Mikrobiol. Z. 2022; 84(6):16-25.
Characteristics of Lelliottia nimipressuralis F9A1 Lipopolysaccharide Obtained by Different Methods
T.V. Bulyhina1, L.A. Pasichnyk1, K.G. Garkava2
1Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Acad. Zabolotny Str., Kyiv, 03143, Ukraine
2National Aviation University
Lubomyr Huzar Ave., Kyiv, 03680, Ukraine
Bacterial wetwood, bacterial dropsy, or bacterial slime is a common disease caused by Lelliottia nimipressuralis, which affects the central core of many conifers and deciduous trees. Representatives of this species have been isolated from a variety of trees showing symptoms of the disease, as well as from water and, less commonly, from clinical samples. Important aspects of pathogenesis is the process of pathogen recognition and the protection mechanisms of bacterial cells from plant’s antimicrobial substances. It is known that lipopolysaccharides (LPS) take an active part in these processes. They provide the barrier function of the outer membrane, helping to protect bacteria from plant antimicrobial compounds, and the attachment of bacteria to plant cells. Therefore, the aim of the work was to study the peculiarities of the chemical composition and functional and biological characteristics of Lelliottia nimipressuralis F9a1 LPS obtained by different methods. Methods. LPS was isolated from dry bacterial mass by phenol-water method (LPS I), extraction method with 0.85% NaCl solution (LPS II), and phenol-water extraction of LPS insoluble in NaCl solution (LPS III). The carbohydrates were analyzed by Dubois method, nucleic acids ‒ by Spirin, protein content ‒ by Lowry and 2-keto-3-deoxyoctonic acid (KDO) ‒ by Osborn. The identification of monosaccharides and fatty acids in LPS preparations was carried out on an Agilent 6890N/5973 inert chromato-mass spectrometry system. The pyrogenicity of LPS was tested keeping the rules of bioethics in rabbits. Serological studies were performed by the Ouchterlony method. Results. LPS II of L. nimipressuralis F9а1 was characterized by low relative yield (2.12%), low content of carbohydrates (9.16%) and nucleic acids (3.7%), and high protein content (26.44%), while the studied preparations of LPS I and LPS III were characterized by a high yield, a rather high content of carbohydrates (46.68 and 38.4%, respectively), an insignificant amount of protein (up to 6.72%) and nucleic acids (up to 4.06%). All LPSs contained up to 0.27% KDO, which is a specific component of the LPS of gramnegative bacteria. The monosaccharide composition indicates that the LPS of the studied L. nimipressuralis strains turned out to be heterogeneous. At the same time, such monosaccharides as fucose, galactose, and glucose were recorded in the LPS of all tested strains. The fatty acid composition of LPS was represented by the presence of fatty acids containing from 12 to 18 carbon atoms. Нydroxylated, saturated, and monounsaturated acids were found. In LPS I and LPS III, the dominant fatty acid was 14:0 (3-OH), which is a kind of marker for the entire family of Enterobacteriaceae. In addition to the marker acid, a 16:1 acid was also predominant in LPS III, whereas in LPS II, 16:1 (32.7%) and 16:0 (22.6%) fatty acids dominated. The pyrogenic effect of L. nimipressuralis LPS studied showed that LPS solutions are pyrogenic. The serological studies showed that tested LPS in homologous systems exhibits antigenic activity. Antisera to L. nimipressuralis F9a1 react with LPS strains IMV 8791, LGK1, and L14b, which may indicate the presence of common antigenic determinants and belonging of these strains to the same serogroup. Conclusions. The LPSs of L. nimipressuralis F9a1 were heterogeneous in both monosaccharide and fatty acid composition, which is explained by the use of different methods for their isolation. To isolate LPS from L. nimipressuralis cells, the water-phenol method is better than sodium chloride extraction since with using the latter, the LPS yield is very low and very contaminated with proteins. At the same time, the isolation method does not affect the serological activity of the studied LPS. The results received during these biological-functional studies of L. nimipressuralis LPS contribute to the biological characteristics of this species.
Keywords: Lelliottia nimipressuralis, lipopolysaccharides, monosaccharide and fatty acid composition, serological and pyrogenic activities.
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