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The interaction of the surface agglutinins of Rhizobium leguminosarum by. viciae 252 with the carbohydrate components of host pea roots alters the beta-glucosidase and proteolytic activities of the agglutinins.

A comparative study of the lipopolysaccharides (LPS) isolated from Sinorhizobium meliloti SKHM 1-188 and two its LPS-mutants (Th29 and Ts22) with sharply decreased nodulation competitiveness was conducted. Polyacrylamide gel electrophoresis with sodium dodecyl sulfate revealed two forms of LPS in all the three strains: a higher molecular-weight LPS1, containing O-polysaccharide (O-PS), and a and lower molecular-weight LPS2 without O-PS. However, the LPS1 content in mutants was significantly smaller than in the parent strain. The LPS of the strains studied contained glucose, galactose, mannose, xylose, three nonidentified sugars--X1 (TGlc 0.53), X2 (TGlc 0.47), and X3 (TGlc 0.43), glucosamine, and ethanolamine, while the LPS of S. meliloti SKHM1-188 additionally contained galactosamine, glucuronic and galacturonic acids, and 2-keto-3-deoxyoctulosonic acid (KDO), as well as fatty acids, such as 3-OH C14:0, 3-OH C15:0, 3-OH C16:0, 3-OH C18:0, nonidentified hydroxy X (T3-OH C14:0 1.33), C18:0, and unsaturated C18:1 fatty acids. The LPS of both mutants were similar in the component composition but differed from the LPS of the parent strain by a lower X2, X3, and 3-OH C 14:0 content and a higher KDO, C18:0, and hydroxy X content. The LPS of all the strains were subjected to mild hydrolysis with 1% acetic acid and fractionated on a column with Sephadex G-25. The higher molecular weight fractions (2500-4000 Da) contained a set of sugars typical of intact LPS and, supposedly, corresponded to the LPS polysaccharide portion (PS1). In the lower molecular weight fractions (600-770 Da, PS2), glucose and uronic acids were the major components; galactose, mannose, and X1 were present in smaller amounts. The PS1/PS2 ratio for the two mutants was significantly lower than for strain SKHM1-188. The data obtained show that the amount of O-PS-containing molecules (LPS1) in the heterogeneous lipopolysaccharide complex of the mutants was smaller than in the SKHM1-188 LPS; this increases the hydrophobicity of the cell surface of the mutant bacteria. This supposedly contributes to their nonspecific adhesion on the roots of the host plant, thus decreasing their nodulation competitiveness.

The functional activity of the exoglycan complex (EGC) polysaccharides from Rhizobium leguminosarum bv. viciae 250a and its nitrogen-resistant mutant M-71 capable of inducing the formation of nitrogen-fixing nodules on pea roots against a high-nitrogen background (4.8 mM NO3-) was studied in vegetation tests. For this purpose, the bacterial inoculum washed free of its own exoglycans was supplemented with EGC of this or another strain grown in the presence of 6 or 20 mM nitrate. The best symbiotic characteristics (nodule number and nitrogenase activity, mass of the roots and aerial parts of plants) were recorded when the inoculum cells and exoglycans were obtained from strain M-71 grown in the presence of 20 mM nitrate. When the plants were inoculated with the cells (grown at 6 mM nitrate) + EGC (obtained at 6 mM nitrate) of this strain, the nodulation characteristics and the effectiveness of symbiosis decreased 1.5-2-fold. Partial recovery of the symbiotic potential of strain M-71 was observed when EGC (obtained at 20 mM nitrate) was substituted for its exoglycans (obtained at 6 mM nitrate). In the presence of exoglycans of the parent strain 250a (obtained at 6 or 20 mM nitrate), the mutant formed a substantially lesser number of nodules with a very low nitrogen-fixing activity. In turn, the mutant exoglycans synthesized in medium with either high or low nitrate nitrogen concentration did not recover the fix+ phenotype of strain 250a capable of forming symbiosis with pea plants only against a low-nitrogen background. When studying the relative content of high-molecular-weight exopolysaccharide components and low-molecular-weight glycans in the exoglycan complex, it was established that, in strain 250a (grown at 6 and 20 mM nitrate), as well as in its mutant M-71 (grown at 6 mM nitrate), exopolysaccharides prevailed, accounting for 72-75% of the sum of both types of glycopolymers, while low-molecular-weight glycans accounted for 25-28%. In contrast, in the EGC of strain M-71 obtained at 20 mM nitrate, which was the most active inducer of the formation of the symbiotrophic system by strain M-71 in the presence of a high mineral nitrogen concentration, low-molecular-weight glycans were the main component, accounting for 61% of total glycopolymers, while the polysaccharide content was 39%. Low-molecular-weight exoglycans are supposed to be involved in maintaining the physiological activity and the symbiotic status of rhizobia under unfavorable environmental conditions.

The influence of lipopolysaccharides (LPS), glucans, and their unseparated complexes on nodulation activity of rhizobia and efficiency of their symbioses with pea plants was studied in vegetation experiments. Two Rhizobium leguminosarum bv. viciae strains which differed in their symbiotic properties were used: strain 31 (fix+, efficient, moderately virulent, moderately competitive), and strain 248b (fix-, inefficient, highly virulent, highly competitive). Preparations of LPS-glucan complex and the respective LPS from the highly virulent strain 248b increased the nodulation activity of both strains by 10-26%. Analogous preparations from a less virulent strain 31 did not have this ability. Unseparated LPS-glucan complexes from these strains increased the productivity of plants infected with the efficient strain by 18-23% but did not change it in plants inoculated with the other, inefficient strain. No significant influence of LPS preparations on the symbiosis productivity was observed. Glucans from both strains enhanced the nodulation ability of the highly virulent strain by 36-56%. In addition, treatment of pea plants with glucan from strain 248b increased nitrogen fixation by root nodules by 27% in plants inoculated with strain 31. In general, the formation and efficiency of the symbiosis of R. leguminosarum bv. viciae with pea plants was more influenced by preparations from strain 248b, highly virulent but deficient in nitrogen fixation, than by preparations from the nitrogen fixation-proficient but less virulent strain 31.

The study of the effect of the periplasmic glucan isolated from the root-nodule bacterium S. meliloti CXM1-188 on the symbiosis of another strain (441) of the same root-nodule bacterium with alfalfa plants showed that this effect depends on the treatment procedure. The pretreatment of alfalfa seedlings with the glucan followed by their bacterization with S. meliloti 441 insignificantly influenced the nodulation parameters of symbiosis (the number of root nodules and their nitrogen-fixing activity) but induced a statistically significant increase in the efficiency of symbiosis (expressed as the masses of the alfalfa overground parts and roots). At the same time, the pretreatment of S. meliloti 441 cells with the glucan brought about a considerable decrease in the nodulation parameters of symbiosis (the number of the root nodules and their nitrogen-fixing activity decreased by 2.5-11 and 7 times, respectively). These data suggest that the stimulating effect of rhizobia on host plants may be due not only to symbiotrophic nitrogen fixation but also to other factors. Depending on the experimental conditions, the treatment of alfalfa plants with the glucan and their bacterization with rhizobial cells enhanced the activity of peroxidase in the alfalfa roots and leaves by 10-39 and 12-27%, respectively.

The effect of the plant growth stimulant bactozole on the growth of Rhizobium leguminosarum bv. viciae 250a and its nitrogen-tolerant mutant M-71 and the synthesis of extracellular carbohydrates was studied. At a low content of nitrate (6 mM) in the medium, all three bactozole concentrations tested (0.001, 0.01, and 0.1%) exerted similar stimulating effects on the growth of the parent strain 250a (about 1.5-fold) and the synthesis of extracellular carbohydrates (about 2-fold). At a high content of nitrate (20 mM) in the medium, when the growth of the parent strain and the synthesis of extracellular carbohydrates were inhibited, bactozole at all three concentrations exerted only a growth-stimulating effect. At the same time, mutant M-71 showed better growth at higher concentrations of bactozole, whereas the ability of the mutant to synthesize extracellular carbohydrates decreased with increasing bactozole concentration. The cell biomass of the mutant accumulated at 20 mM nitrate was 1.8-2.5 times greater than it was at 6 mM nitrate. Bactozole enhanced the symbiosis of legume plants with both parent and mutant strains, raising the mass of plants and enhancing nodulation and the nitrogen-fixing activity of root nodules. The symbiotic parameters of mutant M-71 were better (irrespective of whether bactozole was present or not) when its inoculum was grown at a high nitrogen content (20 mM nitrate), whereas the respective parameters of the parent strain were better when it was grown at 6 mM nitrate. The inference is made that the better physiological characteristics of the mutant in the high-nitrate medium is due to its higher nitrate reductase activity (as compared with the parent strain) in both the free-living state and in legume nodules.

A possibility has been demonstrated to use laser spectroscopy of bacterial glycopolymers by means of measurement of their water solutions fluorescence. Comparative investigations of native lipopolysaccharide (LPS) Ralstonia solanacearum and its structure components permits a supposition to be made that the LPS total spectrum is a result of superposition of the spectrum of O-specific polysaccharide and core oligosaccharide as well as core oligosaccharide and lipid A. The LPS spectrum maximum shift is determined by core oligosaccharide and lipid A luminescence contribution. A decrease as well as complete loss of serological activity as a result of 30 and 60 min UV irradiation of LPS has been established. It has been shown that LPS Rhizobium leguminosarum bv. viciae quenches luminescence of host-plant (pea) lectin depending on the extent of their affinity. Luminescence spectrum of glucan Sinorhizobium meliloti CXM1-188 and two its LPS-mutants differ between themselves both in luminescence intensity and in presence and expression degree of the site 2 with maximum 2.8 eV.

Effect of two plant growth stimulators: bactozol (drug of bacterial origin) and D1 (synthetic analog of phytohormones) on metabolism of pea rhizobia (Rhizobium leguminosarum bv. viciae 2636) and efficiency of their symbiosis with pea plants have been studied. The D1 drug in concentration 0.1% suppressed growth of bacteria. However, bactozol stimulating action on pea rhizobia growth in a pure culture and synthesis of extracellular carbohydrates by them have been established. The trial of three concentrations (0.1%, 0.01%, 0.001%) has shown that bactozol effect has dose-dependent character. The highest effect of stimulation is achieved at concentration 0.1%. Bactozol decreases partially the repressing influence of the mineral nitrogen on growth of rhizobia and their carbohydrate-synthesizing activity under growth of bacteria against a high nitrogen background (20 mM NO3-). Treatment of pea plants by bactozol (0.1%) increases considerably the efficiency of their symbiosis with pea rhizobia, evoking the growth of the overground and root mass of the plants, quantity of nodules and their nitrogenase activity.

Four Tn5-transposon LPS mutants of Sinorhizobium meliloti (Tb9, Tb29, Ts22 and Ts32) have been studied. Each of four mutants has been established to contain a single insertion of Tn5-transposon in its genome. All mutations are located on a chromosome. Nodulation competitiveness (NC) of mutants towards the parent strain of S. meliloti CXM1-188 was investigated by resistant method using coinoculation of mutant and parent strain in the ratio 1:1. It was shown that NC was only 19-31% and 8-10%, for two strains Tb29 and Ts22, respectively which had lost the capability to synthesize higher molecular weight form of lipopolysaccharide (LPS1). Nodulation competitiveness of two other strains (Tb9 and Ts32) which retained the capability to synthesize LPS1 although in modified form varied from 49 to 62% and did not differ from NC of strain CXM1-188. The investigation of nodule formation rate has shown that four LPS-mutants did not differ from the parent strain by the number of root nodules. However the appearance of nodules induced by the mutant Tb29 was registered 7 days later than the nodules formed by other LPS-mutants and CXM1-188 strain. Obtained data concerning a single Tn5-insertion in genome of each of four S. meliloti LPS-mutants testify to the fact that both the disturbance of lipopolysaccharide synthesis and change of nodulation competitiveness in mutants Tb29 and Ts22 are results of a single mutation.

Mycobacterium flavum 158a can produce exopolysaccharides whose quantity varies, depending on the culture age, from 88.2 to 186.8% of the cell biomass weight in a medium with sucrose and from 1.3 to 25.0% in a medium with a polysaccharide synthesized by the oligonitrophilic bacterium Pseudomonas sp. 158a. The absolute and relative content of exopolysaccharides in the cultural broth decreases during the intensive growth of M. flavum 158a. This appears to be caused by their assimilation as a carbon source in the processes of energy and constructive metabolism. The exopolysaccharides of M. flavum 158a contain galactose, glucose, mannose, fucose, xylose, ramnose and a lipophilic sugar X1 at the molar ratio 10:11:11:8:11:12:16 as well as a non-identified sugar X2 with the Rf below than that of ramnose. The exopolysaccharide of M. flavum 158a was shown to be heterogeneous. It is composed of at least 16 fractions differing in molecular mass (from 98,980 to 490 D), quantity (from 2 to 8), composition and percentage of the constituent monosaccharides.

Exopolysaccharides from ten strains of oligonitrophilic bacteria were found to contain galactose, glucose and mannose. Strains belonging to the genus Mycobacterium contained also fucose, ramnose and three sugars that had not been identified: two of them were lipophilic and one had a lower Rf than ramnose. A correlation has been established between the composition of monosaccharides, their molar ratios in exocellular polysaccharides of oligonitrophilic bacteria, and the taxonomy of these microorganisms. Sporeforming bacteria belonging to the genus Bacillus produce polysaccharides which contain galactose, glucose and mannose at a ratio of 10:19: : (13--15). The molar ratio between the same sugars in exopolysaccharides of bacteria belonging to the genera Pseudomonas and Azotomonas is 10:(12--19) : (4--6). The ratio between galactose, glucose, mannose, fucose, ramnose, the unknown sugar and the sum of two lipophilic sugars in exocellular polysaccharides of the genus Mycobacterium is 10:(15--16) : (5--6) : (7--10) : (0.1--9) : (0.3--0.5) : (0.4--0.8).