovars viciae, trifolii, and phaseoli), R. meliloti, and Agrobacterium tumefaciens strains were analyzed and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Differences in one or both profiles allowed us to distinguish all 18 R. leguminosarum strains tested in this study from each other.     

细菌荚膜多糖的分离纯化研究进展

荚膜多糖是细菌的保护性抗原和毒力因子,也是细菌疫苗最重要的靶抗原之一,其分离纯化是制作疫苗的首要步骤。本文从去除菌体、收集总糖、去除菌体核酸和蛋白质、去除内毒素等基本工艺步骤,对现有的工艺和目前的工艺进展进行了综述,重点阐述了中空纤维、深层过滤、超滤、酶水解、柱层析等方法在荚膜多糖分离纯化中的应用进展。     

Bacteriocin small of fast-growing rhizobia is chloroform soluble and is not required for effective nodulation.

Small bacteriocin is a low-molecular-weight bacteriocin which is common in fast-growing rhizobia. As its activity could not be detected in chloroform-sterilized culture supernatants (P.R. Hirsch, J. Gen. Microbiol. 113:219-228, 1979), the bacteriocin could not be purified in order to study its mechanism of action. We report here that small is soluble in chloroform, an observation which led to effective and simple (partial) purification. Other properties of small are its low molecular weight, which is estimated to be between 700 and 1,500, its resistance to proteolytic enzymes, pectinase, and lysozyme, and its heat stability at pH 5.5 but not at pH 7.0. Its bactericidal action on exponentially growing sensitive cells was not detected until 11 h after its addition. The bactericidal action was preceded by inhibition of cell division. To determine whether small activity is required for nodulation or nitrogen fixation, a transposon Tn5-induced small-negative mutant was isolated. The observation that this strain formed normal, acetylene-reducing root nodules showed that small production is not a prerequisite for the formation of effective nodules.