Vol. 56, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1990, p. 431-4350099-2240/90/020431-05$02.00/0Copyright C) 1990, American Society for Microbiology

Copper Resistance Gene Homologs in Pathogenic and SaprophyticBacterial Species from Tomato

DONALD A. COOKSEY,* HAMID R. AZAD, JAE-SOON CHA, AND CHUN-KEUN LIM

Department of Plant Pathology, University of California, Riverside, California 92521-0122

Received 21 August 1989/Accepted 17 November 1989

Copper-resistant strains of Xanthomonas campestris pv. vesicatoria, Pseudomonas cichorii, Pseudomonasputida, Pseudomonasfluorescens, and a yellow Pseudomonas sp. were isolated from tomato plants or seeds. InSouthern hybridizations, DNA from each strain showed homology with the copper resistance (cop) operon

previously cloned from Pseudomonas syringae pv. tomato PT23. Homology was associated with plasmid andchromosomal DNA in X. campestris pv. vesicatoria, P. putida, and the yellow Pseudomonas sp. Homology was

detected only in the chromosomal DNA of P. cichorii and P. fluorescens. Homology with cop was also detectedin chromosomal DNA from copper-sensitive strains of P. cichorii, P. fluorescens, and P. syringae pv. tomato,

suggesting that the cop homolog may be indigenous to certain Pseudomonas species and have some functionother than copper resistance. No homology was detected in DNA from a copper-sensitive X. campestris pv.

vesicatoria strain. Copper-inducible protein products were detected in each copper-resistant bacterium byimmunoblot analysis with antibodies raised to the CopB protein from the cop operon. The role of thehomologous DNA in copper resistance was confirmed for the X. campestris pv. vesicatoria strain by cloning andtransferring the cop homolog to a copper-sensitive strain of X. campestris pv. vesicatoria. The possibility andimplications of copper resistance gene exchange between different species and genera of pathogenic andsaprophytic bacteria on tomato plants are discussed.

Copper resistance in two bacterial pathogens of tomato inplaces where copper compounds are commonly applied tocontrol plant diseases has been previously reported. InFlorida, copper-resistant strains of Xanthomonas campes-tris pv. vesicatoria have been found (16), and resistance isdetermined by large conjugative plasmids (21). In California,copper-resistant strains of Pseudomonas syringae pv. to-mato are widespread (2, 7), and resistance is encoded by ahighly conserved 35-kilobase plasmid designated pPT23D(3). DNA encoding copper resistance was cloned frompPT23D (3), sequenced (18), and shown to be organized asan operon (cop) consisting of four genes controlled by acopper-inducible promoter (19). No homology was detectedin hybridizations between the cop operon and copper resis-tance genes from the Florida strains of X. campestris pv.vesicatoria, suggesting that the two copper resistance sys-tems are not closely related (3). Although the existence ofthese genes in other pathogens of tomato or in saprophyticbacteria found on this plant has not been reported, theperiodic selection pressure imposed by copper spray appli-cations seems to favor a more widespread establishment ofcopper-resistant microbial populations. We thereforewanted to determine whether other copper-resistant mi-crobes occur on tomato plants and whether this resistancewas determined by genes related to the cop operon of P.syringae pv. tomato.

During recent isolations from field-grown tomato plantsand from tomato seeds, we recovered several species ofcopper-resistant bacteria. These included three saprophyticspecies, Pseudomonas putida, Pseudomonas fluorescens,and a yellow Pseudomonas sp., and two pathogenic species,Pseudomonas cichorii and X. campestris pv. vesicatoria.This paper reports the detection of plasmid and chromo-somal DNA with homology to the copper resistance operon(cop) of P. syringae pv. tomato in each of these bacteria and

* Corresponding author.

demonstrates the role of one of the gene homologs in copperresistance.

MATERIALS AND METHODS

Bacterial strains. The sources and characteristics of thebacterial strains used are listed in Table 1. Bacteria wereisolated from diseased tomato leaves with either bacterialspeck symptoms, caused by P. syringae pv. tomato, orbacterial spot symptoms, caused by X. campestris pv.vesicatoria. Samples were from commercial tomato fields inCalifornia. P. putida 08891 was isolated from a commercialtomato seed lot.MIC determination. Bacteria were grown on mannitol-

glutamate agar (13) supplemented with yeast extract at 0.25g/liter (MGY). Cells were suspended in sterile water to about5 x 108/ml and spotted in duplicate (10 ,ul per spot) ontoMGY agar containing different concentrations of cupricsulfate (0 to 3.2 mM). The MIC of cupric sulfate wasexpressed as the concentration that inhibited confluentgrowth of the culture after 3 days at 28°C.

Plasmid and chromosomal DNA isolation. Bacteria weregrown in MGY broth, and plasmids were isolated for purifi-cation on cesium chloride gradients essentially as describedby Currier and Nester (9), but the DNA-shearing step wasomitted. Total genomic DNA was isolated by the sameprocedure, but the alkaline denaturation step was omitted.

Detection of homology to cop. The 4.5-kilobase PstI-PstIfragment from pCOP2 (3), which contains the entire copoperon (18, 19), was gel purified with an NA-45 DEAEmembrane (Schleicher & Schuell, Inc.) as recommended bythe manufacturer and labeled by random primed labelingwith digoxigenin-11-dUTP and the Genius DNA Labelingand Detection Kit, both from Boehringer Mannheim Bio-chemicals. Southern blotting of DNA separated on agarosegels to nylon membranes, hybridization and wash condi-tions, and detection of labeled DNA were done essentially asdescribed by the manufacturer of the detection kit. Posthy-

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TABLE 1. Bacterial strains and characteristics

Organism MIC Homology Sourceand strain (MM) o to copa (reference)

P. syringae pv.tomato

PT23 1.6 P (35), C Tomato (2)PT16 1.6 P (35), C Tomato (2)09885 1.6 P (35), C Tomato (this study)PT11 0.4 C Tomato (2)PT12 0.6 C Tomato (2)PT26 0.6 C Tomato (2)

P. cichorii07881 1.6 C Tomato (this study)028410 0.2 C Chrysanthemum (this

study)

P. fluorescens08892 2.4 C Tomato (this study)078236 1.0 C Vegetable soft rot"

P. putida08891 2.4 P (>200), C Tomato seed (this

study)R5-3 1.8 C Sewage sludge (5)

Pseudomonas sp. 2.8 P (200), C Tomato (this study)strain 07888

X campestns pv.vesicatoria

07882 1.8 P (100), C Tomato (this study)078518 0.2 Tomato (this study)078518(pCOP100) 2.0 P (50) Transconjugant with

copper-resistantcosmid clone fromstrain 07882

a Plasmid-borne homology; C, chromosomal homology. Numbers in paren-theses indicate the approximate sizes (in kilobases) of homologous plasmids.

" Obtained from L. W. Moore, Oregon State University; previously clas-sified as Pseudomonas marginalis.

bridization washes were at relatively low stringencies (0.5 xSSC [1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate] at65°C), unless otherwise specified.Immunoblot analysis of proteins. Whole-cell proteins from

cultures grown for 24 h in MGY broth with 0.5 mM cupric

sulfate or without added copper were separated by gelelectrophoresis on sodium dodecyl sulfate-10% polyacryl-amide gels, as described by Laemmli (15). Proteins were

then electrophoretically transferred to nitrocellulose filters(1). The filters were washed twice with TBST buffer (10 mMTris hydrochloride, 140 mM NaCl, 0.1% Tween 20 [pH 7.4])and incubated with antibodies raised to a CopB-LacZ fusionprotein (J.-S. Cha and D. A. Cooksey, manuscript in prep-aration) diluted 1:500 in HST buffer (10 mM Tris hydrochlo-ride, 1 M NaCl, 0.5% Tween 20 [pH 7.4]). The filters werethen washed twice with TBST, once with HST, and twicewith TBST. The filters were incubated with anti-rabbitantibodies conjugated with alkaline phosphatase (SigmaChemical Co.) that had been diluted 1:1,000 in HST. Afterfurther extensive washes with TBST, HST, and TBS (TBSTwithout Tween 20), alkaline phosphatase was detected withNitro Blue Tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate.

Cloning of copper resistance genes. A cosmid library ofplasmid DNA from X. campestns pv. vesicatoria 07882 wasconstructed in Escherichia coli DH5 with the vectorpLAFR3 (22), essentially as described previously (3). About100 colonies were pooled by washing them off agar plateswith 1 ml of LB broth (GIBCO Laboratories). A 0.25-mlportion of this suspension was mixed with 0.25 ml of E. coliHB101 containing the helper plasmid pRK2013 (11) and 0.25ml of a copper-sensitive, rifampin-resistant strain of X.campestris pv. vesicatoria (078518). The mixture was spot-ted onto a plate of yeast-glucose-calcium carbonate agar (20)and incubated for 24 h at 28°C. Bacteria were suspended in1.0 ml of sterile water and plated onto nutrient agar (DifcoLaboratories) containing cupric sulfate (0.8 mM) and ri-fampin (50 ,ug/ml).

RESULTS

Identification of copper-resistant pathogens and sapro-phytes. The species of Pseudomonas isolated in this studywere identified by the characteristics listed in Table 2.Pseudomonas sp. strain 07888 appeared to be affiliated withthe genus Pseudomonas, but the characteristics listed inTable 2 and the results of over 50 other physiological tests(data not shown) did not correspond closely to those of anyof the described species of the genus (14). X. campestris pv.

vesicatoria 07882 was a gram-negative, oxidase-negative,yellow mucoid bacterium with a single polar flagellum;

TABLE 2. Identification of copper-resistant Pseudomonas species from tomato

Result of test"03 0

a a~~~rE 0 No.of~~~~Q0On EDe 0 flagella/cell

P.cichoni00~~~~~~~~~~~~~~~~~~~~~~~~~0a0~~~~~~~~~~~~~~~

P. cichorii 07881 - + - - - + + - - - -- + >1P. fluorescens 08892 - + - - - + + + - + - - - >1P. putida 08891 - + - - - + + + - - - - - >1Pseudomonas sp. 07888 - - + - - - + - - - + - -

a Test methods were as described in Gerhardt et al. (12) and Schaad (20).b Cells of all strains were rods.c PHB, Poly-,-hydroxybutyrate.

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COPPER RESISTANCE GENE HOMOLOGS 433

1 2 3 4 5 6 7 8 9kb

- 23.1- 9.4

6.6- 4.4

- 2.32.0

FIG. 1. Southern blot hybridization of the cop operon of P.syringae pv. tomato PT23 to plasmid and chromosomal DNA fromcopper-resistant pathogens and saprophytes from tomato. Lane 1,Plasmid DNA from PT23; lane 2, plasmid DNA from Pseudomonassp. strain 07888; lane 3, total DNA from 07888; lane 4, plasmid DNAfrom X. campestris pv. vesicatoria 07882; lane 5, total DNA from07882; lane 6, plasmid DNA from P. putida 08891; lane 7, total DNAfrom 08891; lane 8, total DNA from P. cichorii 07881; lane 9, totalDNA from P. fluorescens 08892. The DNA in each lane was digestedwith EcoRI before electrophoresis. A total of 2 ,ug of DNA per lanewas loaded for each total DNA sample, and 0.5 ,ug of DNA per lanewas loaded for each plasmid sample. kb, Kilobases.

bacterial spot symptoms were reproduced after the inocula-tion of tomato with strain 07882.The levels of copper resistance in some of these bacteria

were high compared with those of previously describedcopper-resistant strains of P. syringae pv. tomato (Table 1).Some bacteria from sources other than tomato, such as P.fluorescens 078236 and P. putida R5-3, had moderate levelsof resistance to copper.

Detection of homology to cop. Homology between the cop

operon from pPT23D and a plasmid of about 100 kilobases inX. campestris pv. vesicatoria and with larger plasmids in P.putida 08891 and the yellow Pseudomonas sp. strain 07888was detected by Southern blot hybridizations. Additionalhomology was detected in the chromosomal DNA of thesethree strains. This additional homologous DNA was identi-fied as chromosomal by comparing the sizes of homologousfragments after EcoRI digests (Fig. 1) or PstI digests (datanot shown) of total genomic DNA with the sizes of homol-ogous fragments after digestion of purified plasmid DNAwith the same enzyme.No plasmids were detected in P. cichorii 07881, and the

single detectable plasmid in P. fluorescens 08892 did notshow homology. However, homology was detected in thechromosomal DNA of P. cichorii 07881 and P. fluorescens08892 (Fig. 1).The strength of hybridization to the cop operon was

similar for each of the new strains but less than the strengthof hybridization of the cop operon to pPT23D, from whichthe operon was originally cloned (Fig. 1). With stringentwashes after hybridization, homology was still detectable inthe plasmid and chromosomal DNA of these bacteria, butthe hybridization strength was less than that to pPT23D (datanot shown).No homology was detected between the cop operon of

pPT23D and the total DNA from a copper-sensitive strain ofX. campestris pv. vesicatoria, but homology was detected inthe chromosomal DNA of copper-sensitive strains of P.cichorii and in the chromosomal DNA of both sensitive andresistant strains of P. syringae pv. tomato. P. fluorescensand P. putida strains from sources other than tomato were

moderately copper tolerant and showed homology to the copoperon in their chromosomal DNA (Table 1).

1 2 3 4 5 6 7 8 9 kDa_-~~~~~~97

CopA- - 86

__. ~~~~~~-46

- 31

-I22FIG. 2. Immunoblot analysis of copper-inducible proteins from

copper-resistant pathogens and saprophytes from tomato. Proteinswere detected with antibodies raised to the CopB protein from P.syringae pv. tomato PT23. Equivalent amounts of total bacterialprotein were loaded in each lane. L..ne 1, PT23; lane 2, Pseudomo-nas sp. strain 07888 grown in the presence of 0.5 mM CuS04; lane3, 07888 grown without added copper; lane 4, P. cichorii 07881grown in the presence of 0.5 mM CuSO4; lane 5, 07881 grownwithout added copper; lane 6, P. fluorescens 08892 grown in thepresence of 0.5 mM CuSO4; lane 7, 08892 grown without addedcopper; lane 8, P. putida 08891 grown in the presence of 0.5 mMCuSO4; lane 9, 08891 grown without added copper. kDa, Kilodal-tons.

Control experiments which used the cloning vectorpRK404 (from which the cop probe was derived) as a probeshowed that the hybridizing bands in the Southern blotsdiscussed above were not due to vector homology (data notshown).Immunoblot analysis of proteins. Each copper-resistant

strain that showed homology with the pPT23D cop operon inSouthern blot hybridizations also produced proteins withdetectable serological relatedness to a protein product of thecop operon (Fig. 2). Related proteins from X. campestris pv.vesicatoria 07882 were detected, but the bands on Westernblots (immunoblots) were faint and are not shown in Fig. 2.The related proteins were detected only when each bacte-rium was grown in the presence of copper, except the yellowPseudomonas sp., which showed some constitutive produc-tion of the proteins. The antibodies used in this analysis wereraised to the CopB protein; these antibodies also reactedwith CopA, which was not unexpected, since the two openreading frames share homologous repeated sequences (18).Similarly, the antibodies detected two or more proteins inthe yellow Pseudomonas sp. strain 07888, P. cichorii 07881,P. putida 08891, and X. campestris pv. vesicatoria 07882,but only one copper-inducible protein was detected in P.fluorescens 08892.

Each copper-sensitive P. syringae pv. tomato strain pro-duced a copper-inducible protein about the size of CopA(data not shown).

Cloning of copper resistance genes from X. campestris pv.vesicatoria strains. A cosmid library of total plasmid DNAfrom X. campestris pv. vesicatoria 07882 was constructed inthe vector pLAFR3. About 100 pooled clones were massmated into a copper-sensitive, rifampin-resistant strain of X.campestris pv. vesicatoria (078518), and copper-resistant,rifampin-resistant transconjugants were selected. Three cop-per-resistant transconjugants were selected for analysis ofplasmid DNA. The cosmid DNA was isolated directly fromstrain 078518 for analysis, since this strain has no detectableindigenous plasmids. Restriction analysis showed that thethree clones were identical and that a portion of each clonehybridized with the cop operon from pPT23D (Fig. 3). Thesizes of the fragments hybridized in EcoRI and PstI digestsof the clones were the same as those hybridized in EcoRIand PstI digests of total plasmid DNA from strain 07882. The

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1 2 3 4 5 6 7 8kb

pLAFR3 -_:2212

- - -- - 4.4

FIG. 3. Southern blot of pLAFR3 cosmid clones containingplasmid DNA from X. cacmpestiis pv. i'esicatoria 07882 that ex-

pressed copper resistance in X. (campestris pv. v,esicatoria 078518.The blot was probed with the cop operon of P. syrintigae pv. tomnatocloned in pRK404 (10). Homology with pLAFR3 is due to homologybetween the two RK2-based vectors, pRK404 and pLAFR3. Lane 1.PstI-digested plasmid DNA from 07882; lanes 2 through 4, Pstl-digested cosmid clones pCOP100 to pCOP102, respectively; lane 5,EcoRI-digested plasmid DNA from 07882; lanes 6 through 8, EcoRl-digested cosmid clones pCOP100 to pCOP102, respectively. kb.Kilobases.

clones were designated pCOP100 to pCOP102. The level ofcopper resistance for the transconjugants was similar to thatof strain 07882 and represented about a 10-fold increase over

that of recipient strain 078518 (Table 1).

DISCUSSION

Several copper-resistant bacterial species were isolatedfrom tomato plants and seeds, and each bacterium containedeither plasmid or chromosomal DNA that was homologousto the cop operon from P. syringae pv. tomato. For X.campestris pv. i'esicatoria 07882, the role of the cop ho-molog in copper resistance was confirmed by cloning andtransferring the homolog to a copper-sensitive strain. The100-kilobase plasmid that contained the cop homolog instrain 07882 represents a new type of copper resistanceplasmid for this species, since it is homologous to cop and ismuch smaller than previously characterized X. campestrispv. vesicatoria copper resistance plasmids from Florida (21).Since this homology was not detected in a copper-sensitivestrain of X. campestris pv. vesicatoria, the cop genes areprobably not a normal component of this species; thissuggests a horizontal transfer of copper resistance genes tothis bacterium, perhaps from a bacterium such as one of thePselidomonas species described in this paper.

The role of the cop homologs in copper resistance has notyet been determined for the other copper-resistant bacteriadescribed in this paper. The presence of such homologousDNA clearly does not confer resistance in copper-sensitivestrains of P. syringae pv. tomato, P. cichorii, and P.flimorescens, suggesting that these genes have some otherfunction and may be indigenous to certain Pseiudomonasspecies. However, computer database searches for homol-ogy to cop have not revealed any strong suggestions foralternate functions of these genes. It is possible that thecopper resistance operon of pPT23D evolved from its chro-mosomal homolog. Cloning and sequencing these homolo-gous genes may provide interesting information on theevolution of copper resistance.Western blot analysis of proteins from the various copper-

resistant bacteria with antibodies raised to CopB demon-strated that two or more copper-inducible proteins werepresent in several species. The serological cross-reactionwith CopA and CopB in P. syriingae pv. tomato PT23 is

probably due to the presence of the homologous repeatedsequences in the c(opA and copB genes (18). This suggeststhat similar homologous regions may be present in the genesof these other copper-resistant species, leading to the pro-duction of multiple serologically related proteins. In the copoperon from pPT23D, these repeated sequences encodepeptides rich in methionine, histidine, and aspartic acid, allof which can serve as copper ligands in known copper-binding proteins (6). The possible interactions of thesepeptides with copper is still under investigation, but thepresence of similar repeated regions in the other copper-resistant bacteria would strengthen the speculation that suchregions are functionally important structures.

Copper-resistant strains of P. syringae pv. tionato arehighly conserved, both in the structure of their copperresistance plasmids (7) and in their genomes (8), suggestingthat they were recently introduced and perhaps spread to thetomato-growing areas of California on tomato seeds ortransplants (4, 17). In addition to such an introduction ofcopper resistance from an outside source, the presence ofcopper-resistant saprophytes on tomato plants suggests an-other possible mechanism for the acquisition of copperresistance genes by pathogenic species; the saprophyticbacteria might provide a reservoir of resistance genes thatcould be acquired by copper-sensitive pathogens. The pres-ence of a copper-resistant P. plutida strain with a plasmid-borne cop homolog in a commercial tomato seed lot alsosuggests that saprophytic bacteria could contribute to thespread of resistant bacterial populations between fields andbetween different geographical areas.

ACKNOWLEDGMENTS

This work was supported by National Science Foundation grantBSR-8717421 and a University of California Biotechnology Re-search and Education Program training grant.

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