Characteristics of Stenotrophomonas rhizophila Lipopolysaccharide, a Representative of Antarctica

Abstract

In 2002, Wolf described a new species of Stenotrophomonas, Stenotrophomonas rhizophila, which is a non-pathogenic plant inhabitant. The defining characteristics of the new species, in contrast to S. maltophilia, were the following: growth at 4 °C, but its absence at 37 °C; the use of xylose as a carbon source; low osmolytic tolerance. S. rhizophila is isolated exclusively from the rhizosphere or from the internal tissues of plants, in particular from the vascular tissues of the root and stem. In 2014, a number of authors, studying cultivated heterotrophic bacteria and yeasts, which dominate in plant samples collected from various terrestrial biotopes near the Ukrainian Antarctic station on Galindez Island in Marine Antarctica, isolated a number of bacterial isolates. The authors’ phylogenetic analysis using only BLAST analysis made it possible to determine the approximate phylogenetic affiliation of the bacterial isolate to the family Gammaproteobacteria (genus Stenotrophomonas, species S. rhizophila). It is known that many processes that determine the biological features of microorganisms, the nature of their relationships between them, as well as micro- and macroorganisms in biocenoses, are carried out with the participation of the surface structures of the bacterial cell, which are in direct contact with the environment. Among them, of particular interest are lipopolysaccharides (LPS), components of the outer membrane of Gram-negative bacteria. However, to date, we have not found any works devoted to the study of S. rhizophila lipopolysaccharides in the literature available to us. The aim of the work was to clarify the phylogenetic position of the 6p5m bacterial strain isolated from the Antarctic region by constructing dendrograms, to study some of its phenotypic properties, to isolate lipopolysaccharides and study chemical and component composition, serological, and biological properties. Methods. Identification was carried out via ribosomal phylogeny. A fragment of the 16S rRNA gene of bacterial strain 6p5m was amplified by PCR using primers 8F and 1492R. The phylogenetic position of the strains was determined by construction of dendrograms, which show the position of the investigated strains among closely related and typical species and type species (programs ClustalX 2.1, Mega v. 6.00). LPS was obtained from cells by water-phenol extraction, electrophoresis was carried out in polyacrylamide gel, monosaccharide and fatty acid composition was determined by chromato-mass spectrometry, antigenic activity was studied by immunodiffusion in agar. Results. Phylogenetic analysis of the nucleotide sequence of the 16S rRNA gene revealed a high level of homology (99.8%) of the bacterial strain 6p5m with typical strain S. rhizophila DSM 14405T from the GenBank database, which allows us to classify it. Comparing phylogenetic analysis with phenotypic data, we can assert that strain 6p5m belongs to the S. rhizophila species. When studying the biopolymer composition of LPS by specific reactions to each component, it was found that the carbohydrate content was relatively low and amounted to 27.42% and 13.34% for LPS1 and LPS2, respectively. The content of characteristic LPS components: heptose and 2-keto-3-deoxyoctonic (KDO) acid was 1.78% and 0.034%, as well as 5.38% and 0.09%, respectively, for LPS1 and LPS2. Analysis of the monosaccharide composition of LPS preparations showed that fucose (72.57%) is present as a dominant monosaccharide in LPS1 and rhamnose (65.04%) in LPS2. The main monosaccharides of the extracellular component were galactose (39.92%) and rhamnose (24.36%). Analysis of the lipid part of the studied LPS indicates significant differences between them. In the composition of lipid A LPS1 and LPS2 of S. rhizophila 6p5m, anti-iso-pentadecanoic acid (a-i-C15:0) was predominant (25.58% and 39.24%, respectively), and 3-hydroxydodecanoic acid (3-OH-C12:0) in LPS2 (26.21%), hexadecanoic acid (С16:0) (27.06% and 10.51%, respectively) for LPS1 and LPS2. Cyclic acids were present only in LPS1, while i-C15:0 and 9-C16:1 only in LPS2. Electrophoretic analysis showed heterogeneity characteristic of an LPS molecule, manifested by a plurality of bands in the form of a «ladder» when the gel was stained with silver ions, which is associated with the presence in the composition of LPS preparations of O-specific polysaccharide structures with different lengths of oligosaccharide chains, which determine their different molecular weight. On the electropherogram of LPS1 and LPS2, bands were not visualized in the upper part of the electrophoretic track, which indicates the absence of S-forms of LPS with long O-specific chains, but a significant predominance of SR-form molecules in the membrane LPS pool. S. rhizophila 6p5m LPS showed significantly higher pyrogenic activity compared to pyrogenal, a pharmaceutical preparation, an active ingredient of which is Shigella typhi LPS. The extracellular polymer did not exhibit pyrogenic activity. In the double immunodiffusion reaction in agar according to Ouchterlony, it was found that the studied LPSs in the homologous system exhibit antigen activity. The antigens present in both LPS1and LPS2 and the extracellular component are identical: the antigen present in each of the wells binds to all antibodies that can interact with the antigen from the other well. It was shown that pre-sowing treatment of seeds with LPS preparations of S. rhizophila 6p5m stimulates the energy of seed germination. LPS1 (+2.82 cm) showed the greatest impact in comparison with the control. A significant stimulatory effect of LPS1, LPS2, and extracellular polymer was observed when studying their effects on mustard seedlings. Conclusions. The taxonomic position of bacterial strain 6r5m of the ecosystems of the polar region (Antarctica) was determined via the phylogenetic analysis taking into account the phenotypic features of the strain. The bacterial strain is represented in the phylum Proteobacteria, class Gammaproteobacteria, family Xanthomonadaceae, genus Stenotrophomonas, species S. rhizophila. From S. rhizophila cells, LPS preparations were obtained, which differed in monosaccharide and fatty acid composition, but showed high pyrogenicity. S. rhizophila represents a promising alternative to S. maltophilia for applications in agricultural biotechnology and biological control due to its ability to both stimulate plant growth and protect roots from biotic and abiotic stresses.