Mikrobiol. Z. 2020; 82(1):33-42. Ukrainian.
Characteristic of Pseudomonas putida Lipopolysaccharide
O.S. Brovarskaya1, L.D. Varbanets1, A.F. Likhanov2
1Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine
2National University of Life and Environmental Sciences of Ukraine
15 Heroes of Defense Str., Kyiv, 03041, Ukraine
Pseudomonas putida, along with representatives of other species such as Pseudomonas syringae and Pseudomonas fluorescens, cause diseases of a significant number of wild and cultivated plants. One of the factors of pathogenicity of gram-negative bacteria are lipopolysaccharides. There is evidence in the literature that, depending on the medium on which the bacteria are grown, the lipopolysaccharides obtained from its may differ in their composition and biological activity. Aim. To conduct a comparative study of the composition and biological activity Pseudomonas putida lipopolysaccharide (LPS) when grown on different media. Methods. In work microbiological, biochemical and serological methods were used. LPS was obtained from cells by waterphenolic extraction, its heterogeneity was determined by SDS-PAAG electrophoresis, the monosaccharide and fatty acid composition was identified by chromatography-mass spectrometry, and serological activity was determined by immunodiffusion in agar. Results. LPS isolated from Pseudomonas putida cells grown on meat-peptone agar (LPS-MPA) or on potato agar (LPS-PA) differ both in their qualitative composition and in the quantitative content of monosaccharides. In contrast to LPS-KA, in LPSMPA identified rhamnose (20.38%) and fucose (13.33%), and also galactose, glucose, mannose and ribose. Saturated and hydroxy acids with a carbon chain length from C12 to C18 were identified as part of the studied LPS preparations. Hexadecanoic acid was the dominant acid in LPS preparations (32.3 and 40.39% respectively for LPS-MPA and LPS-PA). Also, 2-hydroxy-dodecanoic (2-OHC12:0) (8.47 and 18.79%), 3-hydroxy-dodecanoic (3-OH-C12:0) (4.29 and 9.69%), dodecanoic (C12:0) (7.97 and 19.25%) acids (respectively for LPS-MPA and LPS-PA were udentified). The LD50 of LPS-MPA was found to be 29.73 μg/mouse, while LPS-PA was less toxic: its LD50 was 84.09 μg/mouse. The studied LPS preparations was pyrogen-free compared to Pyrogenal. LPS-KA did not show antigenic activity. Conclusions. LPS obtained from P. putida cells grown on various media differ monosaccharide, fatty acid composition, toxicity and serological activity.
Keywords: Pseudomonas putida, lipopolysaccharide, monosaccharide and fatty acid composition, serological activity, toxicity, pyrogenicity.
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- Fernández M, NiquiArroyo JL, Conde S, Ramos JL, Duque E. Enhanced tolerance to naphthalene and enhanced rhizoremediation performance for Pseudomonas putida KT2440 via the NAH7 catabolic plasmid. Appl. Environ. Microbiol. 2012; 78:5104–5110. https://doi.org/10.1128/AEM.00619-12
- Carpenter RJ, Hartzell JD, Forsberg JA, Babel BS, Ganesan A. Pseudomonas putida var wound infection in a US Marine: a case report and review of the literature. J. Infect. 2008; 56:234–240. https://doi.org/10.1016/j.jinf.2008.01.004
- Erol S, Zencirovglu A, Dilli D, Okumuks N, Aydin M, Göl N. Evaluation of nosocomial blood stream infections caused by Pseudomonas species in newborns. Clin. Lab. 2014; 60:615–620. https://doi.org/10.7754/Clin.Lab.2013.130325
- Molina L, Udaondo Z, Duque E, Fernández M, MolinaSantiago C, Roca A. Antibiotic resistance determinants in a Pseudomonas putida strain isolated from a hospital. PLoS ONE. 2014; 9(1):e81604. https://doi.org/10.1371/journal.pone.0081604
- Yoshino Y, Kitazawa T, Kamimura M, Tatsuno K, Ota Y, Yotsuyanagi H. Pseudomonas putida bacteremia in adult patients: five case reports and a review of the literature. J. Infect. Chemotherm. 2011; 7:278-282. https://doi.org/10.1007/s10156-010-0114-0
- Bennett JW, Herrera M L, Lewis JS, Wickes BW, Jorgensen JH. KPC2producing Enterobacter cloacae and Pseudomonas putida coinfection in a liver transplant recipient. Antimicrob. Agents Chemother. 2009; 53:292–294. https://doi.org/10.1128/AAC.00931-08
- Surkina AK, Konnova SA, Fedonenko Yu. Influence of the conditions of cultivation of Azospirillum lipoferum sp59b on the biological activity of their glycopolymers. Saratov University News. New series. Series: Chemistry. Biology. Ecology. 2017; 17(2):177-183. Russian. https://doi.org/10.18500/1816-9775-2017-17-2-177-183
- 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 determination of sugars and related substranses. 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. Russian.
- 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
- Muthannan AR. Determination of 50% endpoint titer using a simple formula. World J. Virol. 2016; 5(2):85-86. https://doi.org/10.5501/wjv.v5.i2.85
- Ouchterlony O. Diffusion in gel methods for immunological analysis. Prog. Allergy. 1962; 6:3-15. https://doi.org/10.1159/000313797
- Yakovleva LM, Makhinya LV, Scherbina NN, Ogorodnik LE. Micrococcus sp. − the agent of leaf necrosis. Mikrobiol. Z. 2013; 741(3):62-67. Ukrainian.
- Drazhnikova A. Monitoring of the condition of chestnut trees in the event of damage by the passing mile Cameraria ohridella and phytopathogenic fungi. Agroecological journal. 2010; 4:99–101.
- Kabanov DS, Zubova SV, Prokhorenko IR. Comparative analysis of BOD in the composition of non-toxic and toxic lipopolysaccharides. Basic glycobiology. Saratov. 2014:112. Russian.
- Varbanets LD, Zdorovenko EA, Brovarskaya OS, Kalinichenko S.V.Characterization of the lipopolyysaccharide of E. coli 126. Microbiology (Moscow). 2017; 86(1):54-61. Russian. https://doi.org/10.1134/S0026261717010167
- Brovarskaya OS, Varbanets LD. Influence of isolation methods on composition and biological properties of Esherichia coli lipopolysaccharides. Mikrobiol. Z. 2019; 81(2):3-13. Ukrainian. https://doi.org/10.15407/microbiolj81.02.003