Structural studies of the O polysaccharides from the lipopolysaccharides of Azospirillum thiophilum BV-ST and Azospirillum griseum L-25-5w-1T.

Diazotrophic bacteria of the genus Azospirillum are known widely, because they are ubiquitous in the rhizosphere and can promote the growth and performance of nonlegume plants. Recently, more Azospirillum species have been isolated from sources other than plants or soil. We report the structures of the O polysaccharides (OPSs) from the lipopolysaccharides of the type strains A. thiophilum BV-ST (1) and A. griseum L-25-5w-1T (2), isolated from aquatic environments. Both structures have a common tetrarhamnan in the repeating-unit, which is decorated with a side xylose in the OPS of A. thiophilum BV-ST.

Characterization of cellular, biochemical and genomic features of the diazotrophic plant growth-promoting bacterium Azospirillum sp. UENF-412522, a novel member of the Azospirillum genus.

Given their remarkable beneficial effects on plant growth, several Azospirillum isolates currently integrate the formulations of various commercial inoculants. Our research group isolated a new strain, Azospirillum sp. UENF-412522, from passion fruit rhizoplane. This isolate uses carbon sources that are partially distinct from closely-related Azospirillum isolates. Scanning electron microscopy analysis and population counts demonstrate the ability of Azospirillum sp. UENF-412522 to colonize the surface of passion fruit roots. In vitro assays demonstrate the ability of Azospirillum sp. UENF-412522 to fix atmospheric nitrogen, to solubilize phosphate and to produce indole-acetic acid. Passion fruit plantlets inoculated with Azospirillum sp. UENF-41255 showed increased shoot and root fresh matter by 13,8% and 88,6% respectively, as well as root dry matter by 61,4%, further highlighting its biotechnological potential for agriculture. We sequenced the genome of Azospirillum sp. UENF-412522 to investigate the genetic basis of its plant-growth promotion properties. We identified the key nif genes for nitrogen fixation, the complete PQQ operon for phosphate solubilization, the acdS gene that alleviates ethylene effects on plant growth, and the napCAB operon, which produces nitrite under anoxic conditions. We also found several genes conferring resistance to common soil antibiotics, which are critical for Azospirillum sp. UENF-412522 survival in the rhizosphere. Finally, we also assessed the Azospirillum pangenome and highlighted key genes involved in plant growth promotion. A phylogenetic reconstruction of the genus was also conducted. Our results support Azospirillum sp. UENF-412522 as a good candidate for bioinoculant formulations focused on plant growth promotion in sustainable systems.

Fourier Transform Infrared (FTIR) Spectroscopic Analyses of Microbiological Samples and Biogenic Selenium Nanoparticles of Microbial Origin: Sample Preparation Effects.

To demonstrate the importance of sample preparation used in Fourier transform infrared (FTIR) spectroscopy of microbiological materials, bacterial biomass samples with and without grinding and after different drying periods (1.5-23 h at 45 °C), as well as biogenic selenium nanoparticles (SeNPs; without washing and after one to three washing steps) were comparatively studied by transmission FTIR spectroscopy. For preparing bacterial biomass samples, Azospirillum brasilense Sp7 and A. baldaniorum Sp245 (earlier known as A. brasilense Sp245) were used. The SeNPs were obtained using A. brasilense Sp7 incubated with selenite. Grinding of the biomass samples was shown to result in slight downshifting of the bands related to cellular poly-3-hydroxybutyrate (PHB) present in the samples in small amounts (under ~10%), reflecting its partial crystallisation. Drying for 23 h was shown to give more reproducible FTIR spectra of bacterial samples. SeNPs were shown to contain capping layers of proteins, polysaccharides and lipids. The as-prepared SeNPs contained significant amounts of carboxylated components in their bioorganic capping, which appeared to be weakly bound and were largely removed after washing. Spectroscopic characteristics and changes induced by various sample preparation steps are discussed with regard to optimising sample treatment procedures for FTIR spectroscopic analyses of microbiological specimens.

Structure of the O-specific polysaccharide from Azospirillum formosense CC-Nfb-7(T).

The lipopolysaccharide was obtained from the cells of Azospirillum formosense CC-Nfb-7(T), a diazotrophic bacterium isolated from agricultural soil. The O-specific polysaccharide (OPS) was released by mild acid hydrolysis of the lipopolysaccharide and was studied by sugar analysis along with 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments, and Smith degradation. The following structure of partially methylated OPS composed of trisaccharide repeating units was established.

Structure of the O-specific polysaccharide of Azospirillum doebereinerae type strain GSF71T.

O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of plant-growth-promoting rhizobacteria Azospirillum doebereinerae GSF71T and studied by sugar analysis along with 1H and 13C NMR spectroscopy, including 2D 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments. It was established that the polysaccharide is linear and consists of tetrasaccharide repeating units with the following structure.

Structure of the O-specific polysaccharide from Azospirillum fermentarium CC-LY743T.

O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of nitrogen-fixing bacterium Azospirillum fermentarium CC-LY743T (IBPPM 578) and was studied by sugar analysis along with 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, and 1H,13C HSQC and HMBC experiments. The polysaccharide was found to be linear and to consist of alterating α-l-fucose and α-d-mannose residues in tetrasaccharide repeating units of the following structure: →2)-α-D-Manp-(1 → 3)-α-L-Fucp-(1 → 3)-α-D-Manp-(1 → 3)-α-L-Fucp-(1→.

[Characterization of the lipopolysaccharides of serogroup II Azospirillum].

Lipopolysaccharides of six Azospirillum strains (A. brasilense SR50, SR80, SR88, SR109, SR111, SR115, and A. lipoferum SR 42) isolated from the rhizosphere of cereal plants of Saratov oblast, Russia and assigned to serogroup II by serological analysis were studied. In the lipid A fatty acid composition, the lipopolysaccharides under study were similar to those of other Azospirillum strains and were characterized by predominance of 3-hydroxytetradecanoic, 3-hydroxyhexadecanoic, and octadecenoic acids. Monosaccharide analysis of the O-specific polysaccharides (including determination of the absolute configurations, methylation analysis, and one- and two-dimensional NMR spectroscopy) revealed the presence of two types of repeating units in varying ratios. High degree of serological similarity between the strains under study was shown to result from the presence of repeating units with identical structure in their O antigens.

Immunochemical characterization of the capsular polysaccharide of Azospirillum irakense KBC1.

The repeating unit structure of Azospirillum irakense KBC1 capsular polysaccharide (CPS) was established and was found to be identical to that of the O polysaccharide of A. irakense KBC1 lipopolysaccharide (LPS). The antigenic heterogeneity of the LPS and the CPS was shown to be related to differences in the macromolecular organization of these glycopolymers. After an immune response activation, R-form CPS molecules were found to be predominant.

Structural peculiarities of the O-specific polysaccharides of Azospirillum bacteria of serogroup III.

Lipopolysaccharides and O-specific polysaccharides were isolated from the outer membrane of bacterial cells of three strains belonging to two Azospirillum species, and their structures were established by monosaccharide analysis including determination of the absolute configurations, methylation analysis, and one- and two-dimensional NMR spectroscopy. It was shown that while having the identical composition, the O-polysaccharides have different branched tetrasaccharide repeating units. Two neutral polysaccharides were found in the lipopolysaccharide of A. brasilense 54, and the structure for the predominant O-polysaccharide was determined. The structural data, together with results of serological studies, enabled assignment of strains examined to a novel serogroup, III. The chemical basis for the serological relatedness among the azospirilla of this serogroup is presumably the presence of a common →3)-α-L-Rhap-(1→2)-α-L-Rhap-(1→3)-α-L-Rhap-(1→ oligosaccharide motif in their O-polysaccharides.

Azospirillum picis sp. nov., isolated from discarded tar.

A polyphasic taxonomic study was performed on a pink-coloured unknown bacterium isolated from discarded road tar. Comparative analysis of the 16S rRNA gene sequence demonstrated that the isolate belongs phylogenetically to the genus Azospirillum with Azospirillum lipoferum, A. melinis and A. rugosum as its closest phylogenetic relatives (96.7, 96.6 and 96.6 % similarity to the respective type strains). The generic assignment was confirmed on the basis of chemotaxonomic data, which revealed a fatty acid profile characteristic for the genus Azospirillum, consisting of straight-chain saturated and unsaturated fatty acids, with C(18 : 1)omega7c as the major unsaturated non-hydroxylated fatty acid, and C(16 : 0) 3-OH as the major hydroxylated fatty acid, and a ubiquinone with ten isoprene units (Q-10) as the predominant respiratory quinone. On the basis of both the phenotypic and molecular genetic evidence, it is proposed that the unknown isolate should be classified within a novel species of the genus Azospirillum, for which the name Azospirillum picis sp. nov. is proposed. The type strain is IMMIB TAR-3(T) (=CCUG 55431(T) =DSM 19922(T)).

[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 LPS(Cd), LPS(Sp59b), LPS(Sp7), LPS(S17), and LPS(KBC1) 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, Ab(Sp59b) and Ab(Cd) 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.

Azospirillum zeae sp. nov., a diazotrophic bacterium isolated from rhizosphere soil of Zea mays.

Two free-living nitrogen-fixing bacterial strains, N6 and N7(T), were isolated from corn rhizosphere. A polyphasic taxonomic approach, including morphological characterization, Biolog analysis, DNA-DNA hybridization, and 16S rRNA, cpn60 and nifH gene sequence analysis, was taken to analyse the two strains. 16S rRNA gene sequence analysis indicated that strains N6 and N7(T) both belonged to the genus Azospirillum and were closely related to Azospirillum oryzae (98.7 and 98.8 % similarity, respectively) and Azospirillum lipoferum (97.5 and 97.6 % similarity, respectively). DNA-DNA hybridization of strains N6 and N7(T) showed reassociation values of 48 and 37 %, respectively, with A. oryzae and 43 % with A. lipoferum. Sequences of the nifH and cpn60 genes of both strains showed 99 and approximately 95 % similarity, respectively, with those of A. oryzae. Chemotaxonomic characteristics (Q-10 as quinone system, 18 : 1omega7c as major fatty acid) and G+C content of the DNA (67.6 mol%) were also similar to those of members of the genus Azospirillum. Gene sequences and Biolog and fatty acid analysis showed that strains N6 and N7(T) differed from the closely related species A. lipoferum and A. oryzae. On the basis of these results, it is proposed that these nitrogen-fixing strains represent a novel species. The name Azospirillum zeae sp. nov. is suggested, with N7(T) (=NCCB 100147(T)=LMG 23989(T)) as the type strain.

[Role of the carbohydrate-binding center of bacterial lectin in the effect on adipocytes under the influence of increased temperature].

The effect of temperature and lectin from bacteria of the genues Azospirillum with blocked activity on human adipose tissue cells has been studied. The temperature used was 43.5 +/- 0.5 degrees C. Comparative results are given for the effect of lectin with the blocked and active carbohydrate-binding centers on adipocytes during heating, and the time course of the structural changes of adipocytes is described. When lectin with the active carbohydrate-binding centers was used for treatment, the heat-treated cells of a healthy obesity-prone subject died on the average in 55 +/- 5 min, whereas cells treated with lectin with the L-fucose-blocked carbohydrate-binding centers died in 80 +/- 5 min. The heat-treated cells of a diabetic obesity-prone patient died in 150 +/- 10 min on average when exposed to both active and inactive lectin. Consequently, when the lectin center is blocked with L-fucose, the effectiveness of lectin action on adipose cells of healthy obesity-prone persons decreases.

Structure of the O-polysaccharide of the lipopolysaccharide of Azospirillum irakense KBC1.

The O-polysaccharide was isolated from the lipopolysaccharide of the plant-growth-promoting bacterium Azospirillum irakense KBC1 and studied by sugar and methylation analyses, Smith degradation and 1H and 13C NMR spectroscopy, including 1H, 13C HSQC and NOESY experiments for linkage and sequence analysis. The following structure of the branched hexasaccharide repeating unit of the O-polysaccharide with an unusually long side chain was established: [carbohydrate structure: see text].

[Relationship between lectin, alpha-, beta-glucosidase, and beta-galactosidase activities of Azospirillum]. / Izuchenie vzaimosviazi lektinovoi, al'fa-, beta-gliukozidaznykh i beta-galaktozidaznoi aktivnostei azospirill.

The activities of alpha-glucosidase, beta-glucosidase, and beta-galactosidase were studied during the isolation and purification of lectins from Azospirillum brasilense Sp7 and Azospirillum lipoferum 59b cells. These enzymatic activities were revealed in crude extracts of surface proteins, protein fraction precipitated with ammonium sulfate or ethanol-acetone mixture, and protein fraction obtained by gel filtration on Sephadex G-75. The distribution of the enzymes between different protein fractions varied among the azospirilla studied. The cofunction of the A. brasilense Sp7 lectin and beta-galactosidase on the cell surface is assumed. A strong interaction between the A. lipoferum 59b lectin and glucosidases was revealed. The lectin from A. lipoferum 59b may possess saccharolytic activity.

Cell-surface lectins of Azospirillum spp.

Cell-surface lectins were screened in seven strains of Azospirillum brasilense and A. lipoferum. The presence of lectins was determined by particle agglutination assays employing latex beads coated with neoglycoproteins and by Western blot with neoglycoproteins labeled with horseradish peroxidase as a probe. Seven strains were agglutinated with the assayed sugar residues. The highest agglutination was with fucose and glucose and to a lesser extent with mannose residues. Cell-wall proteins extracted from two Azospirillum spp. strains exhibit lectin-like activities. We believe that lectins are present in the cell-wall of Azospirillum spp.