Mikrobiol. Z. 2017; 79(5):23-33. Russian.
Lipopolysaccharide of Pantoea agglomerans 7604: Chemical Identification and Biological Activity
Bulyhina T.V., Varbanets L.D., Pasichnyk L.A.
Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine
The goal of the present work was isolation, chemical characterization of the lipopolysaccharide (LPS) of P. agglomerans 7604, investigation of its biological activities and also estimation of serological relationships with other strains of this species. Methods. The amounts of the following components were determined: carbohydrates by the Dubois method; nucleic acids by the method of Spirin; protein by the Lowry method; 2-keto-3-deoxyoctulosonic acid by the Osborn method. Monosaccharide and fatty acid composition were analyzed on an Agilent 6890N/5973 inert chromato-mass-spectrometric system. The sensitivity of microbial cultures of P. agglomerans to polymyxin B was determined by disco-diffusion method. The antigenic activity of LPS was studied by the method of double immunodiffusion in agar according to Ouchterlony. Adhesive activity was determined by express-method for Briles. Results and conclusions. Lipopolysaccharide (LPS) of Pantoea agglomerans 7604 was purified and characterized chemically. The predominant monosaccharides were mannose (69.9 %) and arabinose (17.0 %). The LPS included fatty acids with chain lengths from 12 to 16 carbon atoms. In addition to saturated acids, monounsaturated acids have also been detected: palmitoleic, oleic and its cis- and trans-isomers. Since the studied strain of P. agglomerans was sensitive to polymixin B, it can be concluded that the LPS didn`t contain in the structure of lipid A such a substitute as 4-amino-4-deoxy-L-arabinose. The results of thermometry showed that the investigated LPS displays a pyrogenic effect. The antisera to the test strain reacted with LPS strains of 8606 and 8674, indicating the presence the common antigenic determinants. This may indicate that this strains belong to one and the same serogroup. The studied LPS reduces the index of adhesiveness, suggesting a possible competition between molecules of P. agglomerans 7604 LPS and adhesions of E. coli F-50, involved on the erythrocytes of the rabbit.
Key words: Pantoea agglomerans, lipopolysaccharide, monosaccharide and fatty acid composition, 4-amino-4-deoxy-L-arabinose, adhesion.
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- Down JM, Newman MA., von Roepenack A. The induction and modulation of plant defense responses by bacterial lipopolysaccharides. Ann. Rev. Phytopathology. 2000; 38: 241-261.
- Rietschel ET, Kirikae T, Schade FU, Mamat U, Scmidt G. Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J. 1994; 8(2): 217–225. https://doi.org/10.1096/fasebj.8.2.8119492
- Holst O, Molinaro A. Microbial Glycobiology: Structures Relevance and Applications. San Diego: Elsevier; 2009.
- Gavini FJ, Beji MA, Mielcarek C, Izard D, Kerster K, De Ley J. Transfer of Enterobacter agglomerans (Beijerink 1988) Ewing and File 1972 to Pantoea gen. nov. as Pantoea agglomerans comb. nov. and description of Pantoea dispersa sp. nov. Int. J. Syst. Bacteriol. 1989; 39(3): 337-345.
- Cimmino A, Marchi G, Surico G, Hanuszkiewicz A, Evidente A, Holst O. The structure of the O-specific polysaccharide of the lipopolysaccharide from Pantoea agglomerans strain FL1. Carbohydrate Research. 2008; 343(2): 392-396. https://doi.org/10.1016/j.carres.2007.10.023
- Westphal O, Jann K. Bacterial lipopolysaccharide-extraction with phenol. Methods Carbohydr. Chem. 1965; 5: 83-91.
- Dubois M, Gilles KA, Hamilton JK. Colorimetric method for determination of sugars and related substances. Anal. Chem. 1956; 28(3): 350-356.
- Spirin AS. Spectrophotometric determination of total nucleic acids. Biokhimiia. 1958; 23(5): 656-62.
- Lowry OH., Rosenbrough NJ, Farr AL, Randal RJ. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951; 193(5): 265-275.
- Osborn MJ. Studies on the gram-negative cell wall. I. Evidence for the role of 2-keto-3-deoxyoctonate in the lipopolysaccharide of Salmonella typhimurium. Proc. Nat. Acad. Sci. USA. 1963; 50(3): 499.
- Sawardeker JS, Sloneker JH, Jeans A. A quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography. Anal. Chem. 1965; 37(5): 1602-1603.
- Аlbershein P, Nevis DJ, English P D, Karr A. A method for analysis of sugars in plant cell–wall polysaccharides by gas–liquid chromatography. Carbohydr. Res. 1976; 3: 340-345.
- Performance standards for antimicrobial susceptibility testing; Fifteenth Informational Supplement. Clinical and Standarts Institute. 2005; 25(1): 87-151.
- 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. Exp. Med. 1948; 88(3): 279-284.
- Brilis V, Briline T, Lencner H, Lencner A. Metodika izucheniya adgezivnogo processa mikroorganizmov. Laboratornoe delo. 1968; 4: 210-212. Russian.
- Ouchterlony О. Diffusion in gel methods for immunological analysis Prog. Allergy. 1962; 6: 3-15.
- Varbanets LD, Brovarskaya OS, Bulyhina TN, Garkavaya EG, Zhitkevich NV. Characterisation of Pantoea agglomerans lipopolysaccharide. Microbiology. 2014; 83(6): 754-763. https://doi.org/10.1134/S0026261714060198
- Knirel YA. Structure of O-antigens. In: Chemical Synthesis, Biogenesis and Interactions with Host Cells. Eds. Y.A. Knirel, M.A. Valvano. Wien N.Y.: SpringerVerlag; 2011.
- Silipo A, Molinaro A. Lipid A structure. In: Bacterial Lipopolysaccharides. Structure, Chemical Synthesis, Biogenesis and Interactions with Host Cells. Eds. Y.A. Knirel, M.A. Valvano. Wien N.Y.: SpringerVerlag; 2011.
- Bulyhina TV, Varbanets LD, Pasichnyk L.A., Zhitkevych NV. [Antibiotic resistance of Pantoea agglomerans]. Microbiology and biotechnology. 2016; 1: 68-75. Ukrainian.
- Shubchynskyy VV, Varbanets LD, Brovarskaya OS. [Endotoxic activity of lipopolysaccharide of Pragia fontium]. Modern toxicology problems. 2007; 4: 35-38. Ukrainian.
- Ostapchuk AN, Varbanets LD. [Biological activity of native and modified lipopolysaccharides Rahnella aquatilis]. Mikrobiol Z. 2004; 66(6): 31-36. Ukrainian.