Mikrobiol. Z. 2022; 84(6):16-25.
doi: https://doi.org/10.15407/microbiolj84.06.016

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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  1. Brady C, Cleenwerck I, Venter S, Coutinho T, De Vos P. Taxonomic evaluation of the genus Enterobacter based on multilocus sequence analysis (MLSA): Proposal to reclassify E. nimipressuralis and E. amnigenus into Lelliottia gen. nov. as Lelliottia nimipressuralis comb. nov. and Lelliottia amnigena comb. nov., respectively, E. gergoviae and E. pyrinus into Pluralibacter gen. nov. as Pluralibacter gergoviae comb. nov. and Pluralibacter pyrinus comb. nov., respectively, E. cowanii, E. radicincitans, E. oryzae and E. arachidis into Kosakonia gen. nov. as Kosakonia cowanii comb. nov., Kosakonia radicincitans comb. nov., Kosakonia oryzae comb. nov. and Kosakonia arachidis comb. nov., respectively, and E. turicensis, E. helveticus and E. pulveris into Cronobacter as Cronobacter zurichensis nom. nov., Cronobacter helveticus comb. nov. and Cronobacter pulveris comb. nov., respectively, and emended description of the genera Enterobacter and Cronobacter. Syst Appl Microbiol. 2013; 36(5):309—319. https://doi.org/10.1016/j.syapm.2013.03.005
  2. Iavniy MI, Puzrina NV. Bacterial disease of ulmus glabra huds in stands of kiev polissya of Ukraine. Microbiol Z. 2018; 80(1):66—76. https://doi.org/10.15407/microbiolj80.01.067
  3. Shvets MV. [On the situation of birch plantations in the forests of Zhytomyr Polissia of Ukraine]. Materials intern. Scientific-practical conf. KhNAU. 2015; 8:193—196. Ukrainian.
  4. Shelukho VP, Sidorov VA. [Bacterial dropsy of birch and the effectiveness of measures to combat it in the stands of mixed and deciduous forests]. Bryansk: BGITA. 2009. Russian.
  5. Khodaygan P, Sedaghati E, Sherafati F. Isolation of Enterobacter nimipressuralis associated with bacterial wetwood from elm (Ulmus spp.) in Rafsanjan. Iranian Journal of Plant Pathology. 2011; 47(4):481—482.
  6. Hoichuk AF, Drozda VF, Kulbanska IM. [Tuberculosis of ash-trees in Western Podillya of Ukraine: etiology, symptomatology, pathogenesis]. Naukovi pratsi Lisivnychoi akademii nauk Ukrainy. 2018; 16:31—39. Ukrainian.
  7. Alexander C, Rietschel ET. Bacterial lipopolysaccharides and innate immunity. J Endotoxin Res. 2001; 7:167—202. https://doi.org/10.1179/096805101101532675
  8. Newman MA, Dow JM, Molinaro A, Parrilli M. Priming, inductionand modulation of plant defence responses by bacterial lipopolysaccharides. J Endotoxin Res. 2007; 13:68—79. https://doi.org/10.1177/0968051907079399
  9. Silipo A, Molinaro A, Sturiale L, Dow JM, Erbs G, Lanzetta R, et al. The elicitation of plant innate immunity by lipooli-gosaccharide of Xanthomonas campestris. J Biol Chem. 2005; 280:33660—33668. https://doi.org/10.1074/jbc.M506254200
  10. Westphal O, Jann K. Bacterial lipopolysaccharides: Extraction with phenol-water and further application of the procedure. Methods Carbohydr Chem. 1965; 5:83‒91.
  11. Varbanets LD, Zdorovenko GM, Knirel YuA. [Methods of endotoxin investigations]. Kyiv: Naukova Dumka; 2006. Russian.
  12. Dubois M, Gilles KA, Hamilton JK. Colorimetric method for determination of sugars and related substances. Anal Chem. 1956; 28:350‒356. https://doi.org/10.1021/ac60111a017
  13. Spirin AS. Spectrophotometric determination of total nucleic acids. Biokhimiia. 1958; 23:656662.
  14. Lowry OH, Rosenbrough NJ, Farr AL, Randal RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951; 193:265‒275. https://doi.org/10.1016/S0021-9258(19)52451-6
  15. Osborn MJ. Studies on the gram-negative cell wall. I. Evidence for role of 2-keto-3-deoxyoctonate in the lipopolysaccharide of Salmonella typhymurium. Proc Natl Acad Sci USA. 1963; 50:499. https://doi.org/10.1073/pnas.50.3.499
  16. Albershein P, Nevis DJ, English PD, Karr A. A method for analysis of sugars in plant cell wall polysaccharides by gasliquid chromatography. Carbohyd Res. 1967; 5(3):340—345. https://doi.org/10.1016/S0008-6215(00)80510-8
  17. Bennett IL. A study of the relationship between the fevers caused by bacterial pyrogens and by the intravenous injection of the sterile exudates of acute inflammation. J of Exp Med. 1948; 88:279‒284. https://doi.org/10.1084/jem.88.3.279
  18. Ouchterlony O. Diusion-in-gel methods for immunological analysis. Prog Allergy. 1962; 6:30 ‒154. https://doi.org/10.1159/000391328
  19. Carter CJ. Wetwood of elms. Illinois Natural History Survey Bulletin. 1945; 023(04):407—448. https://doi.org/10.21900/j.inhs.v23.202
  20. Bennett IL, Cluff LE. Bacterial pyrogens. Pharmacological Reviews. 1957; 9:427—475.
  21. Westphal O, Westphal U. The history of pyrogen research. Microbiology. 1977; 1977:221—238.
  22. Beutler B, Rietschel ET. Innate immune sensing and its roots: The story of endotoxin. Nature Reviews Immunology. 2003; 3:169—176. https://doi.org/10.1038/nri1004
  23. Galanos C, Liideritz 0, Westphal 0. Zeitbl Bakt Hyg. 1979; A243—226.
  24. Zdorovenko EL, Ovod VV, Shashkov AS, Kocharova NA, Knirel YA, Krohn K. Structure of the O-Polysaccharide of the Lipopolysaccharide of Pseudomonas syringae pv. garcae ICMP 8047. Biochemistry (Moscow). 1999; 64(7):765—773.
  25. Vinogradov EV, Holst O, Thomas-Oates JE, Broady KW, Brade H. The structure of the O-antigenic polysaccharide from lipopolysaccharide of Vibrio cholerae strain H11 (non-O1). European Journal of Biochemistry. 1992; 210(2):491—498. https://doi.org/10.1111/j.1432-1033.1992.tb17447.x
  26. Rahman MM, Guard-Petter J, Carlson RW. A virulent isolate of Salmonella enteritidis produces a Salmonella typhi-like lipopolysaccharide. Journal of Bacteriology. 1997; 179(7):2126—2131. https://doi.org/10.1128/jb.179.7.2126-2131.1997
  27. Brahmbhatt HN, Lindberg AA, Timmis KN. Shigella lipopolysaccharide: structure, genetics, and vaccine development. Current Topics in Microbiology and Immunology. 1992; 180:45—64. https://doi.org/10.1007/978-3-642-77238-2_3
  28. Ovod VV, Zdorovenko EL, Shashkov AS, Kocharova NA, Knirel YA. Structure of the O-polysaccharide and serological classification of Pseudomonas syringae pv. ribicola NCPPB 1010. European Journal of Biochemistry. 2000; 267(8):2372—2379. https://doi.org/10.1046/j.1432-1327.2000.01250.x
  29. Meredith TC, Aggarwal P, Mamat U, Lindner B, Woodard RW. Redefining the requisite lipopolysaccharide structure in Escherichia coli. ACS Chemical Biology. 2006; 1:33—42. https://doi.org/10.1021/cb0500015