Fragarin, an antibiotic that was isolated and purified from a soluble fraction of strawberry leaves, may be a new type of preformed antimicrobial compound (phytoanticipin). Here, we report that the growth and oxygen consumption of the phytopathogenic bacterium Clavibacter michiganensis were rapidly inhibited after the addition of fragarin to cultures. Also, dissipation of the membrane potential and an increase of cell membrane permeability were observed in the presence of fragarin. The ability of fragarin to dissipate the membrane potential was confirmed with the use of small unilamellar liposomes made with lipids extracted from C. michiganensis. Our results suggest that fragarin is able to act at the membrane level, and that this action is correlated with a decrease in cell viability.Fil: Filippone, María Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Diaz Ricci, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Castagnaro, Atilio Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Farias, Ricardo Norberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentin

Castagnaro, Atilio Pedro

Diaz Ricci, Juan Carlos

Farias, Ricardo Norberto

Filippone, María Paula

Molecular Plant-Microbe Interactions

English

CONICET Digital

Vol. 14, No. 7, 2001 / 925 MPMI Vol. 14, No. 7, 2001, pp. 925–928. Publication no. M-2001-0425-01N. © 2001 The American Phytopathological Society Research Note Effect of Fragarin on the Cytoplasmic Membrane of the Phytopathogen Clavibacter michiganensis M. Paula Filippone, Juan C. Diaz-Ricci, Atilio P. Castagnaro, and Ricardo N. Farías Departamento de Bioquímica de la Nutrición, Instituto Superior de Investigaciones Biológicas (Consejo Nacional de Investigaciones Científicas y Técnicas–Universidad Nacional de Tucumán) and Instituto de Química Biológica “Dr. Bernabé Bloj,” 4000 San Miguel de Tucumán, Argentina Submitted 27 November 2000; Accepted 20 March 2001. Fragarin, an antibiotic that was isolated and purified from a soluble fraction of strawberry leaves, may be a new type of preformed antimicrobial compound (phytoanticipin). Here, we report that the growth and oxygen consumption of the phytopathogenic bacterium Clavibacter michiganen-sis were rapidly inhibited after the addition of fragarin to cultures. Also, dissipation of the membrane potential and an increase of cell membrane permeability were observed in the presence of fragarin. The ability of fragarin to dis-sipate the membrane potential was confirmed with the use of small unilamellar liposomes made with lipids extracted from C. michiganensis. Our results suggest that fragarin is able to act at the membrane level, and that this action is correlated with a decrease in cell viability. Additional keywords: biological membrane, preformed antiphy-topathogenic compounds. It has been demonstrated that secondary metabolites have different functions in plants and many are important compo-nents of plant-defense mechanisms (Harbone 1991). Two categories of secondary metabolites with antibiotic activity were proposed in accordance to how they are produced: induc-ible and preformed compounds. Inducible compounds such as phytoalexins are antimicrobials, which are synthesized de novo in response to biotic or abiotic elicitors (Kuc 1995). Pre-formed compounds such as phytoanticipins, however, can be found in their biologically active form, even in uninjured plants, or as inactive precursors that can be activated by means of specific enzymes (Osbourn 1996). Therefore, the fundamental difference between phytoalexins and phytoan-ticipins is that the former are involved in an active plant re-sponse against a pathogen, whereas phytoanticipins are in-volved in a passive plant-defense response (Van Etten 1994). Recently, we isolated a 316-kDa amphipathic molecule, fra-garin, from a soluble fraction of strawberry leaves. Fragarin shows activity against bacterial and fungal plant pathogens and, according to the biological and chemical features shown by fragarin, we speculate that it may be a new type of phy-toanticipin related to secondary metabolites (Filippone et al. 1999) and could be involved in the defense of strawberry against pathogen attack. The aim of this work is to obtain insight into the antibiotic activity of fragarin. The bacterium Clavibacter michiganensis subsp. sepedoni-cus strain C5 was used in biological assays provided by A. Molina from the Escuela Técnica Superior de Ingenieros Agrónomos (ETSIA) collection, Madrid, Spain. Strawberry (Fragaria ananassa) cultivar Chandler was grown for 20 days in sterilized substrate (3:2:1 parts of soil–sand–perlite, respec-tively), at 25ºC and 70% relative humidity, with a 16-h photo-period. Leaves were collected and frozen immediately in liq-uid nitrogen. Leaf homogenate was obtained from fresh material that was frozen previously and ground in liquid nitro-gen. Fragarin was extracted from leaf powder, as previously reported (Filippone et al. 1999). The purification of fragarin included the direct adsorption of the extract (centrifuged at 16,000 × g for 15 min) on 60 ml of a preparative Mega Bond Elut C8 cartridge (Varian, Palo Alto, CA, U.S.A.), followed by washing with water, water–acetonitrile (50:50 vol/vol), and acetonitrile. The latter fraction containing fragarin was sub-jected to fast protein liquid chromatography with a reverse-phase C1/C8 column (0.5 × 10 cm, ProRPC HR 5/10; Amer-sham Pharmacia Biotech, Piscataway, NJ, U.S.A.) equilibrated with water containing 0.1% trifluoroacetic acid (TFA). The elution was performed for 30 min with a linear gradient (0 to 100%) of water–acetonitrile containing 0.1% TFA (vol/vol) with an additional 15 min of 100% acetonitrile at 0.25 ml per min (detection at 280 nm). Fractions (0.5 ml) were collected and assayed for antibiotic activity with C. michiganensis. Ac-tive fractions that eluted at 100% acetonitrile were pooled, lyophilized, dissolved in water, and rechromatographed with a linear gradient of water–acetonitrile (90 to 100%) and the same column for 15 min. The active fraction eluted as a unique and homogeneous peak at 98% acetonitrile (Filippone et al. 1999). Oxygen consumption was measured polarographically with a Gilson oxygraph (Middleton, WI, U.S.A.) equipped with a Clark-type electrode in a closed, stirred glass vessel of 2.0-ml volume at 30ºC. Fragarin (2.0 µM) was added to a culture of C. michiganensis growing in Luria-Bertani (LB) medium at 30ºC during the exponential-phase optical density (OD) at 600 nm of 0.2 to 0.3. Aliquots of 2 ml were removed at different times and transferred to an oxymeter cell. Oxygen consump-tion was recorded on-line. Corresponding author: R. Farias; Telephone: +54 0381 424 8921; Fax: +54 0381 424 8025; E-mail: farias@unt.edu.ar 926 / Molecular Plant-Microbe Interactions Cytoplasmic membrane permeability was determined with potential-sensitive cyanine dye 3,5′-dipropylthiacarbocyanine iodide (diSC3-5) (Wu et al. 1999). This dye partitions between cells and the medium, depending upon the membrane-potential gradient. When the gradient is high, the dye pene-trates into the cell and quenches its own fluorescence. An increase of the fluorescence resulting from the disruption of the cytoplasmic membrane can be observed after the addition of a membrane-interacting agent (Wu et al. 1999). C. michi-ganensis was grown in LB medium at 30ºC to midexponential phase (OD600 = 0.4 to 0.5). The cells were collected by centrifugation, washed once with 5 mM Na N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES; pH 7.2, 5 mM glucose), and resuspended in the same buffer to obtain OD600 = 1.0. A 100-µl cell suspension was transferred to a spectrofluorometer cuvette containing 2.0 ml of 5 mM Na HEPES buffer (pH 7.2, 5 mM glucose, 100 mM KCl) to ob-tain OD600 = 0.05. After the addition of 0.4 µM diSC3-5, the cuvette was incubated at 37ºC until the dye uptake was maxi-mal (as indicated by a reduction of fluorescence to a stable value, approximately 5 min). The desired concentration of fragarin was added to a suspension of diSC3-5-loaded cells, and the dye fluorescence increase was recorded at 670 nm with a RF-5301PC spectrofluorometer, with excitation set at 622 nm (Shimadzu, Kyoto, Japan). A cuvette containing diSC3-5-loaded cells was used to subtract the background. The effect of the external KCl concentration on the fluores-cence intensity in the presence and absence of valinomycin after the stabilization was determined previously, as indicated by Wu et al. (1999). The antibiotic valinomycin is a K+ selec-tive ionophore that changes the membrane potential. Results indicate that in the presence of 1 µM valinomycin, fluores-cence reached a maximum of 100 mM KCl as a result of a K+ concentration equilibrium established between both sides of the cell membrane. In the absence of valinomycin, the fluo-rescence intensity was not modified by the addition of 100 mM KCl to the assay buffer. In order to prevent any ef-fects resulting from the movement of potassium ions, an ex-ternal concentration of 100 mM KCl was added in all cases to the cell suspension (Wu et al. 1999). Changes in the membrane potential dissipation were studied in liposome, as indicated by Gao et al. (1991). Lipids were extracted from C. michiganensis cells grown previously in LB medium at 30ºC to midexponential phase (OD600 = 0.4 to 0.5) and purified according to Kates (1986). Appropriate quantities of lipids dried extensively under a nitrogen stream were sus-pended by vortexing in 1 ml of 50 mM potassium phosphate buffer, pH 7.0. To obtain small unilamellar phospholipid vesi-cles, the formed multilamellar liposomes were subjected to sonication for 20 min, with intervals of 15 s at 5ºC, with a sonicator (Cole-Parmer, Vernon Hills, IL, U.S.A.). Large par-ticles were eliminated by centrifugation at 10,000 × g in a fixed angle rotor centrifuge for 5 min. Potassium-loaded lipo-somes were diluted 100-fold in 50 mM sodium phosphate buffer, pH 7.0, and 0.4 µM diSC3-5 was added. Afterward, 0.05 µM valinomycin was added to generate a membrane po-tential that was stable for 2 h. The fluorescence intensity was measured, as described above. Experiments were repeated at least three times under identi-cal experimental conditions and identical outcomes were ob-tained. Results of representative experiments are shown in the figures of this paper. In a previous report, it was shown that fragarin is capable of inhibiting the growth of plant-pathogenic bacteria and fungi (Filippone et al. 1999). In the present work, we chose the C. michiganensis strain C5 because it proved to be the most fra-garin-sensitive bacterium (Filippone et al. 1999). When 2 µM fragarin was added to an actively growing culture of C. michi-ganensis, growth stopped and remained constant. After 2 h, the viable cell counts decreased by three orders of magnitude (Fig. 1). We also observed that the oxygen consumption was inhibited by the addition of fragarin. The respiratory rate of C. michiganensis depends upon the cellular concentration in the oxymeter cell (not shown). Under our experimental condi-tions, the oxygen concentration decreased by approximately 35% in 10 min (Fig. 2, line a) in control experiments of cul-tures without fragarin. By contrast, when a culture of C. mi-chiganensis was treated with 2 µM fragarin for 5 min, oxygen consumption was abolished totally (Fig. 2, line b). Because respiratory function is dependent upon an intact and functional cytoplasmic membrane, we evaluated the influence of fragarin on the integrity of the cytoplasmic membrane of C. michi-ganensis by the cyanine dye 3,5′-dipropylthiacarbocyanine  Fig. 1. Effect of 2 µM fragarin on the growth of Clavibacter michi-ganensis.  = Optical density (OD) in the absence of fragarin. ! = OD inthe presence of fragarin. c = CFU/ml in the absence of fragarin. Y = CFU/ml in the presence of fragarin (arrow). Vol. 14, No. 1, 2001 / 927 iodide (diSC3-5) method (Wu et al. 1999). After the addition of 0.15 µM fragarin to dye-loaded cells, an increase of fluo-rescence appeared, reaching a maximum within 1 min (Fig. 3A). As suggested by Wu et al. (1999), this increase in fluo-rescence correlates with the dissipation of the membrane po-tential and increased membrane permeability. In parallel ex-periments, valinomycin was used as a positive control. The fluorescence did not change during the assay time in the ab-sence of fragarin or valinomycin (Fig. 3A). Figure 3B shows the influence of fragarin concentration on the membrane per-meability of C. michiganensis. This outcome agrees with the 50% effective concentration values (0.07 µM) reported previ-ously (Filippone et al. 1999). The ability of fragarin to dissipate the membrane potential also was confirmed with small unilamellar liposomes made with lipids extracted from C. michiganensis. Figure 4 shows that fluorescence decreased after the addition of valinomycin, indicating membrane-potential generation in liposomes (∆ψ, interior negative). Also shown in Figure 4 is the gradual fluo-rescence increase observed upon fragarin addition, indicating that the induced potential is dissipated. The addition of fra-garin to liposomes that were not treated previously with valinomycin had no effect (not shown). In these experiments, the antibiotic nigericin, known for its ability to disrupt mem-brane potential (Rottenberg and Scarpa 1974), was used as a positive control. When potassium-loaded liposomes were di-luted 100-fold in 50 mM potassium phosphate instead of 50 mM sodium phosphate, no effect of fragarin was observed. In this liposome preparation, valinomycin is not able to generate a potential difference (data not shown). In summary, the present paper demonstrates that fragarin is able to alter permeability and disrupt membrane function, resulting in the dissipation of membrane potential in intact cells of the sensitive C. michiganensis strain used. Further-more, the ability of fragarin to dissipate membrane potential was further confirmed with the use of liposomes (Fig. 4). The immediate inhibition of oxygen consumption (Fig. 2) and the immediate dissipation of membrane potential (an increase of fluorescence) (Fig. 3) suggest that the fragarin  Fig. 2. Effect of 2.0 µM fragarin on the oxygen consumption of a Clavi-bacter michiganensis culture growing in Luria-Bertani medium. Oxygen concentration of a control culture (a) and a culture treated with fragarin for 5 min (b). Fig. 3. Fragarin effect on membrane permeability. A, Release of diSC3-5 from loaded Clavibacter michiganensis cells after the addition of 0.15 µM fragarin (!), 1.0 µM valinomycin (), and without fragarin or valinomycin addition (). B, Dose response of fragarin effect on mem-brane permeability. 928 / Molecular Plant-Microbe Interactions effect on the membrane precedes or is simultaneous with cell death (Fig. 1). ACKNOWLEDGMENTS This work was supported partially by Consejo de Investigaciones de la Universidad Nacional de Tucumán (CIUNT); Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); International Foun-dation for Science (IFS), Sweden (grant F/2568-1); and Agencia Na-cional de Promoción Científica y Tecnológica (PMT/SID No: 552). M. P. Filippone is a fellow and J. C. Diaz-Ricci, A. P. Castagnaro, and R. N. Farías are career members of CONICET. LITERATURE CITED Filippone, M. P., Diaz Ricci, J. C., Mamani de Marchese. A., Farias, R. N., and Castagnaro, A. P. 1999. Isolation and purification of a 316 Da pre-formed compound from strawberry (Fragaria ananassa) leaves active against plant pathogens. FEBS Lett. 459:115-118. Gao, F. H., Abbe, T., and Konings, W. N. 1991. Mechanism of action of the peptide antibiotic nisin in liposomes and cytochrome c oxi-dase containing proteoliposomes. Appl. Environ. Microbiol. 57:2164-2170. Harbone, J. B. 1991. Bioactive compounds from plants. Pages 126-139 in: Role of Secondary Metabolites in Chemical Defense Mechanisms in Plants. D. J. Chadwik and J. Marsh, eds. John Wiley and Sons, Chichester, U.K. Kates, M. 1986. Techniques of Lipidology: Isolation, Analysis and Iden-tification of Lipids, 2nd ed. Elsevier Biomedical Press, Amsterdam. Kuc, J. 1995. Phytoalexins, stress metabolism and disease resistance in plants. Annu. Rev. Phytopathol. 33:275-295. Osbourn, A. E. 1996. Preformed antimicrobial compounds and plant defense against fungal attack. Plant Cell 8:1821-1831. Rottenberg H., and Scarpa, A. 1974. Calcium uptake and membrane potential in mitochondria. Biochemistry 13:4811-4817. Van Etten, H. D., Mansfield, J. W., Bailey, J. A., and Farmer, E. E. 1994. Two classes of plant antibiotics: Phytoalexins versus phytoanticipins. Plant Cell 6:1191-1192. Wu, M., Maier, E., Benz, R., and Hancock, E. W. 1999. Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli. Biochemistry 38:7235-7242.   Fig. 4. Effect of fragarin on liposome membrane potential generated by valinomycin. The addition of 0.05 µM valinomycin, 0.4 µM fragarin, and 1.0 µM nigericin are indicated by arrows a, b, and c, respectively. 

Effect of Fragarin on the Cytoplasmic Membrane of the Phytopathogen Clavibacter michiganensis

LAReferencia - Red Federada de Repositorios Institucionales de Publicaciones Científicas Latinoamericanas

Vol. 14, No. 7, 2001 / 925 MPMI Vol. 14, No. 7, 2001, pp. 925–928. Publication no. M-2001-0425-01N. © 2001 The American Phytopathological Society Research Note Effect of Fragarin on the Cytoplasmic Membrane of the Phytopathogen Clavibacter michiganensis M. Paula Filippone, Juan C. Diaz-Ricci, Atilio P. Castagnaro, and Ricardo N. Farías Departamento de Bioquímica de la Nutrición, Instituto Superior de Investigaciones Biológicas (Consejo Nacional de Investigaciones Científicas y Técnicas–Universidad Nacional de Tucumán) and Instituto de Química Biológica “Dr. Bernabé Bloj,” 4000 San Miguel de Tucumán, Argentina Submitted 27 November 2000; Accepted 20 March 2001. Fragarin, an antibiotic that was isolated and purified from a soluble fraction of strawberry leaves, may be a new type of preformed antimicrobial compound (phytoanticipin). Here, we report that the growth and oxygen consumption of the phytopathogenic bacterium Clavibacter michiganen-sis were rapidly inhibited after the addition of fragarin to cultures. Also, dissipation of the membrane potential and an increase of cell membrane permeability were observed in the presence of fragarin. The ability of fragarin to dis-sipate the membrane potential was confirmed with the use of small unilamellar liposomes made with lipids extracted from C. michiganensis. Our results suggest that fragarin is able to act at the membrane level, and that this action is correlated with a decrease in cell viability. Additional keywords: biological membrane, preformed antiphy-topathogenic compounds. It has been demonstrated that secondary metabolites have different functions in plants and many are important compo-nents of plant-defense mechanisms (Harbone 1991). Two categories of secondary metabolites with antibiotic activity were proposed in accordance to how they are produced: induc-ible and preformed compounds. Inducible compounds such as phytoalexins are antimicrobials, which are synthesized de novo in response to biotic or abiotic elicitors (Kuc 1995). Pre-formed compounds such as phytoanticipins, however, can be found in their biologically active form, even in uninjured plants, or as inactive precursors that can be activated by means of specific enzymes (Osbourn 1996). Therefore, the fundamental difference between phytoalexins and phytoan-ticipins is that the former are involved in an active plant re-sponse against a pathogen, whereas phytoanticipins are in-volved in a passive plant-defense response (Van Etten 1994). Recently, we isolated a 316-kDa amphipathic molecule, fra-garin, from a soluble fraction of strawberry leaves. Fragarin shows activity against bacterial and fungal plant pathogens and, according to the biological and chemical features shown by fragarin, we speculate that it may be a new type of phy-toanticipin related to secondary metabolites (Filippone et al. 1999) and could be involved in the defense of strawberry against pathogen attack. The aim of this work is to obtain insight into the antibiotic activity of fragarin. The bacterium Clavibacter michiganensis subsp. sepedoni-cus strain C5 was used in biological assays provided by A. Molina from the Escuela Técnica Superior de Ingenieros Agrónomos (ETSIA) collection, Madrid, Spain. Strawberry (Fragaria ananassa) cultivar Chandler was grown for 20 days in sterilized substrate (3:2:1 parts of soil–sand–perlite, respec-tively), at 25ºC and 70% relative humidity, with a 16-h photo-period. Leaves were collected and frozen immediately in liq-uid nitrogen. Leaf homogenate was obtained from fresh material that was frozen previously and ground in liquid nitro-gen. Fragarin was extracted from leaf powder, as previously reported (Filippone et al. 1999). The purification of fragarin included the direct adsorption of the extract (centrifuged at 16,000 × g for 15 min) on 60 ml of a preparative Mega Bond Elut C8 cartridge (Varian, Palo Alto, CA, U.S.A.), followed by washing with water, water–acetonitrile (50:50 vol/vol), and acetonitrile. The latter fraction containing fragarin was sub-jected to fast protein liquid chromatography with a reverse-phase C1/C8 column (0.5 × 10 cm, ProRPC HR 5/10; Amer-sham Pharmacia Biotech, Piscataway, NJ, U.S.A.) equilibrated with water containing 0.1% trifluoroacetic acid (TFA). The elution was performed for 30 min with a linear gradient (0 to 100%) of water–acetonitrile containing 0.1% TFA (vol/vol) with an additional 15 min of 100% acetonitrile at 0.25 ml per min (detection at 280 nm). Fractions (0.5 ml) were collected and assayed for antibiotic activity with C. michiganensis. Ac-tive fractions that eluted at 100% acetonitrile were pooled, lyophilized, dissolved in water, and rechromatographed with a linear gradient of water–acetonitrile (90 to 100%) and the same column for 15 min. The active fraction eluted as a unique and homogeneous peak at 98% acetonitrile (Filippone et al. 1999). Oxygen consumption was measured polarographically with a Gilson oxygraph (Middleton, WI, U.S.A.) equipped with a Clark-type electrode in a closed, stirred glass vessel of 2.0-ml volume at 30ºC. Fragarin (2.0 µM) was added to a culture of C. michiganensis growing in Luria-Bertani (LB) medium at 30ºC during the exponential-phase optical density (OD) at 600 nm of 0.2 to 0.3. Aliquots of 2 ml were removed at different times and transferred to an oxymeter cell. Oxygen consump-tion was recorded on-line. Corresponding author: R. Farias; Telephone: +54 0381 424 8921; Fax: +54 0381 424 8025; E-mail: farias@unt.edu.ar 926 / Molecular Plant-Microbe Interactions Cytoplasmic membrane permeability was determined with potential-sensitive cyanine dye 3,5′-dipropylthiacarbocyanine iodide (diSC3-5) (Wu et al. 1999). This dye partitions between cells and the medium, depending upon the membrane-potential gradient. When the gradient is high, the dye pene-trates into the cell and quenches its own fluorescence. An increase of the fluorescence resulting from the disruption of the cytoplasmic membrane can be observed after the addition of a membrane-interacting agent (Wu et al. 1999). C. michi-ganensis was grown in LB medium at 30ºC to midexponential phase (OD600 = 0.4 to 0.5). The cells were collected by centrifugation, washed once with 5 mM Na N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES; pH 7.2, 5 mM glucose), and resuspended in the same buffer to obtain OD600 = 1.0. A 100-µl cell suspension was transferred to a spectrofluorometer cuvette containing 2.0 ml of 5 mM Na HEPES buffer (pH 7.2, 5 mM glucose, 100 mM KCl) to ob-tain OD600 = 0.05. After the addition of 0.4 µM diSC3-5, the cuvette was incubated at 37ºC until the dye uptake was maxi-mal (as indicated by a reduction of fluorescence to a stable value, approximately 5 min). The desired concentration of fragarin was added to a suspension of diSC3-5-loaded cells, and the dye fluorescence increase was recorded at 670 nm with a RF-5301PC spectrofluorometer, with excitation set at 622 nm (Shimadzu, Kyoto, Japan). A cuvette containing diSC3-5-loaded cells was used to subtract the background. The effect of the external KCl concentration on the fluores-cence intensity in the presence and absence of valinomycin after the stabilization was determined previously, as indicated by Wu et al. (1999). The antibiotic valinomycin is a K+ selec-tive ionophore that changes the membrane potential. Results indicate that in the presence of 1 µM valinomycin, fluores-cence reached a maximum of 100 mM KCl as a result of a K+ concentration equilibrium established between both sides of the cell membrane. In the absence of valinomycin, the fluo-rescence intensity was not modified by the addition of 100 mM KCl to the assay buffer. In order to prevent any ef-fects resulting from the movement of potassium ions, an ex-ternal concentration of 100 mM KCl was added in all cases to the cell suspension (Wu et al. 1999). Changes in the membrane potential dissipation were studied in liposome, as indicated by Gao et al. (1991). Lipids were extracted from C. michiganensis cells grown previously in LB medium at 30ºC to midexponential phase (OD600 = 0.4 to 0.5) and purified according to Kates (1986). Appropriate quantities of lipids dried extensively under a nitrogen stream were sus-pended by vortexing in 1 ml of 50 mM potassium phosphate buffer, pH 7.0. To obtain small unilamellar phospholipid vesi-cles, the formed multilamellar liposomes were subjected to sonication for 20 min, with intervals of 15 s at 5ºC, with a sonicator (Cole-Parmer, Vernon Hills, IL, U.S.A.). Large par-ticles were eliminated by centrifugation at 10,000 × g in a fixed angle rotor centrifuge for 5 min. Potassium-loaded lipo-somes were diluted 100-fold in 50 mM sodium phosphate buffer, pH 7.0, and 0.4 µM diSC3-5 was added. Afterward, 0.05 µM valinomycin was added to generate a membrane po-tential that was stable for 2 h. The fluorescence intensity was measured, as described above. Experiments were repeated at least three times under identi-cal experimental conditions and identical outcomes were ob-tained. Results of representative experiments are shown in the figures of this paper. In a previous report, it was shown that fragarin is capable of inhibiting the growth of plant-pathogenic bacteria and fungi (Filippone et al. 1999). In the present work, we chose the C. michiganensis strain C5 because it proved to be the most fra-garin-sensitive bacterium (Filippone et al. 1999). When 2 µM fragarin was added to an actively growing culture of C. michi-ganensis, growth stopped and remained constant. After 2 h, the viable cell counts decreased by three orders of magnitude (Fig. 1). We also observed that the oxygen consumption was inhibited by the addition of fragarin. The respiratory rate of C. michiganensis depends upon the cellular concentration in the oxymeter cell (not shown). Under our experimental condi-tions, the oxygen concentration decreased by approximately 35% in 10 min (Fig. 2, line a) in control experiments of cul-tures without fragarin. By contrast, when a culture of C. mi-chiganensis was treated with 2 µM fragarin for 5 min, oxygen consumption was abolished totally (Fig. 2, line b). Because respiratory function is dependent upon an intact and functional cytoplasmic membrane, we evaluated the influence of fragarin on the integrity of the cytoplasmic membrane of C. michi-ganensis by the cyanine dye 3,5′-dipropylthiacarbocyanine  Fig. 1. Effect of 2 µM fragarin on the growth of Clavibacter michi-ganensis. ■ = Optical density (OD) in the absence of fragarin. ! = OD inthe presence of fragarin. c = CFU/ml in the absence of fragarin. Y = CFU/ml in the presence of fragarin (arrow). Vol. 14, No. 1, 2001 / 927 iodide (diSC3-5) method (Wu et al. 1999). After the addition of 0.15 µM fragarin to dye-loaded cells, an increase of fluo-rescence appeared, reaching a maximum within 1 min (Fig. 3A). As suggested by Wu et al. (1999), this increase in fluo-rescence correlates with the dissipation of the membrane po-tential and increased membrane permeability. In parallel ex-periments, valinomycin was used as a positive control. The fluorescence did not change during the assay time in the ab-sence of fragarin or valinomycin (Fig. 3A). Figure 3B shows the influence of fragarin concentration on the membrane per-meability of C. michiganensis. This outcome agrees with the 50% effective concentration values (0.07 µM) reported previ-ously (Filippone et al. 1999). The ability of fragarin to dissipate the membrane potential also was confirmed with small unilamellar liposomes made with lipids extracted from C. michiganensis. Figure 4 shows that fluorescence decreased after the addition of valinomycin, indicating membrane-potential generation in liposomes (∆ψ, interior negative). Also shown in Figure 4 is the gradual fluo-rescence increase observed upon fragarin addition, indicating that the induced potential is dissipated. The addition of fra-garin to liposomes that were not treated previously with valinomycin had no effect (not shown). In these experiments, the antibiotic nigericin, known for its ability to disrupt mem-brane potential (Rottenberg and Scarpa 1974), was used as a positive control. When potassium-loaded liposomes were di-luted 100-fold in 50 mM potassium phosphate instead of 50 mM sodium phosphate, no effect of fragarin was observed. In this liposome preparation, valinomycin is not able to generate a potential difference (data not shown). In summary, the present paper demonstrates that fragarin is able to alter permeability and disrupt membrane function, resulting in the dissipation of membrane potential in intact cells of the sensitive C. michiganensis strain used. Further-more, the ability of fragarin to dissipate membrane potential was further confirmed with the use of liposomes (Fig. 4). The immediate inhibition of oxygen consumption (Fig. 2) and the immediate dissipation of membrane potential (an increase of fluorescence) (Fig. 3) suggest that the fragarin  Fig. 2. Effect of 2.0 µM fragarin on the oxygen consumption of a Clavi-bacter michiganensis culture growing in Luria-Bertani medium. Oxygen concentration of a control culture (a) and a culture treated with fragarin for 5 min (b). Fig. 3. Fragarin effect on membrane permeability. A, Release of diSC3-5 from loaded Clavibacter michiganensis cells after the addition of 0.15 µM fragarin (!), 1.0 µM valinomycin (■), and without fragarin or valinomycin addition (▲). B, Dose response of fragarin effect on mem-brane permeability. 928 / Molecular Plant-Microbe Interactions effect on the membrane precedes or is simultaneous with cell death (Fig. 1). ACKNOWLEDGMENTS This work was supported partially by Consejo de Investigaciones de la Universidad Nacional de Tucumán (CIUNT); Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); International Foun-dation for Science (IFS), Sweden (grant F/2568-1); and Agencia Na-cional de Promoción Científica y Tecnológica (PMT/SID No: 552). M. P. Filippone is a fellow and J. C. Diaz-Ricci, A. P. Castagnaro, and R. N. Farías are career members of CONICET. LITERATURE CITED Filippone, M. P., Diaz Ricci, J. C., Mamani de Marchese. A., Farias, R. N., and Castagnaro, A. P. 1999. Isolation and purification of a 316 Da pre-formed compound from strawberry (Fragaria ananassa) leaves active against plant pathogens. FEBS Lett. 459:115-118. Gao, F. H., Abbe, T., and Konings, W. N. 1991. Mechanism of action of the peptide antibiotic nisin in liposomes and cytochrome c oxi-dase containing proteoliposomes. Appl. Environ. Microbiol. 57:2164-2170. Harbone, J. B. 1991. Bioactive compounds from plants. Pages 126-139 in: Role of Secondary Metabolites in Chemical Defense Mechanisms in Plants. D. J. Chadwik and J. Marsh, eds. John Wiley and Sons, Chichester, U.K. Kates, M. 1986. Techniques of Lipidology: Isolation, Analysis and Iden-tification of Lipids, 2nd ed. Elsevier Biomedical Press, Amsterdam. Kuc, J. 1995. Phytoalexins, stress metabolism and disease resistance in plants. Annu. Rev. Phytopathol. 33:275-295. Osbourn, A. E. 1996. Preformed antimicrobial compounds and plant defense against fungal attack. Plant Cell 8:1821-1831. Rottenberg H., and Scarpa, A. 1974. Calcium uptake and membrane potential in mitochondria. Biochemistry 13:4811-4817. Van Etten, H. D., Mansfield, J. W., Bailey, J. A., and Farmer, E. E. 1994. Two classes of plant antibiotics: Phytoalexins versus phytoanticipins. Plant Cell 6:1191-1192. Wu, M., Maier, E., Benz, R., and Hancock, E. W. 1999. Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli. Biochemistry 38:7235-7242.   Fig. 4. Effect of fragarin on liposome membrane potential generated by valinomycin. The addition of 0.05 µM valinomycin, 0.4 µM fragarin, and 1.0 µM nigericin are indicated by arrows a, b, and c, respectively. 

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Effect of Fragarin on the Cytoplasmic Membrane of the Phytopathogen Clavibacter michiganensis

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