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Effects of Rhamnolipid-Biosurfactant on Cell Surface of Pseudomonas Aeruginosa

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Effects of Rhamnolipid-Biosurfactant on Cell Surface of Pseudomonas Aeruginosa Effects of rhamnolipid-biosurfactant on cell surface of Pseudomonas aeruginosa. A. Sotirova, D. Spasova, E. Vasileva-Tonkova, D. Galabova To cite this version: A. Sotirova, D. Spasova, E. Vasileva-Tonkova, D. Galabova. Effects of rhamnolipid-biosurfactant on cell surface of Pseudomonas aeruginosa.. Microbiological Research, Elsevier, 2009, 164 (3), pp.297-303. 10.1016/j.micres.2007.01.005. pasteur-00748968 HAL Id: pasteur-00748968 https://hal-riip.archives-ouvertes.fr/pasteur-00748968 Submitted on 6 Nov 2012 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. ARTICLE IN PRESS Microbiological Research 164 (2009) 297—303 www.elsevier.de/micres Effects of rhamnolipid-biosurfactant on cell surface of Pseudomonas aeruginosa à A. Sotirova, D. Spasova, E. Vasileva-Tonkova, D. Galabova Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev str., bl.26, 1113 Sofia, Bulgaria Received 23 June 2006; received in revised form 25 January 2007; accepted 25 January 2007 KEYWORDS Summary Biosurfactant; The effect of rhamnolipid-biosurfactant produced by Pseudomonas sp. PS-17 on cell Hydrophobicity; surface structures of Pseudomonas aeruginosa NBIMCC 1390 was studied. The results Lipopolysaccharide; demonstrated that the rhamnolipid at concentrations below and above CMC Outer membrane provoked a multi-component response of the bacterial cells without affecting their proteins; growth and viability. Above CMC, the rhamnolipid caused reduction of total cellular Rhamnolipid LPS content of 22%, which can be associated with an increase in cell hydrophobicity to 31% adherence. The rhamnolipid-biosurfactant at concentration below CMC did not affect the LPS component of the bacterial outer membrane but caused changes in OMP composition of P. aeruginosa. Examination of the OMP profiles revealed that the amount of major proteins (Opr F,Opr D, Opr J and Opr M) markedly decreased. To our knowledge this is the first report on the rhamnolipid-biosurfactant interactions with bacterial cells showing changes in outer membrane proteins of P. aeruginosa.In both concentrations, the biosurfactant caused changes in cell surface morphology. The results indicate that the rhamnolipid-biosurfactant from Pseudomonas sp. PS-17 has a potential application in the relatively new field of biomedicine. & 2007 Elsevier GmbH. All rights reserved. Introduction antibiotics as a result of permeability barrier function of the outer membrane. The outer Pseudomonas aeruginosa is a Gram-negative membrane of Gram-negative bacteria is composed bacterium living in soil and aqueous environments. of lipopolysaccharides (LPS), phospolipids and It is an important opportunistic pathogen, highly lipoproteins, covalently linked to the peptidoglycan resistant to a large number of disinfectants and layer through hydrophobic interactions (Sikkema et al., 1995) and also contains porins and efflux à pump embedded in the LPS layer (Poole, 2001). In Corresponding author. Tel.: +359 2 9793130; fax: +359 2 8700109. P. aeruginosa, four major (OprF, OprP, OprB, OprD) E-mail address: [email protected] (D. Galabova). and two minor (OprC, OprE) outer membrane 0944-5013/$ - see front matter & 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2007.01.005 ARTICLE IN PRESS 298 A. Sotirova et al. proteins (OMPs) were reported to act as porins maintained at 4 1C on Bacto agar (Difco) slants and (Denyer and Maillard, 2002). transferred monthly. A number of studies suggest that it is possible to modify the outer membrane of Gram-negative Culture medium and growth conditions bacteria by mutation (Yoneyama et al., 1995; Nikaido, 2003; Ni and Chen, 2004) or by addition The cells of P. aeruginosa were grown in a of chemical agents that can act as membrane mineral salts medium (MSM) (Spizizen, 1958) permeabilizing agents (Daugelavicˇius et al., 2000; supplemented with CaCl2, 2 mM; casein hydrolysate Denyer and Maillard, 2002; Longbottom et al., (Fluka), 0.5%; maltose, 0.5%; pH 7.2. Inoculum was 2004). These processes result in changes of the cell prepared by transferring the cells from agar slants surface properties as well as in increased perme- to 2 ml of MSM medium in 20-ml flasks and ability and hydrophobicity of the bacterial mem- cultivated for 18 h at 37 1C with agitation at brane. Changes in cell surface properties were 200 rpm. The experimental culture of 20 ml was observed also in presence of surfactants and inoculated with 1% (v/v) inoculum and incubated in biosurfactants, surface active compounds with 250-ml flasks until late exponential phase (20 h). amphiphilic structure (Zhang and Miller, 1994; Growth conditions were the same as those used for Al-Tahhan et al., 2000; Mo¨bius et al., 2001). The preparing the inoculum. The growth was monitored biosurfactants that were studied most extensively by measuring the absorbance at 570 nm. Extracel- in terms of their industrial and environmental lular protein content was determined by the application are the rhamnolipids. Nevertheless method of Bradford (1976). little is known about their interaction with bacter- ial cells, although it is likely that their surface and membrane active properties play an important role Cell surface hydrophobicity (Lang and Wullbtandt, 1999; Singh and Cameotra, 2004). Cell hydrophobicity was measured by microbial The rhamnolipid-biosurfactant PS was produced adherence to hexadecane according to the method by bacterial strain Pseudomonas sp. PS-17 (Shulga of Rosenberg et al. (1980). The cells were washed et al., 2000). The low parameters for surface and twice and resuspended in PUM buffer, pH 7.1 (22.2 g Á interfacial tensions and critical micelle concentra- K2HPO4 3H2O, 7.26 g KH2PO4, 1.8 g urea, 0.2 g Á tion of the rhamnolipid-biosurfactant PS indicate MgSO4 7H2O and distilled water to 1000 ml) to an its high surface activity. initial absorbance of the cell suspension at 550 nm – The aim of this study was to investigate the of 0.5 0.6. The cell suspension (1.2 ml) after the effect of a rhamnolipid-biosurfactant PS produced addition of hexadecane (0.2 ml) was vortexed in a by Pseudomonas sp. PS-17 on (i) growth and total test tube at high speed for 2 min and equilibrated protein release, (ii) cell surface properties, and (iii) for 1 h. The optical density of the bottom aqueous chemical and structural changes in the cell surface phase was then measured at 550 nm. Hydrophobi- of P. aeruginosa NBIMCC 1390. city was expressed as the percentage of cells adhered to the hydrocarbon and calculated as follows: 100  (1 – OD of the aqueous phase/OD of the initial cell suspension). Materials and methods Isolation and analysis of LPS Biosurfactant Total LPS content in whole-cell lysates was The rhamnolipid-biosurfactant PS used in this determined by the thiobarbituric acid assay of 2- study was produced and purified from Pseudomonas keto-3-deoxyoctonic acid (KDO) adapted from the sp. PS-17 in the Laboratory of Biotechnology, method of Osborn et al. (1972). A standard curve Ukrainian Academy of Sciences (Lviv town), and was prepared by using purified LPS obtained from provided by Dr. E. Karpenko and Dr. A. Shulga. P. aeruginosa (Calbiochem). Cells were prepared for KDO assay as follows: 0.1 ml samples of culture were washed twice with distilled water, and the Microorganism cell pellet was lysed by dissolving in 50 ml of lysing buffer and heating at 100 1C for 10 min (Hitchcock The strain P. aeruginosa NBIMCC 1390 (National and Brown, 1983). Bank of Industrial Microorganisms and Cell Cultures) For LPS isolation, a modified version (Skurnik was used throughout this study. The culture was et al., 1995) of the protocol devised by Hitchcock ARTICLE IN PRESS Effects of rhamnolipid-biosurfactant on cell surface 299 and Brown (1983) was used. The cells were grown and then were subjected to electrophoresis at a till late log phase. The cell content of 1.5 ml cell constant current of 25 mA at 4 1C. suspension with an OD at 540 nm of 1.7 was harvested by centrifugation in a microcentrifuge Transmission electron microscopy (TEM) (13,000g; 3 min), and then resuspended in 100 ml lysis buffer (2% deoxycholate [DOC], 4% 2-mercap- The bacterial cells were prepared for electron toethanol, 10% glycerol, and 0.002% bromophenol microscopy using the method of Sabatini et al. (1960) – blue in 1 M Tris HCl buffer (pH 6.8). Lysates were with 6% glutaraldehyde for 2 h and osmium postfixa- 1 heated at 100 C for 10 min. Then, 40 mgof tion overnight at room temperature. This procedure proteinase K was added, and the suspension was was followed by dehydration with alcohol (in 1 incubated at 55 C for at least 2 h. Samples were increasing concentrations) and embedding in Durcu- À 1 stored at 20 C until analyzed by DOC-PAGE. pan. Staining was carried out using the technique of Raynolds (1963). Thin sections were examined with a DOC-PAGE Zeiss electron microscope (model 10C). DOC-PAGE was performed according to the system of Laemmli (1970) modified by Komuro Results and Galanos (1988) with DOC as detergent. The separation gel contained final concentration of Effect of rhamnolipid PS on bacterial growth – 13% acrylamide, 0.5% DOC, and 375 mM Tris HCl and protein release (pH 8.8). The stacking gel contained 4% acrylamide, – 0.5% DOC, and 125 mM Tris HCl (pH 6.8). LPS The effect of biosurfactant concentration on the samples (0.1%, w/v) were prepared in the growth and protein release of P.
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