APPILIED AND ENVIRONMENTAL MICROBIOLOGY, July 1989. p. 1677-16830099-2240/89/071677-07$02.00/0Copyright © 1989. American Society for Microbiology

Analysis of Pectate Lyases Produced by Soft Rot BacteriaAssociated with Spoilage of Vegetables

CHING-HSING LIAO

Easterni Regionail Resear(ch Center, Agriclultutral Research Serv,ice, U.S. Depar tment of Agric iulture,Philadelphia, Pennsylvania 19118

Received 17 January 1989/Accepted 5 April 1989

Isoelectric focusing (IEF) profiles of pectate lyases (PLs) produced by five different groups of soft rot bacteriawere analyzed by using the combined techniques of thin-layer polyacrylamide gel IEF and agarose-pectate

overlay activity staining. Four strains of soft rot Erwinia spp. produced three or more PL isozymes. All of eightPseudomonas viridiflava strains examined produced one single PL with a pl of 9.7. All 10 of Pseudomnotasfluorescens strains produced two PLs; the major one had a p1 of 10.0 and the minor one had a pl of 6.7. A singlePL with a pl of -10.0 was detected in one strain each of Xanthomonas campestris and Cytophaga johnsonae.PLs of six representative strains were purified from culture supernatants by ammonium sulfate precipitationand anion-exchange chromatography. All purified PL samples macerated potato slices, but to different degrees.The Mrs of alkaline PLs produced by P. viridiflava, P. fluorescens, X. campestris, and C. johnsonae were

estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 42,000, 41,000, 41,500, and35,000, respectively. IEF profiles of PLs were distinct among the bacterial species. Profiles of non-Erwiniaspoilage bacteria were considerably simpler than those of Erwinia spp. The PL with an alkaline pl appearedto be the principal or the sole enzymatic factor involved in tissue maceration caused by most strains of soft rotbacteria.

The ability of soft rot bacteria to cause vegetable spoilageresults mainly from their ability to produce massive amountsof pectic enzymes. Pectate lyase (PL; EC 4.2.2.2), whichcleaves polygalacturonates by 3-tri-ans-elimination, is be-lieved to be the principal enzyme responsible for tissuemaceration, electrolyte loss, and cell death (22). Activities ofPL have been detected previously in culture supernatants ofdiverse groups of soft rot bacteria (20), including Erw'iniaspp. (27, 34, 36), Pseililoilnotiias fliuor-escenis (or Pseiudoinlo-n1as inar-ginialis) (7, 9, 10, 23, 37), Pseiuldoinzon)ais51ridflaviai(10, 15), Xcanthomonas calmpestris (17, 24), and Cvtop/hagajohnisonsae (13, 16, 35). The PL produced by Ertl'inia spp. hasbeen studied extensively and is unique for its occurrence as

multiple (-3) isozymic forms (12). In Erwinia chrysantlhemifor example, five PL isozymes with pIs ranging from 4.0 to10.0 have been detected in most strains examined so far (2,29, 34). Several studies indicate that alkaline PL (pl, -9.5) is

more efficient than neutral or acidic PLs in inducing tissuedisintegration or tissue maceration (1, 25, 30, 31, 33) and thatalkaline PL by itself is sufficient to macerate potato tuberand other plant tissues (11, 25, 33). The physiological basisfor the production of more PLs than are required to macerateplant tissue by Erwiinia spp. is not understood. It has beensuggested that some PL activities (or isozymes) may berequired for other pathological functions such as recognitionof the host (1, 33) and induction of disease resistance (5).At present, little is known about the enzymatic mechanism

which controls the soft rot pathogenicity of non-Erwiiniaspoilage bacteria. Two recent studies (15, 32) suggest thatpectic enzyme systems of non-Erninia spoilage bacteria are

not as complex as those demonstrated in Erwinia spp.Schlemmer et al. (32) have shown that P. fliuorescens W51produces a pectin lyase (pl, 9.4) that is required for macer-

ation of plant tissue. Liao et al. (15) have isolated andpurified a single PL (pl 9.7) from culture supernatants of P.iviridiflava SF312 and have shown that the bacterium became

nonpathogenic when the gene encoding for PL synthesis or

secretion was inactivated by Tn5 transposition. It remains tobe determined whether the simple pectic enzyme system, as

demonstrated in the two pseudomonads described above.represents a common feature of non-Er) winia soft rot bacte-ria.The objectives of this study were (i) to examine the

isoelectric focusing (IEF) profiles of PLs produced by fivedifferent groups of soft rot bacteria, including 4 strains ofEriniia spp., 8 strains of P. viriclriflaia, 10 strains of P.fluorescens, and 1 strain each of X. camnpestris and C.joltinsoniuie; and (ii) to determine the pls, M,.s and maceratingabilities of PLs purified from six representative strains.(A preliminary account of the results of this study have

been presented previously [C.-H. Liao, Abstr. Annu. Meet.Am. Soc. Microbiol. 1988, P-28, p. 2781.)

MATERIALS AND METHODS

Bacterial strains. Table 1 lists the origins and sources of

the 25 strains of soft rot bacteria used in this study. Thesestrains were isolated from rotten specimens of variousvegetables. With the exception of P.fluorescens PJ-08-14, allstrains have been shown to macerate potato slices and otherplant tissues by producing large amounts of PLs (18). P.fluorescens PJ-08-14 produced PL intracellularly and exhib-ited little or no tissue-macerating ability. All of the bacterialstrains were maintained on Pseu(doinoizas Agar F (DifcoLaboratories, Detroit, Mich.) and were grown to the late-stationary phase in MMY broth medium at 20°C for enzymepreparation. The MMY medium (pH 7.1) containedK,HPO4, 0.7%; KH2PO4, 0.2%; MgSO4 7H,O, 0.0219:(NH4)2S04' 0.1%/; CaCl, 1 mM; yeast extract, 0.1%; andpectin (grade 1, 0.3%; Sigma Chemical Co., St. Louis, Mo.).

Assay of PL activity. PL activities were determined by a

spectrophotometric method (37). One unit of activity was

defined as the amount of enzyme which produced an in-

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TABLE 1. Bacterial strains used in this study

Stralin" Host origin reference

Er-winia (carotovora subsp.( arot()ovoa

SR319 Potato A. KelmanCU -06-1 Cucumber 18

Erwt'inia (oarotolora subsp. itiro- Potato A. Kelmansepti(! SR8

Ertt'inii (Irnsathllelni EC16 Chrysanthemum A. Chatterjee

Pseuidoinolnais fliuorescuensCY(91 Celery 1817816 Dahlia ATCC"SJ-08-2 Squash 18SJ-08-3 Squash 18PJ-08-30 Pepper 18PJ-08-14 Pepper 18BC-05-1 B Broccoli 18BC-05-2B Broccoli 18LC-04-2B Lettuce 18AJ-06-2A AsparagLis 18HtJ-08-11B Spinach 18

PseiidIoltloliis viridiflavaSF312 Squash 1813223 Bean ATCCSF-05-3 Squash 18PJ--09-6A Pepper 18PJ-08-6B Pepper 18PJ-08-9 Pepper 18PJ-08-16B Pepper 18TU-04-2A Tomato 18

Xanilhotnontis (inmpestli.s CJ091 Cucumber 17

Cytophaga johnsonae PF062 Pepper 16

' The ability tonmace-ate potato slices was demonstrated in all strainsexcept Psi(lsoiidolitisofloo olre,s clens PJ-t)8-14, which produced PL intracellUlarlybtUt did not excrete the enzyme out of the cell.

" ATCC. American Type CultUre Collection. Rockville. Md.

crease of 1.73 units in the A32 per mi at 30°C. An increasein the A232 of 1.73 was considered to represent the formationof 1 p.mol of unsaturated uronides. The reaction mixture (2to 3 ml) contained 100 mM Tris hydrochloride (Tris-HCI)(pH 8.5), 0.25%G sodium polygalacturonate (Sigma), 1 mMCaCL, and enzyme sample.

Preparation of enzyme samples. Culture supernatants ob-tained after centrifugation (10,000 x g, 10 min) were con-centrated and equilibrated with 50 mM Tris-HCI (pH 7.5) byultrafiltration (Centricon 10 microconcentrator; AmiconCorp.. Danvers, Mass.). The concentrated samples contain-ing 0.1 to 0.3 U of PL activity per [Li were used to determineextracellular PL profiles. For the preparation of intracellularPL samples, the following procedures were used. Cell pelletsobtained after centrifugation were washed twice and sus-pended in 50 mM Tris-HCI (pH 7.5) buffer in a 1/100 volumeof the culture medium. Cells were disrupted by ultrasonica-tion, and cell debris was removed by centrifugation (350,000x g. 30 min). The clear supernatant was Llsed directly todetermine intracellular PL profiles.

Purification of PLs. The purification scheme previouslydescribed for the extracellular PL of P. viridciflava wasfollowed (15). Culture supernatants obtained from six repre-sentative strains (P. fluorescens CY091 and BC-05-lB, P.

i,ridifla,a SF312, X. (clampestris CJ091, C. johnisontaePF062, and E. chrvsanthemni EC16) were treated with am-monium sulfate. The precipitate that formed at 50 to 85%saturation was dialyzed against 50 mM Tris-HCl (pH 7.5)buffer and subsequently applied onto a DEAE-cellulosecolumn equilibrated with 50 mM Tris-HCl (pH 8.0). The PLwas eluted stepwise with 50 mM Tris-HCl (pH 8.0) buffercontaining 0.00, 0.05, 0.10, or 0.20 M NaCl.

Thin-layer polyacrylamide gel IEF. lEF techniques were

performed by the procedures described by Ried and Collmer(28, 29). Thin-layer (1 mm) polyacrylamide gel plates (pH 3.5to 9.5) containing a gel concentration of 5% and an Ampho-line concentration of 2.4% were purchased from LKB/Pharmacia Instruments (Piscataway, N.J.). The method ofGuo and Bishop (8) was used to prepare gels, with thealkaline end of the pH gradient extending above pH 9.5. Gelswere run on a Multiphor II 2117 apparatus (LKB), andenzyme samples (3 to 8 [L) were applied directly onto thegel. Prefocusing was carried out at 9 W for 30 min, andfocusing was carried out at 12 W for 40 min after applicationof samples. Phosphoric acid (0.1 M) and sodium hydroxide(0.1 M) were used. respectively, as the anolyte and thecatholyte throughout the study. The wide range of pl mark-ers (LKB/Pharmacia Instruments) were included in eachrun. After electrofocusing, gels were either stained withCoomassie blue or subjected to enzyme activity staining as

described below.Enzyme activity staining. Agarose-pectate overlay stain

techniques for detecting PL activities have been describedpreviously (2, 4. 28, 34). Thin-layer (0.75 mm) agarosesubstrate gels were prepared by casting a melted mixture ofagarose (0.8%). sodium polygalacturonate (0.2%), Tris-HCl(100 mM, pH 8.5), and CaCl, (1 mM) onto an AgaroseGelbond Film (FMC Corp., Marine Colloids Div., Rockland,Maine). The agarose-pectate gel bond on the film supportwas overlayed onto the polyacrylamide gel immediately afterelectrofocusing, and incubation was carried out at roomtemperature for 30 min. PL activity was visualized 1 h aftersubmerging the agarose-pectate gel in 1% mixed alkyltrime-thyl ammonium bromide (Sigma). Alkyltrimethyl ammoniumbromide precipitates pectate and leaves a clear zone wherethe pectate was enzymatically degraded (34).Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresiswas performed by previously described procedures (14).Resolving gels contained 10%, acrylamide, 0.33% bisacryla-mide, and 0.1% sodium dodecyl sulfate. Enzyme samplescontaining 4 to 6 jg of protein or 6 to 12 U of PL activitywere added to each well. The proteirq concentration wasdetermined based on the method of Lowry et al. (19), asdescribed in the Protein Assay Kit (Sigma). Protein molec-ular weight markers purchased from Bethesda ResearchLaboratories (Gaithersburg, Md.) were included in each runto estimate the M,.s of PL samples.

Assay of tissue maceration. Purified or partially purified PLsamples from six representative strains of soft rot bacteriawere compared for their abilities to macerate potato tuber.Potato slices were prepared as described previously (16, 17),except that the slices were treated with 30 pg of kanamycinper ml before they were placed onto 0.6% agar plates. Eachslice was inoculated with 5 pIl of an enzyme sample contain-ing 0.1 to 5.0 U of PL activity. The maceration zone wasexamined after 2 days of incubation at 20°C.

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6 7 8 pllr A bro- 10.6

_W V W W W 4.8FIG. 1. IEF profiles of extracellular PLs produced by E. chry-

santhemi EC16 (lanes 1 and 5), E. carotoivora subsp. carotov'oraSR319 (lanes 2 and 6), E. carotovora subsp. atroseptica SR8 (lanes3 and 7), and E. carotovora subsp. carotovora CU-06-1 (lanes 4 and8). The arrow indicates the sites where the samples were applied.

RESULTS

IEF profiles of extracellular PLs. Concentrated culturesupernatants from 25 strains of soft rot bacteria were sub-jected to thin-layer polyacrylamide gel IEF and subse-quently assayed for PL activity by agarose-pectate overlaystaining techniques. IEF profiles of PLs produced by Er-Winia spp. P. viridiflava, P. fluorescenis, X. cainpestris, andC. johnsonae were distinct. An alkaline PL (pl, -9.7) wasidentified as the sole or the principal PL produced by allgroups of non-Erwinia spoilage bacteria. The specific PLprofiles of each group of organisms are described below.

(i) Erwinia spp. Two strains of E. carotoi'ora subsp.carotovora and one strain each of E. carotoi'ora subsp.atroseptica and E. chr'santhemi were examined. All fourstrains produced at least three PL isozymes (Fig. 1). How-ever, the PL profile of E. chiysanthemni differed greatly fromthat of E. carotovora subsp. atroseptica and E. carotoi'orasubsp. carotovora. E. chrysanthemi EC16 produced onebasic (pl, 10.0), two neutral (pIs, 8.8 to 9.0), and one acidic(pl, 4.2) PLs. E. carotovolra subsp. carotovorla and E.carotovora subsp. atroseptica strains produced three PLisozymes, all of which had alkaline PIs (pl, -9.5). Nodifferences in PL profiles were observed between E. caroto-

2 3 4 5 6 7 8

v'ora subsp. at}roseptica and E. carotov'ora subsp. c ar-oto-vora strains. It should be noted that the acidic PL of E.chrvsantherni EC16 is not visible in Fig. 1. This isozyme isproduced in extremely small quantities (29), and its migra-tion in the IEF gel was interferred with by the enzymesample that was retained at the application site (Fig. 1).

(ii) P. viridiflava. All of the eight P. viridiflai'a strainsincluded in this study produced a single PL at nearlyidentical pIs (9.7) (Fig. 2A). Neutral or acidic PLs were notdetected even when a large quantity of enzyme sample(containing up to 1.6 U of PL activity) was applied (Fig. 2B).The formation of bands with minor activity, similar to thoseshown in lanes 4 and 7 of Fig. 2A, likely resulted from thesample-trailing effect, since these bands did not appearconsistently in every experiment. Bands with irregular ac-tivities were often observed in gel sites where enzymesamples were initially applied (Fig. 2), indicating that aportion of enzyme sample was bound to the gel and remainedimmobilized during electrofocusing.

(iii) P. fluorescens. Ten strains of P. fluorescens isolatedfrom eight different plants were examined (Table 1). Thesestrains differed somewhat in their nutritional and physiolog-ical properties and have been classified and grouped intobiovar II (five strains) or biovar V (five strains) of P.fluiorescens (18). All 10 strains produced, in addition to thealkaline PL (pl, 9.7), a neutral PL with a pl of 6.7. Theproduction of neutral PLs by strains PJ-08-30, HU-08-11B,CY091, and BC-05-lB is shown in Fig. 3A (lanes 5 and 10)and Fig. 3B (lanes 1 and 6). The neutral PL produced bystrains 17816, SJ-08-2, SJ-08-3, BC-05-2B, LC-04-2A, AJ-06-2A is hardly identified in Fig. 3A (lanes 2 to 4 and lanes 7to 9, respectively), but it has been detected in other gels(data not shown). When decreasing amounts of enzymesamples were analyzed by IEF, neutral PL was hardlydetected in samples containing <0.25 U of PL activity (Fig.3B, lanes 4 and 8). Alkaline PL thus constituted the majorproportion whereas neutral PL constituted the minor pro-portion of the total PL produced by P. fluorescens strains.

(iv) C. johnsonae and X. campestris. One strain each of C.johnsonae and X. canmpestris was examined. Both organismsproduced a single PL with a p1 of -10.0. Neutral or acidicisozymes were not detected, even when samples containing2.4 U of PL activity were applied (Fig. 4, lanes 1 and 5).

pl 1 2 3 4 5 6 7 8 pI-10.6 10.6

-8.3

- 7.3

6.5

5.9

-8.3

- 7.3

-6.5

-5.9

-4.8FIG. 2. (A) Extracellular PL profiles of P. viridiflaiva SF312 (lane 1). 13223 (lane 2), SF-05-3 (lane 3), PJ-08-6A (lane 4). PJ-08-6B (lane 5),

PJ-08-9 (lane 6), PJ-08-16B (lane 7), and TU-04-2A (lane 8). (B) Comparison of PL profiles of strains PJ-08-16B (lanes 1 to 4) and SF312 (lanes5 to 8) by using decreasing quantities of PL samples: 1.6 U (lanes 1 and 5). 0.8 U (lanes 2 and 6), 0.4 U (lanes 3 and 7), and 0.2 U (lanes 4and 8). The arrows indicate the sites where the samples were applied.

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IZ 3 4 S 6 7 8 9 10 pl I 2 3 4 5 6 7 8 plr v' 10.6

i83[X 8 . 3~~~~~~~~~8.7.3

6.5~~~~~~~~~~~~~~~~~~~~~.

4 .8 >k-> Z

FIG. 3. (A) IEF profiles of extracellular PLs produced by P. fluorescens CY091 (lane 1). 17816 (lane 2), SJ-08-2 (lane 3), SJ-08-3 (lane 4),PJ-08-30 (lane 5), BC-05-lB (lane 6), BC-05-2B (lane 7), LC-04-2A (lane 8). AJ-06-2A (lane 9) and HU-08-llB (lane 10). (B) Comparison ofextracellular PL profiles of strains BC-05-lB (lanes I to 4) and CY091 (lanes 5 to 8) by using decreasing quantities of PL samples: 2.0 U (lanes1 and 5), 1.0 U (lanes 2 and 6), 0.5 U (lanes 3 and 7), and 0.25 U (lanes 4 and 8). The arrows indicate the sites where the samples were applied.

IEF profiles of intracellular PLs. To determine whether thebacteria produced PL species which were not readily ex-creted into the medium, IEF profiles of PLs prepared fromculture supernatants and from intracellular fluids of sevenrepresentative strains were compared. Results show that theextracellular and intracellular PL profiles of P. iiridiflai'a, P.fluorescens, X. canpestris, and C. jolhnsonae were identical.There was no indication that these organisms produced PLspecies in addition to the PLs that were readily detected inthe culture supernatant. P. fluorescens PJ-08-14 caused verymild or no maceration of potato slices. The PL produced bythis strain could be detected in intracellular fluid but not inculture supernatant. The intracellular PL of strain PJ-08-14was similar, at least in terms of the pl, to the alkaline PL ofsoft rot strains (CY091 and BC-05-IB) of P. fliuore^scens.

Purification of PLs. Extracellular PLs were purified fromculture supernatants of E. (chysanthedini EC16, P. flito-rescens CY091 and BC-05-lB, P. viridiflava SF312, X.campestris CJ091, and C. johnsonae PF062 by ammoniumsulfate precipitation and anion-exchange chromatography.About 40 to 60% of total PL activity was recovered in theprecipitate obtained between 50 and 85% saturation withammonium sulfate. The dialyzed enzyme samples weresubjected to DEAE-cellulose (Cl-) chromatography and

2 3 4 5 6 7 8 p1X.____ -10:.6

-8.3

-7.3

4.) 6 . 5~~~~~6.

t 5 . 9~~~~~~~~~.-0!.t t 9 4.~~~~~48

FIG. 4. Extracellular PL profiles of C. jolhn.sonae PF062 (lanes 1to 4) and X. campestris CJ091 (lanes 5 to 8). The amounts of PLsample used were as follows: 2.4 U (lanes 1 and 5), 1.2 U (lanes 2

and 6), 0.6 U (lanes 3 and 7), and 0.3 U (lanes 4 and 8). The arrowindicates the site where the samples were applied.

were eluted with the Tris-HCl buffer (50 mM, pH 8.0)containing 0.00, 0.05, 0.10, or 0.20 M NaCl. For PLs of E.chrysanthemi EC16, two peaks of enzyme activity wereobserved. The major peak was eluted with the buffer alone,whereas the minor peak was eluted with buffer containing0.10 M NaCl. For PLs of non-Erit'inia strains, the enzymewas eluted as a single peak with buffer containing 0.05 MNaCl.

Purified PL samples were subsequently examined forpurity, M,., and pl by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (Fig. 5) and thin-layer polyacrylamide gelIEF (Fig. 6). PLs produced by two strains of P. fluorescensand one strain of P. viridiflava were purified almost tohomogeneity by the two-step procedures described above(Fig. 5, lanes 5 to 7; Fig. 6, lanes 2 to 4). However, PLsamples prepared from E. (hrysanthemi EC16 (Fig. 5, lanes4, Fig. 6, lane 5), X. campestris CJ091 (Fig. 5, lane 3; Fig. 6,lane 6), and C. johlsolace PF062 (Fig. 5, lane 2; Fig. 6, lane

l 2 3 4 5 6 7 8 MW- 04. -200;0OK

-97.4K

-68.0K

- 43.0K

-25.7K

4i -l 1 8.4 K

FIG. 5. Sodium dodecyl sulfate-polyacrylamide gel electropho-resis of extracellular PLs purified from culture supernatants of C.johnsonae PF062 (lane 2), X. campestris CJ091 (lane 3), E. chiysan-tlielni EC16 (lane 4), P. i'ridifltwa SF312 (lane 5), and P.fluorescensBC-05-lB (lane 6) and CY091 (lane 7). The M, markers (MW [inthousands in the figure]; lanes 1 and 8) used were myosin (200,000),phosphorylase h (97,400), bovine serum albumin (68,000), ovalbu-min (43.000), cx-chymotrypsinogen (25,700), and ,B-lactoglobumin(18,400).

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2 3r a'* 4

_

IN

I&a.=I*_ #

4 5 6,I R. I

7 8 pl_~ _ -IO.6

h-J 8.3

6.5

4.8

FIG. 6. Thin-layer polyacrylamide gel IEF of extracellular PLpurified from culture supernatants of P. fliorescens CY091 (lane 2)and BC-05-lB (lane 3). P. v'iridiflava SF312 (lane 4). E. (cIhlrs(l-thlielii EC16 (lane 5), X. campestris CJ091 (lane 6), and C. jolhnslsotlePF062 (lane 7). The gel was stained with Coomassie blue. The plmarkers (lanes 1 and 8) were cytochrome c (horse heart) (10.6).myoglobin (sperm whale) (8.3), myoglobin (equine) (7.3). myoglobin(procine) (6.5), trifluroacetylated myoglobin (porcine) (5.9). andC-phycocyanin (4.8).

7) were contaminated with other proteins, since severalprotein bands were detected by Coomassie blue staining andonly one of them was shown to contain PL enzyme activityby the agarose-pectate overlay staining technique (28). PLsproduced by the various groups of soft rot bacteria were

different not only in pl but also in molecular mass. The Mrsof PLs produced by P. fluorescens. P. viridiflaval, X.caimpestris, and C. jolhnisonale were estimated to be 41.000.42,000, 41,500, and 35,000, respectively.

Induction of tissue maceration by purified PLs. PLs purifiedfrom culture supernatants of six representative strains were

capable of inducing soft rot of potato slices, but to differentdegrees. The enzyme preparation of E. (clhrysnthleni EC16containing 0.5 U or less of PL activity was sufficient to cause

visible maceration of potato slices. On the contrary, the PLpreparation of C. jolhaisonntie was the least effective in induc-ing tissue maceration. An enzyme preparation containing 5.0U or more of PL activity was required to induce visiblemaceration. The macerating ability of PL produced by P.fluor-escens. P. iviridcflivai, and X. camlpestris was moderate.falling between the PL of E. (clhrsallthwehi and that of C.jolilisoiiae.

DISCUSSION

The data presented in this study demonstrate that PLprofiles of non-Erwiiinia soft rot bacteria are much simplerthan those of Erwtiinia spp. All of the P. ilridiflava, P.flioescenis. X. cainpestris, and C. joIhnzsonlae strains thatwere examined produced one or two PLs, whereas all of theErwt inia strains-that were studied here and elsewhere (2, 27.29, 34, 36) produced three or more. Erwt inia spp. as a wholediffer in their pathological capacity from non-Er-winiia soft rotbacteria, in that the former attack plants both in the field andin storage (26) and the latter usually cause spoilage ofdetached plant products after harvest (16-18. 20). The simplepectic enzyme system detected in non-Er-winiai soft rotbacteria may account, at least in part, for the low efficiencyof these microorganisms to attack actively growing plants.Davis et al. (5) have reported that PLs purified from culturesof E. carotovora are capable of eliciting phytoalexin accu-

mulation in plants. They proposed that soft rot pathogens

must produce large quantities or multiple forms of PL toovercome the host defense mechanism triggered by pecticenzymes or other elicitors. The correlation between the lowPL activity and the inability to infect field plants, as ob-served in non-Erwi-ii soft rot bacteria, seems to provideindirect evidence supporting the above hypothesis. More-over. it is worth noting that the postharvest pathogen X.caimpestris CJ091 examined in this study produced a singlePL. This organism is indistinguishable from the black rotpathogen X. (cimpestris pv. camnpestris in terms of itsphysiological and nutritional properties (17). In contrast tothe single PL detected in strain CJ091, the field pathogen X.camnpestris pv. campestris has been shown to produce atleast three PL isozymes (6).Two lines of evidence suggest that PL isozymes of E.

chlrysintheini may play a role in the recognition of hostplants. Ried and Collmer (29) and Thurn et al. (33) havereported that PL profiles of E. clhrysinthemni strains isolatedfrom different hosts are distinct, but the profiles are similaramong strains isolated from the same host. Barras et al. (1)have shown that the reaction patterns catalyzed by four PLsof E. chIrvsanthelni EC16 are different. The 8 strains of P.'i/ridflavati and the 10 strains of P. fliorescens examined inthis study were isolated from 10 different hosts (Table 1).Despite the difference in host origins and in nutritional andphysiological properties (18). strains of P. viridifltiva and P.fltorescens appear to possess a common PL pattern specificfor either species. The PL genes of P. iiridiflai'ai or P.fliuorescens strains are possibly derived from the same originand are well conserved during evolution. Because of thehomogeneous pattern, it is unlikely that PLs of P. fluo-r.escens or P. viridliflai'a serve a function in differentiatingone host from another.PLs with alkaline pls appear to be the principal or the sole

enzymatic factor involved in tissue maceration caused bymost strains of soft rot bacteria. Results obtained from anumber of recent studies, including the one reported here.strongly support this conclusion. Kotoujansky (12) andCollmer and colleagues (25. 30) have shown that deletion ofalkaline PL genes from the genome of E. (hrystintlhenigreatly diminishes the virulence or macerating ability of theorganism. Chatterjee and colleagues (1, 33) have reportedthat alkaline PL constitutes 40 to 60% of total PLs producedby E. chrvsaIlltIlenhi EC16 and that alkaline PL is moreefficient in inducing tissue maceration. electrolyte loss, andcell death than are neutral or acidic PLs. Acidic PLsproduced by certain strains ofE. chrysantlhemi (1. 11, 12. 30,33) and species of Yer-sinii( (3. 21) and Klebsiellai (3) usuallyexhibit very little or no macerating ability. The biochemicalbasis for the fact that alkaline PLs are more effective ininducing tissue maceration than are neutral or acidic PLs isnot known but warrants further investigation.Because of their simple pectic enzyme systems, non-

Ertwinia soft rot bacteria could provide useful sources for thepreparation of pure PLs. In this study. PLs produced byrepresentative strains of P. viridfflai'a, P. fluorescens, X.clampestris. and C. jo/hn sontie were purified or partiallypurified following two simple steps: ammonium sulfate pre-cipitation and anion-exchange chromatography. Purified orpartially purified PL preparations differed in pls. Mr.s, andtissue-macerating abilities. The enzymological basis onwhich one PL is more efficient in inducing maceration ofpotato slices than another is not understood. Preliminaryresults of a study of enzyme kinetics have indicated that PLsproduced by the aforementioned organisms degrade polyga-lacturonates by the endo mode of action but exhibit some-

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APPL. ENVIRON. MICROBIOL.

what different cleavage patterns (A. L. Hotchkiss, K. B.Hicks, and C. H. Liao, unpublished data). It remains to bedetermined whether the difference in enzymological proper-ties detected in vitro can be correlated with the difference intheir ability to digest the pectic components of various plantcell walls.

Previous studies of PLs from a limited number of strains ofP. fluiorescens (7, 9, 10, 23, 37), P. viridiflav'a (10, 15), X.ccampestris (17, 24), and C. johnsoniae (13, 16, 35) havefocused on the detection and inducibility of the enzymes.Results presented in this study provide further informationabout the isoelectric properties, Mrs, and tissue-maceratingabilities of PLs produced by the organisms listed above. Therelatively simple pectic enzyme system demonstrated innon-Erwinia soft rot bacteria indicates that these microor-ganisms may be used as a model to study how procaryotessynthesize and excrete extracellular proteins at the molecu-lar level.

ACKNOWLEDGMENTS

I thank W. F. Fett and J. S. Huang for reviewing the originalversion of the manuscript.

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