Abstract
The bacterial release of outer membrane vesicles (OMVs) is an important physiological mechanism of Gram-negative bacteria playing numerous key roles. One function of the release of OMVs is related to an increase in surface hydrophobicity. This phenomenon initiates biofilm formation, making bacteria more tolerant to environmental stressors. Recently, it was qualitatively shown for Pseudomonas putida that vesicle formation plays a crucial role in multiple stress responses. Yet, no quantification of OMVs for certain stress scenarios has been conducted. In this study, it is shown that the quantification of OMVs can serve as a simple and feasible tool, which allows a comparison of vesicle yields for different experimental setups, cell densities, and environmental stressors. Moreover, the obtained results provide insight to the underlying mechanism of vesicle formation as it was observed that n-alkanols, with a chain length of C7 and longer, caused a distinct and steep increase in vesiculation (12–19-fold), compared to shorter chain n-alkanols (2–4-fold increase).
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Atashgahi S, Sanchez-Andrea I, Heipieper HJ, van der Meer JR, Stams AJM, Smidt H (2018) Prospects for harnessing biocide resistance for bioremediation and detoxification. Science 360(6390):743–746. https://doi.org/10.1126/science.aar3778
Bauman SJ, Kuehn MJ (2006) Purification of outer membrane vesicles from Pseudomonas aeruginosa and their activation of an IL-8 response. Microbes Infect 8(9–10):2400–2408. https://doi.org/10.1016/j.micinf.2006.05.001
Baumgarten T, Vazquez J, Bastisch C, Veron W, Feuilloley MG, Nietzsche S, Wick LY, Heipieper HJ (2012a) Alkanols and chlorophenols cause different physiological adaptive responses on the level of cell surface properties and membrane vesicle formation in Pseudomonas putida DOT-T1E. Appl Microbiol Biotechnol 93(2):837–845. https://doi.org/10.1007/s00253-011-3442-9
Baumgarten T, Sperling S, Seifert J, von Bergen M, Steiniger F, Wick LY, Heipieper HJ (2012b) Membrane vesicle formation as a multiple-stress response mechanism enhances Pseudomonas putida DOT-T1E cell surface hydrophobicity and biofilm formation. Appl Environ Microbiol 78(17):6217–6224. https://doi.org/10.1128/AEM.01525-12
Bernadac A, Gavioli M, Lazzaroni JC, Raina S, Lloubes R (1998) Escherichia coli tol-pal mutants form outer membrane vesicles. J Bacteriol 180(18):4872–4878
Dubern JF, Diggle SP (2008) Quorum sensing by 2-alkyl-4-quinolones in Pseudomonas aeruginosa and other bacterial species. Mol BioSyst 4(9):882–888. https://doi.org/10.1039/b803796p
Eberlein C, Baumgarten T, Starke S, Heipieper HJ (2018) Immediate response mechanisms of Gram-negative solvent-tolerant bacteria to cope with environmental stress: cis-trans isomerization of unsaturated fatty acids and outer membrane vesicle secretion. Appl Microbiol Biotechnol 102:2583–2593. https://doi.org/10.1007/s00253-018-8832-9
Fernandez-Pinar R, Ramos JL, Rodriguez-Herva JJ, Espinosa-Urgel M (2008) A two-component regulatory system integrates redox state and population density sensing in Pseudomonas putida. J Bacteriol 190(23):7666–7674. https://doi.org/10.1128/JB.00868-08
Hartmans S, Smits JP, Vanderwerf MJ, Volkering F, Debont JAM (1989) Metabolism of styrene oxide and 2-phenylethanol in the styrene-degrading Xanthobacter strain 124x. Appl Environ Microbiol 55(11):2850–2855
Heipieper HJ, de Bont JA (1994) Adaptation of Pseudomonas putida S12 to ethanol and toluene at the level of fatty acid composition of membranes. Appl Environ Microbiol 60(12):4440–4444
Heipieper HJ, Diefenbach R, Keweloh H (1992) Conversion of cis unsaturated fatty acids to trans, a possible mechanism for the protection of phenol-degrading Pseudomonas putida P8 from substrate toxicity. Appl Environ Microbiol 58(6):1847–1852
Heipieper HJ, Loffeld B, Keweloh H, de Bont JAM (1995) The cis/trans isomerisation of unsaturated fatty acids in Pseudomonas putida S12: an indicator for environmental stress due to organic compounds. Chemosphere 30(6):1041–1051
Heipieper HJ, Meinhardt F, Segura A (2003) The cis-trans isomerase of unsaturated fatty acids in Pseudomonas and Vibrio: biochemistry, molecular biology and physiological function of a unique stress adaptive mechanism. FEMS Microbiol Lett 229(1):1–7
Heipieper HJ, Neumann G, Cornelissen S, Meinhardt F (2007) Solvent-tolerant bacteria for biotransformations in two-phase fermentation systems. Appl Microbiol Biotechnol 74(5):961–973. https://doi.org/10.1007/s00253-006-0833-4
Ingram LO (1976) Adaptation of membrane lipids to alcohols. J Bacteriol 125(2):670–678
Ingram LO (1977) Changes in lipid composition of Escherichia coli resulting from growth with organic solvents and with food additives. Appl Environ Microbiol 33(5):1233–1236
Kadurugamuwa JL, Beveridge TJ (1995) Virulence factors are released from Pseudomonas aeruginosa in association with membrane vesicles during normal growth and exposure to gentamicin: a novel mechanism of enzyme secretion. J Bacteriol 177(14):3998–4008
Kadurugamuwa JL, Beveridge TJ (1997) Natural release of virulence factors in membrane vesicles by Pseudomonas aeruginosa and the effect of aminoglycoside antibiotics on their release. J Antimicrob Chemother 40(5):615–621. https://doi.org/10.1093/jac/40.5.615
Kobayashi H, Uematsu K, Hirayama H, Horikoshi K (2000) Novel toluene elimination system in a toluene-tolerant microorganism. J Bacteriol 182(22):6451–6455
Kulp A, Kuehn MJ (2010) Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu Rev Microbiol 64:163–184. https://doi.org/10.1146/annurev.micro.091208.073413
Makin SA, Beveridge TJ (1996) The influence of A-band and B-band lipopolysaccharide on the surface characteristics and adhesion of Pseudomonas aeruginosa to surfaces. Microbiology 142:299–307
Mashburn LM, Whiteley M (2005) Membrane vesicles traffic signals and facilitate group activities in a prokaryote. Nature 437(7057):422–425. https://doi.org/10.1038/nature03925
Mashburn-Warren L, Howe J, Garidel P, Richter W, Steiniger F, Roessle M, Brandenburg K, Whiteley M (2008) Interaction of quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation. Mol Microbiol 69(2):491–502
Mashburn-Warren L, Howe J, Brandenburg K, Whiteley M (2009) Structural requirements of the Pseudomonas quinolone signal for membrane vesicle stimulation. J Bacteriol 191(10):3411–3414. https://doi.org/10.1128/JB.00052-09
McBroom AJ, Johnson AP, Vemulapalli S, Kuehn MJ (2006) Outer membrane vesicle production by Escherichia coli is independent of membrane instability. J Bacteriol 188(15):5385–5392. https://doi.org/10.1128/JB.00498-06
Meers PR, Liu C, Chen R, Bartos W, Davis J, Dziedzic N, Orciuolo J, Kutyla S, Pozo MJ, Mithrananda D, Panzera D, Wang S (2018) Vesicular delivery of the antifungal antibiotics of Lysobacter enzymogenes C3. Appl Environ Microbiol 84(20):e01353-18. https://doi.org/10.1128/AEM.01353-18
Molina-Santiago C, Udaondo Z, Gomez-Lozano M, Molin S, Ramos JL (2017) Global transcriptional response of solvent-sensitive and solvent-tolerant Pseudomonas putida strains exposed to toluene. Environ Microbiol 19(2):645–658. https://doi.org/10.1111/1462-2920.13585
Neumann G, Kabelitz N, Zehnsdorf A, Miltner A, Lippold H, Meyer D, Schmid A, Heipieper HJ (2005) Prediction of the adaptability of Pseudomonas putida DOT-T1E to a second phase of a solvent for economically sound two-phase biotransformations. Appl Environ Microbiol 71(11):6606–6612. https://doi.org/10.1128/AEM.71.11.6606-6612.2005
Neumann G, Cornelissen S, van Breukelen F, Hunger S, Lippold H, Loffhagen N, Wick LY, Heipieper HJ (2006) Energetics and surface properties of Pseudomonas putida DOT-T1E in a two-phase fermentation system with 1-decanol as second phase. Appl Environ Microbiol 72(6):4232–4238. https://doi.org/10.1128/AEM.02904-05
Okuyama H, Okajima N, Sasaki S, Higashi S, Murata N (1991) The cis trans isomerization of the double bond of a fatty acid as a strategy for adaptation to changes in ambient temperature in the psychrophilic bacterium, Vibrio sp strain Abe-1. Biochim Biophys Acta 1084(1):13–20. https://doi.org/10.1016/0005-2760(91)90049-N
Pardo YA, Florez C, Baker KM, Schertzer JW, Mahler GJ (2015) Detection of outer membrane vesicles in Synechocystis PCC 6803. FEMS Microbiol Lett 362(20). https://doi.org/10.1093/femsle/fnv163
Pinkart HC, Wolfram JW, Rogers R, White DC (1996) Cell envelope changes in solvent-tolerant and solvent-sensitive Pseudomonas putida strains following exposure to o-xylene. Appl Environ Microbiol 62(3):1129–1132
Rekker RF, Kort HMD (1979) Hydrophobic fragmental constant - extension to a 1000 data point set. Eur J Med Chem 14(6):479–488
Rijnaarts HHM, Norde W, Lyklema J, Zehnder AJB (1995) The isoelectric point of bacteria as an indicator for the presence of cell-surface polymers that inhibit adhesion. Colloid Surface B 4(4):191–197. https://doi.org/10.1016/0927-7765(94)01164-Z
Rojas A, Duque E, Schmid A, Hurtado A, Ramos JL, Segura A (2004) Biotransformation in double-phase systems: physiological responses of Pseudomonas putida DOT-T1E to a double phase made of aliphatic alcohols and biosynthesis of substituted catechols. Appl Environ Microbiol 70(6):3637–3643. https://doi.org/10.1128/aem.70.6.3637-3643.2004
Sabra W, Lunsdorf H, Zeng AP (2003) Alterations in the formation of lipopolysaccharide and membrane vesicles on the surface of Pseudomonas aeruginosa PAO1 under oxygen stress conditions. Microbiology 149:2789–2795. https://doi.org/10.1099/mic.0.26443-0
Schertzer JW, Whiteley M (2012) A bilayer-couple model of bacterial outer membrane vesicle biogenesis. MBio 3(2). https://doi.org/10.1128/mBio.00297-11
Schertzer JW, Whiteley M (2013) Bacterial outer membrane vesicles in trafficking, communication and the host-pathogen interaction. J Mol Microbiol Biotechnol 23(1–2):118–130. https://doi.org/10.1159/000346770
Schertzer JW, Boulette ML, Whiteley M (2009) More than a signal: non-signaling properties of quorum sensing molecules. Trends Microbiol 17(5):189–195. https://doi.org/10.1016/j.tim.2009.02.001
Schwechheimer C, Kuehn MJ (2015) Outer-membrane vesicles from gram-negative bacteria: biogenesis and functions. Nat Rev Microbiol 13(10):605–619. https://doi.org/10.1038/nrmicro3525
Segura A, Duque E, Mosqueda G, Ramos JL, Junker F (1999) Multiple responses of Gram-negative bacteria to organic solvents. Environ Microbiol 1(3):191–198
Tashiro Y, Ichikawa S, Nakajima-Kambe T, Uchiyama H, Nomura N (2010) Pseudomonas quinolone signal affects membrane vesicle production in not only Gram-negative but also Gram-positive bacteria. Microbes Environ 25(2):120–125. https://doi.org/10.1264/jsme2.ME09182
Toyofuku M, Tashiro Y, Hasegawa Y, Kurosawa M, Nomura N (2015) Bacterial membrane vesicles, an overlooked environmental colloid: biology, environmental perspectives and applications. Adv Colloid Interf Sci 226(Pt A:65–77. https://doi.org/10.1016/j.cis.2015.08.013
Van Loosdrecht MCM, Lyklema J, Norde W, Schraa G, Zehnder AJB (1987) The role of bacterial cell wall hydrophobicity in adhesion. Appl Environ Microbiol 53:1893–1897
Wagner T, Joshi B, Janice J, Askarian F, Skalko-Basnet N, Hagestad OC, Mekhlif A, Wai SN, Hegstad K, Johannessen M (2018) Enterococcus faecium produces membrane vesicles containing virulence factors and antimicrobial resistance related proteins. J Proteome 187:28–38. https://doi.org/10.1016/j.jprot.2018.05.017
Weber FJ, de Bont JAM (1996) Adaptation mechanisms of microorganisms to the toxic effects of organic solvents on membranes. Biochim Biophys Acta 1286(3):225–245
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The financial support by the European Union’s Horizon 2020 research and innovation program under grant agreement no. 633962 for the project P4SB are greatly appreciated.
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Eberlein, C., Starke, S., Doncel, Á.E. et al. Quantification of outer membrane vesicles: a potential tool to compare response in Pseudomonas putida KT2440 to stress caused by alkanols. Appl Microbiol Biotechnol 103, 4193–4201 (2019). https://doi.org/10.1007/s00253-019-09812-0
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DOI: https://doi.org/10.1007/s00253-019-09812-0