V. V. Ignatov

Comparative spectroscopic characterization of different pectins and their sources

The results of atomic absorption spectrometry (AAS) analyses and Fourier transform infrared (FTIR) spectroscopic studies of several pectins obtained from pumpkin and sugar beet, as well as of their vegetable sources, are compared and discussed. Special emphasis is put on the state of carboxylic groups of the polymer backbone and the mineral composition of both the sources and the resulting pectins, including the content of alkaline (Na, K) and alkaline-earth metals (Mg, Ca), as well as traces of heavy metals (V, Fe, Cu, Pb). The pectins were obtained from dried pumpkin pulp by extraction with dilute hydrochloric acid or using a biotechnological process involving the multi-enzyme cell-free culture supernatant from the bacterium Xanthomonas campestris; commercial sugar beet pectin extracted by the standard method of acid treatment was obtained from a sugar beet processing plant in Krasnodar (Russia). For comparison, a sample of commercial acid-extracted citrus pectin (Copenhagen, Denmark) was also studied. The results obtained show that potassium seems to occur as a relatively free constituent, whereas a more specific interaction between sodium ions and pectic substances may be assumed depending on the origin of the pectin and obviously on its properties. Much higher amounts of Mg and, especially, Ca found in pumpkin biopectin as compared to all of the three pumpkin, sugar beet and citrus acid-extracted products correlate with a relatively well exhibited capability of pectins to bind these two cations, which is noticeably suppressed in acidic media. The increased content of Ca (and, probably, Mg) may in principle contribute to poorer gelling properties of pumpkin pectin and, in general, of biopectins as compared to the corresponding acid extracts. The results on the mineral fraction of the samples are compared considering the FTIR spectroscopic data for the pectins studied as well as for their sources featuring, in particular, the state of carboxylic groups responsible for metal binding. It has also been found that lead and copper essentially accumulate in pectins upon extraction, whereas iron does not, being relatively more weakly bound by pectic substances (which may, however, depend on its oxidation state) than other heavy metals; the accumulation process is slightly (for Fe and Pb) or not at all (for Cu) suppressed during acid extraction. Comparing the content of vanadium in the pectins and their sources, it may be concluded that this element occurring in plant tissue obviously in different chemical forms may be partly transferred to pectin during its extraction in a proportion similar to that in which it is bound to pectic substances in the plant cell wall, thus indicating its strong binding not affected by acid treatment.

Structure of the O-specific polysaccharide of the lipopolysaccharide of Azospirillum brasilense Sp245.

An O-specific polysaccharide was isolated from the lipopolysaccharide of a plant-growth-promoting bacterium Azospirillum brasilense Sp245 and studied by sugar analyses along with one- and two-dimensional 1H and 13C NMR spectroscopy, including NOESY. The polysaccharide was found to be a new rhamnan with a pentasaccharide repeating unit having the following structure:-->2)-beta-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-Rhap-(1-->2)-alpha-D-Rhap-(1-->

Azospirillum brasilense colonisation of wheat roots and the role of lectin–carbohydrate interactions in bacterial adsorption and root-hair deformation

The dynamics of adsorption of the nitrogen-fixing soil bacteria Azospirillum brasilense 75 and 80 (isolated from soil samples collected in Saratov Oblast, southern Russia) and A. brasilense Sp245 to the roots of seedlings of common spring wheat was studied in relation to inoculum size, period of incubation with the roots and bacterial-growth phase. The number of root-attached cells increased with increasing size of inoculum and time of contact. The saturation of root-surface adsorption was observed by 24 h of co-incubation for A. brasilense 75, by 6 h for A. brasilense 80, and by 3 h for A. brasilense Sp245. The firmness of bacterial–root attachment increased after extended co-incubation. Differences in the adsorption kinetics of the azospirilla were found that were associated with bacterial-growth phases. Azospirilla attached to the roots of their host cultivar more actively than they did to the roots of a non-host cultivar. Adsorption was partially inhibited when the roots were treated with N-acetyl-D-glucosamine. Maximal inhibition occurred after a 3-h exposure of the roots to the bacteria. Root-hair deformation induced with polysaccharide-containing complexes from the Azospirillum capsular material was inhibited by N-acetyl-D-glucosamine and chitotriose, specific haptens of wheat germ agglutinin. A possible mechanism of the mutual influence of bacteria and plants may involve key roles of wheat germ agglutinin, present on the roots, and the polysaccharide-containing components of the Azospirillum capsule.

[Oil-oxidizing potential of associative rhizobacteria of the genus Azospirillum].

The oil-oxidizing potential of associative rhizobacteria of the genus Azospirillum was studied under laboratory conditions. After screening, A. brasilense strain SR80 was chosen for further investigation. The strain was capable of degrading 56.5% of crude oil (added in a concentration of 1%) over 14 days in a medium containing malate as an additional source of carbon and energy. Studies of associative properties showed that the strain had positive chemotaxis to wheat root exudates, colonized wheat roots, and produced indole-3-acetic acid. The synthesis of indole-3-acetic acid was not inhibited by oil. Under hydroponic conditions, crude oil stimulated growth of A. brasilense SR80, which promoted development of the wheat root system in the presence of oil and enhanced the level of oil degradation by the plant-microbial association.

Fourier transform infrared spectroscopic study of intact cells of the nitrogen-fixing bacterium Azospirillum brasilense

Abstract The data of Fourier transform infrared (FTIR) spectroscopic measurements performed on intact cells of the soil nitrogen-fixing bacterium Azospirillum brasilense grown in a standard medium and under the conditions of an increased metal uptake are compared and discussed. The structural FTIR information obtained is considered together with atomic absorption spectrometry (AAS) data on the content of metal cations in the bacterial cells. Some methodological aspects concerning preparation of bacterial cell samples for FTIR measurements are also discussed.

Enzymic extraction of pectin preparations from pumpkin

Pectin samples have been extracted from pumpkin raw material with 0.1 M HCl and with three enzymic preparations (cellulase from Trichoderma viride, hemicellulase from Aspergillus niger and the glycosidase complex produced by Xanthomonas campestris). The yield of pectin preparations, their monosaccharide composition, degree of acetylation, molecular weight and viscosity were determined. Enzymic methods of extraction gave much bigger yields of pectic substances. Compared to the commercial citrus pectin, all the samples derived from pumpkin raw material had a significantly lower uronide content and a higher content of neutral sugars, notably glucose and galactose. This study proposes that pumpkin pectin molecules may contain a large number of branched regions formed from neutral sugars.

Involvement of the Lipopolysaccharides of Azospirilla in the Interaction with Wheat Seedling Roots

The present study was undertaken to comparatively investigate the attachment capacities of Azospirillum brasilenseSp245 and its lipopolysaccharide-defective Omegon-Km mutants KM018 and KM252, as well as their activities with respect to the alteration of the morphology of wheat seedling root hairs. The adsorption dynamics of the parent Sp245 and mutant KM252 strains of azospirilla on the seedling roots of the soft spring wheat cv. Saratovskaya 29 were similar; however, the attachment capacity of the mutant KM252 was lower than that of the parent strain throughout the incubation period (15 min to 48 h). The mutation led to a considerable decrease in the hydrophobicity of the Azospirillumcell surface. The lipopolysaccharides extracted from the outer membrane of A. brasilenseSp245 and mutant cells with hot phenol and purified by chromatographic methods were found to induce the deformation of the wheat seedling root hairs, the lipopolysaccharide of the parent strain being the most active in this respect. The role of the carbohydrate moiety of lipopolysaccharides in the interaction of Azospirillumcells with plants is discussed.

Structural analysis of the O-polysaccharide from the lipopolysaccharide of Azospirillum brasilense S17

A mixture of two structurally distinct neutral O-polysaccharides was obtained by mild acid degradation of the lipopolysaccharide isolated by the phenol/water extraction from the asymbiotic diazotrophic rhizobacterium Azospirillum brasilense S17. The following structures of the O-polysaccharides were established by composition and methylation analyses, Smith degradation, and 1H and 13C NMR spectroscopy, including a 2D NOESY experiment: [Formula: see text] where L-Rha2Me stands for 2-O-methyl-L-rhamnose and SHb for the (S)-3-hydroxybutanoyl group. The occurrence of two distinct polysaccharides is reported for the first time in Azospirillum spp.

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.

Spectroscopic characterization of cell membranes and their constituents of the plant-associated soil bacterium Azospirillum brasilense

Abstract Structural and compositional features of bacterial membranes and some of their isolated constituents (cell surface lipopolysaccharide, phospholipids) of the plant-growth-promoting diazotrophic rhizobacterium Azospirillum brasilense (wild-type strain Sp245) were characterized using Fourier transform infrared (FTIR) spectroscopy and some other techniques. FTIR spectra of the cell membranes were shown to comprise the main vibration modes of the relevant lipopolysaccharide and protein components which are believed to be involved in associative plant–bacterium interactions, as well as of phospholipid constituents. The role and functions of metal cations in the structural organization and physicochemical properties of bacterial cell membranes are also discussed considering their accumulation in the membranes from the culture medium.