Department of Microbiology, Biology Faculty, Complutense University of Madrid, Madrid 28040, Spain
Correspondence
D. Marquina
dommarq{at}bio.ucm.es
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ABSTRACT |
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INTRODUCTION |
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Since it was first reported (Makower & Bevan, 1963), the killer phenomenon in yeasts has been extensively studied in several genera and species, and its importance is gaining further recognition by industrialists, clinical microbiologists and molecular biologists (Fink & Styles, 1972
; Provost et al., 1995
; Séguy et al., 1996
; Tipper & Bostian, 1984
; Vondrejs et al., 1996
). The food and beverage industries were among the first to explore the ability of toxin-producing yeasts to kill other micro-organisms (Javadekar et al., 1995
). Attention has mainly focused on the characterization of killer toxins from Saccharomyces cerevisiae (Bevan et al., 1973
; Breinig et al., 2002
; Carroll & Wickner, 1995
; Schmitt & Radler, 1988
; Weinstein et al., 1993
; Wickner, 1974
, 1986
) and Kluyveromyces lactis (Niwa et al., 1981
; Young, 1987
), more recently followed by investigation of yeasts such as Zygosaccharomyces bailii (Radler et al., 1993
), Hanseniaspora uvarum (Radler et al., 1990
), Pichia membranifaciens (Santos et al., 2000
), Debaryomyces hansenii (Gunge et al., 1993
; Marquina et al., 2001a
; Santos et al., 2002
), Kluyveromyces phaffi (Ciani & Fatichenti, 2001
) and Schwanniomyces occidentalis (Chen et al., 2000
). These mycocins are proteins or glycoproteins that bind to polysaccharide structures on the yeast cell wall and this property has been used for the production of purified toxin proteins (Hutchins & Bussey, 1983
).
Strains of P. membranifaciens are common contaminants in food-related environments (Heard & Fleet, 1987; Noronha-da-Costa et al., 1995
) and occur with high frequency in fermenting olive brines (Marquina et al., 1992
, 1997
). One of the isolates from such an environment, P. membranifaciens CYC 1106, showed a particularly strong, broad-spectrum, zymocidal activity. Previous biochemical studies of the killer toxin action of P. membranifaciens on sensitive yeast cells have indicated a set of specific cell-surface interactions, including binding to a (1
6)-
-D-glucan (Santos et al., 2000
). The specific binding of this killer toxin to immobilized (1
6)-
-D-glucan was utilized to develop an effective method for the purification of the killer toxin. The objectives of the present work were to purify the killer toxin, characterize it thereby contributing to the understanding and development of a novel fungicidal agent and to conduct a primary investigation about the potential usefulness of this novel killer toxin in the biological control of B. cinerea.
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METHODS |
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For the purpose of killer toxin purification, P. membranifaciens CYC 1106 was cultured in YNB-D, a 0·67 % (w/v) yeast nitrogen base (Difco) medium containing 1 % glucose. The medium was buffered to pH 4·0 with 0·2 M sodium citrate/phosphate and supplemented with 0·01 % (w/v) Brij-58 (polyoxyethylene 20 cetyl ether; Serva).
To improve killer toxin production, cells were inoculated from stock cultures into different media: YMB [1 % (w/v) glucose, 0·3 % (w/v) yeast extract (Difco), 0·3 % (w/v) malt extract (Difco) and 0·5 % (w/v) proteose peptone no. 3 (Difco)]; YPD [1 % (w/v) glucose, 1 % (w/v) yeast extract and 1 % (w/v) proteose peptone no. 3]; modified Gorodkowa medium [1 % (w/v) glucose, 1 % (w/v) proteose peptone no. 3 and 0·5 % (w/v) NaCl]; minimal medium [YNB-D; 1 % (w/v) glucose and 0·67 % (w/v) yeast nitrogen base] and K medium (Marquina et al., 2001b).
Killer toxin assay.
The killer toxin assay was based on the well-tested method in YMA-MB agar plates, as described by Woods & Bevan (1968). A working definition of arbitrary units (AU) has been described previously (Santos et al., 2000
). For calculations of the specific activity, protein contents were estimated using the Bradford method, with bovine serum albumin as standard.
Kinetics of killer toxin production.
Growth of P. membranifaciens was made in 6 l flasks with 3 l of complex medium (YMB) or minimal medium (YNB-D) buffered with 0·2 M citrate/phosphate, pH 4·0. The optical density of the cultures was determined in a spectrophotometer at 600 nm. Samples of 100 ml were taken under sterile conditions to measure killer toxin production. These were filtered through Millipore membranes (0·45 µm pore size) and precipitated with ethanol to a final concentration of 70 % (v/v). After centrifugation (7000 g, 0 °C, 10 min) the pellet obtained was resuspended in 0·2 M sodium citrate/phosphate buffer, pH 4·0 and killer activity was determined as above.
Improvement of killer toxin production.
Killer cells were inoculated from stock culture and grown in 250 ml Erlenmeyer flasks containing 100 ml of different media (YMB, YPD, modified Gorodkowa medium, YNB-D and K medium). These media were buffered to pH 4·0 with 0·2 M citrate/phosphate. The values shown were obtained in one of three independent experiments.
YNB-D medium was selected because it yielded the highest specific killer activity. To improve killer toxin production, it was supplemented with various additives and assayed at different incubation temperatures (20, 25 and 30 °C), pH values (3·0, 3·5, 4·0, 4·5, 5·0, 5·5 and 6·0) and shaking rates (100, 150, 200 and 250 r.p.m.) in a rotary bed shaker. The most representative additives and their concentrations are detailed in Results and Discussion. The experiments were repeated three times and mean values are presented.
To evaluate killer activity, 10 ml aliquots of the cultures at an OD600 of 1·0 were centrifuged (5000 g, 5 min, 4 °C). The supernatants were filtered through a Millipore membrane (0·45 µm pore size) and the protein in the supernatant was precipitated with ethanol to a final concentration of 70 % (v/v). The precipitate was recovered by centrifugation at 7000 g at 0 °C, for 10 min. The pellet thus obtained was then resuspended in 0·2 M sodium citrate/phosphate buffer, pH 4·0 and killer activity was estimated.
Temperature and pH stability of the killer toxin.
Samples of killer toxin (concentrated 75-fold) were incubated at a range of different temperatures: 5, 10, 15, 20, 25, 30 and 35 °C. Aliquots (40 µl) were removed at specific intervals and killer activity was assayed.
For stability to pH, concentrated toxin samples were adjusted with 0·1 M sodium citrate/phosphate buffer at a range of pH values between 3 and 7·5. The solutions were incubated at 20 °C for 1 h and killer activity was determined. The mean values of three independent experiments are presented.
Influence of temperature and pH on killer toxin activity.
Activity plates (YMA-MB, 6 % NaCl) were incubated at 4 °C for 24 h with the killer toxin (40 µl) from concentrated culture supernatants to ensure the complete diffusion. The plates were then seeded with the sensitive strain and incubated at different temperatures up to 35 °C.
To determine activity at different pH values, activity plates adjusted to pH values between 3 and 7 were seeded with the sensitive strain and incubated at 20 °C in the presence of aliquots of the killer toxin (40 µl). The inhibition zone was determined after 3 days of incubation. The mean values of three independent experiments are shown.
Effect of proteolytic enzymes.
The effects of the proteolytic enzymes Pronase, pepsin and papain (Sigma) on the killer activity of P. membranifaciens CYC 1106 were examined as described by Young & Yagiu (1978).
Fractionation of cell wall polysaccharides.
The extraction of cell wall polysaccharides, mannoproteins and glucans from mechanically disrupted cells of the sensitive yeast (Candida boidinii IGC 3430), together with the separation of the different glucan fractions, were accomplished following the conventional procedure reported by Fleet & Manners (1976). The (1
6)-
-D-glucan fraction was used in the subsequent purification of killer toxin.
Purification procedure.
P. membranifaciens CYC 1106 was cultured in YNB-D-Brij-58 medium (3x1 litre, in 2 l Erlenmeyer flasks) for 3 days, at 20 °C and 150 r.p.m. The cells were centrifuged (4000 g, 10 min, 4 °C) and the supernatant was adjusted to a final glycerol concentration of 15 % (v/v). The supernatant was then concentrated (40-fold) to a final volume of 75 ml by tangential ultrafiltration (Filtron Technology Corporation) with a 10 kDa cut-off membrane (Minisette membrane cassette, Omega type). Ice-cold ethanol was added to a final concentration of 45 % (v/v) and, following 30 min incubation at 04 °C, the resulting precipitate was separated by centrifugation (8000 g, 10 min, 0 °C). The proteins in the supernatant were precipitated by further addition of ice-cold ethanol up to a final concentration of 75 % (v/v). The resulting pellet was dissolved in 5 ml 1 mM sodium citrate/phosphate buffer (pH 4·0) and the solution was used for preparative isoelectric focusing in Ultrodex (Pharmacia). Active fractions from preparative isoelectric focusing (see Fig. 3) were then pooled and applied to a column for affinity chromatography in (1
6)-
-D-glucan-epoxy-Sepharose 6B. The eluted fractions (1 ml each) were assayed for killer activity and the active samples were used to further characterize the killer toxin.
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Preparation of (16)-
-D-glucan-epoxy-Sepharose 6B and affinity chromatography of the killer toxin.
Epoxy-Sepharose 6B affinity chromatography was accomplished as described previously with slight modifications (Hutchins & Bussey, 1983). The pre-swollen gel was coupled to (1
6)-
-D-glucan and was then washed several times with a 15 % glycerol solution in 0·01 M citrate/phosphate buffer (pH 3·5) before use. The killer toxin from preparative isoelectric focusing was added to the column (1·9x3·8 cm) and eluted with 10 mM citrate/phosphate buffer (pH 5·0) containing 15 % (w/v) glycerol and 1·5 M NaCl (see Fig. 4
). The fractions were collected, treated and assayed for killer activity as described above.
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Native PAGE.
The active fractions from affinity chromatography were electrophoresed in a discontinuous acid polyacrylamide gel prepared in a 1·5 M acetate/KOH buffer, pH 4·3. The dissolving buffer was 0·25 M acetate/KOH, pH 5·5. Samples were electrophoresed for 12 h, 75 V, at 4 °C. Methyl green was used as the tracking dye. To determine the activity of the killer toxin, half of the gel was cut into slices (2 mm) and these were transferred to YMA-MB plates seeded with the sensitive strain. For the visualization of protein bands, the other half of the gel was stained with Coomassie brilliant blue R-250.
Determination of the isoelectric point.
Isoelectric focusing was performed at 4 °C in polyacrylamide gels (12 cmx6·5 cmx0·4 mm) containing ampholytes (0·5 ml), pH range 2·55·0 (Pharmalyte), 2 ml of acrylamide/bisacrylamide solution (24·25 %/0·75 %), 2 ml glycerol (20 %, v/v), 150 µl ammonium persulphate (10 %, w/v), 35 µl TEMED (N,N,N'N'-tetramethylethylenediamine) and distilled water (5·5 ml). Pre-focusing was carried out for 15 min, using a constant voltage of 125 V. Samples of the purified killer toxin were then added to Whatman no. 3 paper strips applied directly onto the gel surface. Focusing was carried out with the following time/voltage sequences: 15 min/100 V, 15 min/200 V and 45 min/450 V. To determine the isoelectric point of the killer toxin, half of the gel was cut into slices (22·5 mm) and these were transferred to Eppendorf tubes and extracted, overnight at 4 °C, with 1 ml demineralized water. The pH in each tube was measured. For visualization of protein bands, the other half of the gel was stained with Coomassie brilliant blue R-250 as follows. Upon completion of the focusing experiment, the gel was first fixed in 5 % (w/v) sulphosalicylic acid plus 10 % (v/v) trichloroacetic acid for 60 min and then incubated in destaining solution [methanol : acetic acid : distilled water (3 : 1 : 6)] for 30 min. Gel staining was accomplished over 3 h in the same solution containing 0·2 % Coomassie brilliant blue R-250. The gels were then destained until the background had become clear.
Infection on Vitis vinifera.
Disease-free plants were grown under 200 µE m2 s1 white fluorescent light, 16/8 h photoperiod and 20 °C day/night temperature. Infection was implemented in five sets of six plants. Spores, yeast cells and toxin were resuspended in 0·1 M citrate/phosphate buffer, pH 4·8. One set was inoculated with B. cinerea, placing 10 µl of a spore suspension (105 spores ml1) on the back of the leaves. The second set was inoculated with 10 µl of a mixture of B. cinerea (105 spores ml1) and P. membranifaciens CYC 1106 (105 cells ml1). The third set was inoculated with 10 µl of a mixture (105 spores ml1) of B. cinerea mixed with P. membranifaciens CYC 1106 purified killer toxin (576 AU ml1). The fourth set was inoculated with 10 µl of a P. membranifaciens CYC 1106 suspension (105 cells ml1). Growth parameters and the development of infection were compared with those of a fifth set of non-inoculated plants. Three independent experiments were carried out.
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RESULTS AND DISCUSSION |
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Once the protein nature of the toxin produced had been established, the secreted protein was purified from the supernatant of a growing culture of P. membranifaciens at the early stationary phase. Preliminary experiments revealed that the synthetic medium YNB-D supplemented with the non-ionic detergent Brij-58, known to be useful in the isolation of functional membrane complexes, gave the highest specific activity in the supernatant. This was used as a starting point to purify the killer toxin. The killer toxin which accumulated in the culture fluid increased as growth progressed and then levelled off as the culture reached the stationary phase. The final toxin activity yield was twofold higher in cultures in YMB (5·25 AU ml1), which contained yeast extract, malt extract and peptone, than in YNB-D. Similar observations have been reported previously (Palfree & Bussey, 1979; Woods & Bevan, 1968
). The specific growth rates in both cases were different: 0·30 h1 for YMB and 0·18 h1 for YNB-D. The production of killer toxin on complex media (YMB, YPD and modified Gorodkowa medium) and minimal media (YNB-D and K) containing mineral salts, trace elements and growth factors was compared. Although production in the YMB medium (the best complex medium as regards toxin production) was severalfold higher than in YNB-D medium (the best minimal medium as regards toxin production), specific activity was much higher in the latter case (120·2 AU mg1 for YMB and 831 AU mg1 for YNB-D). Accordingly, YNB-D medium was chosen to optimize production, which reached the highest activity at pH 4·0, 20 °C and 150 r.p.m. The production of killer toxins may be strongly affected by the culture conditions and optimal conditions may need to be found empirically. Toxin production depends on the nitrogen source supplied to the growth medium and, in particular, yeast extract may be stimulatory. Thus, we found a much higher production in YMB than in YNB-D medium. The preparations of P. membranifaciens CYC 1106 in minimal medium were sensitive to mechanical shaking, as evident from the decreasing yield of toxin activity when killer cells were cultured with increasing shaking. The addition of glycerol, sorbitol and polyethylene glycol is known to prevent inactivation in active toxin solutions (Ouchi et al., 1978
; Ohta et al., 1984
). The influence of these stabilizing agents, some non-ionic and ionic detergents, and protease inhibitors was studied. The non-ionic detergents Brij-58, Triton X-100, Pluronic F-127 and Tween-80 enhanced the killer toxin activity found in the culture supernatant after growth. By contrast, polyethylene glycol (400, 1500 and 6000), glycerol and sorbitol were less effective. SDS inhibited killer toxin production and growth. The optimal concentration for toxin production was determined in each case. At a concentration of 0·01 % (w/w) Brij-58 was the most effective agent in the enhancement of killer toxin production (351 %).
Many reagents are known to stabilize certain enzymes, including organic solvents (ethanol, butanol, acetone, dimethylsulfoxide, etc.), polyhydric alcohols (ethylene glycol, glycerol, mannitol, etc.), and salts and minerals (ammonium sulphate, Mg2+, Ca2+). The present investigation revealed that glycerol, sorbitol, polyethylene glycol (PEG 400, 1500, 6000) did not significantly improve killer toxin production at the concentrations tested. However, Ouchi et al. (1978) found that glycerol (20 %) was very effective for the production of killer toxin.
The ski5 mutation that results in the loss of PMSF-inhibitable exocellular protease in S. cerevisiae leads these mutants to have a superkiller phenotype (Tipper & Bostian, 1984). In our killer strain, P. membranifaciens CYC 1106, PMSF did not improve killer toxin production significantly at the concentrations tested.
(16)-
-D-Glucan linkages in cell walls are receptors in the binding of the toxin to sensitive cells (Hutchins & Bussey, 1983
; Santos et al., 2000
). Killer cells are immune but nonetheless have these linkages, and it is therefore obvious that some secreted toxin is bound by (1
6)-
-D-glucan linkages and does not appear in the extracellular medium. The kre1 mutants, which largely lack this linkage, secrete an increased amount of toxin and are superkillers. The detergents Brij-58, Triton X-100, Pluronic F-127 and Tween-80 enhanced the killer toxin activity found in the culture supernatant after growth and the specific growth rates in these cases were not significantly different. The effect of these agents on killer toxin production could be attributed to the stabilization of the killer toxin produced during growth or to an interference with some process, such as adsorption [by (1
6)-
-D-glucan linkages] or secretion.
The stability of all killer toxins is strongly dependent on pH and temperature, and mechanical shaking is destructive. These common properties are consistent with the proteinaceous nature of killer toxins, which is also evident from the susceptibility of most toxins to proteolytic enzymes. Most toxins are irreversibly inactivated above pH 5·0 and are stable only in a narrow pH range (Bevan et al., 1973; Chen et al., 2000
; Marquina et al., 2001a
; Middelbeek et al., 1979
; Pfeiffer & Radler, 1984
), although the killer toxin from Hansenula saturnus (Ohta et al., 1984
) has a broad stability range. Moreover, differences in other properties indicate that the toxins of several yeasts are biochemically distinct (Pfeiffer & Radler, 1984
). The killer toxin produced by P. membranifaciens CYC 1106 was stable only within a narrow pH range (3·04·8) (Fig. 1
) and activity was rapidly lost at temperatures above 20 °C in liquid medium (Fig. 2a
). These findings are in accordance with the loss of killer toxin production in pH and temperatures during growth above these values.
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ACKNOWLEDGEMENTS |
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Received 29 January 2004;
revised 7 April 2004;
accepted 22 April 2004.
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