Vol. 171, No. 3

Cloning and Expression in Escherichia coli of Pseudomonas StrainLB400 Genes Encoding Polychlorinated Biphenyl Degradation

FRANK J. MONDELLO

Biological Sciences Laboratory, Research and Development Center, GE Co., Schenectady, New York 12301

Received 27 July 1988/Accepted 7 December 1988

Pseudomonas strain LB400 is able to degrade an unusually wide variety of polychlorinated biphenyls (PCBs).A genomic library of LB400 was constructed by using the broad-host-range cosmid pMMB34 and introducedinto Escherichia coli. Approximately 1,600 recombinant clones were tested, and 5 that expressed 2,3-dihydroxybiphenyl dioxygenase activity were found. This enzyme is encoded by the bphC gene of the2,3-dioxygenase pathway for PCB-biphenyl metabolism. Two recombinant plasmids encoding the ability totransform PCBs to chlorobenzoic acids were identified, and one of these, pGEM410, was chosen for furtherstudy. The PCB-degrading genes (bphA, -B, -C, and -D) were localized by subcloning experiments to a12.4-kilobase region of pGEM410. The ability of recombinant strains to degrade PCBs was compared with thatof the wild type. In resting-cell assays, PCB degradation by E. coli strain FM4560 (containing a pGEM410derivative) approached that of LB400 and was significantly greater than degradation by the originalrecombinant strain. High levels of PCB metabolism by FM4560 did not depend on the growth of the organismon biphenyl, as it did for PCB metabolism by LB400. When cells were grown with succinate as the carbonsource, PCB degradation by FM4560 was markedly superior to that by LB400.

Polychlorinated biphenyls (PCBs) are a family of man-made compounds with excellent thermal stability and dielec-tric properties. Structurally, they are composed of a biphe-nyl molecule carrying from 1 to 10 chlorines. Depending onthe number and position of the chlorines, 209 different PCBcongeners can be produced (23). From 1929 to 1978, approx-imately 1.4 billion lb (635 million kg) of PCBs was produced,and it is estimated that several hundred million pounds havebeen released into the environment (16). The vast majority ofthis material is in the form of commercial mixtures (such as

the Aroclors) which contain 60 to 80 different PCB conge-ners and are thus particularly difficult to biodegrade (23).There are numerous reports in the literature of pure andmixed cultures of microorganisms with the ability to oxidizethe mono-, di-, and trichlorinated biphenyls (1, 5, 11, 12, 20,25, 27). However, there are relatively few reports of bacteriacapable of metabolizing the more highly chlorinated PCBs(4, 6, 8, 14, 23). The development of efficient biodegradationprocesses for the destruction of PCBs will require organismswith superior oxidative ability. These may be either natu-rally occurring strains isolated by enrichment culture or

those created through laboratory selection or recombinantDNA technology. An excellent candidate for use in suchstudies is Pseudomonas strain LB400, an organism capableof oxidizing biphenyl molecules containing up to six chlo-rines (8).PCB metabolism by LB400 involves at least two different

chlorobiphenyl dioxygenase activities. One of these cata-lyzes the insertion of two oxygen atoms at carbon positions2 and 3 (a 2,3-dioxygenase). This is the first enzyme of thepathway leading to the formation of benzoic and chloroben-zoic acids from biphenyl/PCB degradation and results in theproduction of a 2,3-dihydrodiol (Fig. 1) (22). This compoundis then converted by a dihydrodiol dehydrogenase to 2,3-dihydroxybiphenyl, which undergoes a meta cleavage by2,3-dihydroxybiphenyl dioxygenase to produce 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid. A hydrase reactionconverts this compound to the corresponding benzoic acid

(22). This type of attack has been described for otherbiphenyl- and PCB-metabolizing bacteria (1, 11, 23).The second PCB dioxygenase activity present in LB400

inserts an oxygen molecule at carbon position 3,4. Such3,4-dioxygenase activity has been confirmed in both LB400and Alcaligenes eutrophus H850, an organism with verysimilar PCB-degrading ability (4, 22). At present, it is un-known whether a single enzyme is responsible for both typesof dioxygenase activity and whether the two methods ofattack explain the unusually broad congener specificity ofLB400.

This report describes the isolation of the genes responsiblefor PCB biodegradation by LB400. This is the first instancein which recombinant plasmids encoding the entire pathwayfor the conversion of PCBs to chlorobenzoic acids have beenobtained.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions. Thebacterial strains and plasmids used in this study are listed inTable 1. Pseudomonas strain LB400 was grown on PASminimal medium (8). Sterile molten biphenyl (approximately1.0%) was added directly to liquid media or to petri plate lidsfor use as the carbon and energy source. E. coli strains weregrown in either Luria (L) broth (8) or PAS medium contain-ing 0.5% succinate and supplemented with the requiredamino acids and vitamins. Solid media contained 1.5%purified agar (Difco Laboratories, Detroit, Mich.). Whereappropriate, the antibiotics kanamycin and ampicillin wereused for selection at concentrations 30 and 100 ,ug/ml,respectively. Cultures were incubated at 30°C for Pseudo-monas and recombinant E. coli strains and at 37°C for otherE. coli strains. Liquid cultures were incubated in rotaryshaker-incubators at 250 rpm.

Preparation ofDNA for cloning. A 100-ml culture of LB400grown overnight in L broth was harvested by centrifugation(13,000 x g, 10 min). The supernatant fluid was removed,and the cells were suspended in 20 ml of buffer (50 mM Trishydrochloride [pH 8.0], 250 mM EDTA [pH 8.0]). The cells

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xlbphB

OHOH

bphC

0

HO °1 OH

clxxlbph D

Cl K .z OH

FIG. 1. Degradation of biphenyl and chlorobiphenyls by the2,3-dioxygenase pathway in Pseudomonas strain LB400. Genedesignations: bphA, biphenyl 2,3-dioxygenase; bphB, dihydrodioldehydrogenase; bphC, 2,3-dihydroxybiphenyl dioxygenase; bphD,2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (meta-cleavage pro-duct) hydrase.

were lysed by the addition of 1/20 volume of 20% sodiumlauryl sulfate and heating at 65C for 15 min. The lysate wasthen cooled to 50°C, and a 20-mglml solution of proteinase Kwas added to a final concentration of 500 ,ug/ml. Afterovernight incubation at 50°C, the solution was phenol-chloroform extracted three times as described by Maniatis etal. (19). The final aqueous phase was extracted six times withequal volumes of water-saturated ether. The DNA in theextract was then precipitated by the addition of 2 volumes o'f-20°C absolute ethanol, collected by spooling onto a glassrod, and rehydrated in TE buffer (10 mM Tris hydrochloride[pH 7.5], 1.0 mM EDTA).Approximately 200 ,ug of the genomic DNA was partially

digested with restriction enzyme Sau3A. The DNA washeated to 68°C for 5 min and loaded onto a 12-ml 10 to 40%sucrose gradient prepared in 1 M NaCl, 5 mM Tris hydro-chloride (pH 8.0), 1 mM EDTA, and 1 p.g of ethidiumbromide per ml. Centrifugation was conducted for 11 h in anSW41 rotor (Beckman Instruments, Inc., Fullerton, Calif.)(40,000 rpm, 25°C). Genomic DNA fragments 25 to 35kilobases (kb) in size were identified by comparing theirmigration with that of Sail-digested lambda DNA in anidentically prepared and simultaneously run gradient. Frag-ments of the desired size were collected by puncturing thetube with a needle and drawing out the DNA with a syringe.Ethanol precipitation was used to concentrate the DNA (19).Cosmid cloning vector pMMB34 DNA (approximately 10

,ug) was digested to completion with BamHI according to thedirections of the manufacturer and then treated with alkalinephosphatase as described by Maniatis et al. (19).

Ligation, in vitro packaging, and tauection. LB400 DNAfragments (6 ,ug) were joined to pMMB34 (1.5 ,ug), using T4DNA ligase in a total reaction volume of 20 ,ul incubated at

TABLE 1. Bacterial strains and plasmids

StiCSa.Source orStrain or plasmid Relevant characteristicsa rcereference

Bacterial strainPseudomonas strain LB400 Bph+ 8Escherichia coliHB101 recA13 leuB6 proA2 thi-I hsdR hsdM Smr BRLbTB1 A(lac proAB) lacZAM15 hsdR SmT BRLFM4100 HB101(pGEM410) This workFM4110 TB1(pGEM410) This workFM4560 TB1(pGEM456) This work

PlasmidpUC18 Apr BRLpMMB34 Kmr cos Mob' 10pGEM400 Kmr, pMMB34 with Pseudomonas strain LB400 DNA insert, BphA+B+C+ This workpGEM410 Kmr, pMMB34 with Pseudomonas strain LB400 DNA insert, BphA+B+C+D+ This workpGEM411 Apr, pUC18 with Pstl insert, BphB+C+ This workpGEM420 Kmr, pMMB34 with Pseudomonas strain LB400 DNA insert, BphA+B+C+D+ This workpGEM43O Kmr, pMMB34 with Pseudomonas strain LB400 DNA insert, BphB+C+D+ This workpGEM440 Kmr, pMMB34 with Pseudomonas strain LB400 DNA insert, BphAB+C+D This workpGEM451 ApK, pUC18 with EcoRI insert This workpGEM452 Apr, pUC18 with EcoRI insert This workpGEM453 ApT, pUC18 wvith h:coRI insert This workpGEM454 Apr, pUC18 with EcoRI insert, BphB+C+ This workpGEM455 ApT, pUC18 with EcoRI insert, BphB+C+D+ This workPGEM456 Apr, PUC18 With ECORI inSert, BPhA+B+C+ ThiS WorkpGEM457 Apr, PUC18 with EcoRI insert, BPhD+ This workpGEM458 Apr, pUC18 with EcoRI insert, BphA+B+C+ This workpGEM459 Ap , pUC18 with EcoRI insert, BphA+B+C D+ This work

a Bph+, Ability to grOW on biphenyl.b BRL, Bethesda Research Laboratories, Inc., Gaithersburg, Md.

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CLONING OF GENES ENCODING PCB DEGRADATION

5°C for 18 h. In vitro packaging of the recombinant mole-cules was performed by using a commercially preparedextract (Promega Biotech, Madison, Wis.) as recommendedby the manufacturer. Portions of the packaging reactionwere used to transfect cells of E. coli HB101 which had beengrown in L broth containing 0.2% maltose (19). These cellswere then plated onto L agar plates containing 30 jig ofkanamycin per ml to select transfectants.

Subcloning procedures. Plasmid pGEM410 was partiallydigested with EcoRI, and the fragments were treated withalkaline phosphatase. This DNA was ligated to EcoRI-digested vector pUC18, and the products of the reactionwere transformed into cells of E. coli TB1 (19). Cells were

plated onto L agar containing ampicillin (100 ,ug/ml), iso-propyl-p-D-thiogalactopyranoside (IPTG) (140 Vig/ml), and5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside (X-Gal)(125 pgIml). Strains receiving pGEM410 fragments (iden-tified by white colonies) were purified by being restreaked on

the same medium. Purified strains were screened for thepresence of Bph pathway enzymes as described previously(22). The plasmids from the pGEM410 subclones were

isolated by the method of Holmes (15). These DNAs were

digested with EcoRI and analyzed by agarose gel electro-phoresis to determine which pGEM410 fragments they con-

tained.Isolation of plasmid DNA. Plasmid-containing strains were

grown overnight in 1.0 liter of L broth containing an antibi-otic appropriate for selection. The cells were harvested bycentrifugation and then suspended in 36 ml of cold 25%(wt/vol) sucrose prepared in 0.05 M Tris hydrochloride (pH8.0). A freshly prepared solution of lysozyme (6.0 ml, 2.0mg/ml) was added to the cell suspension and mixed slowly at37°C for 30 min. The mixture was placed on ice for 5 min,and 19.5 ml of 0.25 M EDTA (pH 8.0) was added. After thesuspension had become viscous, a 2% (wt/vol) solution ofN-lauroyl sarcosine was added to a final concentration of0.5%, and the mixture was shaken gently at 37°C for 30 min.Chromosomal DNA was precipitated from the lysate byaddition of ice-cold 5 M NaCl to a final concentration of 1 Mand overnight incubation at 4°C. After centrifugation at17,000 x g for 30 min, the lysate was filtered throughsterilized glass wool and the plasmid DNA was purified bycesium chloride-ethidium bromide density gradient centrifu-gation (19).

Detection of enzymatic activities. The expression of bphA(encoding PCB/biphenyl dioxygenase) in recombinantstrains was demonstrated by the ability to degrade any of thecongeners in PCB mixes 1B and 2B by resting-cell assay (4).In addition, strains were tested for depletion of 2,3-dichlo-robiphenyl (100 ,uM), 2,4,4'-trichlorobiphenyl (5 ,uM), and2,5,2',5'-tetrachlorobiphenyl (10 ,uM) in single-congenerresting-cell assays. The ethereal spray technique of Kiyo-hara et al. (18) was used as a rapid screening procedure forbiphenyl metabolism.The presence of 2,3-dihydroxybiphenyl dioxygenase ac-

tivity was used to demonstrate bphC expression in recom-

binant strains. This activity can be detected by the ability toproduce the yellow metabolite 2-hydroxy-6-oxo-6-phenyl-hexa-2,4-dienoic acid (meta-cleavage product) when treatedwith an ether solution containing 1% 2,3-dihydroxybiphenyl(prepared by G. Yeager and K. Longley, GE Co.,Schenectady, N.Y.) (22). In strains expressing bphC, thepresence of bphB activity could be detected by the formationof the meta-cleavage product upon incubation in a solutioncontaining 1 mg of 2,3-dihydro-2,3-dihydroxybiphenyl per

ml. The ability to convert the meta-cleavage product of

2,3-dichlorobiphenyl to 2,3-dichlorobenzoic acid was used totest bphD expression.

Resting-cell assays and GC analysis. PCB depletion studieswith single congeners, mixes 1B and 2B, and Aroclor 1242were conducted by using resting-cell assays (4, 6). Gaschromatography (GC) was performed with a Vista GC 6000(Varian, Palo Alto, Calif.) equipped with an electron capturedetector and splitter-injector, both operated at 300°C, and afused silica capillary column (30 m by 0.25 mm [innerdiameter]) coated with a 0.25-,um bonded liquid phase ofDB-1 (polydimethylsiloxane) (J & W Scientific Inc.). Helium(19 cm/s) was used as the carrier gas, and nitrogen (30ml/min) was the makeup gas. The temperature program formixes 1B and 2B used an initial column temperature of 185°Cheld for 3 min and then raised to 230°C at a rate of 5°C/min.The program for Aroclor 1242 used an initial temperature of160°C, which was increased by 2°C/min to 200°C and then by8°C/min to 240°C. The extent of Aroclor 1242 depletion wasdetermined after normalization of nondegradable peak areasin experimental chromatograms with those from controlsamples of mercury-killed cells (4, 6).

Metabolite isolation and GC-mass spectrometry analysis.Metabolites were isolated from the transformation of indi-vidual PCB congeners at a concentration of 250 ,uM byresting cells (optical density at 615 nm of 5.0). Reactionswere conducted in five 35-ml glass vials, each containing 10ml of cells and sealed with foil-lined caps. After 24 h ofincubation at 30°C, each vial was extracted with 15 ml ofether on a horizontal reciprocal shaker for 18 h. For isolationof benzoic acids, 100 [lI of perchloric acid (final concentra-tion, 0.7%) was added before extraction. After extraction,the ether phase was removed and dehydrated with anhy-drous sodium sulfate. The solution was passed through a no.1 filter (Whatman, Inc., Clifton, N.J.) and allowed to evap-orate to dryness. GC-mass spectrometry analysis was con-ducted as described previously (4).DNA hybridization. Southern blots (26) were performed by

using a GeneScreen hybridization transfer membrane (Du-pont, NEN Research Products, Boston, Mass.) according tothe recommendations of the manufacturer. Nick translationswere carried out by using a reagent kit (Bethesda ResearchLaboratories, Inc., Gaithersburg, Md.) according to theinstructions of the manufacturer. DNA was labeled with32P-labeled dCTP (800 Ci/mmol; Amersham Corp., ArlingtonHeights, Ill.).

RESULTS

Cloning and expression of the bph genes. The wide-host-range, mobilizable cosmid pMMB34 (13.75 kb) (10) was usedas a vector in the construction of more than 5,500 recombi-nant E. coli strains containing LB400 DNA sequences.Approximately 1,600 of these were tested for 2,3-dihydrox-ybiphenyl dioxygenase. This enzyme is the product of bphCand is responsible for the conversion of 2,3-dihydroxybi-phenyl to a yellow meta-cleavage product (13). Colonies ofrecombinant cells expressing bphC were detected by theiraccumulation of visible quantities of the yellow product afterbeing sprayed with a solution containing 2,3-dihydroxy-biphenyl. Five strains with bphC activity were isolatedand purified. The recombinant plasmids conferring this ac-tivity were designated pGEM400, pGEM410, pGEM420,pGEM430, and pGEM440. Each of the strains containingthese plasmids was found to have additional Bph pathwayactivities (Table 2), which suggested that the genes forPCB/biphenyl catabolism in LB400 are grouped together.

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TABLE 2. PCB-biphenyl-degrading functions encoded byrecombinant plasmids

Activity' in gene:Plasmid

bphA bphB bphC bphD

pGEM400 + + + NTpGEM410 + + + +pGEM420 + + + +pGEM430 - w + wpGEM440 + + + -pMMB34 - - - -

a NT, Not tested; w, weakly positive.

Preliminary data indicated that FM4100 (containingpGEM410) had the highest stability and PCB-degradingability; therefore, this strain was chosen for further study.Resting-cell assays were conducted by using the individualPCB congeners 2,3-dichloro-, 2,5-dichloro-, and 2,2'-dichlo-robiphenyl. After 24-h incubations, the substrates wereconverted to metabolites with GC retention times identicalto those of 2,3-dichloro-, 2,5-dichloro-, and 2-chlorobenzoicacid, respectively. GC-mass spectrometry analysis con-firmed that the product formed from 2,3-dichlorobiphenylwas 2,3-dichlorobenzoic acid. These results demonstratedthat pGEM410 encoded all of the genes required to convertPCBs to chlorobenzoic acids and that these genes wereexpressed in E. coli.

Degradation of 2,5,2',5'-tetrachlorobiphenyl by LB400produced 3,4-dihydroxy-3,4-dihydro-2,5,2',5'-tetrachlorobi-phenyl (3,4-PCB-dihydrodiol) as the major metabolite,which demonstrated that this strain has a 3,4-dioxygenaseactivity (22). Metabolism of 2,5,2',5'-tetrachlorobiphenyl byFM4100 resulted in production of the same metabolite, asshown by comparing GC retention times with those ofpurified 3,4-PCB-dihydrodiol (provided by D. T. Gibson).These results demonstrated the ability of FM4100 to degradePCBs by both the 2,3- and 3,4-dioxygenase pathways.

Hybridization. Southern blotting experiments were used toconfirm that the genes encoding PCB metabolism onpGEM410 were derived from strain LB400. PurifiedpGEM410 DNA labeled with 32P and used as a probehybridized to LB400 genomic fragments corresponding insize to EcoRI-digested pGEM410 DNA and two end frag-ments (Fig. 2). No hybridization of the vector to LB400DNA was detected (data not shown).

Subcloning of the bph genes. Digestion of pGEM410 DNAwith restriction enzyme EcoRI produces nine fragmentswhose sizes and relative order are 15.7 (13.7-kb vector and2.0-kb insert), 6.1, 2.9, 0.8, 2.0, 2.9, 6.7, 2.3, and 0.5 kb. Theproducts of a partial EcoRI digest of pGEM410 were sub-cloned into pUC18, and the region associated with PCBmetabolism was identified (Fig. 3).

Expression of the 2,3- and 3,4-PCB dioxygenase activitiesrequired both the adjacent 2.9- and 6.7-kb EcoRI fragmentsfrom pGEM410. Plasmids lacking either of these fragments(pGEM453, -454, and -455) were unable to mediate thedegradation of any congener in mix 1B or 2B. This resultsuggests that either a structural or regulatory function re-quired for dioxygenase activity is located near the junctionof the 2.9- and 6.7-kb EcoRI fragments. The 6.7-kb fragmentwas found to encode both bphB and bphC. Further subclon-ing of the 6.7-kb fragment with PstI produced pGEM411,which contains a 2.0-kb PstI fragment encoding bphB and-C. As expected, strains containing plasmids with both the2.9- and 6.7-kb fragments (e.g., pGEM456) expressed bphA,

FIG. 2. Autoradiogram of Southern blot showing hybridizationof pGEM410 DNA probe to genomic LB400 DNA. Lanes: A,pGEM410; B, E. coli C600; C, Pseudomonas strain LB400.

-B, and -C and accumulated meta-cleavage products whenincubated with biphenyl or a variety of chlorinated biphe-nyls. No chlorobenzoic acid metabolites were formed bythese strains, which indicated a lack of bphD. Strains withplasmids containing only the 2.3- and 0.5-kb EcoRI frag-ments ofpGEM410 (e.g., pGEM457) expressed bphD (meta-cleavage product hydrase). Both fragments are required forexpression, since elimination of either resulted in loss ofbphb activity (e.g., pGEM451, -452, and -458) (Fig. 3).Restriction endonuclease mapping indicated that at least 2.0kb of DNA separates bphD from the PstI fragment encodingbphB and -C.PCB degradation. The PCB-degrading abilities of recom-

binant strains FM4110 and FM4560 were compared with thatof LB400. FM4560 consists of E. coli TB1 containing plas-mid pGEM456. This plasmid is a derivative of pGEM410 inwhich the 2.9- and 6.7-kb EcoRI fragments (encoding bphA,-B, and -C) have been cloned onto vector pUC18 (Fig. 3).The restriction endonuclease map of pGEM456 is shown inFig. 4. FM4110 was produced by transformation of pGEM410 into E. coli TB1.The ability of FM4110 and FM4560 to degrade PCBs was

examined by using 24-h resting-cell assays with PCB mixes1B and 2B (Table 3). The recombinant strains were grownwith succinate as the carbon and energy source since theyare unable to grow on biphenyl. Unlike LB400, FM4110 wasunable to totally deplete each of the tetrachlorinated biphe-nyls tested and was almost totally inactive against the

A B C

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CLONING OF GENES ENCODING PCB DEGRADATION

pGEM410

2.9E E

6.7

...bphA.. bphBIC

2.3 .5E EEI Il I

... bphD ...

E EE E

E E

E

E

E

EJ1

E E

p p

E E

E E

ENZYME ACTIVITYbphA bphB bphC bphD

± + + +

+ + + +

+ + + _

+ + + -

- + + +

+ + _

+ + _

_~~ +

E EpGEM452 _ - - - -

E EpGEM451 i _ _ _

FIG. 3. Localization the bph genes on pGEM410. Top line shows arrangement of the EcoRI fragments of pGEM410 containing bph genesas determined by subclone analysis. Subclones were produced by using pUC18 as vector and introduced into E. coli TB1. The regions ofpGEM410 contained in the subclones are shown along with the presence (+) or absence (-) of enzymatic activities of the bph pathway. Therelative bphB-bphC gene order has not yet been established. Thick lines represent LB400 DNA; thin lines represent pMMB34 regions.Restriction site designations: E, EcoRI; P, PstI.

pentachlorobiphenyls or those PCBs containing chlorineatoms at both para ring positions.PCB degradation by FM4560 was found to be much

greater than that by FM4110 for the congeners in the two

FIG. 4. Partial restriction map of pGEM456 indicating the loca-tion of bphA, -B, and -C. The relative order of bphB and bphC hasnot been established. Thick line represents LB400 DNA; thin linerepresents pUC18. Arrow shows direction of transcription for theampicillin resistance gene (AMP). Arrowhead indicates the locationand direction of transcription for the lac promoter. Restriction sitedesignations: C, ClaI; E, EcoRI; Ev, EcoRV; H, HindIll; N, NdeI;P, PstI; Pv, PvuII; S, SstI; Sc, ScaI; X, XhoI; Xm, Xmnl.

TABLE 3. Degradation of PCB congeners in mixtures 1B and 2Bby FM4110, FM4560, and LB400

% Depletion'

Congener LB400bFM4110 FM4560

+Bp -Bp

2,3 100 100 100 1002,2' 100 100 100 1002,4' 100 100 100 1002,5,2' 100 100 100 1002,5,4' 100 100 100 682,3,2',3' 44 100 100 892,3,2',5' 51 100 100 842,5,3',4' 44 100 100 612,5,2',5' 66 100 100 952,4,5,2',5' 16 87 100 472,3,4,2',5' 0 45 100 62,4,5,2',3' 0 40 75 04,4' 0 28 47 02,4,4' 0 68 86 02,4,3',4' 0 15 59 02,4,2',4' 0 22 94 03,4,3',4' 0 3 21 02,4,5,2',4',5' 0 11 40 02,4,6,2',4" 0 0 0 0

" Combined data from resting-cell assays on PCB mixtures 1B and 2Bperformed individually.

b +Bp, Biphenyl used as the only carbon source; -Bp, succinate used asthe only carbon source.

Nondegradable internal standard used in both mixtures.

1kblHl

pGEM459

pGEM458

pGEM456

pGEM455

pGEM454

pGEM411

pGEM453

pGEM457

bL-

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Aroclor 1242, FM4560 was able to degrade 56% of the PCBs,compared with 68% for LB400.

Control

FM4560

LB400

FIG. 5. Biodegradation of Aroclor 1242 by E. coli FM4560 andPseudomonas strain LB400. (Top) Aroclor 1242 incubated withmercury-killed cells; (middle and bottom) Aroclor 1242 (10 ppm)incubated at 30°C for 24 h with cells (optical density at 615 nm of 1.0)of FM4560 and LB400, respectively. FM4560 was grown on succi-nate; LB400 was grown on biphenyl.

mixtures. FM4560 demonstrated significant degradative ac-tivity against a wide variety of tetra-, penta-, and double-para-substituted PCBs. Although the degradative ability ofthis strain was not as great as that of LB400, its substraterange and the extent to which congeners were depleted inthese assays were very similar. In control experiments, PCBdegradation by the host strain TB1 was no greater than thatof mercury-killed cells (<1%).The PCB-degrading ability of FM4560 was superior to that

of LB400 when both organisms were grown with succinateas the carbon source. Under these conditions, PCB metab-olism by LB400 was significantly reduced for tetra- andpentachlorobiphenyls, and the organism was unable to de-grade congeners with chlorines at both para ring positions(Table 3). This reduction in PCB-degrading ability occursunless LB400 is grown with biphenyl as the sole source ofcarbon and energy and is not overcome by the addition ofbiphenyl to media containing other carbon sources (L. H.Bopp, personal communication).Degradation of Aroclor 1242. The vast majority of environ-

mental PCB contamination involves the commercial mix-tures known as Aroclors. FM4560 was tested for ability todegrade Aroclor 1242 in resting-cell assays. The patterns ofdepletion produced by FM4560 and LB400 were nearlyidentical (Fig. 5), which further demonstrated the ability ofFM4560 to degrade the same wide range of PCB congenersas LB400. After a 24-h incubation with Aroclor 1242 at 10ppm (10 jxg/ml), FM4560 and LB400 showed comparablePCB depletion (85 and 91%, respectively). At 50 ppm of

DISCUSSION

Pseudomonas strain LB400 is able to catabolize a widervariety of PCB congeners than any known bacterial strainexcept A. eutrophus H850 (4, 5, 8, 22). The superior abilityof these organisms to degrade highly chlorinated PCBs couldresult from their possession of a 3,4-dioxygenase activity inaddition to a 2,3-dioxygenase pathway found in other organ-isms (4, 22).

This report describes the cloning of the genes responsiblefor PCB biodegradation in strain LB400 and their expressionin E. coli. The genes encoding PCB metabolism by the2,3-dioxygenase pathway (bphABCD) and for 3,4-dioxy-genase activity were found to be encoded within a 12.4-kbregion of LB400 DNA. Furukawa and Miyazaki have previ-ously described the cloning of bphABC from Pseudomonaspseudoalcaligenes KF707, which degrades several mono-,di-, and trichlorinated biphenyls via a 2,3-dioxygenase path-way (13). As in KF707, the PCB-degrading genes of LB400are clustered, presumably on a fragment of chromosomalorigin. No plasmids have been detected in LB400 despiterepeated attempts using a wide range of protocols (unpub-lished data). The clustering of genes for aromatic hydrocar-bon metabolism is not unusual and has been previouslydemonstrated for compounds such as toluene, naphthalene,and benzene (9, 17, 24, 29). An examination of the PCB-degrading ability of E. coli FM4110 (containing recombinantplasmid pGEM410) demonstrated its ability to degrade di-,tri-, tetra-, and pentachlorinated biphenyls. Especially inter-esting is the degradation of 2,5,2',5'-tetrachlorobiphenyl, aPCB in which all 2,3 positions are chlorinated. The initialstep in the degradation of this congener by LB400 is a3,4-dioxygenase attack resulting in the formation of 3,4-PCB-dihydrodiol (22). The production of this metabolite byFM4100 and FM4560 demonstrates that at least one gene hasbeen cloned from what may be an alternate pathway ofPCBmetabolism. It has yet to be determined whether the 2,3- and3,4-dioxygenase activities result from a single enzyme; how-ever, attempts to separate these activities by using cellextracts (21), mutagenesis, and subcloning have thus farbeen unsuccessful (data not shown).The PCB-degrading ability of strain FM4560 (containing

pGEM456) was found to be significantly better than that ofFM4110 (containing pGEM410). It is possible that thisimprovement is the result of copy number differences be-tween the cloning vectors used in the two constructs.Plasmids derived from the RSF1010 replicon (such aspGEM410) are usually maintained at 15 to 20 copies per cell,whereas those from pUC vectors (such as pGEM456) may bepresent at 50 to 60 copies per cell (2, 7). A greater number ofbph genes in a strain may result in increased enzyme levelsand, thus, improved PCB-degrading ability. A similar effectwould be observed if expression of the bph genes onpGEM456 were under the control of the efficient lac pro-moter in the pUC vector (3). If this is the case, thenplacement of pGEM456 into a lacIq strain should result in arepression of PCB degradation which would be reversible bythe addition of IPTG. The addition of IPTG to cultures ofFM4560 (a non-laclq strain) did not increase PCB metabo-lism (data not shown).The degradative competence of FM4560 was found to be

similar to that of LB400 both in congener specificity and inthe extent of degradation for PCB mix 1B, mix 2B, and

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CLONING OF GENES ENCODING PCB DEGRADATION

Aroclor 1242. The results with Aroclor 1242 were especiallyencouraging since this compound contains some 60 differentPCB congeners and is a widespread environmental contam-inant.Bedard et al. (4) have examined the PCB-metabolizing

ability of 25 environmental isolates obtained from contam-inated sites. As measured by resting-cell assays against PCBmixes 1B and 2B, the degradative activity of FM4560 isgreater than for all of the natural isolates except LB400 andA. eutrophus H850. Although the PCB competence ofFM4560 is nearly identical to that of LB400 and H850, thelatter organisms are better able to degrade PCBs havingchlorines at both para positions. This may be due to theinability of FM4560 to be grown on biphenyl, a conditionthat is required for optimal PCB degradation by LB400 andH850. Growth of LB400 on medium that does not containbiphenyl as the only carbon source severely decreases thecompetence of the strain, especially for double-para-substi-tuted PCBs. Since FM4110 and FM4560 must be grown onsubstrates other than biphenyl, it is possible that the degra-dative abilities of these strains are even greater than havebeen demonstrated. If this is the case, then the introductionof a pathway for benzoic acid metabolism might enableFM4110 to grow on biphenyl and thereby increase its abilityto degrade PCBs. For FM4110, FM4560, and LB400, theaddition of biphenyl to growth medium containing succinatedid not increase the activity of the organisms (unpublisheddata).When both organisms were grown with succinate as a

carbon source, FM4560 was found to be significantly supe-rior to LB400 in ability to degrade a wide variety of the PCBcongeners in mixes 1B and 2B. Under these same condi-tions, FM4560 should also be superior to LB400 in degrada-tion of Aroclor 1242, as all but two of the congeners presentin mixes 1B and 2B are in this Aroclor. Thus, for situationsin which it would be impractical to grow an organism onbiphenyl or in which other carbon sources would be present,a strain such as FM4560 might be more effective than LB400for bioremediation.DNA-DNA hybridization studies indicate that the similar-

ities in PCB metabolism between LB400 and H850 reflecttheir possession of closely related bph clusters which are notfound in strains of lesser degradative capability (28). Plas-mids containing this genetically distinct class of bph clustercan therefore be used as DNA probes to distinguish strainscontaining the LB400/H850-type genes for PCB degradationfrom those with other types (28). This ability should facilitate(i) quantitation and isolation of other highly competentstrains from environmental samples and (ii) determination ofthe fate of recombinant strains or plasmids containing theLB400 or H850 bph genes in the environment. The genesencoding PCB metabolism from H850 have been cloned (F.Mondello, manuscript in preparation), and studies are cur-rently under way to further examine their relationship tothose of LB400.

Isolation of the bph genes of LB400 is an important steptoward understanding the ability of this strain to degradehighly chlorinated PCBs and developing organisms witheven greater degradative competence for use in bioremedi-ation processes.

ACKNOWLEDGMENTSThis work was supported by grant CR812727 from the U.S.

Environmental Protection Agency, Office of Research and Devel-opment, Hazardous Waste Engineering Research Laboratory, Cin-cinnati, Ohio.

I thank P. R. Sferra, U.S. Environmental Protection Agencyproject officer, for his interest, support, and suggestions. Thanks arealso given to Herman Finkbeiner, Donna Bedard, and James R.Yates for reviews of the manuscript; David T. Gibson for providing3,4-PCB-dihydrodiol; James R. Yates, Larry Bopp, and Ron Unter-man for useful suggestions and assistance; Gary Yeager and Kath-ryn Longley for 2,3-dihydroxybiphenyl; Ralph May for GC-MSanalysis; and K. N. Timmis for pMMB34.

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