Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 240613 .... North Dakota State University, Fargo) .... The bar at the upper right.
Vol. 167, No. 1
JOURNAL OF BACTERIOLOGY, JUlY 1986, p. 279-284
0021-9193/86/070279-06$02.00/0
Requirement for Two or More Erwinia carotovora subsp. carotovora Pectolytic Gene Products for Maceration of Potato Tuber Tissue by Escherichia colit DANIEL P. ROBERTS,1 PHYLLIS M. BERMAN,2 CAITILYN ALLEN,3 VERLYN K. STROMBERG,3 GEORGE H. LACY,3* AND MARK S. MOUNT2 Department of Plant Pathology, University of Georgia, Athens, Georgia 30602,1 Department of Plant Pathology, University of Massachusetts, Amherst, Massachusetts 01003,2 and Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 240613 Received 1 August 1985/Accepted 15 April 1986
Several genes encoding enzymes capable of degrading plant cell wail components have been cloned from Erwinia carotovora subsp. carotovora EC14. Plasmids containing cloned EC14 DNA mediate the production of endo-pectate lyases, exo-pectate lyase, endo-polygalacturonase, and cellulase(s). Escherichua coil strains containing one of these plasmids or combinations of two plasmids were tested for their ability to macerate potato tuber slices. Only one E. coli strain, containing two plasnids that encode endo-pectate lyases, exo-pectate lyase, and endo-polygalacturonase, caused limited maceration. The pectolytic proteins associated with one of these plasmids, pDRI, have been described previously (D. P. Roberts, P. M. Berman, C. Allen, V. K. Stromberg, G. H. Lacy, and M. S. Mount, Can. J. Plant Pathol. 8:17-27, 1986) and include two secreted endo-pectate lyases. The second plasmid, pDR30, contains a 2.1-kiloabse EC14 DNA insert that mediates the production of an exo-pectate lyase and an endo-polygalacturonase. These enzymes are similar in physicochemical properties to those produced by EC14. Our results suggest that the concerted activities of endo-pectate lyases with endo-polygalacturonase or exo-pectate lyase or both cause maceration.
information has been presented [D. P. Roberts, P. M. Berman, G. H. Lacy, and M. S. Mount, Proceedings of the 6th International Conference on Plant Pathogenic Bacteria, College Park, Md., 2 to 7 June, 1985].)
Erwinia carotovora subsp. carotovora causes soft rot on many plants. Soft rot results from the activity of extracellular enzymes (11). Extracellular enzymes produced by E. carotovora subsp. carotovora capable of degrading plant cell wall and plant cell membrane components include the following: endo-pectate lyase, endo-polygalacturonase, cellulase, protease, phosphatidase C, and phosholipase A (2, 8, 9, 18, 20, 22). In an effort to clarify the role of Erwinia pectic enzymes in pathogenesis, several laboratories have isolated genes encoding enzymatic pathogenic determinants from Erwinia spp. (1, 6, 10, 17a, 23). Pectolytic enzymes are key factors in soft rot pathogenesis (for a review, see reference 5); however, the individual roles of the six or more pectolytic enzymes produced by E. carotovora subsp. carotovora EC14 (17a, 18) are not fully understood. Purified EC14 endo-pectate lyase macerates potato (Solanum tuberosum L.) tuber slices, but a strain of Escherichia coli that secretes this endo-pectate lyase does not macerate potato tuber slices (17a). In contrast, an E. coli strain which produces an Erwinia chrysanthemi pectate lyase does macerate potato tuber slices (10). We sought to determine whether the combined activities of EC14 cell wall-degrading enzymes would enable E. coli to macerate tissue. We report here the interaction of E. coli strains harboring plasmids that encode EC14 pectolytic enzymes with potato tuber slices, the cloning and linkage of the EC14 intracellular exo-pectate lyase and endo-polygalacturonase on a 2.1kilobase (kb) fragment, and the cloning of the gene(s) mediating cellulase activity. (A preliminary report of some of this
MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids constructed or used or both constructed and used in this research are listed in Table 1 and in Table 2, respectively. Strain names with the prefix L- indicate designations for strains in the culture collection of G. H. Lacy. Chromosomal DNA isolation. Chromosomal DNA was isolated from strain EC14 as previously reported (17a). Cloning and construction of recombinant strains. Strain EC14 chromosomal DNA was partially digested with BamHI; ligated into dephosphorylated, BamHI-cleaved pBR322 DNA; and transformed into E. coli HB101 by procedures described previously (17a). Transformants with chimeric plasmids had ampicillin-resistant, tetracyclinesensitive phenotypes when screened on YT agar medium (13) containing ampicillin (30 jig/ml) or ampicillin and tetra-
cycline (10 ,ug/ml). Transformants producing pectolytic or cellulolytic enzymes were detected on ampicillin-amended PEC-YA medium (19) or M9 cellulase medium (la), respectively. Strains containing plasmid combinations were constructed by transforming E. coli L-643 (containing pDR1 mediating tetracycline resistance) with pDR30, pDR40, or pDR70 (all mediating ampicillin resistance); they were selected on YT medium containing both antibiotics. The phenotypes and genotypes of these strains were confirmed by plating on enzyme detection media and by plasmid analyses with agarose gel electrophoresis.
* Corresponding author. t Contiibution number 535 from the Department of Plant Pathology, Physiology, and Weed Science, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061.
Plasmid isolation and characterization. Plasmid DNA was isolated by the procedure of Birnboim and Doly (3) as
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TABLE 1. Bacterial strains used Strain (other
EC14a (ICPPB.-EC14b, CUCPB-550c,
Description (source or reference or both) E. carotovora subsp. carotovora EC14 (14)
L-543d) HB101 (L-482) L-630 L-643 L-645 (ICPPB-3739) L-669 L-670 L-673 L-757 L-758 L-761 L-781
E. coli hsdR hsdM recA supE44 lac Y leuB6 proA2 thiBI rpsL20 (R. B. Sparks, Jr., North Dakota State University, Fargo) E. coli DH1(pPL7) (10) E. coli HB1O1(pDR1) (17a) Kleb-siella pneumoniae (A. K. Chatterjee, Kansas State University, Manhattan) E. coli HB101(pDR30) (this report) E. coli HB101(pDR40) (this report) E. coli HB101(pDR70) (this report) E. coli HB101(pDR1, pDR30) (this report) E. coli HB101(pDR1, pDR40) (this report) E. coli HB1O1(pDR1, pDR70) (this report) E. coli HB1O1(pDR31) (this report)
a Strain EC14 was isolated from yellow calla lily (Calla palustris L.) by A. P. Ark in California, placed in W. H. Burkeholder's collection in March 1946, and provided to us by R. S. Dickey (Cornell University, Ithaca, N. Y.). b ICPPB, International Collection of Plant Pathogenic Bacteria, University of California, Davis. c CUCPB, Cornell University Collection of Plant Pathogenic Bacteria, Ithaca, N. Y. d Strain names with the prefix L-, collection of G. H. Lacy, Virginia Polytechnic Institute and State University, Blacksburg.
described by Maniatis et al. (12). Restriction endonuclease analyses of isolated hybrid plasmid DNA were performed with BamHI, BglII, EcoRI, HincII, HindIII, HpaI, NruI, PstI, and SalI under the conditions suggested by the suppliers (Bethesda Research Laboratories, Gaithersburg, Md.; Boehringer Mannheim Biochemicals, Indianapolis, Ind.; and New England BioLabs, Inc., Beverly, Mass.). Hybrid plasmids and restriction fragments were analyzed by agarose gel electrophoresis. Transposon mutagenesis with lambda NK467 was performed by the method of de Bruijn and Lupski (7). Plasmid pDR31 was constructed by recircularization of BglII-digested pDR30. Southern hybridization analyses of complete BamHI digests of total EC14 chromosomal DNA were performed with BamHI-digested pDR30 or pDR70 as the probe. Probe DNA was biotin labeled by nick translation under the conditions recommended by the supplier (Bethesda Research Laboratories). Prehybrndization and hybridization on nylon membranes (Pall Corporation, Glen Cove, N.Y.) were carried out at 42°C in 45% formamide (12). Hybridized probe was detected with the DNA Detection System (Bethesda Research Laboratories) used as recommended by the manufacturer. The 2.1-kb PstI-EcoRI fragment of pDR1 was used as the probe and hybridized to pDR40 in Southern blots as described above; Enzyme fractionation and characterization. Intra- and extracellular pectolytic enzymes produced by E. coli L-669 were extracted by procedures reported previously (17a). The protein in these extracts was purified and characterized by DEAE-cellulose column chromatography, column isoelectric focusing, flatbed isoelectric focusing, and thinlayer chromatography as described previously (17, 17a, 18). Enzyme activity was analyzed by the thiobarbituric acid (TBA) assay and Nelson's reducing sugar analysis (15, 17a, 21). Tuber slice maceration. Tuber slice maceration tests were
J. BACTERIOL.
performed by the eye-of-the-needle method of Roberts et al. (17a) and the well method of Keen et al. (10) with E. coli strains containing plasmids pPL7, pBR322, pDR1, pDR30, pDR40, and pDR70 and E. coli strains containing plasmid pDR1 in combination with pDR30, pDR40, or pDR70. RESULTS Cloning and plasmid characterization. Approximately 3,400 ampicillin-resistant, tetracycline-sensitive colonies were isolated. Of these, two were pectolytic and one was cellulolytic on the appropriate enzyme detection medium. Restriction endonuclease analyses of plasmids pDR30 and pDR40 isolated from the pectolytic clones indicated that they contained 2.1- and 8.7-kb BamHI EC14 DNA inserts, respectively. Plasmid pDR70, isolated from the cellulolytic clone, contained a 7.1-kb EC14 DNA insert. The same phenotype was associated with pDR30, pDR40, or pDR70 upon retransformation of HB101 with each of these plasmids. Restriction maps of the EC14 DNA inserts from plasmids pDR30, pDR40, and pDR70 are shown in Fig. 1. The EC14 DNA inserts from plasmids pDR30 and pDR70 hybridized to 2.1- and 7.1-kb EC14 chromosomal DNA BamHI fragments, respectively. The EC14 DNA region of pDR1 hybridized to pDR40 but not to pBR322. Restriction endonuclease analyses of plasmids pDR1 and pDR40 indicated that both plasmids con'tained 3.4-kb EC14 DNA PstI fragments; the restriction map of pDR1 was determined previously (17a); Therefore, pDR40 contains the EC14 DNA of pDR1 and flanking regions of the EC14 chromosome. Enzyme characterization. DEAE-cellulose DE53 elution profiles and isoelectric focusing profiles from intra- and TABLE 2. Plasmids used Plasmid
pPL7 pDR1
pDR30
pDR30T4 pDR30T11
pDR31
pDR40
pDR70
Sie AntibioticDecito resistnce (source or reference or both) (kb)
Tcr
Encodes an endo-pectate lyase cloned from E. chrysanthemi EC16 with a pl of 8.8 (10) Encodes two pectate lyases 7.8 Tcr cloned from E. carotovora subsp. carotovora EC14 with pls of 9.5 and 7.5 (17a) 6.4 Apr Encodes a pl 7.8 exo-pectate lyase and an endopolygalacturonase cloned from EC14 (this report) 12.1 Apr, Kmr Transposon TnS mutant of pDR30; no pectate lyase activity associated with this plasmid (Fig. 4; this report) 12.1 Apr, Kmr Transposon Tn5 mutant of pDR30; no pectate lyase activity associated with this plasmid (Fig 4; this report) A subclone of pDR30 missing the 6.0 Apr 0.43-kb BglII fragment; no pectate lyase activity associated with this fragment (Fig. 4; this report) 13.0 Apr Contains the EC14 pDR1 insert and flanking regions; encodes pectolytic enzymes cloned from EC14 (this report) 11.4 Apr Encodes cellulolytic enzyme(s) cloned from EC14 (this report)
>40
VOL. 167, 1986
ERWINIA SPP. GENE PRODUCTS REQUIRED FOR MACERATION
281
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FIG. 1. Restriction maps of the E. carotovora subsp. carotovora EC14 DNA insert region of plasmids pDR30, pDR40, and pDR70. The approximate distance in base pairs from the BamHI site closest to the EcoRI site of plasmid pBR322 is indicated below each restriction site. The insert in pDR30 contains no EcoRI, HindIII, or Sall sites; the pDR7i insert contains no PstI or HindIlI sites. The bar at the upper right indicates 1 kilobase.
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FIG. 2. DEAE-cellulose (DE53) column chromatography elution profile of the intracellular extract from E. coli L-669. Fractions were incubated with L.0o sodium polypectate in 0.05 M Tris hydrochloride for 8 h. The relative pectate lyase activities were determined by the TBA assay (A54g) on reactions performed at pH1 8.5 in the presence of 0.15 mM CaC12. Relative polygalacturonase activities (-) were determined by the Nelson reducing sugar assay (Am) at pH 6.0 in the presence of 0.5 mM EDTA.
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10
2.0
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Fraction Number FIG. 3. Isoelectric focusing profile of the pooled void volume fractions eluting from DEAE-celiulose columns of the intracellular extract from E. coli L-669. Fractions were incubated with 1.0%o sodium polypectate in 0.05 M Tris hydrochloride for 8 h. The relative pectate lyase activities (-) were determined by the TBA assay (As") on reactions performed at pH 8.5 in the presence of 0.15 mM CaC12. No polygalacturonase activity was detected by the Nelson reducing sugar assay (A5m) at pH 6.0 in the presence of 0.5 mM EDTA. -----, pH of the fractions.
extracellular protein extracts of E. coli L-669 were similar. Polygalacturonase and pectate lyase were detected in the void volumes from DEAE-cellulose columns (Fig. 2). ColTABLE 3. Reaction product analyses of proteins extracted from E. coli L-669 Analysis Relative Fraction (pH) bCtlVityb Reaction productsc or assay"actvt
PEAE-celiulose peak 8.5 6.0
TBA
6.0
Nelson's
>2.20 0.00 1.95
TBA TBA Nelson's
1.70 0.00 0.00
pl 7.8 peak 8.5 6.0 6.0
TBA
uDimer, utrimer NDd Trimer-hexamer (traces of udimer and utrimer)
uDimer, utrimer ND ND
Analysis of reaction products was by the TBA assay in the presence of 0.15 mM CaC2 for pectate lyase or by Nelson's reducing sugar analysis in the presence of 0.5 mM EDTA for polygalacturonase. b A unit of activity is defined as the As" as determined by TBA assay or the As_ as determined by Nelson's analysis for 800 ml of bacterial culture suspension adjusted turbidimetrically to 109 CFU/ml from which the proteins wefe extracted. C uDimer and utrimer, Unsaturated dimer and unsaturated trimer were detected as reaction products. Trimer-hexamer, saturated trimer, tetramer, pentamer, and hexamer were detected among the reaction products. Reaction products were identified by chromatographic mobility of the degradation products formed from the reaction of sodium polypectate with fractions eluted from DEAE-ceilulose or by isoelectric focusing. The methods were described by Stack et al. (18). d ND, Reaction products were not determined since no enzyme activity was detected. I
umn isoelectric focusing resolved a pectate lyase with an isoelectric point of 7.8 (Fig. 3). Strain EC14 and E. coli L-669 produced pectate lyases which comigrated on flatbed isoelectric focu'sing gels. The polygalacturonase was not stable during isoelectric focusing; no activity was detected in any fraction. Reaction product analyses were performed on the pI 7.8 protein at pH 8.5 in the presence of Ca2+ by TBA assay and thin-layer chromatography. This enzyme is an exo-pectate lyase since it produced unsaturated dimer and unsaturated trimer from sodium polypectate (Table 3). Analyses of polygalacturonase reaction products were conducted on pectolytic fractions from the void volume owing to the' instability of this enzyme during isoelectric focusing. These reactions were conducted at pH 6.0 in the presence of EDTA. No pectate lyase activity was detected in 8-h reactions; however, trace levels of pectate lyase activity were detected by TBA assay in 16-h reactions. In both TABLE 4. Cation preference of the isoelectrically focused pl 7.8 enzyme produced by E. coli L-669 Iona
pH
Sp +actSD)b (mean
Mn2+ Ca2+ EDTA
8.5 8.5 8.5 6.0 6.0
153.4 105.4 29.0 1.7 2.2
Mn2+ Ca2+ a A 0.5 mM
+ 4.7 ± 4.6
± 2.4 ± 0.3 ± 0.2
concentration of EDTA was added to substrates to chelate
endogenous ions, and then 0.1 mM Ca2 , Mn2 , or no ion was added. b Specific activity is expressed in micromoles of unsaturated uronide product formed per milligram of protein and was determined by the TBA assay.
ERWINIA SPP. GENE PRODUCTS REQUIRED FOR MACERATION
VOL. 167, 1986
Ir x90 Q
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283
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FIG. 4. Transposon Tn5 mutagenesis and subclone analyses of plasmid pDR30 which encodes endo-pectate lyase and endopolygalacturonase activity. Transposon Tn5 insertions T4 (in plasmid pDR3OT4) and Tll (in plasmid pDR3OT11) map (V) at base pairs 1981 and 1764, respectively. A subclone, plasmid pDR31, contained pDR30 EC14 DNA deleted for the 430-base-pair BglII-BglII fragment. Plasmids pDR31, pDR3OT4, and pDR3OT11 no longer expressed pectate lyase activity.
reactions, high levels of reducing sugars were detected, suggesting polygalacturonase activity. Trace amounts of unsaturated dimer and trimer and large amounts of saturated trimeric, tetrameric, pentameric, and hexameric galacturonic acid were detected by thin-layer chromatography (Table 3), indicating endo-polygalacturonase activity. The trace levels of unsaturated products were probably the result of residual exo-pectate lyase activity. We concluded that this 2.1-kb fragment mediates the production of both endo-polygalacturonase and exo-pectate lyase. The pl 7.8 exo-pectate lyase preferred Mn2+ over Ca2+ at pH 8.5 and showed trace levels of activity at pH 6.0 (Table 4), corresponding to the intracellular exo-pectate lyase PDII from EC14 described by Stack et al. (18). However, unlike PDII, this enzyme showed limited activity in the presence of TABLE 5. Maceration of potato tuber slices by E. carotovora subsp. carotovora EC14 and E. coli HB101 strains harboring plasmid pBR322 or chimeric plasmids containing cloned EC14 DNA Well maceration assay Bacterial straina
EC14 HB101(pBR322) L-630 L-643 L-669 L-670 L-673 L-757 L-758 L-761
Score (no.
positive/no. tested)b 6/6 0/8 8/8 1/18 0/8 0/8 0/8 8/12 0/8 0/9
CFU/ml of inclm inoculum
6.23 4.30 3.42 1.30 4.19 4.79 7.43 1.00 1.31 3.00
x x
107 107
x x x x x x x x
107 106 106 106 104 106 106
106
No. of CFU in tissuec
ND ND ND ND 1.40 x 107 3.60 x 106 2.54 x 107 1.36 x 106 3.93 x 107 1.30 x 107
a See Table 1 for descriptions of the bacterial strains and Fig. 1 and Table 2 for descriptions of the plasmids. b Results shown in this column are cumulative data from several experiments in which results are scored as the number of macerated tuber slices per total number of inoculated tuber slices. In each of four experiments, 2 tuber slices were inoculated per treatment, except for the slices inoculated with strains L-643 and L-757; for these treatments, 12 and 4 slices, respectively, were inoculated in the third experiment. c Strains L-669, L-670, L-673, L-757, L-758, and L-761 were plated onto plate count agar containing the appropriate antibiotic(s) 48 h after potato tuber slice inoculation to determine plasmid stability. All strains sampled were stable in tuber tissue for the duration of the experiment. ND indicates that the number of CFU in potato tuber tissue was not determined. However, the ability of strains EC14, HB101(pBR322), and L-643 to colonize potato tuber slices has been described previously (17a).
FIG. 5. Maceration of potato tuber slices inoculated by the well method of Keen et al. (10). Wells (2.0-mm diameter) cut in whole tuber slices were filled with 50 RI1 of LB medium (13) containing the appropriate antibiotic(s) plus (A) E. carotovora subsp. carotovora EC14, (B) E. coli L-630, (C) E. coli L-643, (D) E. coli L-669, (E) E. coli L-757, (F) E. coli HB101(pBR322), and (G) sterile antibioticamended broth. After incubation at 30°C for 48 h, blocks of tuber tissue surrounding the wells were cut out and photographed.
EDTA. This may be due to incomplete chelation of endogenous cations at pH 8.5 or to trace contamination with the
polygalacturonase. Transposon mutagenesis and subclone analyses. These studies indicate that the exo-pectate lyase gene spans the BglII site at base pair 1400 and the TnS insertion site at base pair 1981 on the pDR30 EC14 DNA insert. Neither plasmid pDR30 containing transposon inserts T4 and Tll nor plasmid pDR31 (Table 2; Fig. 4) continued to produce pectate lyase, as determined by TBA analysis of intra- and extracellular protein extracts from HB101 containing these plasmids. Tuber slice maceration. When strains EC14, HB101 (pBR322), L-630, L-643, L-669, L-670, L-673, L-757, L-758, and L-761 were tested for maceration of potato tuber slices by the eye-of-the-needle method, only EC14 caused maceration as described previously (17a). When the well method was used, strain EC14 and strain L-630 macerated tuber slices as reported previously (10, 17a). Strain L-757 also macerated potato tuber slices, although to a lesser extent (Table 5; Fig. 5). DISCUSSION E. coli L-757, carrying plasmid pDR1 which encodes endo-pectate lyases and plasmid pDR30 which encodes endo-polygalacturonase and exo-pectate lyase, consistently macerated potato tuber slices. It is not known why strain L-643 macerated potato tuber slices in only 1 of 18 trials; however, E. carotovora subsp. carotovora and pectolytic clostridia are normal tuber microflora, and rotting caused by these indigenous organisms cannot be ruled out (16). Because E. coli L-669 and L-643 did not consistently macerate tuber slices, we conclude that the combination of endopectate lyase with either endo-polygalacturonase or exopectate lyase or both increases the incidence of maceration. However, other factors may be involved in pathogenesis since (i) the degree of maceration caused by L-757 was considerably less than the maceration caused by L-630 or EC14, (ii) unnaturally high levels of inoculum were used, and (iii) maceration occurred only when the test of Keen et al. (10) was used.
284
ROBERTS ET AL.
The well maceration assay of Keen et al. (10) is apparently less stringent for pathogenicity than the eye-of-the-needle assay of Roberts et al. (17a). It is possible that with the well method (i) the immersion of potato tuber slices in water impairs host defense mechanisms or (ii) the use of LB medium in inoculations provides the bacteria with an exogenous nutrient source, resulting in the increased ability of E. coli strains to cause maceration. It is significant that pDR30, containing a 2.1-kb BamHI insert, mediated the production of two pectolytic enzymes. This linkage indicates that some genes related to pathogenicity are clustered. Although we have not demonstrated coordinate regulation of the intracellular exo-pectate lyase PDII with the endo-polygalacturonase on the cloned fragment, until flanking sequences are studied overall regulation cannot be ruled out. Hypothetically, in EC14, coordinate regulation would allow the endo-polygalacturonase to degrade plant cell wall pectic polymers to soluble oligogalacturonic acids. In turn, the intracellular exo-pectate lyase PDII would reduce these oligosaccharides to 4,5unsaturated di- and trigalacturonic acids (5, 18), Unsaturated digalacturonic acids may act as inducers of endo-pectate lyase in E. carotovora subsp. carotovora (P. M. Berman and M. S. Mount, unpublished observations), as well as in E. chrysanthemi (4). Because we were able to obtain maceration by using strain L-757, we may have reconstructed, in E. coli, part of the pectolytic mechanism for tissue degradation by allowing the major extracellular endo-pectate lyase of EC14, PDIa, to interact with endo-polygalacturonase and PDII. J. W. Willis and A. K. Chattejee (Proc. 6th Int. Conf. Plant Path. Bact., 1985) have provided evidence for a similar linkage of pectolytic enzymes on a DNA fragment isolated from E. carotovora subsp. carotovora EC71. ACKNOWLEDGMENTS We gratefully acknowledge the gift of plasmid pPL7 from N. T. Keen (Department of Plant Pathology, University of California, Riverside). We also appreciate the critical review of this manuscript by J. K. Sheehan, S. A. Tolin, and L. D. Moore. This research was supported in part by a USDA CRGO grant (00034034), a grant (BIO-85-018) from the Center for Innovative Technology, Herndon, Va., and a grant (6124370) from the Hatch Project.
LITERATURE CITED 1. Allen, C., V. K. Stromberg, F. D. Smith, G. H. Lacy, and M. S. Mount. 1986. Complementation of an Erwinia carotovora subsp. carotovora protease mutant with a protease-encoding cosmid. Mol. Gen. Genet. 202:276-279. la.Andro, T., J.-P. Chambost, A. Kotoujansky, J. Cattaneo, Y. Bertheau, F. Barras, F. Van G"segem, and A. Coleno. 1984. Mutants of Erwinia chrysanthemi defective in secretion of pectinase and cellulase. J. Bacteriol. 160:1199-1203. 2. Beraha, L., and E. D. Garber. 1971. Avirulence and extracellular enzymes of Erwinia carotovora. Phytopathol. Z. 70:335-344. 3. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. 4. Collmer, A., and D. F. Bateman. 1982. Regulation of extracel-
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