G. L. Burygin

Isolation and characterization of a glyphosate-degrading rhizosphere strain, Enterobacter cloacae K7.

Plant-growth-promoting rhizobacteria exert beneficial effects on plants through their capacity for nitrogen fixation, phytohormone production, phosphate solubilization, and improvement of the water and mineral status of plants. We suggested that these bacteria may also have the potential to express degradative activity toward glyphosate, a commonly used organophosphorus herbicide. In this study, 10 strains resistant to a 10 mM concentration of glyphosate were isolated from the rhizoplane of various plants. Five of these strains--Alcaligenes sp. K1, Comamonas sp. K4, Azomonas sp. K5, Pseudomonas sp. K3, and Enterobacter cloacae K7--possessed a number of associative traits, including fixation of atmospheric nitrogen, solubilization of phosphates, and synthesis of the phytohormone indole-3-acetic acid. One strain, E. cloacae K7, could utilize glyphosate as a source of P. Gas-liquid chromatography showed that E. cloacae growth correlated with a decline in herbicide content in the culture medium (40% of the initial 5mM content), with no glyphosate accumulating inside the cells. Thin-layer chromatography analysis of the intermediate metabolites of glyphosate degradation found that E. cloacae K7 had a C-P lyase activity and degraded glyphosate to give sarcosine, which was then oxidized to glycine. In addition, strain K7 colonized the roots of common sunflower (Helianthus annuus L.) and sugar sorghum (Sorghum saccharatum Pers.), promoting the growth and development of sunflower seedlings. Our findings extend current knowledge of glyphosate-degrading rhizosphere bacteria and may be useful for developing a biotechnology for the cleanup and restoration of glyphosate-polluted soils.

Chemical and serological studies of liposaccharides of bacteria of the genus Azospirillum

The analysis of the lipopolysaccharides (LPS) of nine strains of azospirilla revealed the presence of the characteristic components of these glycopolymers: carbohydrates, hydroxylated fatty acids, and 2-keto-3-deoxyoctonic acid (KDO). SDS electrophoresis revealed the heterogeneous nature and the strains differences in the ratio of the molecular S and R forms present in the LPS. Polyclonal rabbit antibodies (Ab) were obtained against the isolated LPSCd, LPSSp59b, LPSSp7, LPSS17, and LPSKBC1 preparations. Based on the results of the serological studies of the LPS, the bacterial strains investigated in the work were divided into two main serogroups. Based on the immunoblotting data, AbSp59b and AbCd were found to be formed in response to both the S and R forms of the LPS molecules, whereas all the rest formed in response to the S forms only. It was shown that the heterogeneity of the antigenic determinants is typical of the second LPS group. It was suggested that rhamnose plays one of the key roles in the specific interactions between the azospirillum membrane LPS and Ab.

Identification of an O-linked repetitive glycan chain of the polar flagellum flagellin of Azospirillum brasilense Sp7.

This is the first report to have identified an O-linked repetitive glycan in bacterial flagellin, a structural protein of the flagellum. Studies by sugar analysis, Smith degradation, (1)H and (13)C NMR spectroscopy, and mass spectrometry showed that the glycan chains of the polar flagellum flagellin of the plant-growth-promoting rhizobacterium Azospirillum brasilense Sp7 are represented by a polysaccharide with a molecular mass of 7.7 kDa, which has a branched tetrasaccharide repeating unit of the following structure:

[Analysis of DNA, lipopolysaccharide structure, and some cultural and morphological properties in closely related strains of Azospirillum brasilense].

We studied closely related Azospirillum brasilense strains Sp7 and Cd. For probing of their genomes, the fragments of 85 MDa (p85) and 120 MDa (p120) from A. brasilense Sp245 plasmids were hybridized with 115-MDa (p115) and 90-MDa (p90) plasmids of strain Sp7, respectively. Strain Cd was found to lose the 115-MDa plasmid and one of the two EcoRI restriction fragments of the total DNA (localized within p115 and the chromosome) that was homologous to an EcoRI-generated p85 fragment of 2.4 kb. On the contrary, in the total DNA of strain Sp7-S, in spite of the previously established disappearance of the 115-MDa replicon, two fragments homologous to p85 were revealed, as with strain Sp7. It is suggested that the Sp7-S genome contains the total p115 DNA or at least a certain part of it. Strains Sp7 and Cd were found to differ in size and morphology of colonies on solid and semisolid media, in the levels of resistance to the cation surfactant cetavlon, and in the antigen structure of lipopolysaccharides.

Biofilm Formation by Paenibacillus polymyxa Strains Differing in the Production and Rheological Properties of Their Exopolysaccharides

We evaluated the ability of several strains of the rhizobacterium Paenibacillus polymyxa, differing in the yield and rheological properties of their exopolysaccharides, to form biofilms on abiotic surfaces. Of these strains, P. polymyxa 1465, giving the highest yield of extracellular polysaccharides and the highest kinematic viscosity of the culture liquid and of aqueous polysaccharide solutions, proved to be the most active in forming biofilms on hydrophobic and hydrophilic surfaces. Enzyme-linked immunosorbent assay with rabbit polyclonal antibodies developed to isolated exopolysaccharides of P. polymyxa 1465 and 92 was used to detect P. polymyxa’s polysaccharidic determinants in the composition of the biofilm materials.

Study of immunochemical heterogeneity of Azospirillum brasilense lipopolysaccharides

A comparative immunochemical analysis of lipopolysaccharides (LPS) in Azospirillum brasilense model strains Sp7 and Sp245 and in mutants with altered somatic antigens has been performed. According to the results of a complex of various immunochemical methods, including studies with polyclonal antibodies against the LPS these bacteria, their LPS consist of an assembly of macromolecules with different antigenic characteristics. Two types of O-specific polysaccharides (O-PS) are present in the LPS of every strain of A. brasilense under study. The major difference between the two O-PS is the antigenic heterogeneity of one of them. This heterogeneous O-PS has been shown to possess at least two O-factors (antigenic determinants) different in their structure. Meanwhile, according to all the tests performed, the other O-PS in every strain is immunochemically homogeneous and identical to one of the determinants revealed in the more diversified O-PS. The LPS heterogeneity among the given strains may be due to the pattern of O-specific polysaccharide synthesis, one of the O-PS being an intermediate in the synthesis of the other.

Atypical R-S dissociation in Azospirillum brasilense

It was found that atypical R–S dissociation in the type strain A. brasilense Sp7 is not accompanied by drastic changes in the polysaccharide moieties of bacterial lipopolysaccharides but is rather due to different contributions of two O-specific polysaccharides (found in both R and S dissociants) to the age-dependent architectonics of the cell surface.

Detection of a sheath on Azospirillum brasilense polar flagellum

The presence of a polysaccharide sheath on the surface of the polar flagellum of Azospirillum brasilense was revelted by immunoelectron microscopy and immunodiffusion analysis with strain-specific antibodies to lipopolysaccharides (LPS). The antigenic identity of A. brasilense Sp245 sheath material and one of the two O-specific polysaccharides of its somatic LPS was demonstrated. The screening effect of the sheath in respect to flagellin was determined by agglutination tests and by the inhibition of azospirilla motility in liquid and semisolid agarized media caused by strain-specific antibodies to LPS; no pronounced effect of genus-specific antibodies to flagellin was observed.

Capsular polysaccharide of the bacterium Azospirillum lipoferum Sp59b: Structure and antigenic specificity

Antigenic differences were revealed between the cell wall outer membrane lipopolysaccharides and the capsular high molecular weight bioglycans for a typical strain of the nitrogen-fixing rhizobacterium Azospirillum lipoferum Sp59b using antibodies prepared against the homologous lipopolysaccharide and lipopolysaccharide-protein complex. From the capsular lipopolysaccharide-protein and polysaccharide-lipid complexes of A. lipoferum Sp59b, polysaccharides were isolated and their structure was for the first time established in Azospirillum by monosaccharide analysis which included determination of the absolute configurations, methylation, O-deacetylation, and one- and two-dimensional NMR spectroscopy. The polysaccharides of the capsular complexes were shown to have identical structure of the branched tetrasaccharide repeating unit, which differs from the structure of the O-specific polysaccharide within the outer membrane lipopolysaccharide of this strain.

Chemical composition and immunochemical characteristics of the lipopolysaccharide of nitrogen-fixing rhizobacterium Azospirillum brasilense CD

The chemical composition of the lipopolysaccharide of the associative diazotrophic rhizobacterium Azospirillum brasilense Cd has been studied. Among the main components of the hydrophobic part of the lipopolysaccharide, we identified 3-hydroxytetradecanoic, hexadecenoic, 3-hydroxyhexadecanoic, hexadecanoic, octadecenoic, and nanodecanoic fatty acids; the carbohydrate part contained rhamnose, galactose, and mannose. Polyclonal antibodies against the preparation under study were raised in rabbits. Serological relations between A. brasilense Cd and other strains of Azospirillum spp. were studied using double radial immunodiffusion and enzyme-linked immunosorbent assay.