JOURNAL OF BACTERIOLOGY, OCt. 1985, p. 390-3960021-9193/85/100390-07$02.00/0Copyright C) 1985, American Society for Microbiology

Vol. 164, No. 1

recA Is Required in the Induction of Pectin Lyase and Carotovoricinin Erwinia carotovora subsp. carotovorat

RICHARD T. ZINK,: JUDY K. ENGWALL, JAMES L. McEVOY, AND ARUN K. CHATTERJEE*

Department of Plant Pathology, Kansas State University, Manhattan, Kansas 66506

Received 13 May 1985/Accepted 23 July 1985

Pectin lyase (PNL) and the bacteriocin carotovoricin (CTV) were induced in Erwinia carotovora subsp.carotovora 71 by the DNA-damaging agents mitomycin C, nalidixic acid, and UV light. To determine whetherthe recA product was involved in the expression of these damage-inducible phenotypes, we cloned the E.carotovora subsp. carotovora recA+ gene, inactivated it by TnS insertion, and constructed an E. carotovorasubsp. carotovora recA::TnS strain by gene replacement via homologous recombination. The RecA- strain wasmore sensitive to methyl methanesulfonate, nitroquinoline oxide, and UV light than its RecA+ parent. The recAmutation did not affect the production of pectate lyase, polygalacturonase, cellulase, and protease or the abilityto cause soft rot of potato tubers. With this mutant, unlike with the RecA+ parent strain, PNL and CTV werenot induced by mitomycin C or detected in potato tuber tissue. The RecA+ phenotype, including the inducibilityof PNL and CTV, could, however, be restored in the mutant in trans by the recA+ gene from either E.carotovora subsp. carotovora or Escherichia coli. We conclude that, in E. carotovora subsp. carotovora, the recAproduct is required in the induction of PNL and CTV.

In Escherichia coli, the recA product is required in DNArepair as well as in recombination. An additional role of therecA product is the induction of SOS functions in responseto DNA damage (see references 20 and 34 for reviews). Inthe latter case, the RecA protein, under the appropriateinducing signal, becomes proteolytic and cleaves the lexAproduct, which is the repressor of various SOS functionsincluding colicin (9, 12) and cloacin synthesis (33). Similarly,the repressors of such temperate bacteriophages as lambda,P22, and +80 can be cleaved by the activated recA product(27, 28).Among the soft-rot Erwinia spp. (E. carotovora subsp.

atroseptica, E. carotovora subsp. carotovora, and E.chrysanthemi), the pectinolytic enzyme pectin lyase (PNL)was induced by DNA-damaging agents such as mitomycin C,nalidixic acid, bleomycin, and UV light (13, 14, 16, 17, 32).Moreover, in strains of E. chrysanthemi and E. carotovorasubsp. carotovora, PNLs were coordinately induced alongwith either a bacteriocin or a temperate phage by theseagents (13, 32). These coinductions are reminiscent of theinduction of the SOS functions in E. coli which are mediatedby RecA. Whether or not the E. carotovora subsp.carotovora recA product was involved in the expression ofthese damage-inducible phenotypes was heretofore un-known. In this report, we describe the cloning of the recAgene of E. carotovora subsp. carotovora and the construc-tion of an E. carotovora subsp. carotovora recA::TnS strain.Furthermore, we present data on the role of the recAproduct in the induction of PNL and the bacteriocincarotovoricin (CTV).

(Preliminary accounts of some of this work have beenpublished [R. T. Zink and A. K. Chatterjee, Abstr. 2nd Int.Symp. of the Molecular Genetics of the Bacteria-Plant

* Corresponding author.t Contribution no. 85-447-J from the Department of Plant Pathol-

ogy, Kansas Agricultural Experiment Station, Kansas State Univer-sity, Manhattan.

4: Present address: Northwest Branch Experiment Station, Uni-versity of Minnesota, Crookston, MN 56716.

Interaction, Ithaca, N.Y., abstr. no. 139, 1984; R. T. Zink,J. K. Engwall, J. L. McEvoy, and A. K. Chatterjee, Abstr.6th Int. Conf. on Plant Pathogenic Bacteria, College Park,Md., p. 75, 1985]).

MATERIALS AND METHODS

Bacterial strains, plasmids, bacteriophages, and culturemedia. Bacterial strains, plasmids, and bacteriophages usedin this study are listed in Table 1. L, XYM, YGC, minimalsalts, and cellulase media were as described previously (3, 5,35). Nutrient gelatin medium contained gelatin (30 g/liter),nutrient broth (Difco) (8 g/liter), and agar (15 g/liter). Whenrequired, media were supplemented with amino acids,purines, or pyrimidines (50 ,ug/ml) or the followingantimicrobial agents: kanamycin, 50 ,ug/ml; tetracycline, 10jig/ml; chloramphenicol, 10 ,ugIml; streptomycin, 100 ,ug/ml;gentamicin sulfate, 10 ,ug/ml; ampicillin, 50 pLg/ml (for E. coli)or 1 mg/ml (for E. carotovora subsp. carotovora); nitro-quinoline-1-oxide (NQO), 10 ,ug/ml; or methyl methane-sulfonate (MMS), 2.5 to 15 mM.DNA techniques. Total DNA was isolated as previously

described (39). The procedures for the isolation of plasmidDNA, restriction digestion, ligation, transfer of DNA tonitrocellulose, nick translation, and hybridization have beendescribed by Maniatis et al. (22).

Cloning of the recA genes of E. carotovora subsp. carotovoraand E. coli. Total DNA of E. carotovora subsp. carotovora71 was partially digested with SalI and size fractionated on asucrose gradient to obtain 30- to 45-kilobase fragments (22,38). Sall-cut pHC79 was then combined with the sizedgenomic DNA at a ratio of 10:1 (vector to genomic DNAmolecules) for a final concentration of 400 ,ug of DNA perml. The DNAs were ligated with T4 DNA ligase and pack-aged into lambda particles which were used to transduce E.coli HB101. Apr transductants potentially carrying the recA+gene were obtained by screening for resistance to 254-nmUV light (300 ergs/mm2) from a model R-52 lamp (Ultra-violet Products, Inc., San Gabriel, Calif.). The Rec+ pheno-type of Apr UVr colonies was confirmed by recombinationproficiency and resistance to MMS.

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TABLE 1. Bacterial strains, plasmids, and bacteriophages

Strain, plasmid, or bacteriophage Relevant characters Source or reference

E. coliHB101 pro leu thi lacY Strr recA hsdR hsdM 5K-12 (ICPB 2195) Wild type 31RR1 Same as HB101 except recA+ R. R. RodriguezGM13 HfrH Valr thi A(lac-pro) G. L. Marchin2174 met prolGmr (pPH1JI) E. W. NesterAC504 HB1l1/Apr E. carotovora subsp. Transformation of HB101 with

carotovora RecA + pAKC207AC505 HB1l1/Apr Kmr RecA- (E. carotovora Transformation of HB101 with

subsp. carotovora recAl::Tn5) pAKC208AC508 HB1O1/Cmr Apr E. coli RecA+ Transformation of HB101 with

pAKC401E. carotovora subsp. carotovora

26 Wild-type serogroup V S. H. DdBoer71 Wild-type serogroup III S. H. DeBoerAC5117 E. carotovora subsp. carotovora 71 Gene replacement mutant of E.

recAl::TnS (Kmr) carotovora subsp. carotovora 71AC5118 AC5117/Apr Cmr E. carotovora subsp. AC5117 transformed with pAKC210

carotovora RecA+AC5119 Same as AC5117 AC5118 cured of pAKC210AC5120 AC5117/Apr Cmr E. coli RecA+ AC5117 transformed with pAKC401

PlasmidpAKC207 E. carotovora subsp. carotovora recA Cosmid cloning of E. carotovora subsp.

region in pHC79 carotovora genome in Sall site ofpHC79

pAKC208 pAKC207 recAl::Tn 5 TnS mutagenesis of pAKC207pAKC210 E. carotovora subsp. carotovora recA Cloning of 9.8-kilobase Sall fragment of

region in pBR329 pAKC207 into Sall site of pBR329pAKC401 E. coli recA region in pBR329 Cloning of E. coli genome into BamHI

site of pBR329pBR329 ColEl Apr Cmr Tcr F. Bolivar (8)pHC79 ColEl cos Apr Tcr N. T. Keen (11)pPHlJI IncP group plasmid conferring Gmr 10pRZ102 ColE1::TnS 39

PhagePlvir Wild type D. W. MountX467 b221 rex::TnS c1857 Oam8 Pam29 N. Kleckner

The E. coli recA+ gene was cloned as described by Keeneret al. (18). Briefly, genomic DNA from E. coli K-12 wasdigested with BamHI and ligated into the BamHI site ofpBR329. The ligated DNAs were transformed (22) into E.coli HB101. Transformants that were Cmr and NQOr werepurified and tested for resistance to MMS and UV and forrecombination proficiency.

Transposon mutagenesis of the cloned E. carotovora subsp.carotovora recA' gene. E. coli strain AC504 [HB101(pAKC207); Table 1] was grown to 5 x 108 cells per ml inXYM broth containing 10 mM MgCl2, concentrated 10-fold infresh broth, and infected with the TnS vector X467 at amultiplicity of infection (MOI) of 0.1. The culture wasincubated without shaking for 30 min at 37°C and thendiluted 1:20 with L broth supplemented with ampicillin andkanamycin. After overnight incubation at 330C, plasmidDNA was isolated from the culture and used to transformHB101. One Apr Kmr transformant, AC505, that was UV'and MMSS was retained for plasmid characterization.

Sensitivity to UV, MMS, and NQO. Cultures were grown inL broth to approximately 5 x 108 cells per ml at 33°C for E.coli and 28°C for E. carotovora subsp. carotovora strains.The cells were harvested, washed in 55 mM potassiumphosphate buffer (pH 7.4), and suspended to their original

volume in the same buffer. All subsequent steps wereperformed in the dark. Five-milliliter samples of washedcells, dispensed into 4-cm-diameter petri dishes, were irra-diated with UV light while being stirred. After UV irradia-tion, the cells were serially diluted and plated on L agar.After 16 h of incubation at 33°C, the cell survival wasdetermined.

Sensitivities to MMS and NQO were determined by usingcells prepared as described above. Washed cells were seri-ally diluted and spread directly on L agar containing MMS orNQO. Cell survival was determined as stated above.Measurement of recombination proficiency. Hfr matings

were done in L broth at 37°C for 1 h with a donor-to-recipientratio of 1:2 and an approximate cell concentration of 7 x 107per ml. Mating mixtures were spread on minimal glucosemedium supplemented with streptomycin; donors and recip-ients were counterselected by drug sensitivity and auxo-trophy, respectively. Crosses to determine plasmid transferfrequency were performed in L broth with log-phase cells,with a donor-to-recipient ratio of 1:4. The donor strain2174(pPH1JI) was counterselected by auxotrophy, and re-cipient strains were counterselected by sensitivity to genta-micin. Transduction by bacteriophage Plvir was done at anMOI of 0.2, as described by Roth (29).

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Mitomycin C treatment and preparation of assay samples.Mitomycin C treatment of cultures was performed essen-tially as described by Tsuyumu and Chatterjee (32), with thefollowing exception. During early-exponential-phase growthin L broth at 28°C, mitomycin C was added to a finalconcentration of 0.5 jig/ml. After 8 to 9 h of incubation in thedark, 1-ml samples were centrifuged (12,100 x g, 10 min,4°C), and the supernatants were stored at 4°C. The cellpellets were suspended in 1 ml of 10 mM Tris (pH 7) andsonicated. The supernatants and cell sonicates were assayedfor enzymatic activities and CTV. A separate 0.5-ml culturesample was assayed for protein concentration.

Inoculation of potato tuber tissue for analysis of CTV andPNL production. Potato tubers were washed thoroughly withsoap and water and surface disinfected by soaking in 0.5%(wt/vol) sodium hypochlorite for 10 min, followed by 30 minof rinsing in tap water. These tubers, after air drying, wereinoculated by inserting toothpicks, containing bacterial cellsfrom 24-h plate cultures, 2 cm into their midsection. Petro-leum jelly was used to cover the wound sites and retardmoisture loss. The inoculated tubers were placed in moistchambers and incubated for 48 h at 28°C in the dark; themacerated tissue was then removed. To each gram ofmacerated tissue, 0.25 ml of 10 mM Tris (pH 7) was added.After being mixed and centrifuged at 12,100 x g (10 min,4°C), the supernatants were assayed for enzymatic activitiesand CTV, as described below.CTV assays. CTV was assayed on exponentially growing

cells of E. carotovora subsp. carotovora 26. Titers weredetermined by serially diluting samples in L broth andspotting 5 RI onto a soft agar overlay seeded with theindicator strain. Plates were incubated for 16 h at 30°C andscored for absence of growth of the indicator lawn.Enzyme and protein assays. PNL and pectate lyase (PEL)

activities were assayed by measuring the increase in A235with a Gilford DU2 spectrophotometer. The reaction mix-ture (0.6 ml) for PNL contained 0.24 ml of a 2.0-mg/mlconcentration of 98% esterified Link pectin (23), 0.26 ml of 1mM EDTA in 50 mM Tris (pH 8.0), and 0.1 ml of enzymeplus water. For assaying PEL, the reaction mixture (0.6 ml)contained 0.24 ml of a 5.75-mg/ml concentration of sodiumpolygalacturonate, 0.26 ml of 0.78 mM' CaCl2 in 230 mM Tris(pH 8.5), and 0.1 ml of enzyme plus water. One unit of lyaseactivity is defined as the amount of enzyme that produces anincrease in A235 of 1.0 per min at 30°C.

Polygalacturonase (PEH) activity was determined from2-ml reaction mixtures (consisting of enzyme, 0.5%polygalacturonic acid, 0.2 M NaCl, and 2 mM EDTA in 0.05M sodium acetate buffer [pH 5.0]) incubated at 30°C. Atvarious time intervals, 0.2-ml samples were removed, andthe increase in reducing groups was determined by using thearsenomolybdate method of Nelson (25) as modified bySomogyi (30). One unit of PEH activity is defined as theamount of enzyme that produced 1 p.mol of galacturonic acidper min at 30°C.The reaction conditions for each specific enzyme pre-

vented or minimized interference from other enzymes in thecrude preparations. For example, PNL was assayed withoutinterference from PEL by using Link pectin as the substratein the presence of 0.43 mM EDTA. For PEL assays, thesubstrate was polygalacturonate, which is poorly cleaved byPNL (13). PEH activity was measured at a pH of 5.0 and inthe presence of 2 mM EDTA to minimize the interferencefrom PEL (7, 24, 37).

Protein concentration was estimated by the method ofLowry et al. (21) after precipitation in 10%'trichloroacetic

2

UV DOSE (eg mm-9 mM MMSFIG. 1. UV and MMS sensitivity curves. Cells were prepared

and treated as described in text. Panels A and B, UV and MMSsurvival curves, respectively, for E. coli strains RR1 (Rec+) (M),HB101 (Rec-) (O), AC504 (Rec-/E. carotovora subsp. carotovoraRec + (a), and AC505 (Rec-/E. carotovora subsp. carotovora Rec-)(0). Panels C and D, UV and MMS survival curves, respectively,for E. carotovora subsp. carotovora strains 71 (Rec+) (U), AC5117(Rec-) (O), AC5118 (Rec-/E. carotovora subsp. carotovora Rec+)(a), and AC5120 (Rec-/E. coli Rec') (*).

acid (31). Bacterial colonies were scored for cellulase andprotease activities as previously described (5, 6).

Construction of a RecA- strain of E. carotovora subsp.carotovora. E. carotovora subsp. carotovora 71 was trans-formed with pAKC208 (E. carotovora subsp. carotovorarecAI::TnS) to Apr Kmr by the procedure of Chakrabarty etal. (2). One drug-resistant transformant was purified andgrown at 35°C in L broth from a dcnsity of 106 to 108 cells perml to promote plasmid loss. Derivatives of this culture thatwere Kmr, Aps, and UVS were considered RecA-.

RESULTSCloning and characterization of the E. carotovora subsp.

carotovora and E. coli recA+ genes. From gene libraries of

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TABLE 2. Recombination and plasmid transfer frequencies in E. coli strainsa

Recipiet.P1 transductionRecipient Relevant phenotype Hfr cross (Pro') pPHlJI transfer (Gmr)strain Leu + Pro+

HB101 RecA- <3 x 10-8 <3 x 10-8 <2 x 10-8 <1 x 10-4RR1 RecA+ 3 x 10-7 7 x 10-7 7 x 10-7 2 x 10-4AC504 RecA-/E. carotovora subsp. 5 x 10-7 8 x 10-7 5 x 1o-7 1 X 10-4

carotovora RecA'AC505 RecA-IE. carotovora subsp. <2 x 10-9 <2 x 10-9 <5 x 10-8 <3 x 10-4

carotovora RecA-AC508 RecA-/E. coli RecA+ 8 x 10-7 1 X 10-6 NDb 1 X 10-O

a The frequencies of marker acquisition are expressed per recipient cell. The frequencies marked < were cases in which no recombinants were observed.b ND, Not determined.

approximately 1,000 colonies, 11 were found to carry E.carotovora subsp. carotovora, and 3 carried the E. coliputative recA+ gene. Isolated from two of these colonieswere the plasmids pAKC207, carrying 9.8 kilobases of E.carotovora subsp. carotovora DNA, and pAKC401, carry-ing 3.2 kilobases of E. coli DNA. When transformed into E.coli HB101 (recA), these plasmids conferred resistance toUV and MMS (Fig. 1). In P1 transductions, prototrophicrecombinants were obtained with the E. coli AC504[(HB101)(pAKC207)] or AC508 [(HB101)(pAKC401)]. Fre-quencies of recombinant formation were comparable tothose observed with the Rec+ strain RR1 (Table 2).Prototrophic recombinants of AC504 were also obtained inHfr crosses (Table 2). Conversely, prototrophic recombi-nants were not detected by using strain AC505 (Table 2)carrying pAKC208, the recA::Tn5 derivative of pAKC207(Fig. 2). In all cases, however, no significant difference wasnoted in the transfer frequency of the plasmid pPH1JI,regardless of the phenotype of the recipient (Table 2),indicating a similar mating efficiency of the strains. Thesedata confirmed that Rec functions were encoded bypAKC207 and pAKC401 and that the heterologous E.carotovora subsp. carotovora recA gene complemented therecA mutation of HB101.

Construction and characterization of a RecA- E. carotovorasubsp. carotovora. By gene replacement via homologousrecombination, we constructed a RecA- mutant (AC5117) ofE. carotovora subsp. carotovora 71. AC5117 was Kmr Apsand, compared with the parent E. carotovora subsp.carotovora 71, sensitive to UV and MMS (Fig. 1). Tophysically localize the recA::Tri5 in AC5117, we analyzedthe genomic DNA. Agarose gel electrophoresis showed thatthe strain did not harbor pAKC208. We then restricted thegenomic DNA with Sall and localized the Tn5 and recAsequences by Southern hybridization. Our rationale for thiswas as follows. Sall does not cut the 9.8-kilobase insertDNA on pAKC207 (Fig. 2) but has a site within the Tn5DNA (15). Thus, the chromosomal recA region of AC5117,

S

_-CE C BA11 .I II

B1H EC

IT

01kb4 pAKC208FIG. 2. Restriction map of the recA region of E. carotovora

subsp. carotovora. Abbreviations used for restriction enzymes are

as follows: A (AvaI), B (BglII), C (ClaI); E (EcoRI), H (HindIII),and S (Sall). The arrow below the line indicates the location of theTnS insertion which inactivates the Rec functions.

when cut with Sall, will produce two fragments whichshould hybridize with both TnS DNA and the recA+ insertDNA of pAKC207. It also follows that only one Sallfragment of E. carotovora subsp. carotovora 71 (RecA+parent) should hybridize with pAKC207 DNA, and none ofthe fragments should hybridize with the Tn5 DNA. Ourresults (Fig. 3) indicate that this was the case. Based uponthis evidence and the resistance pattern of this strain to-wards antimicrobial agents (see above), we concluded thatthe Rec- phenotype of AC5117 resulted from the site-directed replacement of the E. carotovora subsp. carotovorarecA gene with the recA::TnS region of pAKC208 via ho-mologous recombination. To further support this, we testedfor complementation of the Rec- phenotype with the E.carotovora subsp. carotovora recA+ gene (pAKC210) andthe E. coli recA+ gene (pAKC401). Strains AC5118 andAC5120, constructed by transformation with pAKC210 andpAKC401, respectively, were considerably more resistant toMMS and UV than AC5117 (Fig. 1), suggesting the comple-mentation of the recA mutation. Furthermore, curingAC5118 of pAKC210 gave rise to AC5119, which wasindistinguishable from AC5117 with respect to CTV andPNL production (see below and Table 3) and the location ofTnS (Fig. 3).

Production of PNL and CTV in Rec+ and Rec- E.carotovora subsp. carotovora strains. The level of PNL wasabout 180 times and that ofCTV about 104 times higher in theE. carotovora subsp. carotovora 71 (RecA+) culture withmitomycin C than in cultures grown without the drug (Table3). A similar response was observed with cultures irradiatedwith UV or treated with nalidixic acid (data not shown).When the bacterium was grown in minimal medium contain-ing citrus pectin or polygalacturonate, the pectolytic en-zymes PEL and PEH were induced, but not PNL or CTV(data not shown). These observations indicated that PNLand CTV were induced in the E. carotovora subsp.carotovora strain 71, as in other E. carotovora subsp.carotovora strains (13, 14), by DNA-damaging agents andnot by the substrates that induced the pectolytic enzymes.

In the Rec- strain AC5117, PNL was not induced in Lbroth containing mitomycin C or in potato tuber tissue;however, a basal level of the activity was present (Tables 3and 4). In contrast, CTV was not detected in cultures of theRec- strain grown in the presence or absence of the drug orin tuber tissue (Tables 3 and 4). We did not observe anydifference between E. carotovora subsp. carotovora 71 andAC5117 in the production of protease, PEL, PEH, andcellulase in the absence of mitomycin C or in the ability tocause soft rot in potato tubers (data not shown). In thestrains AC5118 and AC5120, carrying the recA+ genes of E.carotovora subsp. carotovora and E. coli, respectively, both

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PNL and CTV were induced by mitomycin C, althoughAC5118 and AC5120 consistently produced approximately48 and 35% of the PNL activity of the wild-type E.carotovora subsp. carotovora 71, respectively (Table 3). Incontrast to the broth culture assays, in potato tuber tissue,AC5120 produced 20 times the PNL and 10 times the CTVactivity of E. carotovora subsp. carotovora 71 and AC5118(Table 4).

DISCUSSION

In this study, we cloned and characterized the E.carotovora subsp. carotovora recA+ gene and demonstratedthat a functional recA+ product was required in the inductionofPNL and CTV in E. carotovora subsp. carotovora. The E.carotovora subsp. carotovora recA+ gene complementedtwo Rec-associated phenotypes (recombination and DNArepair) in the E. coli strain HB101. Likewise, the E. coli

M:!. A

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El

1 ';

FIG. 3. Autoradiograph of Southern blots of genomic DNA fromE. carotovora subsp. carotovora strains digested with Sall. Papel A,Hybridization of pRZ102 DNA containing TnS. Lane 1, E.carotovora subsp. carotovora 71 (recA+); lane 2, strain AC5117(recA::TnS); lane 3, AC5119 (recA::TnS). Panel B, Hybridization ofpAKC207 DNA containing the E. carotovora subsp. carotovorarecA gene. Lane 1, E. carotovora subsp. carotovora 71; lane 2,strain AC5117. Shown at the left are molecular weight markers inkilobases (kb) obtained by a HindIlI digest of bacteriophage lambdaDNA.

TABLE 3. The effect of mitomycin C on PNL and CTVproduction in E. carotovora strains

Strains Relevant phenotype Mitomycin C Sp act (U)(mg/ml) PNLa CTVb

71 RecA+ 0 1.0 55500 179.0 105

AC5117 RecA- 0 0.2 <1C500 0.5 <1

AC5118 RecA-/E. carotovora 0 0.8 57subsp. carotovora 500 86.0 104RecA+

AC5119 RecA- 0 0.2 <1500 0.2 <1

AC5120 RecA-/E. coli RecA+ 0 2.5 47500 62.5 104

a Specific activity of PNL = units of activity (1 U of actlvity produces achange in absorbance of 1.0 at 235 nm per min) per mg of protein.

b Specific activity of CTV = units of activity (reciprocal of the highestdilution which caused clearing of the indicator lawn) per mg of protein.

c The CTV specific activities marked < were cases in which no clearing ofthe indicator lawn was detected with undiluted samples.

recA+ gene restored the Rec-associated functions as well asthe inducibility of PNL and CTV in strain AC5117(recAl::TnS), although the induced levels of PNL and CTVdiffered somewhat from those of wild-type E. carotovorasubsp. carotovora 71. The complementation of the recAmutation in E. carotovora subsp. carotovora by the E. colirecA was expected since recA in enterobacteria appears tohave been conserved (26, 36). Indeed, the restriction map ofthe E. carotovora subsp. carotovora recA gene (Fig. 2)appears quite similar to those of other enterobacteria (18).Although PNL and CTV were induced in the comple-

mented strains and E. carotovora subsp. carotovora 71, in Lbroth cultures containing mitomycin C, AC5118 (RecA-/E.carotovora subsp. carotovora RecA+) and AC5120(RecA-/E. coli RecA+) consistently produced less PNL andCTV than did the wild-type E. carotovora subsp. carotovora71. Contrary to the broth culture assays, we detected morePNL and CTV activity in potato tuber tissue infected withAC5120 than with any other strain tested. These differences,and the differences between the basal levels of PNL inAC5120 and E. carotovora subsp. carotovora 71, may be dueto a copy number effect of recA and, in the case of AC5120,the properties of the E. coli recA product, including thedegree of RecA activation by various inducing agents (i.e.,mitomycin C and the unidentified inducer in potato tubers).In this context, Keener et al. (18) recently reported func-tional differences between the recA products of various

TABLE 4. Production of CTV and pectinolytic and pectolyticenzymes by E. carotovora subsp. carotovora in potato tuber

tissue

Activity/g of macerated tissueaStrain Relevant phenotype

CTV PNL P~EL PEH

71 RecA+ 25 1.8 1.7 0.13AC5117 RecA- 0 0.2 1.6 0.13AC5118 RecA-/E. carotovora 25 1.7 1.5 0.19

subsp. carotovoraRecA+

AC5120 RecA-/E. coli RecA+ 250 36.4 1.4 0.11a See Materials and Methods and footnotes of Table 3 for a description of

the units of CTV, PNL, PEL, and PEH.

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ienterobacteria, including E. carotovora subsp. carotovoraand E. coli.The data presented here and the results of previous studies

(13, 14, 32) have established that PNL is induced along withCTV by DNA-damaging agents and not by pectin or pectate.These findings prompted the hypothesis that PNL and CTVwere regulated by the recA product in a manner similar tothe induction of SOS functions in E. coli (20, 34). While ourobservations with the recA::Tn5 mutant of E. carotovorasubsp. carotovora demonstrate a role of the recA product,we do not yet know the molecular basis for this effect. It ispossible that the activated recA product allows the expres-sion ofpnl and ctv genes by cleaving their repressor(s) in thesame way as the cleavage of LexA and repressors of thelambdoid bacteriophages in E. coli (19, 27, 28). As anextension of that hypothesis, we have entertained the pos-sibility that pnl and ctv constitute a single transcriptional unitregulated by a common repressor. Such a genetic organiza-tion, however, appears unlikely since we have obtained Tn5insertion mutants that are either Pnl+ Ctv- or Pnl- Ctv+(J. L. McEvoy, H. Murata, J. K. Engwall, and A. K.Chatterjee, Abstr. 6th Int. Conf. on Plant Pathogenic Bac-teria, College Park, Md., p. 47, 1985). The lack of a polareffect of Tn5 insertions strongly suggests that these genes areindependent transcriptional units. However, whether thesegenes share a common regulator molecule remains an openissue.The ecological significance of the unusual induction of

PNL awaits elucidation. Several lines of evidence, however,suggest the recA-mediated expression of pnl in plants. Forexample, an array of DNA-damaging agents occur in plantsunder both normal and stress conditions (1). Moreover, wedetected the production of both PNL and CTV in potatotuber tissue with Rec+ E. carotovora subsp. carotovorastrains but not with the Rec- strain (Table 4). Also, produc-tion of the temperate phage Erch-12 (4) and PNL in a Rec+E. chrysanthemi (EC183) was observed in infected planttissue and in culture medium supplemented with plant ex-tracts believed to contain putative DNA-damaging agents (S.Tsuyumu, T. Funakubo, Y. Takikawa, and M. Goto, Abstr.Ann. Phytopathol. Soc. Jpn. 50:410, 1984). Thus, the avail-able data suggest that the expression of Erwinia genes forpectolytic enzymes (PEL and PEH) and the pectinolyticenzyme (PNL) is modulated by host factors. When in thepresence of uronate derivatives, these bacteria produce thepectolytic enzymes that act as virulence factors. However,when the host presents DNA-damaging agents, the bacteriarespond by inducing recA and activating its product. Thisresponse allows the pathogen to repair DNA damage andconcomitantly produce PNL, an additional virulence factor.Confirmation of this hypothesis would demonstrate a novelmechanism by which a pathogen can overcome an inimicalenvironment presented by the host plant.

ACKNOWLEDGMENTS

This investigation was supported by the National ScienceFoundation (grants PCM-8022003 and PCM8022003/Amendment No.01), the Science and Education Administration of the U.S.Department of Agriculture (grant 59-2201-1-1-686-0 from theCompetitive Research Grants Office), and the Kansas AgriculturalExperiment Station, Manhattan.We wish to acknowledge the assistance of K. K. Thurn in some

phases of this study and thank F. Bolivar, S. H. DeBoer, N. T.Keen, N. Kleckner, G. L. Marchin, D. W. Mount, E. W. Nester,and R. R. Rodriguez for providing us with bacterial strains, plas-mids, and bacteriophages.

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