Characterization of Azospirillum brasilense Lipopolysaccharides

Abstract

Azospirillum brasilense is a gram-negative, nitrogen-fixing bacterium that colonizes the rhizosphere of various types of grasses and cereals. Lipopolysaccharides (LPS) are a class of complex glycolipids present in the cell membrane of gramnegative bacteria and mediate plant-bacteria interactions. Although the effects of LPS of pathogenic plant bacteria on the induction of plant defense mechanisms have been characterized, the role of LPS of beneficial rhizobacteria on plant growth is less clear. Therefore, a very important point is the study of the chemical, biological, and functional activities of A. brasilense LPS, which was the aim of this work. Methods. A. brasilense LPSs were isolated from dry bacterial mass by the phenol-water method. The carbohydrates were analyzed by the Dubois method, nucleic acids — by Spirin, protein content — by Lowry and 2-keto-3-deoxyoctonic acid (KDO) — by Osborn. Pyrogenicity of LPS was tested observing the rules of bioethics in rabbits. Serological studies were performed by the Ouchterlony method. The identification of monosaccharides and fatty acids in LPS preparations was carried out on an Agilent 6890N/5973 inert chromatomass spectrometry system. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAAG electrophoresis) was performed according to Laemmli. Results. LPS of 3 strains of A. brasilense were isolated from dry bacterial mass and purifi ed from nucleic acids by ultracentrifugation. The purified LPSs were characterized by different relative yields from 2.44% to 4.75%, which is slightly higher than other strains of the A. brasilense (1—3%). The studied preparations were characterized by a rather high content of carbohydrates from 50.1% to 72.1%. All LPS contained up to 0.17% KDO, which is a specific component of the LPS of gram-negative bacteria. Analysis of the monosaccharide composition indicates that the LPSs of the studied A. brasilense strains turned out to be heterogeneous. At the same time, such monosaccharides as mannose, galactose, glucose, and heptose were recorded in the LPS of all tested strains. The study of the fatty acid composition of LPS showed the presence of fatty acids containing from 14 to 18 carbon atoms. Нydroxylated, saturated, and monounsaturated acids and their cis isomers were found. In the investigated LPS, the dominant fatty acids were 16:0, 18:1, 14:0(3-OH), and 16:0(3-OH), which coincides with the literature data. The research of the pyrogenic effect of LPS of A. brasilense studied strains showed that LPS solutions are apyrogenic. The double immunodiffusion reaction in Ouchterlon agar showed that all tested LPS in homologous systems exhibited ancultitigenic activity. Serological cross-reactions can be used as an approach in classifying different bacteria. Thus, we found that antisera to A. brasilense 18-2 and 61 react with all LPSs of the studied strains, which may indicate the presence of common antigenic determinants in them and that these strains belong to the same serogroup. The electrophoretic distribution data indicate that A. brasilense produces S-forms of LPS which differ in the length of O-specifi c polysaccharide chains. Conclusions. For the first time, LPS were isolated from cells of A. brasilense 10/1, 18-2 and 61. A characteristic feature of these LPS is their heterogeneity in monosaccharide and fatty acid composition, all of them were apyrogenic. The results obtained during biological-functional studies of three strains of A. brasilense LPS contribute to the biological characteristics of this species.