Vol. 170, No. 4JOURNAL OF BACTERIOLOGY, Apr. 1988, p. 1445-14510021-9193/88/041445-07$02.00/0Copyright C) 1988, American Society for Microbiology

Cloning of the egl Gene of Pseudomonas solanacearumand Analysis of Its Role in PhytopathogenicityDANIEL P. ROBERTS,1'2 TIMOTHY P. DENNY,2 AND MARK A. SCHELL'i2*

Department of Microbiology1 and Department ofPlant Pathology,2 University of Georgia, Athens, Georgia 30602

Received 22 June 1987/Accepted 23 December 1987

The egl gene of Pseudomonas solanacearum was cloned on a cosmid and expressed in Escherichia coli.Restriction endonuclease mapping, transposon mutagenesis, and subclone analysis showed that the egl genewas located on a 2.7-kilobase XhoI-Salf P. solanacearum DNA fragment. Immunoabsorption experiments andsodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis showed that the egl gene encodes the43-kilodalton endoglucanase that is the major excreted endoglucanase of P. solanacearum. In E. coli, the eglgene appeared to be expressed from its own promoter, but its product was restricted to the cytoplasm. Thecloned egl gene was mutagenized with TnS and used to specifically mutate the chromosomal egl gene of P.solanacearum by site-directed mutagenesis. The resultant mutant was identical to the wild-type strain inproduction of extracellular polysaccharide and extracellular polygalacturonase as well as several other excretedproteins but produced at least 200-fold less endoglucanase. This mutant strain was significantly less virulent ontomato than the wild-type strain in plant bioassay experiments. Virulence of the endoglucanase-deficient strainwas restored to near wild-type levels by complementation in trans with the cloned egl gene, indicating that theegl gene is important but not absolutely required for pathogenesis.

Pseudomonas solanacearum, a phytopathogenic bacte-rium that is distributed worldwide, causes a lethal wiltingdisease on over 200 different host plants including theimportant crops potato, tobacco, tomato, and banana (4).The mechanisms by which this bacterium produces diseaseremain uncertain. Husain and Kelman (10) concluded thatextracellular polysaccharide slime was the primary factorinvolved in producing wilt symptoms. It was also demon-strated that culture filtrates from this pathogen containedpectinmethylesterase, polygalacturonase, and cellulase ac-tivities (9, 13). The authors suggested that these enzymesmay also have important roles in disease by facilitating thepenetration and breakdown of host tissues. However, theseconclusions were based on studies of spontaneous pleio-tropic, nonpathogenic mutants and biochemical tests withrelatively crude preparations.The advent of molecular genetics has led to approaches

that allow the investigation of specific mechanisms of patho-genesis. Boucher et al. (1, 2) have used random TnS muta-genesis to inactivate the P. solanacearum genes that areapparently required for wilting tomato plants. Most of theinsertions were clustered on a megaplasmid (2) and reducedthe invasiveness of the mutant strains in tomato stems (28).It may take substantial effort to determine the exact role ofthese genes in pathogenesis.

In contrast, we have chosen an approach that involves thecloning and characterization of genes that encode moleculeslikely to have a role in pathogenesis and their selectiveinactivation in P. solanacearum by site-directed mutagene-sis (22). Plant bioassays with the resultant mutant strains canbe used to determine the importance of the mutated genes inpathogenesis. The egl gene of P. solanacearum was em-ployed in such an approach because some circumstantialevidence supports a role for its gene product, endoglucanase(EGL), in wilt disease: (i) EGL is a P-1,4-endoglucanase thatcleaves soluble cellulose, apparently releasing cellobiose(23); (ii) EGL is excreted at high levels by several virulent

* Corresponding author.

strains of P. solanacearumn isolated from various geograph-ical regions (23); and (iii) EGL is one of several geneproducts that are produced at very low levels in spontaneousavirulent, pleiotropic mutants (25).We report here the use of a directed mutagenesis approach

in creating a strain of P. solanacearun that was deficientonly in EGL production. Plant virulence studies showed thatthe EGL-deficient mutant was less virulent than the wild-type parent on tomato, and that full virulence was restoredby complementation in trans with the wild-type egl gene.These results demonstrate that EGL is an important factor inwilt disease and illustrate the potential of this technique todetermine the relative contributions of virulence factors inplant disease.

MATERIALS AND METHODS

Materials. Medium ingredients were from Difco Labora-tories, Detroit, Mich. All enzymes and biochemicals em-ployed in DNA manipulations were from Boehringer Mann-heim Biochemicals, Indianapolis, Ind.; Bethesda ResearchLaboratories, Gaithersburg, Md.; New England Biolabs,Beverly, Mass.; or United States Biochemical Corp., Cleve-land, Ohio. Carboxymethylcellulose (CMC; C-8758) wasfrom Sigma Chemical Co., St. Louis, Mo. All other reagentswere reagent grade.

Bacterial strains and plasmids. Descriptions of the bacte-rial strains and plasmids used are listed in Table 1.Media and culture conditions. Unless otherwise indicated,

Escherichia coli and P. solanacearum strains were grown tothe stationary phase at 37 and 30°C, respectively, at 250 rpm.The following media were used: Luria broth (LB) (18);nutrient broth containing 0.5% glucose (NBG); and basalsalts medium [BSM; 50 mM sodium-potassium phosphate(pH 7.0), 15 mM (NH4)2SO4, 0.8 mM MgCl2, 2 ,uM FeSO4,0.2 mM CaCl2, 8 ,uM Na2MoO4, 5 ,uM MnCI2] or M9 salts(18), both supplemented as described below. Unless other-wise indicated, the antibiotic levels used to maintain orselect strains and plasmids were 50 ,ug/ml for kanamycin, 100

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TABLE 1. Bacterial strains and plasmids

Strain or plasmid Descriptiona Reference or source

StrainP. solanacearum AW Wild-type pathogen isolated from tomato S. M. McCarter (23)P. solanacearum PS6 egl::Tn5 Kmr This workE. coli HB101 hsdS20 recA13 ara-14 proA2 lacYI rpsL20 16E. coli JM83 ara A(lac-proAB) rpsL 4)80 lacZ A(M15) J. MessingE. coli JM107 endAl gyrA96 thi hsdR17 A(Iac-proAB) (F' traD36 proAB laCIq Z AM15) R. Geever

PlasmidspLAFR3 Tcr, pLAFR1 derivative J. Jones, AGSb (8)pRK404 Tcr G. Ditta (6)pKR2013 Kmr G. Ditta (7)R751 Tpr A. Summers (11)pHE3 egl+ Tcr (Fig. 1) This workpTD29, pDR250, pDR251 egl+ Apr (Fig. 1) This workpPA14 Aegl Apr (Fig. 1) This workpJD1 egl::TnS Tcr Kmr This worka Tcr, Kmr, Apr, and Tpr designate resistance to tetracycline, kanamycin, ampicillin, and trimethoprim, respectively; egl+, produces EGL activity.b AGS, Advanced Genetic Sciences, Oakland, Calif.

,ug/ml for ampicillin, 20 jig/ml for tetracycline, and 200 ,ug/mlfor trimethoprim.

Cloning, restriction mapping, transposon mutagenesis, andSouthern hybridization. P. solanacearum AW chromosomalDNA was isolated by a modification of the method ofMarmur (17), partially digested with Sau3AI, and fraction-ated on a sucrose gradient (16). Fragments 25 to 35 kilobases(kb) in size were ligated into BamHI-digested, dephosphory-lated pLAFR3. Ligated DNA was packaged by the methodof Silhavy et al. (27) and transfected into E. coli JM107, andcells containing recombinant plasmids were selected onLuria agar plates with tetracycline. Recombinants that pro-duced EGL (EGL+) were detected by replica plating oncellulase plates (LB agar plus 0.3% CMC) developed with0.1% Congo red and 1 M NaCl (30).

Plasmid pTD29 was constructed by ligating EcoRI-di-gested pHE3 with EcoRI-digested pUC9 and isolating AprEGL+ colonies after transformation of E. coli JM83. Plas-mids pDR250 and pDR251 were constructed by ligating the2.7-kb XhoI-SaIl fragment from pTD29 into the SalI site of

pUC9. Plasmid pPA14 was constructed by ligating the 2.2-kbSmaI fragment from pTD29 into the SmaI site of pUC9 (Fig.1). Plasmid pJD1 was constructed by ligating EcoRI-digestedpTD299 (Fig. 1) with EcoRI-digested pRK404 and selectingTcr Kmr Aps colonies.

Transformation of E. coli with plasmid DNA was bystandard procedures (16). Restriction mapping of cosmidpHE3 was performed by a modification of the SouthernCross method (New England Nuclear Corp.), whereas allother plasmids were mapped by standard procedures (16).Transposon TnS mutagenesis with A NK467 was as de-scribed by deBruijn and Lupsky (5). Preparation of the3.3-kb Tn5 HindlIl fragment (12) and the 1.45-kb pTD29 PstIfragment as probes by nick translation, transfer of EcoRIdigests of P. solanacearum AW and PS6 chromosomal DNAto nitrocellulose filters, prehybridization, and hybridizationwere performed by standard procedures (16).

Bacterial matings. Cultures were grown to the midlogphase in NBG or LB for P. solanacearum or E. coli strains,respectively, plus the appropriate antibiotics. Donors (0.05

R S

pHE 3RB R 8

I1 i

pTD 29S LI I

pDR250IOapI

BR B

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0

z C

LSZ

LR

61 9

p L IP

S R

pPA 14

EGL

FIG. 1. Physical maps of plasmid inserts containing the cloned egl gene. Abbreviations: MCS, multicloning site; B, BamHI; C, ClaI; H,HindIII; L, Sall; P, PstI; R, EcoRI; S, SmaI; X, XhoI; and Z, SphI. Cloning vectors used were pLAFR3 for pHE3 and pUC9 for pTD29,pDR250, and pPA14. Symbols: position of Tn5 insertions which inactivate EGL activity (V); position of TnS insertion not affecting EGLactivity (V). The designations 0, 1, 6, and 9 above each triangle indicate the pTD29 derivatives pTD290, pTD291, pTD296, and pTD299,respectively, which contain that TnS insertion at this location. The dashed arrow at the base of the figure indicates the probable extent anddirection of transcription of the egl gene.

L,H Kb

Kb

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ml) and recipients (0.1 ml) were washed, spotted on nutrientagar, mixed together by pipetting, and incubated at 30°C for2 days. Transconjugants were selected by streaking ontoBSM plates containing the appropriate antibiotics followedby incubation at 30°C for 7 days. Single colonies weretransferred with toothpicks onto a second BSM-antibioticplate and incubated at 30°C for 5 days, and cells were storedat -70°C. Plasmid pHE3 was mobilized from E. coli JM83into P. solanacearum PS6 by a triparental mating with E.coli HB101(pRK2013) (7) followed by Kmr Tcr selection toconstruct P. solanacearum PS6(pHE3).

Site-directed mutagenesis. The EGL-deficient strain P.solanacearum PS6 was constructed by first mobilizing pJD1(egl: :TnS) from E. coli JM83 into P. solanacearum AW in atriparental mating with E. coli HB101(pRK2013) (7) followedby Kmr Tcr selection on BSM plates. To select for strainsthat had undergone recombination events between egl::TnSand the chromosomal egl gene, plasmid R751 (Tpr), which isincompatible with the pRK404 replicon of pJD1, was conju-gated into P. solanacearum AW(pJD1). The resultant P.solanacearum(R751) strain containing egl::TnS on the chro-mosome was selected on BSM-trimethoprim-kanamycinplates.

Preparation of crude EGL enzyme and excretion analysis.Bacterial cultures in LB (for E. coli) or NBG (for P.solanacearum) were centrifuged at 9,000 x g for 20 min at4°C. The culture supernatant (i.e., extracellular fraction) wasused as the P. solanacearum enzyme preparation. The E.coli pellet was suspended in 100 mM Tris hydrochloride (pH7.0), broken by sonication, and centrifuged at 9,000 x g for10 min. The cell-free supernatant, hereafter called the intra-cellular extract, was frozen until use. For excretion analysisof EGL, E. coli JM83(pDR250) was grown in M9 supple-mented with 0.1% dialyzed yeast extract, 0.1% dialyzedCasamino Acids, 100 ,ug of ampicillin per ml, and 0.5%glycerol, whereas P. solanacearum AW was grown in BSMsupplemented with 0.1% dialyzed yeast extract, 0.1% dia-lyzed Casamino Acids, and 0.5% glycerol. Midlog- or sta-tionary-phase cells were fractionated into cytosolic, peri-plasmic, and extracellular fractions as described byYanagida et al. (31).Enzyme assays. To determine EGL activity enzyme prep-

arations were incubated with substrate (400 pAl of 0.2% CMCin 100 mM potassium phosphate, pH 7.0) at 40°C, andreducing sugars released were quantified by the method ofNelson (20). One unit of EGL activity was defined as theamount of enzyme that releases 1 ,umol of cellobiose-reducing equivalent per min. Polygalacturonase activity wasdetermined as described for EGL, except 0.2% sodiumpolygalacturonate in 50 mM sodium-potassium phosphate(pH 6.5) was used as the substrate. Assays for P-lactamas(and glucose-6-phosphate dehydrogenase were as describedpreviously (15, 26). Protein concentrations were determinedby the method of Bradford (3) with bovine serum albumin asthe standard.

Gel electrophoresis of extracellular proteins. Culture super-natants from P. solanacearum strains grown in BSM sup-plemented with 0.1% dialyzed yeast extract, 0.1% dialyzedCasamino Acids, and 0.3% succinate were lyophilized todryness, dissolved in 1 ml of water, dialyzed (1/1,000)overnight against 10 mM Tris (pH 7.5), lyophilized, anddissolved in 0.1 ml of water. Protein preparations wereanalyzed on 10% sodium dodecyl sulfate-polyacrylamidegels (14) stained with Coomassie brilliant blue or isoelectricfocusing gels prepared and run as described by Ried andCollmer (21).

Virulence assay. P. solanacearum strains were streakedonto the appropriate antibiotic plates from storage at -700Cimmediately before each experiment. Single fluidal colonieswere grown in NBG plus the appropriate antibiotics for 12 to24 h at 30°C. Inocula for plants (109 or 106 cells per ml) wereprepared by centrifugation and suspension in sterile water.Tomato (Lycopersicon esculentum cv. Marion) seed was

germinated in vermiculite, grown to 2 clp in height, andtransplanted into 4-cm pots with a soil-peat mix. Plants 15 to20 cm in height were inoculated by stabbing a 200-,udisposable pipette tip containing 20 pul of 109 or 106 cells perml into the third leaf axil down from the plant apex.Inoculated plants were placed in a 300C growth chamber,and the pipette tips were removed after the entire bacterialsuspension entered the stem. Five plants were inoculatedwith each strain at each concentration and were watereddaily during the course of the experiment.The inoculated plants were coded and scored in a single

blind experiment for the development of disease utilizing anindex modified from Winstead and Kelman (28): 0, no leaveswilted; 1, 1 to 25% of leaves wilted; 2, 26 to 50% of leaveswilted; 3, 51 to 75% of leaves wilted; and 4, 76 to 100% ofleaves wilted. All plants from the same treatment werescored daily, and the average disease index was computed.

RESULTS

Cloning and analysis of the egl gene. A cosmid library(1,100 clones) of genomic DNA fragments (average size, 30kb) was constructed in pLAFR3. Upon screening this librarytwo E. coli clones producing low levels of CMCase activityon cellulase plates were detected. The cosmids from thesetwo clones were isolated and shown by restriction endonu-clease analysis to contain identical inserts. Southern hybrid-ization analysis indicated that they contained unaltered P.solanacearum AW chromosomal DNA (24). One of thesecosmids, designated pHE3 (Fig. 1), was chosen for furtherstudy.The egl gene on pHE3 was mapped and isolated by

shotgun subcloning with cellulase detection plates. CosmidpHE3 was completely digested with EcoRI and ligated intothe EcoRI site of pUC9. Plasmid pTD29 (Fig. 1), containinga 10.5-kb EcoRI fragment, was the only plasmid detectedproducing CMCase (EGL) in E. c6li. Plasmid pPA14, whichcontained the 2.2-kb SmaI fragment of pTD29, did notproduce EGL in E. coli. However, plasmid pDR250, ob-tained by cloning the 2.7-kb XhoI-SalI fragment of pTD29into the Sall site of pUC9, did produce EGL in E. coli. Sincethe 2.7-kb XhoI-SalI pTD29 fragment contains the 2.2-kbSmaI fragment of pPA14 and an additional adjacent 500-base-pair (bp) region, this 500-bp region is necessary forEGL activity and probably contains a terminus of egl (Fig.1).Plasmid pDR251 contained the same XhoI-SalI fragment

as pDR250 inserted in the opposite orientation into pUC9.Plasmid pDR251 appeared -to mediate the production ofsomewhat less EGL in E. coli than pDR250. Recent exper-iments showed that deletion of all P. solanacearum DNAsequences in pDR250 between the middle SphI site and theSall site in front of the lac promoter of pUC9 resulted in aplasmid with isopropyl-,-D-thiogalactopyranoside-inducibleegl expression (data not shown), whereas deletion of thesame P. solanacearum sequences from pDR251 resulted inloss of all expression in E. coli. This strongly suggests thatthe expression on pDR250 was due to the lac promoter,whereas that observed from pDR251 probably resulted from

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a P. solanacearum promoter upstream (i.e., to the right) ofthe middle SphI site. Therefore, the probable direction of egltranscription is as indicated in Fig. 1. However, furtherpromoter mapping experiments must be performed to pre-cisely locate the egl promoter and transcription start site.

Plasmid pTD29 was subjected to transposon TnS mutagen-esis. Mapping the insertion positions of TnS in the resultingderivatives pTD290, pTD291, pTD296, and pTD299 locatedall TnS insertions within the 2.7-kb XhoI-SalI fragmentshown by subcloning to contain the entire egl gene (Fig. 1).Plasmids pTD291, pTD296, and pTD299 did not mediate theproduction of EGL in E. ccli JM83, whereas E. coliJM83(pTD290) did produce EGL (Fig. 1).The TnS insertion in pTD290 was approximately 200 bp

from the XhoI site of pTD29 within the 500-bp region of the2.7-kb XhoI-SalI fragment shown by subcloning to be nec-essary for EGL activity. Therefore, one terminus of the eglgene is between 200 and 500 bp from the XhoI site of pTD29(Fig. 1). Plasmid pTD299, which does not mediate theproduction of EGL in E. coli JM83, contained a TnS inser-tion 1.3 kb from the pTD29 XhoI site. This places the otherterminus of egl between the TnS insertion site of pTD299 andthe SalI site closest to the multicloning site of pTD29 (Fig.1).

Characterization of the egl gene product. P. solanacearumAW extracellular fractions (i.e., culture supernatants) con-tained 0.06 and 0.38 EGL units per ml of culture in themidlog (optical density at 600 nm, 0.6) and stationary phase(optical density at 600 nm, 2.4), respectively. Intracellular P.solanacearum extracts contained only 0.001 and 0.005 EGLunits per ml of culture. Therefore, EGL is a protein excretedby P. solanacearum, since >97% of total EGL activity wasfound in the culture supernatant. In contrast, the majority of'EGL activity produced by E. coli JM83(pDR250) was con-tained within the cytoplasm in the midlog (optical density at600 nm, 0.6) and stationary (optical density at 600 nm, 1.6)phases (Table 2). Very low levels of EGL activity weredetected in the periplasmic and extracellular fractions in E.coli JM83(pDR250) stationary-phase cultures. However, thepresence of P-lactamase, a periplasmic marker enzyme, inthe E. coli JM83(pDR250) stationary-phase culture superna-tant indicated that this probably was due to nonspecificleakage of EGL into the culture medium. The E. coliJM83(pDR250) stationary-phase intracellular extract con-tained 0.05 EGL units per ml of culture. This was 13% of theactivity in the P. solanacearum extracellular fraction (0.38 Uper ml of culture), indicating that EGL was produced much

TABLE 2. Localization of EGL in E. coli JM83(pDR250)

ActivityaGrowth Enzymephase Cytoplasm Periplasm Culture-

supernatant

Midlog EGL 0.009 UND UNDG-6-P-DH 5 UND UNDP-Lactamase 0.6 9.1 1.4

Stationary EGL 0.052 0.002 0.004G-6-P-DH 1.9 UND UNDP-Lactamase 3.0 6.9 22.0

a Activity is given as follows: units per milliliter cell culture for EGL;decrease in A570 per minute per milliliter of cell culture for 13-lactamase, theperiplasmic enzyme marker; and increase in A340 per minute per milliliter ofcell culture for glucose-6-phosphate dehydrogenase (G-6-P-DH), the cytoplas-mic enzyme marker. UND, Undetectable levels of EGL (<10-5 U ml-') orglucose-6-phosphate dehydrogenase (<lo- U mL').

TABLE 3. Immunoabsorption of EGL activitya

% of residual EGL activityl'Additions (,ul)

P. solanacearum E. coli

None 100 100EGL antiserum (6) <1 7Preimmune serum (6) 89 93

a P. solanacearum AW extracellular fraction (25 p.1) or E. coliJM83(pDR250) intracellular fraction (25 j±l) was mixed with 200 ,ul of 50 mMTris hydrochloride (pH 7.5)-0.15 M NaCI containing EGL antiserum orpreimmune serum, incubated at 4°C for 15 h, mixed with 20 ,ul of 10% proteinA cells (Sigma) in 50 mM Tris hydrochloride (pH 7.5)-0.15 M NaCl, incu-bated at 4°C for 1 h, and centrifuged at 13,000 x g for 2 min; then 80 1d ofsupernatant was assayed for EGL activity at 30°C.

b Percentage of the residual EGL activity in reactions with EGL antiserumin comparison with reactions without antiserum.

less efficiently in E. coli. Immunoadsorption with antiserumproduced against the purified 43-kilodalton (kDa) EGL fromP. solanacearum AW (23) removed most of the EGL activityfrom both P. solanacearum AW and E. coli JM83(pDR250)enzyme preparations (Table 3); very low levels of EGLactivity were immunoadsorbed when preimmune serum wasadded. Therefore, the 43-kDa EGL is encoded by the eglgene.

Construction and characterization ofP. solanacearum EGL-deficient strains. For site-directed mutagenesis of the eglgene, a 16-kb EcoRI fragment from pTD299 containingegl::Tn5 was cloned into pRK404 (Tcr) to construct pJD1,which was subsequently mobilized into P. solanacearumAW. Homologous recombination events between the plas-mid-borne egl: :TnS gene and the chromosomal egl gene wereselected by conjugating R751 (Tp), a plasmid which isincompatible with pJD1, into the egl+legl::TnS mnerodiploidfollowed by Kmr Tpr selection. All P. solanacearum KmrTpr exconjugants were Tcs and were replica plated ontocellulase detection plates. The more than 75 coloniesscreened produce copious quantities of EPS but had verylow, if any, activity on cellulase plates. One colony, desig-nated PS6, was chosen for further study.

P. solanacearum PS6 intra- and extracellular proteinextracts produced <0.002 EGL units per ml of cell culture,at least 200-fold less than the wild-type strain, which pro-duced 0.38 EGL U ml-'. Analysis of extracellular proteinsfrom P. solanacearum PS6 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that P. solana-cearum PS6 produces very little, if any, 43-kDa EGL poly-peptide (Fig. 2). With the exception of the absence of theEGL band, the P. solanacearum PS6 extracellular proteinprofile was identical in pattern and quantity of protein perband with the wild-type virulent strain. Isoelectric focusingwith a polygalacturonase activity overlay of extracellularprotein preparations from P. solanacearum AW and PS6showed that the polygalacturonase profiles associated withthese two strains were identical (data not shown). Thissuggests that P. solanacearum PS6 contains a single muta-tion in the egl gene.

Southern hybridization was employed to confirm that P.solanacearum PS6 contained a single TnS insertion. The3.3-kb HindlIl fragment from TnS, which contained a por-tion of ISSOR and IS5OL and the internal region of TnS (12),and the 1.45-kb PstI fragment from pTD29, which containeda portion of the egl gene (Fig. 1), were used to probecomplete EcoRI digestions of total chromosomal DNA fromP. solanacearum strains AW and PS6. The HindIII fragmentfrom Tn5 and the PstI fragment from pTD29 both hybridized

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A B C

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43

30

FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel electropho-retic analysis of culture supematants from P. solanacearum AW andPS6. Lanes: A, purified EGL; B, 50 jig of protein from P. solana-cearum PS6 culture supernatant; and C, 50 p.g of protein from P.solanacearum AW culture supematant. The arrow indicates theposition where EGL migrates.

only to the same 16-kb P. solanacearum PS6 EcoRI chro-mosomal DNA fragment, whereas only the PstI fragmentfrom pTD29 hybridized to a 10.5-kb P. solanacearum AWEcoRI fragment (data not shown). These results indicatedthat (i) a single copy of TnS was in the same position in theegl gene of P. solanacearum PS6 as the original TnS con-taining insert in pTD299 (Fig. 1), (ii) no independent IS50insertions occurred, and (iii) the site-directed mutagenesistechnique was capable of accurately mutating the P. solana-cearum AW genome.Plasmid pHE3 was transferred into P. solanacearum PS6

resulting in an egi::TnSIeg1+ merodiploid strain. The P.solanacearum PS6(pHE3) merodiploid had restored activityon cellulase plates and produced 0.35 EGL units per ml.Sodium dodecyl sulfate-polyacrylamide gel electrophoresisof extracellular protein extracts from strain PS6(pHE3) alsoindicated that the production of the 43-kDa EGL was re-stored in this strain (data not shown). The remainder of theprotein profile of this strain was identical to that of P.solanacearum AW.

Analysis of the importance of EGL in plant pathogenicity.The EGL-deficient strain P. solanacearum PS6 was studiedby plant bioassay to determine the importance of EGL inplant pathogenesis. Tomato plants inoculated with 2 x 107and 2 x 104 cells of P. solanacearum PS6 reached a diseaseindex rating of 4 at 11 days after inoculation. In comparison,tomato plants inoculated with the same number of cells of P.solanacearum AW wilted to a disease index rating of 4 in -

days. P. solanacearum PS6(pHE3), the egi::TnSIeg1+ mero-diploid, wilted plants to a disease index rating of 4 in 8 daysat both inoculation levels (Fig. 3; results are representativeof the other two independent experiments performed). In allexperiments the EGL-deficient strain, P. solanacearum PS6,took at least 40% longer than the wild-type strain to com-pletely wilt tomato plants. Complementation of the egl: :TnSmutation with the wild-type egl gene in trans restoredvirulence to very near wild-type levels.The 2.7-kb XhoI-SaII fragment of pDR250 containing the

egl gene and promoter was cloned onto pRK404 and trans-ferred into P. solanacearum PS6. The resultant strain hadEGL activity identical (0.35 U ml-') to that found for boththe wild-type strain AW and PS6(pHE3), indicating that thisfragment contains the entire egl gene and promoter. Analysis

of the virulence of this strain showed that its ability to wiltand kill tomato plants was nearly identical to that of thewild-type parent AW and strain PS6(pHE3). This confirmsthe previous suggestion that the reduced virulence caused bythe PS6 mutation results from a lack of only the egl geneproduct.

Analysis of in planta stability of P. solanacearum PS6 andPS6(pHE3) was performed. The transposon (Kmr) waspresent in 99% of the colonies isolated on nutrient agar froma tomato plant inoculated with P. solanacearum PS6. South-ern hybridization analysis, sodium dodecyl sulfate-poly-acrylamide gel electrophoresis analysis, and enzyme assaysof an ex planta P. solanacearum Kmr colony indicated that(i) it was still EGL deficient, (ii) neither transposon TnS northe IS50 elements transposed, and (iii) the extracellularprotein production was identical to that of P. solanacearumPS6 before inoculation (data not shown). This indicates thatTnS and the phenotype of P. solanacearum PS6 were stablein planta. Similarly, the cosmid pHE3 (Tc9 was stable in P.solanacearum PS6 in planta since 92% of the coloniesisolated from inoculated tomato plants were Tcr. The dou-bling times of log-phase P. solanacearum strains AW, PS6,and PS6(pHE3) in M9 salts supplemented with 0.05% Cas-amino Acids and 0.05% sucrose were 1.4, 1.4, and 1.9 h,respectively. These results suggest that the decreased viru-lence ofP. solanacearum PS6 was not due to an alteration ofits growth rate.

DISCUSSIONA 2.7-kb DNA fragment from P. solanacearum encoding

CMCase activity was cloned and expressed in E. coli.Antibody experiments demonstrated that the cloned DNAfragment contains the egl gene that encodes the 43-kDapolypeptide which is the major endoglucanase excreted bymany P. solanacearum strains (23).

4

3

2

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a)Incs

Ca,U)0._

A

48

3-

2-

o0 2 6 8 I I I

o 2 4 6 8 lo 12Day

FIG. 3. Plant bioassay for virulence of P. solanacearum strains.Marion tomato plants were inoculated with 2 x 107 cells (A) or 2 x104 cells (B) of P. solanacearum AW (0), PS6 (0), or PS6(pHE3)(A). Plants were scored daily by utilizing a plant disease index.Graph points represent the average plant disease index of five plantsinoculated with each strain.

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1450 ROBERTS ET AL.

A possible role for the 43-kDa EGL in phytopathogenicityis implied by its potential to degrade plant cell wall compo-nents (i.e., glucans). However, the ability to produce en-zymes that can destroy plant cell components is insufficientevidence to conclude that such enzymes are involved in orare necessary for pathogenesis (32). Other data that suggestEGL has a role in pathogenesis have come from analysis ofspontaneous, pleiotropic, avirulent P. solanacearum mu-tants. These avirulent mutants produce greatly reducedquantities of EGL (25); however, a number of other mole-cules potentially involved in pathogenesis are also producedat markedly reduced levels by these mutants (19, 25).Therefore, the existing data implicating the involvement ofEGL in pathogenesis have been largely circumstantial.To demonstrate involvement of EGL in pathogenesis,

site-directed mutagenesis of the P. solanacearum chromo-somal egl gene was used to derive a strain that was deficientonly in EGL production, apparently without affecting theexpression of any other genes. The egl mutant produced atleast 200-fold less endoglucanase than the wild-type strain,indicating that other glucanases, if present, are produced atvery low levels. This EGL-deficient strain was significantlyless virulent than the wild-type strain on tomato plants. Adelay in the time before onset of wilting as well as a delay inthe time required to kill the plant resulted from the eglmutation. Complementation of the egl mutation restoredvirulence to very near wild-type levels. Therefore EGLappears to be directly involved in disease produced by P.solanacearum.The precise role of EGL in pathogenesis is not known, but

it is probably involved with degradation of plant cell wallglucans. Digestion of plant cell wall components by EGLmay directly facilitate bacterial penetration of plant tissue,ease penetration and degradation of cell wall material byother enzymes (e.g., polygalacturonases, protease), and/oraid in the release of plant cell components that could beutilized for nutrition of the phytopathogen. Since we circum-vented the natural infection route by stem inoculation, ourexperiments cannot rule out an essential role of EGL in theroot infection process. Root infection experiments withwild-type and mutant strains would be necessary to clarifythe role of EGL in this process.The EGL-deficient strain was capable of killing tomato

plants, albeit after a prolonged period of time, indicating thatvirulence genes other than egl are sufficient for pathogene-sis. This suggests that although EGL is not absolutelyrequired for disease production under the conditions of ourassay, it apparently increases the speed of disease develop-ment. Recent site-directed mutagenesis experiments re-sulted in a P. solanacearum mutant strain that was deficientin the production of the major excreted P. solanacearumpolygalacturonase (pglA). Like the EGL-deficient strain,this polygalacturonase mutant was decreased in virulencebut still capable of killing tomato plants after a delayedperiod of time (M. A. Schell, D. P. Roberts, and T. P.Denny, submitted for publication). The egl and pglA genesapparently belong to a class of genes that are not absolutelyrequired in disease under all conditions but may in somemanner enhance the ability of P. solanacearum to cause thedisease. A second class of P. solanacearum genes that areabsolutely required in pathogenesis may exist, since Bou-cher et al. (1) isolated TnS mutants that are avirulent. Theseresults suggest that pathogenicity is a complex polygenicphenotype involving genes of varied importance.

Plant-pathogenic bacteria typically employ a complexarray of enzymes and other virulence factors when produc-

ing disease. Site-directed mutagenesis coupled with theproper plant bioassay can be a powerful tool in determiningwhich genes are involved in pathogenesis and possibly theirrelative importance to the process.

ACKNOWLEDGMENTS

We thank S. M. McCarter for P. solanacearum strains and P. deAbramoff, C. Benner, J. Huang, and J. C. Albertin for technicalassistance.

This research was supported in part by a grant from the Univer-sity of Georgia program in Biotechnology and Biological ResourceRecovery and U.S. Department of Agriculture grant 86-CRCR-1-2242.

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