Lipopolysaccharide of Pseudomonas mandelii, Isolated from Antarctica

Abstract

Representatives of the Pseudomonas mandelii species are able to exist and multiply in places where the temperature is constantly low. The optimum growth temperature for P. mandelii is 25–30°C, although this bacterium can grow at 4°C but not at 37°C. Therefore, P. mandelii is an excellent example of psychrotolerant bacterium which like psychrophilic bacteria is characterized by a number of structural and functional adaptations that facilitate survival at low temperatures. To understand these microorganisms’ role in Antarctica the characterization of its biopolymers is vital. One of these biopolymers is lipopolysaccharide (LPS), composition and structure of which are diagnostically significant. This determines the aim of the work – to isolate lipopolysaccharides from the cells of Antarctic strain of P. mandelii, grown at different temperatures, to characterize them chemically, and to study their functional and biological activity. Methods. The object of the study was Pseudomonas sp. U1, isolated from moss on Galindez Island in Antarctica. Lipopolysaccharides were extracted from dried cells by 45% phenol water solution at 65–68°С by Westphal and Jann method. The amount of carbohydrates was determined by phenol-sulfuric method. Carbohydrate content was determined in accordance to the calibration curve, which was built using glucose as a standard. The content of nucleic acids was determined by Spirin, protein − by Lowry method. Serological activity of LPS was investigated by double immunodiffusion in agar using the method of Ouchterlony. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAAG electrophoresis) was performed according to Laemmli. Results. As a result of phylogenetic analysis (programs ClustalX 2.1, Tree view, Mega v. 6.00) it was shown that the Antarctic bacterial strain Pseudomonas sp. U1 associated with green moss has a 99.4% homology with the type strain from the GenBank database NR024902 P. mandelii CIP 105273T. According to these data and proximity to the corresponding cluster of species, the studied isolate can be identified as P. mandelii. A characteristic feature of LPS isolated from P. mandelii cells, grown at different temperatures (20°C and 4°C) is their heterogeneity. This is evidenced by the data of the monosaccharide composition, electrophoretic distribution, which showed that P. mandelii produces S- and SR-forms of LPS, differed in the length of the O-specific polysaccharide chains. The R-form of LPS is also present, which does not contain an O-specific polysaccharide chains. Structural heterogeneity is also inherent in LPS lipid A. This is evidenced by the data of the fatty acid composition. In LPS grown at 4°C no unsaturated fatty acids were found, while such ones are synthesized in LPS of other bacteria grown in the cold, in response to a decrease in growth temperature. The study of the immunochemical properties of LPS was carried out using polyclonal O-antisera as antibodies, and LPS as antigens indicated that in homologous systems LPS exhibited serological activity. LPS obtained from P. mandelii U1 cells, grown at 20°C, had a complex antigenic composition and gave two clear lines of precipitation at a concentration of 1 mg/mL. LPS obtained from P. mandelii U1 cells, grown at 4°C, gave one line, which indicates their serological homogeneity. Conclusions. For the first time lipopolysaccharides were isolated from cells of P. mandelii U1, grown at 4°C and 20°С. A characteristic feature of these LPS is their heterogeneity. This is evidenced by the data of the monosaccharide and fatty acid composition, electrophoretic distribution, which showed that P. mandelii produces S- and SR-forms of LPS, differed in the length of the O-specific polysaccharide chains. LPS, obtained from cells, grown at different temperatures, are differed by serological activity.