Biological properties and spectrum of activity of tolaasin, a lipodepsipeptide toxin produced by the mushroom pathogen Pseudomonas tolaasii

Physiological and Molecular Plant Pathology (1991) 39, 57-70

57

Biological properties and spectrum of activity of tolaasin, a lipodepsipeptide toxin produced by the mushroom pathogen Pseudomonas tolaasii PAUL B . RAINEYt, CATHERINE

L . BRODEY+ and KEITH , JOHNSTONE

Bolanv School, University of Cambridge, Downing Street, Cambridge CB2 3EA, U .K (Accepted fbr publication May 1991)

She biological properties and spectrum of activity of tolaasin, a lipodepsipeptide toxin produced by the mushroom pathogen Pseudomonas tolaasii, were investigated . An erythrocyte lysis bioassay was developed and used to assay tolaasin activity . Tolaasin-induced haemolysis was dose dependent, maximal between pH 6 .0 and 7 . 0, increased with temperature and was inhibited by the addition of divalent metal ions . Tolaasin production commenced during exponential growth ofP. tolaasii and continued into stationary phase . Culture filtrate from wild type P. tolaasii was able to pit mushroom tissue and lyse erythrocytes, whereas culture filtrate from a tolaasin defective mutant had no lytic activity . Ultrastructural studies showed disruption of the Agaricus bisporus plasma membrane and vacuole membranes by both P. tolaasii and tolaasin, but not by a tolaasin defective mutant . Pre-incubation of tolaasin with multilamellar liposomes resulted in loss of lytic activity and osmotic protectants prevented tolaasin-induced haemolysis, demonstrating that tolaasin partitions into membranes, forms pores in erythrocyte membranes and causes lysis by a colloid osmotic mechanism . Tolaasin was phytotoxic when infiltrated into leaves of Vicotiana tabacum and was shown to be active against a range of basidiomycetes and Gram-positive bacteria . Gram-negative bacteria were resistant to tolaasin, but became susceptible when treated concomitantly with sub-minimal inhibitory concentrations of polymyxin B . P . tolaasii was resistant to tolaasin even in the presence of polymyxin B .

INTRODUCTION

Paine is the causal organism of the economically significant brown blotch disease of the cultivated mushroom Agaricus bisporus (Lange) Imbach [31] . Colonization of mushroom basidiocarps by the bacterium results in unsightly brown lesions which render affected mushrooms unmarketable . P . tolaasii produces an extracellular toxin [18] which was partially purified from culture filtrates by Peng [22] . Peng reported that the toxin is a heat stable polypeptide with a molecular weight of less than 10000 and demonstrated that addition of partially purified toxin to mushroom basidiocarps reproduced the disease symptoms of the intact organism . Peng [22] also showed that the toxin was responsible for precipitating the white line inducing principle in the diagnostic white line in agar test [34] . T Present address of senior author to whom correspondence should be addressed : NERC Institute of Pseudomonas tolaasii

Virology and Environmental Microbiology, Mansfield Road, Oxford OXI 3SR, U .K . ; $SERO, Polaris House, North Star Avenue, Swindon, Wiltshire, SN2 IET, U .K . Abbreviations used in text : PAF, Pseudomonas Agar F ; CMM, compost malt medium ; PBS, phosphate buffered saline ; PEG, poly(ethylene glycol ; ; MIC, minimal inhibitory concentration .

0885-5765/91/070057+14 $03 .00/0

rc, 1991 Academic Press Limited '



58

P . B . Rainey

et al .

he structure of the toxin, tolaasin . has recently been determined 119 J and n a lipodepsipeptide (mol wwt = 1985i consisting of 18 amino acid residues with a /3octanoie acid group' at the 4'-terminus . A sequence of 'n-amino acids causes part of the molecule to form a novel left handed a-helical structure in a SI)S solution J171 . T

Tolaasin bears some similarity to low molecular weight membrane active toxins, such as nisin [121 and alamethicin [20] and possesses attributes typical of membrane penetrating compounds, in particular a hydrophobic JV-terminus and a central amphipathic a-helix [32 . 101 . Electron microscopy has shown that 1' . tolaasii causes disruption of A . bisporus plasma membranes [8] and recent studies have revealed the ability of tolaasin to form voltage-gated, cation-selective, ion channels in planar lipid bilayers [7] . We report here on the biological properties and spectrum of activity of tolaasin .

MATERIALS AND METHODS Strains, media and culture conditions P. tolaasii NCPPB 1116 and P . reactans NCPPB387 were obtained from the National Collection of Plant Pathogenic Bacteria, Harpenden, U .K . PT106 is a tolaasinnegative strain derived from NCPPB 1116 by transposon Tn5 mutagenesis using Escherichia coli donor strain S17-1 containing the suicide vector pSUP1011 [241 . Tolaasin-defective mutants were detected using the white line in agar test [34] . PT106 contains a single copy of Tn5 in a large gene cluster located on a 640 kb

Pacl

chromosomal fragment . PT106 synthesizes a truncated form of TL1, one of three high molecular weight (approx . 500 kD) proteins involved in tolaasin biosynthesis (P . B . Rainey & C . L . Brodey, unpublished data) . Bacillus subtilis 1604, B . megaterium KM, Micrococcus lysodeikticus, Escherichia coli HB101, E. coli ED8767, Pseudomonas putida PMS118, P. tolaasii PMS117, P. tolaasii 255/2 and Xanthomonas campestris were obtained from the culture collection of the Microbial Physiology Group, Botany School, University of Cambridge . Bacteria were cultured in Pseudomonas Agar F (PAF) broth, or L broth at 28 ° C, or 37 °C . Basidiomycete cultures were obtained from the culture collection at the Institute of Horticultural Research, Littlehampton, West Sussex, U .K . Agaricus bisporus U3, A . bitorquis W19, Coprinus cinereus R17, Flammulina velutipes R44, Lentinus edodes R127 . Pleurotus cornucopiae R38, P . cystidiodies R45, P. ostreatus R155, P. sqJor-caju R139 and Volvariella bombycina R83, were grown on compost malt medium (CMM) [23] in 9 cm diameter Petri dishes at 25 ° C . Toxin preparation Tolaasin was isolated and purified by a modified method of Peng [22] which is reported in detail elsewhere [19] . The toxin (tolaasin) preparation used for the work described here contained 50 % tolaasin by weight (determined by HPLC) and was obtained by partial purification of the crude toxin preparation by ion exchange chromatography using a Dowex-1 anion column [19] . Fractionation of the ion exchange toxin preparation by reverse-phase HPLC using a S5P (phenyl) column revealed a number of peaks [19 ] . Assay of these fractions using mushroom tissue and erythrocytes revealed that the only active component was tolaasin [19] . Tolaasin was prepared fresh before



Biological properties of tolaasin

59

each experiment by dissolving 1 mg of toxin preparation in 1 ml of sterile distilled water . Production of tolaasin during growth of P . tolaasii in liquid culture P . tolaasii and P. tolaasii PT106 were each cultured in 3 litre flasks containing 1 litre PAF in an orbital incubator at 25 ° C . The number of viable bacteria were determined at each sampling time using the drop method of Miles & Misra [14] . The turbidity of the solution was also recorded at each sampling time by measuring the absorbance at 600 nm . Samples (1 ml) were taken from the flasks at regular intervals, the cells removed by centrifugation (16000 g, 30 s, room temperature) and filtration (0 . 22 µm filter), and the supernatant lyophilized . Known volumes of sterile distilled water were added to the lyophilized culture filtrates and 5 µl samples applied to blood agar plates agar, 50 ml of defibrinated horse [10 mm phosphate buffer (pH 7 . 0), 0 . 9 % NaCl, 1 . 5 blood per litre] to determine the haemolytic activity of the concentrated culture filtrates . Following incubation (18 h, 37 ° C) the amount of tolaasin was estimated by comparing the degree of haemolysis caused by the culture filtrates, with haemolysis caused by standards containing known amounts of tolaasin dissolved in liquid PAF . Controls of 20 times concentrated PAF were used . The activity of the culture filtrates was also verified on mushroom tissue using the mushroom pitting and browning tests described below . Erythrocyte assay A 0 . 2 ml sample of defibrinated horse blood was suspended in 0 .8 ml of phosphate buffered saline (PBS ; 10 mm phosphate, 0. 9 °% NaCl), briefly vortex-mixed, and the erythrocytes collected by centrifugation in a micro-centrifuge (16000 g, 10 s, room temperature) . Washing was repeated three times before suspending the cells in PBS to an absorbance at 600 nm (A,,,o ) of between 0 . 090 and 0 .095 . The assay was conducted at 37 ° C unless stated otherwise . Lysis was monitored by observing the change in optical density at 600 nm using a Pye Unicam PU 8600 spectrophotometer . Results are expressed as the change in Asoo min' . The effect of monovalent and divalent metal ions on toxin activity was determined by adding the metal ion to erythrocytes suspended in 10 mm Tris-HCI (pH 7 . 0), 0 . 9 % NaCl, to a final concentration of 10 mm . No significant pH change was observed upon addition of the metal ions at the concentrations tested . Osmotic protection experiments The method used was a modification of Weiner et al . [33] as described by Knowles & Ellar [13] . Erythrocytes were suspended in 10 mm phosphate buffer (pH 7 . 0) containing 300 mosmol sugar or poly(ethylene glycol) (PEG) (BDH, Mr 1000 or 1500) . Tolaasin was added and cell lysis assessed as described above . Pore size was estimated from a standard curve of rate of lysis v . the viscometric radius of the sugar . Effect of'plasma membrane lipids on tolaasin activity Liposomes were prepared from chromatographically pure lipids by the method of Thomas & Ellar [30] . Phosphatidyl choline and cholesterol were obtained from BDH, sphingomyelin and dicetylphosphate from Sigma Chemical Co ., and phosphatidyl ethanolamine from Lipid Products Ltd .



60

P . B . Rainey et al.

Lipid, cholesterol and dicetylphosphate were mixed in molar ratios of 4 : .`) : I, respectively . Dicetylphosphate way used to impart a net negative charge to the liposome which was considered appropriate given that tolaasin is positively charged at neutral pH . The mixture was dried as a thin film in vacuo in a 100 ml round bottomed flask and then resuspended with gentle shaking in 3 ml of 10 mm Tris-HCl (pH 7 - W . Liposomes were left to swell for 30 min at room temperature before sonicating for 1 min to give an even dispersion of vesicles . Liposomes were examined microscopically before use . Tolaasin was incubated with liposomes for 30 min (room temperature] at a tolaasin : phospholipid molar ratio of approx . 1 : 30 . Liposomes were removed from the solution by centrifugation (16000g, 20 min, room temperature) and the activity of the supernatant determined on erythrocytes and mushroom tissue to determine whether tolaasin had partitioned into the liposomes .

Transmission electron microscopy Freshly cut basidiocarps of A . bisporus were inoculated with 10 µl of a suspension of P. tolaasii, P . tolaasii PT 106 (1 x 10' cells ml - ', in sterile distilled water), or 10 ld of 'a 1 mg ml - ' tolaasin solution . Basidiocarps were incubated in humid chambers at 25 ° C for 18 h before cutting segments of tissue (2 x 2 x 4 mm) which were washed twice in sterile distilled water and fixed in 3 °' ;, glutaraldehyde, 0 . 075 M phosphate buffer (pH 7 . 2) for 3 h at 20 ° C . Samples were washed in phosphate buffer, post-fixed in I °,') Os0 4 in phosphate buffer (pH 7 . 2) for 3 h, washed again in buffer and dehydrated in an acetone series . Dehydrated material was infiltrated and embedded in Spurr's lowviscosity resin and ultra-thin specimens were cut using a L .K .B . `Nova' Ultrotorne . Specimens were stained with lead citrate and examined in a Philips EM 301 electron microscope at an accelerating voltage of 60 kV .

Mushroom pitting test The effect of P. tolaasii and tolaasin on cut mushroom tissue was determined by placing a 5 µl drop of an aqueous solution of tolaasin, or a 5-µl drop of' distilled water containing 10 s cells ml - ' of P. tolaasii, onto a block of mushroom tissue (10 x 10 x 2 mm) cut from fresh basidiocarps [34] . Pitting of tissue within 10 mill was recorded as a positive result .

Mushroom browning test The effect of tolaasin and P . tolaasii on mushroom caps was determined by placing either the toxin or the bacterium (in a 5 sl drop) onto the surface of the intact cap . The caps were incubated for 16 h at 28 ° C in a humid chamber and were examined for the appearance of browning and pitting .

Effect of P . tolaasii on growth of basidiomycetes Individual fungal cultures were grown on CMM until the colony diameter was 30 mm . The Petri dishes were then inoculated with P . tolaasii and tolaasin-negative mutant P. tolaasii PT106 on opposite sides of the dish . Plates were reincubated at 25 ° C and the effect of the bacterium on hyphal growth noted after a further 7 days growth .



Biological properties of tolaasin

61

Effect of tolaasin on growth of bacteria Microtitre plates were seeded with bacteria at I x 10 4 cells per well in L broth . Each horizontal row of the microtitre plate received a two fold serial dilution of tolaasin from 0 to 100 µg ml -r . Plates were incubated for 24 h at the temperature optimal for each bacterium before determing the minimal inhibitory concentration (MIC) of tolaasin . The effect of polymyxin B (Sigma, Poole, U .K ., 8000 U .S .I . units mg') was determined in microtitre plates as described above . In order to test the synergistic effects of tolaasin with polymyxin B, polymyxin B (0-2 .tg ml -') was added to each -t vertical column of the microtitre plate and tolaasin added to each row (0-100 lig ml ) . The MIC of tolaasin in the presence of polymyxin B was determined after 24 h of incubation .

Infiltration of leaf intercellular spaces with tolaasin Determination of the activity of tolaasin against leaf cells was made by infiltrating the intercellular spaces of leaves of Nicotiana tabacum and Solanum tuberosum with 100 µ1 volumes of an aqueous solution of tolaasin (0-1 mg ml -t ) . Infiltration was accomplished by forcing the solution from a syringe (without needle) into the underside of the leaf .

RESULTS

Production of tolaasin during growth of P . tolaasii and its effect on mushroom tissue Growth of P. tolaasii and P. tolaasii PT106 and production of tolaasin is shown

in Fig 1(a) and (b) . The sudden decrease in the number of viable cells (and corresponding

11-0 (a)

(b)

10. 0

4-0

9-0

3. 0

8-0

N 2 .0 0

C

C

I-0 a0

7-0 6. 0

0. 0 0 10 20 30 40 50 60 70 80 10 20 30 40 50 60 Time (h)

70 80

J

FIG . 1 . Growth of P. tolaasii NCPPB 1116 and production of tolaasin in PAF broth culture (a) and growth of P. tolaasii PTI06 and production of tolaasin in PAF broth culture (b) . Cell growth !colony forming units : cfu) (0 tolaasin (0 ; .

decrease in optical density results not shown) coincided reproducibly with the onset of tolaasin production by the wild type strain and toxin production continued exponentially in proportion to cell growth . An increase in the amount of tolaasin was noted during the stationary phase . Culture filtrates from the wild type organism caused rapid pitting of cut mushroom tissue and blotching of mushroom caps . No haemolytic



P . B . Rainey et al.

62

2

aw

0 .6

N T

0 0.4 6)

0 .2

0

1 .0 2 .0 3 . 0 4 .0 5 .0 6 . 0 7 .0 Tolaasin concentration (µg ml - ') FIG . 2 . Effect of tolaasin concentration on haemolysis of erythrocytes and dependence on erythrocyte concentration . (s), 1 x 106 cells ml-1 ; (A), 2 x 10 6 cells ml -t ; 4 x 10 6 cells ml t . Data are means+S E . of five replicates .

activity was detected in the culture filtrates from PT 106 and the filtrates did not induce rapid pitting, or blotching of mushroom tissue . Inoculation of intact P. tolaasii onto mushroom caps and cut mushroom tissue resulted in pitting and browning of the tissue . P . tolaasii PT 106 failed to cause any damage to either mushroom caps, or cut mushroom tissue (results not shown) . In fact, none of our 37 Tn5 generated tolaasin-defective mutants, or culture filtrates from them, are able to pit, or brown, mushroom tissue . Erythrocyte assay The effect of tolaasin concentration on lysis of horse erythrocytes is shown in Fig . 2 . The rate of lysis was directly proportional to toxin concentration and tolaasin activity was dependent upon erythrocyte concentration (Fig . 3) . Lysis was most rapid at an 0 .5

O

c) 0 0 .1 W

1 1 1 I 0.2 0.4 0 .6 0 .8 Optical density (A600) FIG . 3 .

tolaasin .

I •0

Effect of erythrocyte concentration on haemolysis of erythrocytes induced by

4

pg ml -



Biological properties of tolaasin

63

erythrocyte concentration giving an A600 of 0.4-0. 6 (4. 0-5 . 0 x 10 8 cells ml- '), however, sensitivity was greatest at low concentrations of erythrocytes (Asoo of 0. 1 ; 1 x 10 8 cells m1 - ') . Lysis occurred within 1-5 s of toxin addition and the lowest detectable level of activity was 1 . 5 .tg ml -1 (0 . 125 µM) tolaasin . Smaller amounts of tolaasin failed to cause detectable haemolysis, however, sequential addition of 0 . 25 .tg ml -1 aliquots of tolaasin revealed a marked threshold effect where activity was observed once the tolaasin concentration reached 1 . 0-1 . 5 pg ml -1 . Haemolytic activity was reduced by 90" 0 after storage of tolaasin in water 1 mg ml-1 ) at room temperature for 24 h and activity was eliminated by a single freeze--thaw cycle . The activity of tolaasin on erythrocytes was greatest at pH 6 . 5-7 . 0 and both increasingly acidic and alkaline conditions reduced lytic activity (Fig . 4) . Temperature 0-4

0. 0

5. 0

6-0

7.0 pH

8 .0

9 .0

FIG . 4 . Effect of pH on haemolysis of erythrocytes induced by 4 pg m1 - ' tolaasin . Data are means + S . E . of five replicates .

also affected the rate of tolaasin-induced erythrocyte haemolysis which was most rapid at high temperatures and eliminated at 5 ° C (Fig . 5) . Divalent metal ions were effective inhibitors of erythrocyte haemolysis . The most potent inhibitor was Zn" followed by Mn", Coe+ Mg` and Ca" . Zn" prevented haemolysis of tolaasin treated erythrocytes (4 µg ml - ' tolaasin) when present at a concentration of 10 mm (Fig . 6) . Neither monovalent metal ions, nor the chloride, or iodide ions, affected haemolysis of erythrocytes . To determine whether inhibition of toxin activity by Zn" was due to binding of the cation to the toxin molecule, or binding of Zn` to the blood cells, tolaasin and erythrocytes were washed in PBS and water, respectively, containing 10 mm ZnCl 2 . Unbound zinc was separated from tolaasin using a Sephadex G-10 column (5 x 50 mm) and excess zinc was removed from erythrocytes by pelleting the cells in a microfuge and washing with PBS . Pre-incubation to tolaasin with Zn` did not affect its ability to lyse erythrocytes and cells pre-treated with Zn" were no less susceptible to tolaasininduced lysis than untreated cells . Inhibition of lytic activity by Zn" was only observed when both tolaasin and the cation were present together in solution .



0

10

20 30 40 50 60 Temperature (°C)

Fce . 5 . Effect of temperature on haemolysis of erythrocytes induced by 4 pg ml - ' tolaasin . Data are means + S .E . of five replicates . 0 .3

0.0

O C 0 U

0 U Z Y

Y

U 0 U

U U U 0

U

U U C C N

Fic . 6. Effect of monovalent and divalent metal ions on tolaasin-induced haemolysis of erythrocytes. Salts were added to a final concentration of 10 mm and tolaasin was added at 4 pg ml - ' . Data are means ± S .E . of five replicates . Effect of osmotic protectants on erythrocyte haemolysis

Experiments reported elsewhere [7] have shown that tolaasin is an ion channel forming peptide . In order to gain evidence of pore formation by tolaasin in erythrocyte membranes and to obtain an estimate of the size of the pore, osmotic protection experiments were conducted using a range of varying molecular weight compounds . Colloid osmotic lysis can be prevented by compounds which are unable to penetrate induced membrane leaks and instead block the leak, preventing influx of ions and water which would ordinarily result in cell lysis . Protection against tolaasin-induced haemolysis was shown to be a function of molecular weight (Fig . 7) . PEG 1000 was the smallest molecule providing complete protection against haemolysis indicating that pores formed by tolaasin in erythrocyte membranes have a radius of 0 . 6-1 . 0 nm [13] .



65

Biological properties of tolaasin 2 .0

E 0 0 a 4 N

I .0

T 0

a) O

0 .0 0 . 2 0.4 0 .8 1 .0 0.6 Viscometric radius (nm)

1 .2

FIG . 7 . Relationship between tolaasin-induced haemolysis of erythrocytes in the presence of colloid osmotic protectants and the viscometric radii of these products . A, arabinose ; G, glucose ; S, sucrose ; L, lactose ; R ., raffinose ; P, poly (ethylene glycol) 1000 ; Q) poly (ethylene glycol) 1500 . Lysis was induced by the addition of 4 .tg ml - ' tolaasin and data are means of three replicates .

To further demonstrate the colloid osmotic nature of tolaasin-induced haemolysis and to determine whether tolaasin bound irreversibly to the cells, erythrocytes were pre-treated with tolaasin in the presence of PEG 1000, and the toxin removed by pelleting the erythrocytes . When the erythrocytes were resuspended in PBS without PEG 1000, lysis occurred, demonstrating both the irreversibility of tolaasin binding and the colloid-osmotic nature of tolaasin-induced haemolysis . The small size of the tolaasin molecule indicates that pores formed in membranes are most likely due to aggregation of monomers . The involvement of more than a single molecule in pore formation is also indicated by the threshold effect described above . It is possible that the diameter of the pore depends upon the number of tolaasin monomers which aggregate and this may be dependent on the concentration of tolaasin . To examine this we added up to 60 µg ml - ' tolaasin to erythrocytes incubated in the presence of PEG 1000, but failed to observe lysis which suggests that the diameter of the pores formed by tolaasin do not exceed a radius of 0 . 6-1 . 0 nm . Interaction of tolaasin with membranes Pre-incubation of tolaasin with sonicated dispersions prepared solely from phosphatidyl choline, phosphatidyl ethanolamine, or sphingomyelin was sufficient to eliminate toxin activity . Multilamellar liposomes were made in an attempt to provide a lipid structure indicative of that found in plasma membranes . Liposomes comprising lipid (phosphatidyl ethanolamine, phosphatidyl choline, or sphingomyelin) plus cholesterol and dicetylphosphate also effectively neutralized both the haemolytic activity of tolaasin and its ability to pit mushroom tissue . Treatment of mushroom caps with tolaasin led to marked disruption of the plasma membrane and vacuole membranes [Figs 8(a) and (b)] and collapse of the cells walls . This damage was identical to that caused by P. tolaasii [Fig . 8(c)] . P. tolaasii PT 106 did not cause any visible ultrastructural change to the hyphae [Fig . 8(d) ] . MPP .39



66

P . B . Rainey et al

FIG . 8 . Eff t oftoh .sin ~a, b` 1' . tolaa . ,, NCPPB 1116 :ci and 1' . Iola, ii NCPPB 1116 : 'I 'n5 mutant PT106 (d ; on .-1 . bispor___ ap hyphae . HW . hyphal wall, P'1', P. tola .___i. Note membrane disruption ;arrows) and loss of hyphal contents caused by tolaasin and P. tolaasii, but not by 1'n5 mutant PT106 . Scale bars = I pm .

TABLE. I .Synergism between polymy.zin B and tolaasin

MIC in combinationa Polymyxin B MIC (µg ml -t l

Tolaasin MIC (µg m1'

Polymyxin B (µg ml -'`

Tolaasin pg ml - ' .

Gram negat e P . tolaasii P . reaclan)

P . putida

E. coli

> 200 0 .9

2.0

> 200 > 200

> 200

0 .4

> 200

NA NA

6. 25 3.5 6 . 25

1, 0. 6 0. 7 0. 8 1.4 1.6 1.8 0. 2 0. 25 0. 3

125 6 . 25 3-13 100

25 1-5 12 . 5 3-13 1 .5

Gram positive B. subtilis B . megaterium Al.lysodeikticus

NA

NA NA NA

N A NA NA

NA, not applicable . 'MIC of tolaasin in the presence of different concentrations of polymyxin B °Growth of P. tolaasii was not inhibited by 30 µg ml - ' tolaasin and 30 pg ml -' polymyxin B .



Biological properties of tolaasin

67

Effect of P . P . tolaasii

tolaasii, P . tolaasii PT106 and tolaasin on micro-organisms and leaf tissue inhibited the growth of all fungal cultures examined, but the tolaasin defective mutant P. tolaasii PT 106 failed to impede hyphal growth . The inhibition zone resulting from the production of tolaasin extended 5-10 mm from the margin of the P. tolaasii colony . The effect of tolaasin and polymyxin B on growth of bacteria is shown in Table 1 . Tolaasin inhibited the growth of Gram-positive bacteria, but was ineffective against the Gram-negative bacteria E . coli, P . tolaasii, P . putida and P . reactans . Polymyxin B inhibited the growth of all Gram-negative bacteria tested, except P . tolaasii, which grew in the presence of 200 ltg ml - ' . Gram-negative bacteria, with the exception of P . tolaasii, were sensitive to tolaasin when polymyxin B was included in the growth medium at a sub-MIC concentration (Table 1) . The minimum concentration of tolaasin causing cytolysis of Nicotiana tabacum and Solanum sp . leaf cells was 10 tg ml - ' . Necrosis was evident by clearing of regions infiltrated by tolaasin and was distinct from damage caused by water controls .

DISCUSSION The inability of P . tolaasii PT 106 and culture filtrates from P . tolaasii PT 106 to cause haemolysis of erythrocytes, browning and pitting of mushroom tissue, or disruption of the A . bisporus plasma membrane, indicates that tolaasin is solely responsible for both haemolysis of erythrocytes and disease symptoms on mushrooms . Further evidence for the decisive role of tolaasin is provided by the ability of the purified toxin to lyse erythrocytes and reproduce the symptoms of brown blotch disease in situ . Toxin production by P. tolaasii during growth in liquid culture was estimated by Nair & Fahy [18] by the degree of browning of mushroom tissue blocks caused by culture filtrates . These authors showed that toxin was produced during the early part of exponential growth, but ceased during late exponential growth . Results from our work show that production of tolaasin by P. tolaasii NCPPB 1116 was not induced until the mid-exponential phase and tolaasin export continued to increase throughout the stationary phase . The cause of the sudden bacterial lysis during exponential growth is not known . Our inability to observe this phenomenon in other P. tolaasii strains (PMS 117 and 255/2 and the normal growth curve depicted by Nair & Fahy [18], suggest that the correlation between cell lysis and the onset of tolaasin production may not be significant . Recently, Sonnen et al . [25] described the occurrence of marked cell lysis in Brevibacterium flavum cultures and showed that an inducible phage particle was responsible . The erythrocyte assay proved to be a reliable and simple means of investigating aspects of tolaasin activity in vitro . The dependence of lysis upon erythrocyte concentration suggests that lysis (at constant tolaasin concentrations) is dependent upon the rate at which tolaasin molecules insert into, and/or aggregate within, membranes . The reduced rate of lysis at high erythrocyte concentrations is probably due to a reduced population of tolaasin molecules per erythrocyte and indicates that a minimum number of molecules are required per erythrocyte to induce lysis . This -z



68

P . B . Rainey et al.

result also suggests that tolaasin molecules are unable to dissociate from the membrane once inserted . Insertion of the tolaasin molecule into a membrane and its activity within the membrane is likely to be dependent on the charge of individual residues on the molecule . Tolaasin does not contain any groups ionizable over the pH range 6 . 0-8 . 5 [19], and therefore the pH dependent nature of erythrocyte lysis probably reflects differences in ionizable groups on the erythrocyte surface . The apparent exponential increase in erythrocyte haemolysis with increasing temperature is probably the result of a combination of increased membrane fluidity and more rapid diffusion of tolaasin at elevated temperatures . The inhibitory effect of divalent metal ions on the activity of pore forming haemolytic agents, such as certain membrane disrupting bacterial and animal toxins, has been well documented [3, 4, 21 ] . The ability of divalent metal ions to inhibit the activity of tolaasin on erythrocytes strongly suggests that tolaasin-induced erythrocyte haemolysis results from the formation of pores in erythrocyte plasma membranes . Divalent metal ions are thought to exert their inhibitory effect by binding to negatively charged groups on the extracellular side of the plasma membrane, near the site of pore formation [3, 4] . Zn" is frequently found to be the most effective protector against membrane damage by haemolytic agents, followed by Ca" and Mg t + [3] . A similar trend was observed with tolaasin, although Mg 2- afforded slightly better protection than Ca" . It was interesting to note that Zn 21 was only ever effective at preventing tolaasin-induced haemolysis when present in solution concomitantly with the toxin . This suggests that the inhibitory effect of Zn 21 cannot be due to the prevention of tolaasin binding, but a result of the closure of already formed pores . This is consistent with the hypothesis of Bashford et al . [4] to account for the protective effect of divalent metal ions . Osmotic protection experiments demonstrated the colloid-osmotic nature of tolaasininduced erythrocyte lysis . The estimated size of the pore formed by tolaasin is similar to that reported for the P . aeruginosa cytotoxic protein [33] and for several Bacillus thuringiensis 6-endotoxins [13] . The mechanism of action of tolaasin in vivo has not been directly examined, and the constraints afforded by the A . bisporus cell wall may not permit swelling, a necessary prerequisite for colloid-osmotic lysis . It is possible that cellular damage may result from disruption of the electrochemical gradients which would lead to a nett loss of salt, loss of turgor and collapse of cells and hyphae . Alternatively, cellular disruption may be caused by complete perforation of cellular membranes . Irrespective of the mechanism of action in vivo, Figs 8(a) and (b )) and the electron micrographs of Cole & Skellerup [8] show that the A . bisporus plasma membrane and vacuole membranes are sites of tolaasin action in vivo . The ability of plasma membrane lipids to neutralize tolaasin activity provides further evidence that plasma membranes constitute the cellular target of tolaasin . The ability of three different lipids and cholesterol to effectively inhibit haemolytic activity and mushroom pitting may be indicative of the broad spectrum of tolaasin activity . Tolaasin bears some structural similarity to other low molecular weight peptide toxins produced by plant pathogenic pseudomonads [19], such as phaseolotoxin [16], tabtoxin [26], tagetitoxin [15], syringomycin [9], and syringotoxin [2], although the relatively high molecular weight of tolaasin and the small size of its lactone macrocycle



Biological properties of tolaasin 69 render it unique [19] . Like tolaasin, syringomycin and syringotoxin also display a broad spectrum of activity and are active against a range of organisms including certain bacteria, fungi and plants [1, 11] . Both syringomycin and syringotoxin exert their primary effect through disruption of cell membranes [1, 5, 6, 28] however, unlike tolaasin, they are not ion channel forming peptides [6] and syringomycin is thought to exert its influence through regulatory effectors, or covalent modification of ATPase [6], or through alterations in Ca" transport [29] . The difference in structure between Gram-positive and Gram-negative bacterial cell walls probably accounts for the observed differences in sensitivity of bacteria to tolaasin . The ability of low concentrations of polymyxin B, an antibiotic that disrupts the barrier properties of the Gram-negative outer membrane [27], to cause tolaasin sensitivity in Gram-negative bacteria, suggests that tolaasin resistance is a function of the outer membrane . The extreme resistance of P . lolaasii to polymyxin B suggests that its outer membrane has an atypical composition . We are grateful to J . C . Nutkins, R . J . Mortishire-Smith and Dr D . H . Williams for helpful discussions and Mr B . Chapman for assistance with electron microscopy . Fungal cultures were kindly provided by M . Challen . We thank the Gatsby Charitable Foundation for a Research Studentship (C .L .B .) and the AFRC for a project grant 'PG8/509) . The work was performed under provisions of licence number PHF 174A/91(28) issued by the Ministry of Agriculture, Fisheries and Food under the Plant Health (Great Britain) Order 1987 . This work was conducted within the SERC Cambridge Center for Molecular Recognition .

REFERENCES 1 . BACKMAN, P .A . & DE VAY, J .

syringomycin .

E . (1971) . Studies on the mode of action and biogenesis of the phytotoxin 215-233 .

Physiological Plant Pathology 1,

2 . BALLIO, A ., BOSSA, F ., COLLINA, A ., GALLO, M ., IACOBELLIS, N . S ., PACT, M ., PUGCI, P ., SCALONI, A ., SECRE, A . & SIMMACO,

M . (1990) . Structure of syringotoxin, a bioactive metabolite of Pseudomonas 377-380 .

syringae pv syringae . FEBS Letters 269,

3.

BASHFORD, C . L., ADLER, G . M ., MENESTRINA, G ., MICKLEM, K . J ., :MURPHY, J . J . & PASTERNAK, C . A .

1986) . Membrane damage by hemolytic viruses, toxins, complement, and other cytotoxic agents . 9300-9308 . 4 . BASHFORD, C . L ., RODRIGUES, 1 . & PASTERNAK, C . A . (1989) . Protection of cells against membrane damage by haemolytic agents : divalent cations and protons act at the extracellular side of the plasma membrane . Biochimica et Biophysica Acta 983, 56-64 . 5 . BIDwAI, A . P ., TAKEMOTO, J . Y . (1987) . Bacterial phytotoxin syringomycin induces a protein kinase mediated phosphorylation of red beet plasma membrane polypeptides . Proceedings of the National Academy of Sciences of the USA 84, 6755-6759 . 6 . BIDWAI, A . P ., ZHANG, L . & BACHMANN, R . C . (1987) . Mechanism of action of Pseudomonas syringae phytotoxin syringomycin : stimulation of red beet plasma membrane ATPase activity . Plant Physiology 83, 39-43 . 7 . BRODEY, C . L ., RAINEY, P . B ., TESTER, M . & JOHNSTONE, K . (1991) . Bacterial blotch disease of the cultivated mushroom is caused by an ion-channel forming lipodepsipeptide toxin . Molecular Plant-Microbe Interactions (in press .' . 8 . COLE, A. L . J . & SKELLERUP, M . V . (1986) . Ultrastructure of the interaction of Agaricus bisporus and Pseudomonas tolaasii . Transactions of the British Mycological Society 87, 314-316 . 9 . FIKUCHI, N ., 1sOGAI, A ., YAMASHITA, S ., SUYAMA, K ., TAKEMOTO, J . Y . & SUZUKI, A. (1990) . Structure of phytotoxin syringomycin produced by a sugar cane isolate of Pseudomonas syringae pv . syringae . Tetrahedron Letters 31, 1589-1592 . 10 . GIERASCH, L . M . (1989) . Signal sequences . Biochemistry 28 . 923-930 . Journal of Biological Chemistry 261,



70

P . B . Rainey

et al .

11 . GONZALEZ, C . F ., DE VAv,,J . 1: . &''VAKEMAN, R . J . 19811 . Syringotoxin : a phytotoxin unique to gurus isolates of Pseudomonas syringae . Physiological Plant Pathology 18, 41 50 . 12 . GROSS, E . & MOREL .L, ,J . 1 . . ; 1971 fhe SO UCIure of nisin . journal of the American C. 'heinn a! Soricti' 93, 4634--4635 . 13 . KNOwLES, B . H . & ELLAR, D . J . ( 1987 S . Colloid osmotic Ivsis is a general feature of the in(-( hanism (it action of Bacillus thuringien .sis delta endotoxins with different insect specificity . Btochimica et Biop/iysisa Acta 924, 509-518 . 14 . MILES, A . A . & MISRA, S . S . (1938 ) . The estimation of the bactericidal power of the blood . Journal nj Hygiene, Cambridge 38, 732 749 . 15 . MrIUHELL, R . E ., CoDDINGTON . J . M . & YOUNG, H . 1989 A revised structure for tagetitoxin . Tetrahedron Letters 30, 501-504 . 16 . MOORE, R . E., NIEMCZURA, W . P ., KwoK, 0 . C . H . & PATI,. S . S . t 1984 ; . Inhibitors of' ornithine carbamoyltransferase from Pseudomonas ._svringae pv . paseolicola . Revised structure of phaseolotoxin . Tetrahedron Letters 25, 3931-3934 . 17 . MORTISHIRE-SMITH, R . J ., DRAKE, A . R ., NU'rKINS, J . C . & WILLIAMS, D . H . 11991 ) . Left-handed a-helix formation by a bacterial peptide . EBBS Letters 278, 244-246 . 18 . NAIR, N . G . & FAHY . P . C . (1973 . Toxin production by Pseudomonas lolaasii Painc Australian 7ou7nal o/ Biological Sciences 26, 509-512 . 19 . NCTKINS, J . C ., MORTISHIRE-SMITH, R . J ., PACKMAN, L . C ., BRODEY, C . L ., RAINEY, P . B ., JOHNS'toNL, K . & WILLIAMS, D . H . ; 1991 l . Structure determination of tolaasin, an extracellular lipodepsipcptid( , produced by the mushroom pathogen Pseudomonas lolaasii Paine . Journal of the American Chemical Society 113, 2621--2627 . 20 . PANDEY, R . C ., COOK, J . C . & RINEHARI . K . L . ! 1977) . High resolution and field desorption mass spectrometry studies and revised structures of' alamethacin I and II . journal of the American Chemical Society 99, 8469-8483 . 21 . PAS'iERNAK, C . A . (1986 ; . Virus, toxin, complement : common actions and their prevention by calcium or zinc . BioEssays 6, 14--19 . 22 . PEND, J . T . !1986) . Resistance to disease in Agaricus bisporus (Lange ; Imbach . Ph .D . thesis . Cnisersity of Leeds . 23 . RAINEY, P. B . (1989, . A new laboratory medium for the cultivation of Agaricus bisporus . .Aere: Zealand Vatural Sciences 16, 109-112 . 24 . SIMON, R ., PRIEFER, I ; . & PUHLER, A . i 19831 . A broad host range mobilization system for in rho genetic engineering : transposon mutagencsis in Gram-negative ba( teria . Biotechnology 1, 784-791 . 25 . SONNEN, H ., SCHNEIDER, J . & KUTZNER, H . J . (1990) . Characterization of (DGal, an inducible phage from Brevibacterium flavum . journal of General _blicrobiology 136, 567 571 . 26 . STEWART, W . W . (1971 ; . Isolation and proof of structure of wildfire toxin . ,-Vature 229, 174--179 . 27 . STORM, D . R ., ROSENTHAL, K . S . & SWANSON, P . E . (1977' . Polymyxin and related peptide antibiotics . Annual Review, of Biochemistry 46, 723 763 . 28 . SuRico, G. & DE VAY, J . E . (1982) . Effect of syringomycin and syringotoxin produced by Pseudomonas syringae pathosar syringae on structure and function of mitochondria isolated from holcus spot resistant and susceptible maize lines . Physiological Plant Pathology 21, 39-53 . 29 . I AKEMOTO, J . Y ., GIANNINI, J . L ., V ASSEY, 1 . & BRISKIN, D . P . ( 1989) . Syringomycin effects on plasma membrane Ca t- transport . In : Phytotoxins and Plant Pathogenesis (A . Graniti, R . D. Durbin & A . Ballio, eds), pp . 165--175 NATO ASI Series . Springer-Verlag, Berlin . 30 . THOMAS, LV . F . & ELLAR, D . J . (1983 ; . Mechanism of action of Bacillus thuringiensis van israe/ensis insecticidal delta-endotoxin . FEBS Letters 154, 362-368 . 31 .'1OLAAS, A . G . 1915) . A bacterial disease of cultivated mushrooms . Phytopathologv 5, 51 54 . 32 . Vox HEIJNE, G . (1981'1 . Membrane proteins : the amino acid composition of membrane penetrating segments . European .our zal of Biochemistry 120, 275-278 . 33 . WEINER, R . N ., SCHNEIDER, E ., HAEST, C . W . M ., DEUTIGKE, B., BENZ, R . & PRIMMER, M . 1985' . Properties of the leak permeability induced by a cytotoxic protein from Pseudomonas aeruginosa r PACTT in rat erythrocytes and black lipid membranes . Btochimica et Biophysica Acta 829 . 173--182 . 34 . WONG, W . C . & PREECE,'I' . F' . (1979 ; . Identification of Pseudomonas lolaasii : the white line in agar and mushroom tissue block rapid pitting tests . Journal of Applied Bacteriology 47, 401-407 .