The occurrence and characterization of a syringomycin-macromolecular complex in cultures of Pseudomonas syringae pv. syringae

Physiological and Molecular Plant Pathology

91

(1992) 40, 91-105

The occurrence and characterization of a syringomycin-macromolecular complex in cultures of Pseudomonas syringae pv . syringae N . S . IACOBELLISt, P . LAVERMICOCCAt, G . SURICO+ and R . D . DURBIN§ t Istituto Tossine e Micotossine da Parassiti vegetali, CA -R,

V . le L . Einaudi, ARS. USDA and

apologia forestale e agraria, Universitd di Firenze, Italy and § L'niz-ersity of Wisconsin, U.S.A .

51, 70126 Ban, + Istituto di Patologia e Department of Plant Pathology .

(Accepted for publication January 1992;

The findings obtained in this study indicate that syringomycin 'SR) and syringotoxin (ST ; are present mainly in the growth medium of Pseudomonas syringae pv. syringae strains complexed with macromolecular substances . Almost all the syringomycin-like activity present in the culture filtrate and/or in acetonetreated whole culture (cells plus filtrate) was recovered in (NH 4 ) 2 SO 4 precipitates (AS-P) . Moreover, the syringomycin-like activity remained inside the dialysis tube after dialysis of the culture filtrate and/or the water phase after acetone treatment of whole culture . Acetone treatment of the whole culture significantly increased the recovery of antimicrobial activity . Evidence that syringomycin was the active component of AS-P was obtained by applying the standard procedures for extraction and purification of syringomycin to the macromolecular preparations . Acidic extraction with water-saturated n-butanol dissociated syringomycin from the complex . These extracts, analysed by electrophoresis, showed a band corresponding to authentic syringomycin . Moreover, the HPLC profiles of syringomycin preparations from AS-P and their amino acid content were consistent with those of syringomycin prepared in the usual manner . The toxic activities of complexed and pure, free syringomycin were comparable in plants, fungi and yeasts, whereas the sensitivity of bacteria, including P . s . pv . syringae strains, was different toward the two preparations .

INTRODUCTION The majority of virulent strains of the wide-host-range plant pathogen, Pseudomonas syringae pv . syringae, produce in culture syringomycin (SR), a low molecular weight phytotoxin reported to have a role in symptom expression in the diseases caused by various ecotypes of this bacterium [5, 12, 13, 24 j . SR is generally isolated by treating the whole bacterial culture (cells and filtrate) with 50 0,o cold acetone at pH 2 . This mixture is incubated overnight at 4 ° C and then centrifuged . The resulting supernatant is flash evaporated to eliminate the acetone and the water phase is extracted with water-saturated n-butanol . Homogeneous SR by polyacrylamide gel electrophoresis is obtained by purification of the extract by cationAbbreviations used in the text : AS-P, ammonium sulphate-precipitate ; SR, syringomycin ; ST . syringotoxin . 0885-5765/92/020091+15 503 .00/0

n 1992 Academic Press Limited

92

N . S . lacobellis et al.

exchange chromatography 1111 . Further purification of SR by HPLC has recc iiiv shown that the toxin preparation is a complex mixture [31 composed of structurdl ,r related lipodepsipeptides . syrin,gomycins [9, 201 and svringopeptins 1 1,I . Citrus strains produce a toxin similar to SR called ss ringotoxin S]" . ascertained to be a complex mixture of structurally related Ii podepsipeptides, syringotoxin [2 and syringopeptins [4] . Besides their phytotoxic activity SR and ST preparations are biocidal toward several micro-organisms [5, 10, 21 1 . In a previous study [22], it was determined that preparations, obtained by treatment of culture filtrates of P . s . pv . syringae bean isolates with ;NH A j z SO r , followed by exhaustive dialysis of the precipitate solutions, were toxic on bean trifoliate leaves and biocidal toward Geotrichum candidum . Moreover, when the macromolecular preparations were maintained for a few minutes at alkaline pH, a condition in which SR loses its biological activity [211, they lost antimicrobial activity while phytotoxicity on bean leaves remained unaltered . 'l here findings suggested that SR, though dialysable [1], might be present in the ammonium sulphate precipitates 'AS-P in a macromolecular form and that in addition to SR, other phytotoxic substances which are not sensitive to alkaline pHs might also be present in the preparations . The aim of our study was to ascertain the occurrence and nature of SR in P . s. pv . syringae cultures and to search for the presence of other phytotoxic substances besides SR . Here we report evidence that SR mainly occurs in cultures of various strains of P . IV . pv . syringae as a high molecular weight complex . Preliminary results have been reported [15, 16) .

MATERIALS AND METHODS Bacterial strains Seven selected Pseudomonas syringae pv . syringae strains, six producing SR and one S'F, were used in this study (Table 1 ; . Most of the work was done with strains B359, B3A

I ABLL I Strains

ojPseudomonas syringae pr . syringae

used in this study

Strain

Host

Toxin produced

Sourcc

Y37 B3Aa B301a B359a B427" B366 B392

Bean Peach Pear Millet Lemon Sugar beet Sorghum

SR SR SR SR ST SR SR

IMAT` ~ . E . DeVay ~ . E . DeVay J . E . DeVay J . E . DeVay J . E . DeVay ,J . E . DeVay

a Strains used for production, purification and structural studies of syringomycins and syringopeptins [3, 4, 20] . 1 Strain used for production, purification and structural studies or syringotoxin and syringopeptins [2, 4] . e IMAT, Istituto di Microbiologia, agraria e tecnica, Cniversita di Bari, Italy .

Syringomycin-macromolecular complex

93

and Y37, isolated, respectively, from millet, peach and bean . Bacteria were stored in sterile distilled water [14] . For routine work they were grown on NGA (nutrient glycerine agar'[ slants and maintained at 4 ° C .

Syringomycin complex production syringae strains were grown in 1 1 Roux bottles at 25 ° C in still culture, in 150 ml P . s . pve potato dextrose broth (PDB), PDB plus 0 . 4 °=o casamino acids (PDB/CA) [11 ] or in a synthetic medium IMM [23] . Bacterial inocula were produced on NGA . Cultures were grown for 36-48 h at 26 °C, washed twice with sterile water, and resuspended in sterile water to give 0 . 3 optical density (about lO s efu ml - ') ; 1 ml per flask used as inoculum . After 6-12 days incubation in still culture, depending on the medium, the cells were removed by centrifugation and the supernatant was treated with (NH 4 ) 2 SO 4 (66 °,-,, saturation) . The resulting precipitate (AS-P) was taken up in Tris-phosphate buffer (0 . 01 M, pH 6 . 5) and exhaustively dialysed at 4 ° C against the same buffer (Union Carbide dialysis tube, porosity 24 A and cut-off 1 . 7 x 104 Da) . The resulting preparations were then lyophilized . SR was extracted and purified following the method of Gross and DeVay [111 with minor modifications .

Bioassays The antimicrobial activity of the preparations was determined against Geotrichum candidum or Rhodotorula pilimanae using the PDA plate assay [11 ], and expressed in units of SR [21] . The phytotoxic activity was determined by injecting the solutions in the mesophyll of tobacco leaves with a syringe or in bean trifoliate leaves with a pressure gun, as described by Surico and lacobellis [22] . The results were recorded 24-48 h after injection .

Analytical methods The protein and carbohydrate content of AS-P were determined by the method of Lowry et at. [18] and Scott and Melvin [19], respectively . Bovine serum albumin and glucose were used as standards . AS-P preparations were analysed by electrophoresis on polyacrylamide (15,) gel slabs following the method of Gross and DeVay [11] with minor modifications . The gels, stained with Coomassie blue, were then scanned at 560 nm with a DU-65 Beckman spectrophotometer equipped with an automated gel scanner . Unstained gels were sliced using a razor blade and the resulting slices (2 mm) were placed in sequence onto PDA plates for bioassay with G . candidum . Pure SR and SR isolated from AS-P were analysed by HPLC ; the resulting fractions were evaluated for their amino acid content as previously reported [3] .

Recovery of syringomycin The recovery of syringomycin in the macromolecular preparations from the culture filtrate and whole culture (cells plus filtrate) was determined using strain B359 that had been grown in IMM for 6 days in still culture . The cells were pelleted and the culture

94

N . S . lacobellis et al.

filtrate was reduced to one tenth of the original volume by lyophilization . I'herraiter .. the active solution was divided into three parts . One part was exhaustively dialysed at 4 ° C against distilled water, the second portion precipitated with (NH,, ; 2 SO, and then exhaustively dialysed against distilled water and the third part extracted with scatersatw ated n-butanol at pH 2 . The whole culture (filtrate plus cells, was then adjusted to pH 2, cold acetone added 1 : 1) and the suspension maintained overnight at 4 ° C . After centrifugation, the supernatant was flash evaporated to eliminate the acetone and the resulting water phase was reduced to one tenth of the original volume by lyophilization and treated as above for the culture filtrate . The active macromolecular preparations from the above processes were analysed by polyacrylamide gel electrophoresis and extracted at pH 2 with n-butanol. The resulting active extracts were purified by cation-exchange chromatography and then analysed by HPLC in comparison to free SR prepared by the usual manner [11 J .

Fractionation and sizing of AS-P AS-P from peach isolate B3A (200 mg, specific activity 0 . 08 units per µg) was fractionated on a Sephadex G100 column (2 . 5 x 50 cm) eluted with 0 . 02 M ammonium acetate at a flow rate of 0 . 63 ml min -' . The elute was monitored at 280 nm . The 7 ml fractions collected were evaluated for their antimicrobial activity, as well as for protein and carbohydrate content . AS-P (1 mg, specific activity 0 . 7 units per µg) from strain B359 was applied on chromatography columns (60 x 2 cm) packed with Agarose Bio-gel A-5 or A-l •5 (Biorad) . The columns were eluted with Tris-HCl buffer (0 . 05 M, pH 5 . 5) containing 0 . 145 M NaCl at flow rates of 0 . 76 and 0 . 56 ml min - ' respectively . Gel filtration standard kit (Bio-Rad), containing vitamin B12, myoglobulin, ovalbumin, thyroglobulin, and Dextran Blue 2000 were applied to the columns as molecular size markers . The sizing of biocidal activity present in the free-cell broth culture of strain B359 was done using the same procedure . In this case, 1 ml (800 units) of supernatant was used . The optical absorbance of each fraction was monitored at 280 nm and its antimicrobial activity determined .

AS-P characterization AS-P preparations lyophilized and stored at -20 ° C were checked for up to I year for antimicrobial activity and analysed by polyacrylamide gel electrophoresis . AS-P from strain B359 (specific activity 0 . 7 units per µg) was taken up in acetate buffer (0 . 1 M, pH 4) and 1 ml samples (700 units per ml) were passed through Amicon XM ultrafiltration membranes with cutoffs of 3 x 10 5 , 1 x 10 5 , 5 x 10 4 and 1 x 10' Da . Four I ml washes with the buffer were passed through the membrane and combined with the filtered solution . The membranes were then washed three times 5 ml total with distilled water for recovery of the fraction retained by the membrane . For each treatment, the fractions passed through and retained by the membranes were assayed for antimicrobial activity . Furthermore, the AS-P preparation was taken up in acetate buffer containing several dissociating agents, either (a) agitated for 15 min at room

95

Syringomycin-macromolecular complex

) agitated for 15 min at pH 2, and temperature, (b) agitated for 5 min at 60 ° C, or then passed through the ultrafiltration membranes . The agents used were 0 . 3 M NaCl, 8 M Urea, 2 % sodium dodecyl sulphate, 1 °:o ethvlenediaminetetraacetic acid sodium salt, 2 . 5 °, , dithiothreitol or 1 °;o triton X100 . Solutions of AS-P and free SR [111 were brought to pH 10 with 0 . 1 M NaOH, maintained at that value for 10 min, and then the pH was lowered to neutrality with 0 . 1 M HCl . A distilled water control received the same treatment . The solutions were then assayed for their antimicrobial and phytotoxic activities . To assess the rate of alkaline denaturation, AS-P and free SR were taken up in distilled water or in 0 . 1 M (NH 4 ) 2 CO 3 (pH 7 . 8) and maintained at 37 ° C for 24 h . The antimicrobial activity was periodically monitored up to 24 h after the treatment . Biospectrum of AS-P Sensitivity to AS-P and free SR of some fungi, yeasts and bacteria, including several P . s . pv . syringae ecotypes with different SR or ST production abilities, was compared . Solutions of AS-P and free SR isolated from strain B359, both containing 51 200 units per ml, were diluted in a two-fold series and 10 pl drops were applied onto PDA plates . After the droplets were dry, the plates were poured with 3 ml water-agar (0-7" 0 ) seeded with 100 pl suspensions containing about 10 s cells per ml of the test organism . The results were recorded 24-48 h after application of the test micro-organisms . 6-day-old cultures in IMM Co assess the level of biocide production (SR or ST %I,

were added to cold acetone (1 :1) and maintained at 4 °C for 4 h . Then, the mixture was centrifuged, the supernatant flash evaporated to eliminate the acetone and the resulting water phase assayed for antimicrobial activity .

RESULTS Syringomycin complex production Culture filtrates of all the P . s . pv . syringae strains inhibited the growth of R . pilimanae, although the level of activity was variable among the strains (Table 2) and depended on the medium used for the bacterial growth (data not shown) . All the macromolecular preparations obtained by precipitation of broth cultures of the strains used with (NH 4 ) 2 SO 4 followed by exhaustive dialysis of their solution (AS-P) were biocidal toward the test micro-organisms and phytotoxic on bean and tobacco leaves . All the activity originally present in the P . s . pv . syringae culture filtrates was apparently recovered in the AS-P (Table 2) . By contrast, neither the ammonium sulphate soluble phase nor the dialysate of AS-P solutions had any antimicrobial activity . The AS-Ps isolated from the selected strains had different specific activities toward R . pilimanae and their protein and carbohydrate content were also variable (Table 2) . When analysed by electrophoresis, AS-Ps did not contain a band corresponding to free SR, but did have several Coomassie blue stained bands with electrophoretic mobility less than that of SR (Fig . 1) . In addition, the unstained gel sliced and assayed toward G . candidum produced a broad zone of antibiotic activity that started at the point of deposition of samples in the wells and included the zone of the gel corresponding to the Coomassie blue stained bands (Fig . 2) . NIP P 40

96

N . S . lacobellis et al . l - ut .t : 2

Rhodotni iia pilimanae, in /lu° 'of,,, mlani, syringae odlims, and its re,ovol m the rnacrorru'lerulnr /nr/,arn(tons .1S-P?

Presence of start onttun-like a,lisily, de(o pined on

Pseudomonas syringae

pt .

Strains

Activity'` in the broth units',

Activity-" ri ((151 red in AS-P units,

Sugar'' outent (& AS- 1) pg pg t,

Protein' content ()1 A,-,- P pg pg '

Y37 B301 B359 B3A B427 B366 B392

14850 9900 59400 > I000 29970 158400 78480

17280 :0 . 3'" 11064 ;03 62464 121 1566 10-025 ; 28080 i04 ; 138240 1 6, 103680 1-61

0089 0069 0105

0340 0 . 250 0540 0260 0370 0360

0 152

007 : 0091 0107

0 -'130

Total units of syringomycin-like activity contained in 100 nil of cell-free supernatants of P . s . pv . syringae strains cultures grown for 6 clays in 1MM J2/ . " Total units of syringomycin-like activity recovered in AS-P obtained from (tV'H, t 1 z SO, precipitates of 100 ml of culture supernatants and exhaustivelc dialysed . pg of protein and carbohydrate per pg of AS-P . " Values in parentheses arc the specific activity units pci pg ; of AS-P preparations . A

0 .8

0. 6 (a) SR r

0 . 48

- 0 . 64

BF 0-48

0 . 36 E

E C 0

0 m

0 .32 <

a 0 . 24

0 . 12

0

- 0 . 16

1

2 Origin

3 cm

4

5- + Origin

I

2

3

4

0 5-

cm

FIG . 1 . Comparison of electrophoretic mobility of purified syringomycin and AS-P from strain B3A . The biocidal preparations were developed on 15",, polyacrylamide gel slabs, stained with Coomassie blue and then scanned at 560 mm with a DU-65 Beckman spectrophotometer equipped with an automated gel scanner . Basic fuchsin (BF ; was added to the SR sample as a tracking dye . (a scan (upper) and original stained gel flower' of purified syringomycin ; lb) scan fupperl and original stained gel (lower) of AS-P .

Syringomycin-macromolecular complex

97

FIG . 2 . Distribution of antimicrobial activity diffused from polyacrylamide sliced gels of purified syringomycin (a) and AS-P (b , from strain B3A . Gel slices were placed in sequence onto PDA plates and numbered clockwise from the arrowhead with central slices numbered 10, 20, 30, respectively . The test micro-organism Geotrichum candidam is inhibited around gel slices nos 23 to 26 in purified syringomycin gel and around gel slices nos 1 to 20 in AS-P gel .

AS-Ps injected into bean and tobacco leaves induced necrosis of the treated tissues, the intensity of the necrotic symptoms paralleled the SR-like antimicrobial activity contained in the solutions (Fig . 3) .

a

C

. 3 . Phytotoxic symptoms on bean trifoliate leaves (abaxial surfaces are shown) 48 h after ; Ft( infiltration with (a) purified syringomycin, (bj AS-P (solutions both containing 400 syringomycinlike units ml -I ), (c) distilled water .

When AS-P solutions were extracted with water-saturated n-butanol at pH 2, all the biocidal activity was recovered in the organic phase . No antimicrobial or phytotoxic activities were present in the residual water phase . Further purification of the active extracts by cation-exchange column chromatography gave rise to an active fraction which comigrated with authentic SR [111 in polyacrylamide gels . Moreover, HPLC

N . S . lacobellis et al .

98

profiles of purified SR from AS-P and the amino acid content of the HPLC-acti ,, C fractions were consistent with those of SR prepared following the usual procedure [ .'))I (Fig . 4j . 100

0

100

1 .0

(0)

B

a

2

50

0 .5

'50

0 0b

a I

Q

3 (4

2

1 `

4

I

I

0.0

0-0 0

10

20

0 0

10

20

Time (min)

Fic . 4 . Reverse phase HPLC of purified syringomycin preparations of Pseudomonas syringae pv . syringae strain B359 obtained with the standard procedure (al and isolated from AS-P (h) . Elution was performed by solvent gradient obtained by mixing solvent A (0-2",, trifluoracetic acid in water) with solvent B (0 . 1 °„ trifluoracetic acid in acetonitrile : isopropanol 4 : 1, v/v' at the flow rate of2'8 ml min t . Absorbance at 214 rim (-- ) ; percentage ofsolvent B in the elution mixture are shown . 1, syringomycin A t : 2, syringomycin B : 3, syringomycin C ; 4, syringomy(in 1) ; 5, syringomycin E ; 6, syringomycin C . Recovery of syringomycin

All the antimicrobial activity originally present in the culture filtrate of' millet strain B359 was recovered in the macromolecular fractions contained in the tube after dialysis of the filtrate or after (NH 4) 2 SO 4 precipitation of the filtrate followed by dialysis of the resulting AS-P (Table 3) . No activity was observed in the fractions outside the dialysis tube . The extraction of the culture filtrate with water-saturated n-butanol leads to a recovery of antimicrobial activity, corresponding to free SR, similar to that obtained from the above macromolecular preparations (Table 3) . Parallel results were obtained when the different treatments were applied to the whole culture (cells plus filtrate) . However, in this case acetone treatment gave a recovery of bioactivity about two or three times higher compared with that from the culture filtrate (Table 3) . In the case of dialysis of acetone treated whole culture, however, a small part of activity corresponding to free SR (about 4° :0), was found in the fraction outside the dialysis tube .

Syringomycin-macromolecular complex

99

TABLE 3 Recovery of antimicrobial activity, determined on Rhodotorula pilimanae, t he macromolecular fractions obtained by different treatments of the filtrate and whole culture (cells plus filtrate) of Pseudomonas syringae pv . syringae strain B359

Treatment Filtrate plus (NH,t ) 2 SO 4 and dialysis of AS-P Filtrate dialysis Filtrate extraction with n-butanol at pH 2 Whole culture plus' !NH4) 2 SO 4 and dialysis of AS-P Whole culture' dialysis Whole culture' extraction with n-butanol at pH 2

Initial' activity units l

Activity" recovered unitsi

Yield

96000

105000

109

96000 96000 192000

86400 96000`' 236000

89 100 123

192000 192000

185000 256000"

96 M

a Total units ofsyringomycin present in 300 ml of cell-free supernatant or culture (cells plus filtrate) of P. s . pv . syringae cultures grown for 6 days in IMM [23] . ° Units of syringomycin recovered in the macromolecular fractions . 'The antimicrobial activity recovered is due to free SR [II ] . The whole culture (filtrate plus cells) was adjusted to pH 2, cold acetone added (1 : 1) and the suspension then maintained over-night at 4 °C . After centrifugation, the supernatant was flash evaporated to eliminate the acetone ; the resulting water phase was reduced to one tenth of the original volume by lyophilization and treated as indicated .

TABLE 4 Ultrafiltration sizing (Amicon XM membrane)' of the antimicrobial acti / against Rhodotorula pilimanae of AS-P treated with dissociating agents

Antimicrobial activity XM300 Treatment None NaCl (0 . 3 M Urea (8 M) Heat (60 ° C, 5 min ; SDS (1 °-a) EDTA (1°,0) DTT (2 5 Triton X100 (1 °,) pH2

IN°

XM100

XM50

1N

TH

IN

+°

+

-

+

+

+

+

+

+

+

+

+

-

+

+

+

-

+ + + +

+ + + +

+ + + +

-

TH°

TH

+

-

SDS, sodium dodecyl sulphate ; EDTA, ethylendiaminotretracetic acid, sodium salt ; DTT, dithiothreitol . a XM300 : 3 x 10 5 Da cut-off ; XMIOO : l x 105 Da cut-off ; XM50 : 5 x 10' Da cut-off. " IN : fraction retained by the membrane ; TH : fraction that passed through the membrane . +, antimicrobial activity ; -, no antimicrobial activity .

100

N . S . lacobellis et al Sensitivitt o bacteria . fungi and teasts to free SR and SR-eorrtples i :IA-/'! isolated from str ,Iia B3Sft 'nits SR rill ' needed fbr complete inhibition" 'test organism I' . s. pv . tabaci 2706 P . s . pv . phaseolicola 2571 P . s. pv . glycinea 2652 P . s, pv . tomato 2563 1' . s . pv . savastanoi 317 Rrecinia herbicola 656 .Igrobacterium tumefaciens 2405 Rhodococcus fascians 2511 Bacillus megaterium 1008 B . subtilis 1009 Geotrichum candidum F260 Aspergillus flauus 8 . ustus 109 Penicillium e.xpansum 510 21 P . chryogenum 450 Rhodotorula pilimanae 26423 Saccharomyces cerevisiae 403 Kluyveromyces fragilis 397

Source NCPPB NCPPB NCTPB NCPPB JIM NCPPB -NCPPB NCPPB VIM 1' FM J . E . DeVay FFV1 FPM FFM JIM .ACC CBS CBS

SR 4800 4800 4800 25600 6400 12800 12800 19200 400 6100 100 400 200 800 100 25 50 < 50

\S-I' NA" 100 100 NA NA NA' NA` 19200 1600 25 600 10)) 400 20O 800 10(0 25 5 S0 < 50

NCPPB, -National Collection Plant Pathogenic Bacteria ; A FCC, American I'vpc Culture Collection ; CBS, Centraal Bureau voor Schimmelculture, ; I' FM, Istitutn 'Iossine c Micotossine da Parassiti vegetali collection . A unit of toxin is the amount in an end-point dilution that completely inhibits the growth of G . candidum in the area of application of a 10 pl droplet on PDA [21 ] . NA : Solution containing 51 200 units per ml ofsyringomvcin proved not to he inhibitor\ toward the test organism . In this case, the highest concentration of AS-P tested was 12800 SR units per ml . HPLC profiles of SR extracted and purified from AS-P of the culture filtrate and whole culture were comparable to that of SR obtained by the usual procedure [31 ; however, variations in the relative content of the minor metabolites were observed . AS-P fractionation and sizing When AS-P from peach isolate B3A was applied on a Sephadex G100 column, all the antimicrobial activity toward G . candidum eluted with the void volume . The active fractions co-eluted with a broad UV-absorbing peak which contained most of the proteins and carbohydrates of AS-P . The biocidal activity of AS-P from strain B359 applied on Biogel AI-5 and A5 agarose columns eluted, respectively, with the void volume and with Dextran 2000, suggesting that the molecular weight of the active component(s) was about 2 x 10 6 Da . The same elution pattern was obtained when the culture filtrate of the same strain was applied on the above agarose columns . However, in both separations a small amount of antimicrobial activity was present in the fractions where authentic free SR emerges .

101

Syringomycin-macromolecular complex SABLE 6

o/ representatire Pseudomonas syringae pr . syringae strains to,/i( ,e SR and SR-complex (ASP) isolated from strain 8359

Sensit

Strain B359 B382 B397-1 B397-2 B475-A B301 B3A B366 B426 B448 B427" 2842 1242 2843 2106 2264 2326" S29 S5 V33 V37 S38B H345

Host Millet Maize Foxtail Foxtail Oat Pear Peach Sugar beet Walnut Unknown Lemon Wheat Panicum Wheat Sorghum Maize Lemon Cherry Pear Bean Bean Lilac Lettuce

source

J. J. J. J. J. J. J. J. J. J. J.

E . DeVay E . DeVay, E . DeVay E . DeVay E . DeVay E . DeVay E . DeVay E . DeVay E . DeVay E . DcVay E . DeVay NCPPB NCPPB NCPPB NCPPB NCPPB NCPPB IM-IATe I M M IMAT IMAT IMAT IM XI'

Biocide' production units ml- ' l 1200 400

SR 9600 9600 12800 19200 6400 3200 1 600 12800 9600 19200 4800 3200 6400 3200

150 200 1000 1600 2000 100 100 1200 100 1600 800 1600 200 800 400 1600 1200 100

Production of SR or ST i units per ml ; by P. s . py .

Units SR ml - ' needed for the complete inhibition"

sotingae

AS-P NA` NA NA NA 100 3200 6400 100 100 NA 4800 100 31200 100

1600

100

12800 19200 6400 9600 9600 9600 4800 9600

51200 NA 1600 NA 100 400 25600 NA

strains grown for 6 days in I MIM

(231 . A unit of toxin is the amount in an end-point dilution that completely inhibits the growth of G . candidum in the area of application of 10 p1 droplet on PDA [211 . NA : solution containing 51 200 SR units per ml was not inhibitory to the test strain . P . s. pv . syringae strains which produce ST . IMAs, Istituto di Microbiologia, agraria e tecnica, University di Bari, Italy .

('haraclerization of AS-P

AS-P from different strains stored at -20 ° C for up to 1 year, maintained its original biocidal activity toward the test organisms ; moreover, no change was observed in the electrophoretic mobility of other AS-P components and in particular no free SR was observed . The antimicrobial activity of AS-P from strain B359 passed through the Amicon XM type membrane was retained by the membrane, as expected (Table 4) . Moreover . in the presence of high ionic strength and treated with urea, the SR-complex did not seem to dissociate since the biocidal activity was retained by the XM300 membrane Nominal cutoff 3 x 10 ° Da) . In contrast, when AS-P was treated with SDS, DTT, Triton X100, heated or maintained at pH 2, the antimicrobial activity was present in the fraction that passed through the membrane . Furthermore, when the latter active

102

N . S . lacobellis et al.

fractions were passed through an XM100 membrane activity was retained by the membrane .

x 10 5 I)a nominal cutoff , tb( ,

After the alkali treatment pH 10, 10 mini, AS-P and free SR lost their antimicrobial activity but maintained their phytotoxicity, although the intensity of necrosis was slightly reduced . The mild alkali treatment pH 7 . 8, 37 °Ci of free SR and AS-P also caused a loss of antimicrobial activity toward G . candidum . However, the biocide activity of the preparations was lost at different rates : complete loss from free SR and AS-P required 8 or 24 h incubation, respectively . I

Biospectrum of AS-P The growth of all the micro-organisms utilized in this study was inhibited by free SR and AS-P, although a different behaviour was observed for fungi, yeast and bacteria . The sensitivity of fungi and yeasts to free SR and AS-P was comparable, although yeasts were more sensitive, especially R . pilimanae strain ATCC26423 (Table 5i . In contrast, the sensitivity of bacteria to the two biocide preparations was different . In particular, all the bacterial species except P . s . pv, phaseolicola and P . c . pv . glycinea were more sensitive to free SR than AS-P Table 5) . Also different was the sensitivity of' P. s . pv . syringae strains to free SR and AS-P (Table 6) . The amount of free SR necessary for the complete growth inhibition of P. s . pv . syringae strains ranged from 1 600 to 19 200 units per ml . On the other hand, 10 out of 23 strains were insensitive to high quantities of AS-P ( >~ 51 200 units per ml) while eight others were highly sensitive to AS-P (100--400 units per ml) . No apparent correlation was observed between the sensitivity of the strains and their host origin, toxin produced (,SR or ST ; or level of biocide accumulation in nitro Table 6) .

DISCUSSION In this study we have reported several lines of evidence indicating that syringomycin and syringotoxin, produced by Pseudomonas syringae pv . syringae, are present in cultures complexed with macromolecules . All the phytotoxic and anti-microbial activities in the culture supernatants were recovered in the (NH 4 ) 2 SO 4 precipitates AS-P) . Because we never found any biological activity in either the AS-P soluble phases or the dialysates, we suggest that free syringomycin does not occur in cultures of P .s . pv . syringae . 'Phis proposition was strengthened when culture filtrates were exhaustively dialysed against distilled water and almost all the antimicrobial activity remained in the dialysis tubes instead of moving out as would be expected if SR was not complexed . Moreover, polyacrylamide gel electrophoretic analysis of AS-P showed no band corresponding to free SR . The diffused antimicrobial activity shown by unstained gel slices of AS-P can be attributed to possible partial dissociation of the macromolecular substance(s) under the action of the electrical field or to diffusion of the active high molecular weight substance(s) in the low porosity gel utilized . The fractionation of AS-P by gel filtration demonstrated that the SR-complex has a molecular weight of about 2 x 10 6 Da and, moreover, that in culture filtrates a very small amount of free SR was present besides the SR-complex . After treatment of culture filtrates with (NH 4 ) 2 SO 4, the absence of free syringomycin, either in the AS-P or the fraction outside the dialysis tube, may have been due, in part, to a trapping of

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free SR by substances in the dialysis tube . In this regard, we have observed that free SR can be adsorbed onto the dialysis membrane (;unpublished observation), probably because of the capacity of the toxin to interact with polyhydric surfaces [1] . This leaves open the question regarding the level of binding specificity of SR and ST with microbial or plant constituents [6] . On the basis of the serological behaviour of SR preparations, Backman and DeVay [I I suggested that the toxin was an haptenic group of cell wall antigenic macromolecules and, furthermore, that it may be a product of cell wall metabolism . Since AS-P prepared from cultures of various strains contained carbohydrates and proteins and is compartmentalized in the cells, the biocide may be complexed to proteins, glycoprotein or polysaccharides, possibly of cell wall origin . When the native SR-complex is heated, maintained at pH 2 or treated with SDS, EDTA, DTT or Triton X1 .00, it was apparently transformed into another complex of lower molecular weight . However, these results must be interpreted with caution since the complex may interact with various surfaces . Also, water-saturated n-butanol extraction of AS-P dissociates SR from the complex . However, acetone treatment significantly increased the recovery of antimicrobial activity if cells were present . Alkali-treated AS-P and free SR lost their antimicrobial activity but maintained their phytotoxicity . This result is explained by the fact that what has been called SR is really a complex mixture [3] containing two groups of lipodepsipeptides : the syringomycins and syringopeptins [3, 4] . It appears that the principal component of the syringopeptins, syringopeptin 25A, is mainly responsible for phytotoxic activity characteristic of the unfractionated toxin mixture of P . s . pv . syringae whereas syringomycins are mainly responsible for antimicrobial activity [17] . In an alkaline environment, the syringomycins lose both bioactivities whereas syringopeptin A25 maintains almost all its phytotoxicity [17] . However, macromolecules with which SR is associated, partially protect the alkali-labile sites of syringomycin . The activities of free SR and complexed SR, both containing syringomycins and syringopeptins, were comparable when assayed for phytotoxicity on plants and for antimicrobial activity toward fungi and yeasts . But this was not the case with bacteria, including strains of P . s . pv, syringae . Obviously, both plants and micro-organisms can release the toxic components of the complex, although bacteria appear less capable of doing this . All the findings in this study indicate that SR occurs in P . s . pv . syringae cultures as a complex or, at least, that free SR has a strong tendency to bind specifically or nonspecifically to macromolecules, as previous studies have suggested [11, 21 ] . The significance of the SR-complex to the biology of the producing bacterium and the establishment of disease symptoms is still unknown . From the perspective of the bacterium, the production of an SR-complex may be more important from an ecological than a pathological viewpoint . In fact, the phytotoxic activity of free and complexed SR is practically identical, even though the antimicrobial activity is significantly different . This means that the binding of free SR to macromolecular substances affects syringomycins rather than syringopeptins . Therefore, the SRcomplex probably has only a secondary, if any, role in pathogenesis, but it might be significant in the self-protection [7] of P . s . pv . syringae or . more generally, in the competition and survival of the bacterium in nature, as already suggested by other

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authors [81 . P . s . pv . syringae is a bacterium which can ,urvivc for extended periods rt time on the plant surface and the production of anti microbial substances mus inilueucc this saprophytic phase or even minimize secondary invaders in infected tissues . 1'Ve now know that SR is a mixture of'substances with antimicrobial or phytotoxic activities, and that these substances are released into the culture complexed to macromolecules . These findings should help plant physiologists investigating the precise activity and effects of toxins on host plants . This work was partly supported by a grant from the Italian National Council '(AR), special ad hoc programme "Chimica fine II", subproject 3 . We are grateful to Dr J . E . DeVay and Professor G . L . Ercolani for providing several Pseudomonas syringae pv . syringae strains and Dr M . Simmaco for his valuable assistance with HPLC and amino acid analyses . The valuable technical assistance

of Mrs S . L Lonigro arid Mr 'I .

Uchytil, and the assistance of Mr F . DeNfarzo in processing the photographs arc gratefully acknowledged .

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Pseudomonas syringae . Physiological Plant Pathology 11 : 1-11 . 13 . Hrabak EM, Rich JJ, Kennedy CJ, Willis DK . 1989 . A locus required for lesion formation by Pseudomonas syringae pv . syringae on bean affects syringomycin production in vitro . Ph_ytopathology 79 : 1178 . 14. lacobellis NS, DeVay JE . 1986 . Long-term storage of plant pathogenic bacteria in sterile distilled water . Applied and Environmental Vicrobiology 25 : 525-526 . 15 . lacobellis NS, Lavermicocca P, Surico G, Durbin RD . 1988. Occurrence of a syringomycin-high molecular weight complex in Pseudomonas syringae pv . syringae . In : Graniti A, Durbin RD, Ballio A, eds . Phytotoxins and Plant Pathogenesis, NATO ASI Series, Vol H27, pp. 429-431, Berlin : Springer-A%erlag .

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16 . lacobellis NS, Lavermicocca P, Surico G, Uchytil TF, Durbin RD . 1990 . Properties of a syringomycin-macromolecular complex produced by Pseudomonas syringae pv . syringae . In Plant Pathogenic Bacteria, Proceedings of the 7th International Conference on Plant Pathogenic Bacteria, Budapest, June 11-16 1989, Ed . by Z . Klement, pp . 93-97 . Akademiai Kiado, Budapest . 17 . lacobellis NS, Lavermicocca P, Grgurina I, Simmaco M, Ballio A . 1992 . Phytotoxic properties of Pseudomonas syringae pv . syringae toxins . Physiological and Molecular Plant Pathology 40 : 107-1 16 . 18 . Lowry OH, Rosebrough NJ, Farr AL, Randall RJ . 1951 . Protein measurement with the Folin phenol reagent . Journal of Biological Chemistry 193 : 265--275 . 19 . Scott TA, Melvin EH . 1953 . Determination of dextran with anthrone . Analytical Chemistrv 25 : 1656-1661 . 20 . Segre A, Bachmann RC, Ballio A, Bossa F, Grgurina I, lacobellis NS ., Marino G, Pucci P, Simmaco M, Takemoto JY . 1989. The structure of syringomycins A r , F and G . FEBS Letters 255 1) : 27-31 . 21. Sinden SL, DeVay JE, Bakman PA . 1971 . Properties of syringomycin, a wide spectrum antibiotic and phytotoxin produced by Pseudomonas syringae, and its role in the bacterial canker disease of peach trees . Physiological Plant Pathology 1 : 199-213 . 22 . Surico G, Iacobellis NS . 1977 . Conlronto di alcuni indict di virulenza di isolati di Pseudomonas syringae van Hall da fagiolo . Phytopathologia Mediterranea 26 (2-3t : 79---86 . 23 . Surico G, Lavermicocca P, lacobellis NS . 1988 . Produzione di siringomicina e di siringotossina in culture di Pseudomonas syringae pv . syringae . Phytopathologia Mediterranea 27 : 163-168 . 24 . Xu GW, Gross DC . 1988 . Evaluation of the role of syringomycin in plant pathogenesis by using'] n5 mutants o£ Pseudomonas syringae pv . syringae defective in syringomycin production . Applied and Environmental Microbiology 54 161 : 1345-1353 .