Mikrobiol. Z. 2018; 80(4):28-40. Russian.
The Influence of Рseudomonas chlororaphis subsp. chlororaphis UCМ В-106 Cultivation Temperature
on Composition and Biological Properties of Lipopolysaccharides
Brovarskaya O.S., Varbanets L.D.
Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine
The purpose of this work was to conduct a comparative study of the chemical composition of Pseudomonas chlororaphis subsp. chlororaphis UCM B-106 lipopolysaccharides (LPS), isolated from bacteria grown at different temperature regimes (10, 28 and 37 °C). Methods. Phenol-water extraction of LPS, electrophoresis in polyacrylamide gels, chromato-mass spectrometric determination of monosaccharide and fatty acid compositions, study of the antigenic structure by immunodiffusion in agar by Ouchterlony. Results and conclusions. It is shown that an increase in the temperature of cultivation causes changes in the biochemical, immunochemical and biological properties of LPS obtained from cells growing at different temperatures (LPS-10, LPS-28 and LPS-37). The yield of LPS from the “cold” variant was almost 5 times higher than that of cells cultivated at 37 °С. One of the reasons for the low yield of LPS-37 (2.3 %), as compared to 11.3 % of LPS-10 yield, may be the presence of the “thermal” variant of LPS with short O-polysaccharide chains (as we showed by PAGE) and which because of their hydrophobicity poorly extracted by phenol-water procedure. Analysis of monosaccharide composition of LPS showed that the preparations are similar in quality composition. At the same time, with increasing cell growth temperature, the percentage of heptose, a characteristic component of core oligosaccharide, increased (47.5, 52.5, 52.9 % for LPS-10, LPS-28, LPS-37, respectively). The results of the determination of the fatty acid composition of the studied LPS showed that the qualitative composition of fatty acids is very close, and their quantitative content varies significantly. The dominant were 3-OH-C14 : 0 (35.5, 47.2, 32.4 %), and also C14 : 0 (31.6, 34.4, 42.1 %, respectively for LPS-10, LPS-28, LPS-37) the content of which increases with an increase in the growing temperature. Of unsaturated acids, C16: 1 (12.0%) was detected only in LPS-10. The toxicity study showed that the LD50 values decreased in the range of LPS-10, LPS-20 and LPS-37 (2.0, 20.0 and 35.56 mg/kg animals, respectively). That is, the greatest toxicity was shown by LPS “cold variant”, and the least toxic - “thermal” (LPS-37). When studying the antigenic structure by immunodiffusion in Ouchterloni agar, the presence of common antigenic determinants in the composition of the LPS studied was shown. Summary. The increasing of cultivation temprerature of cells growing causes the changes in biochemical, immunochemical and biological properties of LPS.
Keywords: Рseudomonas chlororaphis subsp. chlororaphis УКМ В-106, different temperatures of cultivation, lipopolysaccharide, monosaccharide, fatty acid composition, biological activity.
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- Westphal O, Luderitz O, Rietschel E, Galanos C. Bacterial lipopolysaccharide and its lipid A component: some historical and some chemical aspects. Biochem. Soc. Trans. 1981; 9(3): 191-195. https://doi.org/10.1042/bst0090191
- Westphal O, Jann K. Bacterial lipopolysaccharides – extraction with phenol. Methods Carbohydr. Chem. 1965; 5: 83-91.
- Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric method for determinationn of sugars and related substrances. Anal. Chem. 1956; 28(3): 350-356. https://doi.org/10.1021/ac60111a017
- Lowry OH, Rosenbrough NJ, Farr LA, Randal RJ. Protein measurement with the Folin reagent. J.Biol.Chem. 1951; 193(5): 265-275.
- Spirin AS. [Spectrophotometric determination of total nucleic acids]. Biochemistry. 1958; 23(5): 656-662. Russian.
- Varbanets LD, Zdorovenko GM, Knirel YuA. [Methods of endotoxin investigations]. Kyiv: Naukova Dumka, 2006; 237. Russian.
- Albershein P, Nevis DJ, English PD, Karr A. A method for analysis of sugars in plant cell wall polysaccharides by gas-liquid chromatography. Carboh. Res. 1976; 5(3): 340-345. https://doi.org/10.1016/S0008-6215(00)80510-8
- Laemmli UK. Cleavage of proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227: 680-685. https://doi.org/10.1038/227680a0
- Tsai CM, Frash CE. A sensitive silver stain for detecting lipopolysaccharides in polyacrilamide gels. Anal. Biochem. 1982; 119: 115-119. https://doi.org/10.1016/0003-2697(82)90673-X
- Galanos C, Freudenberg MA, Reuter W. Galactosamineinduced sensitization to the lethal effects of endotoxin. Proc.Natl. Acad. Sci. USA. 1979; 76:5939-5943. https://doi.org/10.1073/pnas.76.11.5939
- Nowotny A. Basic Exercises in Immunochemistry. A laboratory Manual. Berlin; Heidelberg; New York:Springer-Verlag. 1979; 303-305. https://doi.org/10.1007/978-3-642-67356-6_94
- Ouchterlony O. Diffusion in gel methods for immunological analysis. Prog. Allergy. 1962; 6:3-15 https://doi.org/10.1159/000391328
- Bakholdina SI, Krasikova IN, and Solov'eva TF. [The Effect of the culturig method and the growth phase on the lipopolysaccharide composition of Versinia pseudotuberculosis]. Journal of Bioorganic Chemistry. 2001; 27(2): 151-155. Russian.
- Zubova SV. [Effect of lipopolysaccharide composition on bacterial cell wall characteristics]. Abstract dis. cand. Biol. Sciences: 03.00.04 Pushchino, 2006; 95. Russian.
- Dentovskaya SV, Bakhteeva IV, Titareva GM, Shaikhutdinova RZ, Anisimov AP, Kondakova AN, Bystrova OV, Knirel YA, Lindner B. [Structural diversity and endotoxic activity of the lipopolysaccharide of Yersinia pestis]. Biochemistry. 2008; 73(2): 237-246. Russian.
- Knirrel YA, Kochetkov NK. [The structure of lipopolysaccharides of gram-negative bacteria. I. General characteristics of lipopolysaccharides and lipid A structure]. Biochemistry. 1993; 58(2): 166-188. Russian.
- Pieretti G, Carillo S, Lanzetta R, Parrilli M, Merino S, Tomás JM, Corsaro MM. Structural determination of the O-specific polysaccharide from Aeromonas hydrophila strain A19 (serogroup O:14) with S-layer. Carbohydr. Res. 2011; 346: 2519-2522. https://doi.org/10.1016/j.carres.2011.08.003
- Kropinski AM, Lewis V, Berry D. Effect of growth temperature on the lipids, outer membrane proteins, and lipopolysaccharides of Pseudomonas aeruginosa PAO. J. Bacteriol. 1987; 69: 1960-1966. https://doi.org/10.1128/jb.169.5.1960-1966.1987
- Makin SA, Beveridge TJ. Pseudomonas aeruginosa PAO1 ceases to express serotypespecific lipopolysaccharide at 45 degrees C. J. Bacteriol. 1996; 178: 3350-3352. https://doi.org/10.1128/jb.178.11.3350-3352.1996
- Carty SM, Sreekumar KR, Raetz CR. Effect of cold shock on lipid A biosynthesis in Escherichia coli. Induction At 12 degrees C of an acyltransferase specific for palmitoleoylacyl carrier protein. J. Biol. Chem. 1999; 274: 9677-9685. https://doi.org/10.1074/jbc.274.14.9677
- Burtnick MN, Woods DE. Isolation of polymyxin B-susceptible mutants of Burkholderia pseudomallei and molecular characterization of genetic loci involved in polymyxin B resistance. Antimicrob. Agents Chemother. 1999; 43: 2648-2656.
- Rebeil R, Ernst RK, Jarrett CO, Adams KN, Miller SI, Hinnebusch BJ. Characterization of late acyltransferase genes of Yersinia pestis and their role in temperature-dependent lipid A variation. J. Bacteriol. 2006; 188(4): 1381-1388. https://doi.org/10.1128/JB.188.4.1381-1388.2006