Mikrobiol. Z. 2018; 80(2):56-66. Ukrainian.
doi: https://doi.org/10.15407/microbiolj80.02.056
Lipopolysaccharide of Pantoea agglomerans 9649: 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 Pantoea agglomerans 9649, investigation its biological activities and also to establish 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 Osbourne method. Monosaccharide and fatty acid composition was 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 of P. agglomerans 9649 was purifed and characterized chemically. The predominant monosaccharides were rhamnose (36.47 %), mannose (20.17 %), heptoses (14.87 %) and glucose (13.57 %). In the lipid part of the LPS studied were identified saturated, monounsaturated and hydroxy acids with chain lengths from C12 to C16. Since the studiet strain of P. agglomerans were resistant to polymixin B, it can be assume that the LPS 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 display a pyrogenic effect. The antisera against to the tested strain reacted with LPS strain of П1а, indicating the presence of the common antigenic determinants belonging to one of the same serogroup. The studied LPS reduces the index of adhesiveness, suggesting a possible competition between molecules of P. agglomerans 9649 LPS and E. coli F-50 adhesin, involved in the erythrocytes of the rabbit.
Keywords: Pantoea agglomerans, lipopolysaccharide, monosaccharide and fatty acid composition, 4-amino-4-deoxy-L-arabinose, serological and adhesive activity.
Full text (PDF, in Ukrainian)
- Albershein 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.
- 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 infammation. J Exp Med. 1948; 88(3):279-284. https://doi.org/10.1084/jem.88.3.279
- Brilis V, Briline T, Lencner H, Lencner A. Metodika izucheniya adgezivnogo processa mikroorganizmov. Laboratornoe delo. 1968; 4:210-212. Russian.
- Cimmino A, Marchi G, Surico G, Hanuszkiewicz A, Evidente A, Holst O. The structure of the O-specifc 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
- 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. https://doi.org/10.1146/annurev.phyto.38.1.241
- Dubois M, Gilles KA, Hamilton JK. Colorimetric method for determination of sugars and related substances. Anal Chem. 1956; 28(3):350-356. https://doi.org/10.1021/ac60111a017
- Ewing WH, Fife MA. Enterobacter agglomerans (Beijerineck) comb. nov. (the herbicola-lathyri bacteria). Int J Syst Bacteriol. 1972; 22:4–11. https://doi.org/10.1099/00207713-22-1-4
- Gavini F, Mergert J, Beji A, Mielcarek C, Izard D, Kersters K, et al. Transfer of Enterobacter agglomerans (Beiernick 1888) Ewing and Fife 1972 to Pantoea gen. nov. as Pantoea agglomerans comb. nov. and description of Pantoea dispersa sp. nov. Int J Syst Bacteriol. 1989; 39:337–345. https://doi.org/10.1099/00207713-39-3-337
- Holst O, Molinaro A. Microbial Glycobiology: Structures Relevance and Applications. San Diego: Elsevier. 2009.
- Knirel YA, Valvano MA, Wien NY. Structure, Chemical Synthesis, Biogenesis and Interactions with Host Cells. Springer Verlag. 2011.
- Leben C. Epiphytic microorganisms in relation to plant disease. Annu Rev Phytopathol. 1965; 3:209–230. https://doi.org/10.1146/annurev.py.03.090165.001233
- 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 role of 2-keto-3-deoxyoctonate in the lipopolysaccharide of Salmonella typhymurium. Proc Nat Acad Sci USA. 1963; 50(3):499. https://doi.org/10.1073/pnas.50.3.499
- Ostapchuk AN, Varbanets LD. [Biological activity of native and modified lipopolysaccharides R. aquatilis] Mikrobiol Z. 2004; 66(6):31-36. Ukrainian.
- Ouchterlony O. Diffusion in gel methods for immunological analysis. Prog Allergy. 1962; 6:3-15.
- Performance standards for antimicrobial susceptibility testing; Fifteenth Informational Supplement. Clinical and Standarts Institute. 2005; 25(1):87-151.
- 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
- 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. https://doi.org/10.1021/ac60231a048
- Shubchynskyy VV, Varbanets LD, Brovarskaya OS. [Endotoxic activity lipopolysaccharide Pragia fontium]. Modern toxicology problems. 2007; 4:35-38. Ukrainian.
- Silipo A, Molinaro A. Bacterial Lipopolysaccharides. Structure, Chemical Synthesis, Biogenesis and Interactions with Host Cells. Springer Verlag. 2011.
- Spirin AS. [Spectrophotometric determination of total nucleic acids]. Biokhimiia. 1958; 23(5):656-62. Russian.
- 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
- Westphal O, Jann K. Bacterial lipopolysaccharide − extraction with phenol. Methods Carbohydr Chem. 1965; 5:83-91.