Vol. 173, No. 15JOURNAL OF BACTERIOLOGY, Aug. 1991, p. 4587-45940021-9193/91/154587-08$02.00/0Copyright © 1991, American Society for Microbiology

Genetic Organization and Regulation of a meta Cleavage Pathwayfor Catechols Produced from Catabolism of Toluene, Benzene,

Phenol, and Cresols by Pseudomonas pickettii PKO1JEROME J. KUKOR AND RONALD H. OLSEN*

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0620

Received 26 November 1990/Accepted 20 May 1991

Plasmid pRO1957 contains a 26.5-kb BamHI restriction endonuclease-cleaved DNA fragment cloned fromthe chromosome ofPseudomonas pickettu PKO1 that allows P. aeruginosa PAQlc to grow on toluene, benzene,phenol, or m-cresol as the sole carbon source. The genes encoding enzymes for meta cleavage of catechol or3-methylcatechol, derived from catabolism of these substrates, were subcloned from pRO1957 and were shownto be organized into a single operon with the promoter proximal to tbuE. Deletion and analysis of subclonesdemonstrated that the order of genes in the meta cleavage operon was tbuEFGKIHJ, which encoded catechol2,3-dioxygenase, 2-hydroxymuconate semialdehyde hydrolase, 2-hydroxymuconate semialdehyde dehydroge-nase, 4-hydroxy-2-oxovalerate aldolase, 4-oxalocrotonate decarboxylase, 4-oxalocrotonate isomerase, and2-hydroxypent-2,4-dienoate hydratase, respectively. The regulatory gene for the tbuEFGKIHJ operon,designated tbuS, was subcloned into vector plasmid pRO2317 from pRO1957 as a 1.3-kb PstI fragment,designated pRO2345. When thuS was not present, meta pathway enzyme expression was partially derepressed,but these activity levels could not be fully induced. However, when tbuS was present in trans with tbuEFGKIHJ,meta pathway enzymes were repressed in the absence of an effector and were fully induced when an effector waspresent. This behavior suggests that the gene product of tbuS acts as both a repressor and an activator. Phenoland m-cresol were inducers of meta pathway enzymatic activity. Catechol, 3-methylcatechol, 4-methylcatechol,o-cresol, and p-cresol were not inducers but could be metabolized by cells previously induced by phenol orm-cresol.

We have recently reported a novel pathway for catabolismof toluene and benzene (15) in which a monooxygenaseinitially hydroxylates toluene to m-cresol or benzene tophenol. The genes encoding this novel catabolic pathwaywere cloned in Pseudomonas aeruginosa PAO1c as a26.5-kb BamHI restriction endonuclease DNA fragment,designated pRO1957, from the chromosome of Pseudomo-nas pickettii PKO1. We previously reported (17) on thephenol/cresol hydroxylase component of this pathway. Inthis report, we describe the organization of the genes encod-ing enzymes for meta cleavage of catechol or methylcate-chols (Fig. 1) produced from hydroxylation of phenol orcresols. We also provide evidence that regulation of themeta cleavage operon is controlled by a repressor/activatormechanism.

MATERIALS AND METHODS

Bacterial strains and plasmids. The strains and plasmidsused are described in Table 1. For most of the experimentsdescribed, P. aeruginosa PAO1.93, a catA mutant, wasused. Use of this mutant allowed for analysis of P. pickettiiPKO1 cloned catechol catabolic genes in the heterogeneticbackground of P. aeruginosa without confounding effectsfrom the chromosomally encoded catechol catabolic path-way that occurs in P. aeruginosa PAO1c.Media and growth conditions. Bacteria were routinely

cultured on either plate count complex medium (TNA; 23) oron the minimal medium of Vogel and Bonner (VBG; 30)supplemented with Casamino Acids (Difco Laboratories,

* Corresponding author.

Detroit, Mich.) to a final concentration of 0.3%. Selection ortesting for the ability to utilize aromatic substrates was doneby using a minimal basal salts medium (MMO) describedpreviously for this purpose (28). Tetracycline, carbenicilHin,trimethoprim, streptomycin, or gentamicin was used inselective media at 50, 500, 600, 250, or 5 ,ug/ml, respectively.When grown for enzyme assays, bacteria were cultured withaeration at 37°C in 100 ml of MMO medium supplementedwith Casamino Acids to a final concentration of 0.3% andwith aromatic carbon substrates, where appropriate, to afinal concentration of 0.05%. Catechol or methylcatechols,when supplied as inducing substrates, were added to liquidmedia to a final concentration of 0.02%.

Genetic techniques. Isolation of plasmid DNA, restrictionendonuclease or exonuclease digestion, molecular cloning,and transformations were done as described previously (22).Measurement of enzyme activity. Cells were grown for

enzyme assays to a density that gave an apparent A425 of 1.0to 1.5 (Spectronic 21 spectrophotometer; Bausch & Lomb,Inc., Rochester, N.Y.) and were harvested by centrifugationat 10,000 x g for 15 min. The cell pellets were washed twicein 50 mM sodium phosphate buffer (pH 7.6) and then weredisrupted sonically by four 15-s bursts with a Braun-Sonic2000 apparatus. Cellular debris was removed by centrifuga-tion at 100,000 x g at 5°C for 1 h, and the clear supernatantsolution was used immediately for enzyme assays.Phenol hydroxylase (PHH) activity was assayed by mea-

suring the decrease in A340, using NADPH as the cosub-strate, according to previously described procedures (17).Catechol 2,3-dioxygenase (C230), 2-hydroxymuconate

semialdehyde hydrolase (HMSH), and 2-hydroxymuconatesemialdehyde dehydrogenase (HMSD) activities were as-sayed by using previously described procedures (1). The

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RIR2

C230 (tbUE) 02

HMSD (tbuG) /

R2 COO- HMS

401 (tbuH) I

R2 CO-

> 0

40D (tbu

Co2 R Cl°

t ~,OH

,H (tbuF)

RICOOH

| OEH (tbuJ)

HO<O

HOA (tbuK)

R2CH2CHO + CH3C-OCOOH

FIG. 1. Pathway for catabolism of catechol (R1, R2 = H), 3-me-thylcatechol (R1 = CH3, R2 = H), and 4-methylcatechol (R1 = H,R2 = CH3) in P. pickettii PK01. The gene designation is shown inparentheses for each enzyme.

substrate for HMSH and HMSD assays, 2-hydroxymu-conate semialdehyde, was prepared enzymatically fromcatechol, using an extract of P. aeruginosa PA04032(pR01940), which constitutively produces the C230 en-coded by the xylE gene of the TOL plasmid pWWO (31). Theproduction of formate in the HMSH assay was detected byits reduction to formaldehyde in the presence of magnesium,as described by Grant (9).

4-Oxalocrotonate isomerase (401) was determined bymeasuring the initial rate of decrease in A295, using a freshlyprepared aqueous solution of 4-oxalocrotonate as describedpreviously (25) except that 50 mM sodium phosphate buffer(pH 7.6) was used as the reaction buffer. 4-Oxalocrotonatewas prepared by the method of Lapworth (19) from thepotassium salt of diethyl 2,4-hexadiene-5-hydroxy-1,6-dio-ate, which was obtained from condensation of diethyl ox-alate and ethyl crotonate in the presence of potassium metalin toluene, as described by Wiley and Hart (34). Theconcentration of the enol and keto forms of 4-oxalocrotonatein aqueous solution was calculated from extinction coeffi-cients of the two forms in equilibrium state, using theequations of Harayama et al. (11). 4-Oxalocrotonate decar-boxylase (40D) was assayed by determining the rate ofdecrease in A235 caused by the disappearance of the ketoform of 4-oxalocrotonate (1).

2-Hydroxypent-2,4-dienoate hydratase (OEH) was as-

sayed by using previously published procedures (1) except

that the reaction buffer was 50 mM sodium phosphate (pH7.0). The substrate for the hydratase assay was preparedenzymatically from DL-allylglycine according to the methodof Collinsworth et al. (4).

4-Hydroxy-2-oxovalerate aldolase (HOA) activity was de-termined indirectly from pyruvate production by measuringthe rate of oxidation of NADH in the presence of excesslactate dehydrogenase (25). The substrate for the HOA assaywas prepared by mild alkaline hydrolysis of 4-methyl-2-oxobutyrolactone as described by Dagley and Gibson (7).The lactone was synthesized by the method of Rossi andSchinz (24).

Protein was determined by the method of Bradford (3),with bovine serum albumin as the standard. Enzyme specificactivities are reported as nanomoles of substrate utilized orproduct produced per minute per milligram of protein. UVabsorbance spectra were measured on a Shimadzu UV-160spectrophotometer.Chemicals and reagents. All chemicals, enzymes, and

reagents used were of the highest purity commercially avail-able. Catechol, phenol, cresols, 4-methylcatechol, formalde-hyde, ethyl crotonate, and diethyl oxalate were obtainedfrom Aldrich Chemical Co., Inc. (Milwaukee, Wis.). 3-Me-thylcatechol was purchased from Pfaltz and Bauer, Inc.(Waterbury, Conn.). Formic acid was obtained from Merck& Co., Inc. (Rahway, N.J.). Pyruvate, lactate dehydroge-nase, DL-allylglycine, and L-amino acid oxidase were pur-chased from Sigma Chemical Co. (St. Louis, Mo.).Enzymes and reagents used for DNA manipulations were

purchased from Bethesda Research Laboratories, Inc.(Gaithersburg, Md.) or Boehringer Mannheim Biochemicals(Indianapolis, Ind.) and were used as suggested by thesupplier. Tetracycline and trimethoprim were obtained fromSigma. Streptomycin and disodium carbenicillin were ob-tained from Pfizer, Inc. (New York, N.Y.), and gentamicinwas from Elkins-Sinn, Inc. (Cherry Hill, N.J.). Vitamin-freeCasamino Acids, as well as all other bacteriological mediumcomponents, were purchased from Difco.

RESULTS

Identification and subcloning of meta pathway genes frompRO1957. We have previously shown (17) that pRO1957(Fig. 2), which contains a 26.5-kb BamHI DNA fragmentcloned from the chromosome of P. pickettii PKO1, allowedcells of P. aeruginosa PAO1c to grow on phenol or m-cresolas a sole carbon source. We also observed that cells of P.aeruginosa PA01.93, a catA mutant, carrying pR01957were able to grow on phenol or m-cresol minimal mediumand that such cells had enzymes for meta cleavage ofcatechol. In contrast, PA01.93 carrying pR01959, a Hindllldeletant of pRO1957 (Fig. 2), was unable to grow on phenolor m-cresol minimal medium, and enzymes for meta cleav-age of catechol were not detectable. These results indicatedthat the genes encoding meta pathway enzymes were locatedon the 11.4-kb BamHI-HindIII fragment (map coordinates 0to 11.4 kb; Fig. 2) of pRO1957. This fragment was subclonedinto vector plasmid pRO1727 and was designated pRO1996.Plasmid pRO1996 was mapped with restriction endonu-cleases, and the results are shown in Fig. 3.

Localization and regulation of meta pathway enzyme expres-sion. We have recently shown that pRO1957 allows P.aeruginosa to utilize toluene or benzene as the sole carbonsource (15) and that the PHH and meta pathway are integralcomponents in catabolism of these substrates. Therefore, forconsistency in nomenclature, we have renamed the PHH

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TABLE 1. Bacterial strains and plasmids

Strain or plasmid Relevant marker(s)a Reference or derivation

P. aeruginosa strainsPAO1c Prototroph 12PAO1.93 catA 18PA04032 catA met-9020 nar-9011 mtu-9002 tyu-9030 Matsumotob

PlasmidspRO1727 Cb' Tcr, cloning vector 5pRO1769 Smr Gmr, cloning vector 6pRO2317 Cbr Tcr, cloning vector 35pRO2321 Tpr Tcr, cloning vector 35pRO1940 Cbr xylS xyIE 31pRO1957 Cbr Phl+ 17pRO1959 Cbr Phl+ 17pRO1984 Cbr SphI deletion of pRO1996pRO1985 Cbr SstI deletion of pRO1996pRO1987 Cbr Sall deletion of pRO1996pRO1988 Cbr EcoRV deletion of pRO1996pRO1989 Cbr BstEII deletion of pRO1996pRO1990 Cbr ApaI deletion of pRO1996pRO1991 Cbr XhoI deletion of pRO1957pRO1992 Cbr HindIll cleaved pRO1996, digested with Bal31 and religatedpRO1993 Cbr Partial XhoI deletion of pRO1957pRO1994 Cbr XhoI deletion of pRO1996pRO1995 Cbr BglII deletion of pRO1996pRO1996 Cbr BamHI-HindIII digest of pRO1957, cloned into BamHI-

HindlIl-cleaved pRO1727pRO1998 Cbr HindIII-NotI digest of pRO1996, cloned into HindIII-

XmaIII-cleaved pRO1727pRO2345 Tcr PstI digest of pRO2346, cloned into the PstI site of

pRO2317pRO2346 Tpr EcoRI digest of pRO1959, cloned into the EcoRI site of

pRO2321pRO2347 Tpr HindIII deletion of pRO2346pRO2348 Tpr SstI deletion of pRO1957, subsequently digested with

BamHI and cloned into the BamHI site of pRO2321a Abbreviations: Cbr, Tcr, Smr, Gmr, and Tpr, resistance to carbenicillin, tetracycline, streptomycin, gentamicin, and trimethoprim, respectively.b Matsumoto collection, received from P. Phibbs.

structural gene, previously designated phiA, as tbuD and itsregulatory gene, previously designated phiR, as tbuR. Thenew gene designation, tbu, denotes toluene and benzeneutilization.

Regulation of meta pathway enzyme expression is inde-pendent of that reported previously (17) for expression ofPHH encoded by tbuD. meta pathway enzyme expressionwas inducible by phenol, but not by catechols, in cells of P.aeruginosa PA01.93 carrying pRO1957. Growth in the pres-ence of phenol resulted in elevated activity levels for each ofthe meta pathway enzymes as well as for PHH (Table 2).However, cells carrying either pRO1996 or pRO1993, whichis a partial XhoI deletant of pRO1957 (Fig. 2), did not exhibitregulated expression of meta pathway enzyme synthesis(Table 2). Such constructs showed partial elevation of metapathway enzymatic activity, but these activity levels couldnot be fully induced. This behavior is similar to that reportedpreviously by us for regulation of tfdB-encoded 2,4-dichlo-rophenol hydroxylase expression by its cognate regulatorygene, tfdS (16).We had previously shown (17) that a regulatory locus,

designated phiR (now designated tbuR), that controlsexpression of the phenol hydroxylase structural gene, tbuD,was present on pRO1957, and this regulatory gene wasfurther localized when subcloned as an EcoRI fragment onplasmid pRO2346 (Fig. 2). When pRO2346 was present intrans together with either pRO1996 or pRO1993, meta path-

way enzyme synthesis was regulated (Table 2). To determinewhether it was tbuR that was also regulating expression ofthe meta pathway genes, a HindlIl deletant of pRO2346 wasmade. This deletant, designated pRO2347 (Fig. 2), stillregulated expression of the meta pathway when present intrans with either pRO1996 or pRO1993 (Table 2), but nowPHH activity was not detected when pRO2347 was presentin trans with pRO1993 (Table 2). Furthermore, a 1.3-kb PstIsubclone of pRO2347, designated pRO2345 (Fig. 2), alsoregulated meta pathway gene expression but did not regulatethe PHH gene when present in trans with pRO1993 (Table2). These results suggest that genes encoding meta pathwayenzymes are regulated by a locus different from that whichcontrols transcription of the gene encoding PHH. We havedesignated the regulatory locus that controls expression ofthe meta pathway as tbuS (Fig. 2).

Organization and localization of meta pathway genes. De-letion subcloning was used to determine the organization ofthe meta pathway structural genes on pRO1996 (Fig. 3).Plasmid pRO1985 was made by SstI deletion of pRO1996,and this deletant had only C230 activity. Plasmid pRO1984was made by digestion of pRO1996 with SphI, with subse-quent ligation to the SphI site in vector plasmid pRO1727.This deletant lacked C230 activity; therefore, tbuE, whichencodes C230, could be placed between the SphI site at mapcoordinate 10.7 kb and the SstI site at map coordinate 10.0kb (Fig. 3). Plasmid pRO1990, an ApaI deletant of pRO1996,

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0 2 4 6 8 10 12 14 16 18 20 22 24 26 kbIII I I I I I I I I 1.1 II

BEG x; 9) FI lGl I ?( f F I Elp

tbuE tbuD tbuR IbuS

X X BlI

UQ IM f

B

B H

B X1991 1 1

B1992 1

B S2348 L48.J

H EI l

J J

X BLI .j

S

FIG. 2. Restriction endonuclease map of a P. pickettii PK01chromosomal DNA fragment, and subclones derived therefrom,which allows growth on phenol in P. aeruginosa PA01c. Details ofthe plasmid constructions are provided in the text and in Table 1.Abbreviations: B, BamHI; E, EcoRI; G, BgIII; H, HindIII; J, PstI;S, SstI; X, XhoI.

contained HMSH activity, whereas plasmid pR01985, anSstI deletant, lacked HMSH activity; therefore, tbuF, whichencodes HMSH, could be placed between the SstI site atmap coordinate 10.0 kb and the ApaI site at map coordinate8.5 kb. An XhoI internal deletion made in plasmid pR01996,resulting in plasmid pR01994, contained, in addition to

C230 and HMSH activity, HMSD activity. However,BstEII (pRO1989; Fig. 3) and ApaI (pRO1990; Fig. 3)deletions of pRO1996 lacked HMSD activity. Therefore,tbuG, which encodes HMSD, must span the BstEII andApaI sites at map coordinates 8.5 and 8.6 kb, respectively.The XhoI deletion of pRO1994 inactivated HOA, whereas anEcoRV deletion (pRO1988; Fig. 3) retained HOA activity.Therefore, tbuK, which encodes HOA, could be placed atthe XhoI site at map coordinate 8.1 kb. An internal BglIIdeletion in pRO1996, resulting in pRO1995, contained 40Dactivity, whereas the EcoRV deletant (pRO1988) lacked40D activity. Therefore, tbuI, which encodes 40D, could beplaced between the EcoRV site at map coordinate 8.0 kb andthe BglII site at map coordinate 7.0 kb. Plasmid pRO1987was made by digestion of pRO1996 with SalI with subse-quent ligation to a Sall site in vector plasmid pRO1727.Plasmid pRO1987 contained 40I activity, whereas plasmidpRO1995, a BglII deletant, lacked this activity. Therefore,tbuH, which encodes 401, could be placed at the BglII site atmap coordinate 7.0 kb. Precise placement of tbuJ, the geneencoding OEH, has not yet been accomplished. However,based on data discussed below, it is likely that tbuJ is locatednear the SalI site at map coordinate 6.7 kb. PlasmidpRO1998 (Fig. 3), which was derived by digestion ofpRO1996 with HindIlI and NotI followed by subcloning ofthis fragment into HindIII-XmaIII-cleaved vector plasmidpR01727, expressed all of the meta pathway enzymes.

Operonic arrangement of meta pathway genes. The genes ofthe meta pathway in P. pickettii PKO1 are organized into asingle operon, with the promoter of this operon locatedproximal to tbuE (map coordinate 11.4 kb; Fig. 2 and 3).Evidence for this was obtained from an analysis of plasmidpRO1991, which was derived by digestion of plasmidpR01957 with XhoI (Fig. 2). When cells of P. aeruginosaPAO1.93 carrying both plasmids pRO1991 and pR02345were grown in the presence of phenol, meta pathway enzy-matic activities were not detected (Table 3), even thoughtbuH, which encodes 401, and tbuJ, which encodes OEH,are present on this plasmid (Fig. 3), as described in the

0 1 2 3 4 5 6 7 8 9 10 11 12 kb

DM G L E TB |jV E eS r IITGYIXVI S PHIIIII I I 1 I 1 1 I

1996

1984S

1985 1A

1990 -

1989

1994V

1988 J

1995 3

1987

1998

tbuH SbuK tbuF tbuE

tbuJ tbul tbuGpL- <10 <0.1 <0. I <10 <0.1

SS 252

A240

tL 240x x

V

234

C230 HMH HMSD 44 D.OMEH HQA

230 14 19 581 74 622 21

<10 <0.1

<0.1 <0.1 <i0 <0.1 <10 <0.1

14 <0.1 <10 <0.1 <10 <0.1

14 <0.1 <10 <0.1 <10 <0.1

15 19 <10 <0.1 <10 <0.1

245 14 20 <10 <0.1 <10 21

L1 244 15 19 569 73 <10 22

253 15 19 569 74 583 22N

230 14 20 <10 77 <10 20

FIG. 3. Restriction endonuclease map of plasmid pR01996, and subclones derived from it, with the corresponding meta pathwayenzymatic activities for each plasmid. Enzymatic activities are given as milliunits (as defined in the text) per milligram of protein. Each valuerepresents the mean of at least three independent determinations. Cells of P. aeruginosa PA01.93 carrying each of the indicated plasmids (aswell as pR02345 in trans) were grown in 0.3% Casamino Acids plus 0.05% phenol. Abbreviations: A, ApaI; B, BamHI; E, EcoRI; G, BglII;H, HindIll; L, SalI; M, XmaIII; N, NotI; P, SphI; S, SstI; T, BstEII; V, EcoRV; X, XhoI.

1957

1959

1993

2346

2347

2345

1996

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TABLE 2. Regulation of PHH and meta pathway enzyme expression for cells of P. aeruginosa PAO1.93carrying pRO1957 or its subclones

Enzyme activityaPlasmid(s) Inducer

PHH C230 HMSH HMSD 401 40D OEH HOA

pRO1957 None <10 <10 <0.1 <0.1 <10 <0.1 <10 <0.1Phenol 180 230 14 19 581 74 622 21

pRO1996 None ND 44 3 4 98 10 114 3Phenol ND 45 3 4 101 9 114 3

pRO1993 None <10 44 3 4 107 20 105 4Phenol <10 45 3 4 99 21 107 4

pRO1996, pRO2346 None ND <10 <0.1 <0.1 <10 <0.1 <10 <0.1Phenol ND 247 15 21 561 72 618 21

pRO1993, pRO2346 None <10 <10 <0.1 <0.1 <10 <0.1 <10 <0.1Phenol 172 254 15 19 596 76 631 21

pRO1996, pRO2347 None ND <10 <0.1 <0.1 <10 <0.1 <10 <0.1Phenol ND 245 15 20 563 74 601 21

pRO1993, pRO2347 None <10 <10 <0.1 <0.1 <10 <0.1 <10 <0.1Phenol <10 234 15 20 604 78 603 20

pRO1996, pRO2345 None ND <10 <0.1 <0.1 <10 <0.1 <10 <0.1Phenol ND 250 14 20 567 72 601 20

pRO1993, pRO2345 None <10 <10 <0.1 <0.1 <10 <0.1 <10 <0.1Phenol <10 245 15 19 551 72 595 20

a Values are milliunits of enzyme activity per milligram of protein. Each value represents the mean of at least three independent determinations. Cells weregrown in 0.3% Casamin-o Acids plus 0.05% phenol, where indicated. Activities for cells carrying either pR02346, pRO2347, or pRO2345 singly were below thedetection limits for each enzyme assayed. ND, not determined.

foregoing section. To further localize the promoter region ofthe meta pathway operon, we cleaved plasmid pRO1996with Hindlll and digested this cleaved DNA with exonucle-ase Bal31 to yield plasmid pRO1992 (Fig. 2). Restrictiondigest mapping of pRO1992 demonstrated that the exonucle-ase digestion had removed approximately 200 bp of DNA,abolishing the HindIII site at map coordinate 11.4 kb butretaining the SphI site at map coordinate 10.7 kb (Fig. 3).When cells of P. aeruginosa PA01.93 carrying plasmidspR01992 (the deletant) and pRO2345 (tbuS+) were grown inthe presence of phenol, no meta pathway enzymatic activi-ties were detected (Table 3); therefore, the operon promoterhad been deleted.

Additional evidence for the operonic organization of themeta pathway was obtained from analyses using plasmidpRO2348. Plasmid pRO2348 was made by SstI digestion ofplasmid pRO1957 followed by transfer of this deletant intovector plasmid pRO2321 (Fig. 2). Phenol-grown cells of P.aeruginosa PA01.93(pRO2348) had C230 activity, but noneof the lower meta pathway enzymes were detected (Table 3).When cells ofPA01.93 carrying both plasmids pRO1992 andpRO2348 were grown in the presence of phenol, C230activity was present (Table 3); furthermore, such cultures

TABLE 3. meta pathway enzyme activity for cells ofP. aeruginosa PAO1.93 carrying the indicated plasmidsand grown on 0.3% Casamino Acids plus 0.05% phenol

Enzyme activityaPlasmid

C230 HMSH HMSD 401 40D OEH HOA

pRO1991, pRO2345 <10 <0.1 <0.1 <10 <0.1 <10 <0.1pRO1992, pRO2345 <10 <0.1 <0.1 <10 <0.1 <10 <0.1pRO1992, pR02348 246 <0.1 <0.1 <10 <0.1 <10 <0.1pR02348 234 <0.1 <0.1 <10 <0.1 <10 <0.1

a Values are milliunits of enzyme activity per milligram of protein. Eachvalue represents the mean of at least three independent determinations.Activity levels for cells carrying either pR01991, pRO1992, or pRO2345 singlywere below detection limits for each of the enzymes assayed.

turned bright yellow as a result of accumulation of 2-hy-droxymuconate semialdehyde; however, none of the lowermeta pathway enzymatic activities were present.

Inducers and substrates of the meta pathway. Growth of P.aeruginosa PA01.93 carrying both pRO1996 and pRO2345in the presence of phenol or m-cresol resulted in induction ofall of the meta pathway enzymes (Table 4). However,growth of cells in the presence of o-cresol, p-cresol, cate-chol, or methylcatechols did not induce meta pathwayenzymatic activity.We had previously shown (17) that the PHH encoded by

tbuD would hydroxylate phenol to catechol, o-cresol andm-cresol to 3-methylcatechol, or p-cresol to 4-methy1cate-chol. To determine whether these catechols were accommo-dated by the meta pathway cloned from P. pickettii PK01,cells of P. aeruginosa PA01.93 carrying pRO1985, whichhas C230 but lacks the lower meta pathway enzymes (Fig.3), were assayed for their ability to convert catechol ormethylcatechols following prior growth in the presence ofeither phenol or m-cresol. The results presented in Table 5indicate that the C230 encoded by tbuE could utilize cate-

TABLE 4. Inducers of meta pathway enzymes in cells ofP. aeruginosa PAO1.93 carrying both pRO1996 and pRO2345

Enzyme activityaInducer

C230 HMSH HMSD 40I 40D OEH HOA

Phenol 249 14 20 579 73 618 21m-Cresol 230 14 20 570 75 620 20o-Cresol <10 <0.1 <0.1 <10 <0.1 <10 <0.1p-Cresol <10 <0.1 <0.1 <10 <0.1 <10 <0.1Catechol <10 <0.1 <0.1 <10 <0.1 <10 <0.13-Methylcatechol <10 <0.1 <0.1 <10 <0.1 <10 <0.14-Methylcatechol <10 <0.1 <0.1 <10 <0.1 <10 <0.1

a Values are milliunits of enzyme activity per milligram of protein. Eachvalue represents the mean of at least three independent determinations. Cellswere grown in 0.3% Casamino Acids plus 0.05% phenol or cresols or in 0.02%catechols.

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TABLE 5. C230 activity for cells of P. aeruginosaPA01.93(pRO1985) grown on 0.3% Casamino

Acids plus 0.05% phenol or m-cresol

Inducer Assay substrate C230 activitya

Phenol Catechol 2543-Methylcatechol 2454-Methylcatechol 248

m-Cresol Catechol 2443-Methylcatechol 2354-Methylcatechol 240

a Values are milliunits of enzyme activity per milligram of protein. Eachvalue represents the mean of at least three independent determinations.

chol, 3-methylcatechol, or 4-methylcatechol as a substrate.Furthermore, the muconate semialdehyde and methylmu-conate semialdehydes produced by action of C230 on cate-chol or methylcatechols were further metabolized by down-stream enzymes, as indicated by growth of cells of P.aeruginosa PA01.93(pR01996) on minimal medium contain-ing 0.05% 3-methylcatechol or 4-methylcatechol as a pri-mary carbon source and 0.005% phenol as an inducer (datanot shown).

DISCUSSION

Previous work on catabolism of phenol and cresols viameta cleavage of catechol or methylcatechols by Pseudomo-nas putida U (2) has suggested that the genes encoding theenzymes of the meta cleavage pathway are organized into asingle operon. In the present work, molecular genetic as wellas biochemical analyses have shown that the meta pathwaygenes from P. pickettii PKO1 are also organized into a singleoperon and that the promoter for this operon is proximal totbuE, the gene encoding C230. Furthermore, deletion sub-cloning analysis has allowed us to determine that the order ofgenes for the meta pathway operon is tbuEFGKIHJ, whichencode C230, HMSH, HMSD, HOA, 40D, 401, and OEH,respectively. This gene order shows some similarity to thatsuggested for the meta pathway in P. putida U, in which thegene encoding C230 is followed by the genes encodingHMSD and then HMSH (32); however, the order for theother meta pathway genes was not determined in P. putidaU. Likewise, the meta pathway gene order for P. pickettiiPKO1 has features similar to those found in the metacleavage operon of the TOL plasmid pWWO. The metacleavage operon from plasmid pWWO comprises 13 genes(10), xylXYZLTEGFJQKIH. The enzymes encoded by xylXYZ and xylL are toluate oxygenase and cyclohexadienecarboxylate dehydrogenase, which are not present in themeta cleavage operon of P. pickettii PKO1. The function ofxylT from plasmid pWWO is unknown. Next in order onplasmid pWWO are xylEGF, which encode C230, HMSD,and HMSH. This gene arrangement is similar to that found inP. putida U but differs somewhat from that in P. pickettiiPKO1 in that the genes encoding HMSD and HMSH are inreverse order in the latter strain. The arrangements, i.e.,gene orders, of the genes encoding HOA, 40D, and 40I areidentical in P. pickettii PKO1 and the TOL plasmid pWWO;however, in plasmid pWWO the gene encoding OEH occursin the middle of the operon, whereas in P. pickettii PKO1 theanalogous gene is at the end of the operon. Such similaritiesin meta cleavage pathway gene arrangements between P.pickettii PKO1 and the TOL plasmid pWWO suggest that thetwo operons are similar in organization and perhaps share a

considerable degree of DNA homology. However, differ-ences between these two operons suggest parallel evolutionof the two pathways. Further research using DNA hybrid-ization and sequence analyses should elucidate the relation-ship between the two operons.Our previous work on the PHH from P. pickettii PKO1

(17), which is further extended by this study of the metacleavage pathway from this strain, has demonstrated thattbuD, the gene encoding PHH, is in a separate operon fromthat containing tbuEFGKIHJ, which encode the enzymes ofthe meta cleavage pathway. Furthermore, transcriptionalcontrol of these two operons is regulated by separate genes:tbuR, which regulates tbuD, and tbuS, which regulatestbuEFGKIHJ. The physical arrangement of the PHH andmeta cleavage structural genes into separate operons in P.pickettii PKO1 is similar to the physical arrangement re-ported previously for P. putida U (2); however, the mode ofregulation of these operons appears to differ between the twostrains. Wigmore et al. (33) identified a single locus thatcontrolled expression of both the PHH and meta cleavageoperons in P. putida U, whereas we have identified two loci,tbuR and tbuS, which are closely linked on pRO1957 buthave been separately subcloned and shown to independentlyregulate their respective operons when present in trans.The meta cleavage pathway cloned from P. pickettii PKO1

could accommodate catechol, 3-methylcatechol, and 4-me-thylcatechol as substrates; however, these compounds didnot function as inducers of enzyme activity. Phenol andm-cresol were the only effective inducers of meta pathwayenzyme activity found in our studies; moreover, these werethe only two compounds found in our previous work (17) toinduce PHH activity. Such a narrow range of inducingcompounds for catabolism of phenol and cresols via metacleavage of catechols in P. pickettii PKO1 contrasts mark-edly with the broad range of inducers that has been reportedfor phenol and cresol catabolism in other gram-negativebacteria (8, 13, 14). However, as has been reported for othermeta cleavage pathways, control of meta pathway genetranscription in P. pickettii PKO1 is induced by upstreamprimary substrates, not by the ring fission substrate itself (8,26). This induction pattern contrasts with the product-induced ortho cleavage pathway for catechol catabolism thatoccurs in P. aeruginosa PAO1c, reported previously by us(18).We have recently shown that cells of P. aeruginosa

carrying pRO1957 are capable of growth on toluene orbenzene as the sole source of carbon and energy (15) viahydroxylation of toluene to m-cresol and benzene to phenol.The PHH and catechol meta cleavage pathways encoded bytbuD and tbuEFGKIHJ, respectively, on plasmid pRO1957are integral components for catabolism of these hydrocar-bons and represent a novel genetic organization for utiliza-tion of toluene and benzene.TbuS protein, the product of the meta cleavage operon

regulatory gene tbuS, appears to behave as a repressor aswell as an activator of gene transcription. As suggestedpreviously for 2,4-dichlorophenoxyacetic acid metabolism(16), a model for such behavior (Fig. 4) that is consistentwith results presented in this study predicts that in theabsence of the effector phenol or m-cresol, ThuS proteinrepresses transcription of the tbuEFGKIHJ operon, whereasin the presence of an effector, it forms a transcriptionactivator complex which elevates transcription of the metacleavage genes. This model (Fig. 4) predicts that in theabsence of tbuS, expression of the catechol meta cleavagegenes would no longer be fully repressed, nor would these

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P. PICKETTII CATECHOL meta CLEAVAGE PATHWAY 4593

SE.-I ,#I I

, I SE-<--- E*

/RE+--RTf t~~~~~~~~f

_EJI _i111~ ~~1 1

-HE HR

I III I I 1XEX H E H X

5 10

I

E

15

I I 11E X EB

20 25 kb

FIG. 4. Model of tbuD regulation by tbuR and tbuEFGKIHJ regulation by tbuS. The effector (E*) phenol or m-cresol interacts with TbuRto form a complex (RE*) that activates transcription of tbuD. In the absence of effectors, TbuS represses transcription of tbuEFGKIHJ,whereas when an effector is present, the TbuS-effector complex (SE*) acts as a transcriptional activator of the tbuEFGKIHJ operon.

Abbreviations: B, BamHI; E, EcoRI; H, Hindlll; X, XhoI.

genes be fully inducible. We have reported on this type ofgene regulatory pattern previously for the regulation of tfdBby tfdS in the 2,4-dichlorophenoxyacetic acid catabolicplasmid pJP4 (16). Transcriptional regulation in which a

protein functions as both a negative and a positive regulatorhas also been documented for the role of MerR in mediatingmercuric ion resistance (20, 21), for AraC regulation of thearabinose operon (27), and for OxyR-controlled expressionof genes induced by oxidative stress in Salmonella typhimu-rium and Escherichia coli (29). Based on the disparateorigins of tbuS, tfdS, merR, araC, and oxyR, one mightexpect to observe additional examples of catabolic pathwaysin which transcriptional control is mediated by repressor/activator regulators.

ACKNOWLEDGMENTS

This research was supported by NIEHS Superfund Research andEducation grant ES-04911 and by the Office of Research andDevelopment, U.S. Environmental Protection Agency, under grantR-815750-01-0 to the Great Lakes and Mid-Atlantic HazardousSubstance Research Center. Partial funding of the research activi-ties of the center is provided by the State of Michigan Department ofNatural Resources.

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