Vol. 170, No. 10

Cloning and Expression of the catA and catBC Gene Clustersfrom Pseudomonas aeruginosa PAO

JEROME J. KUKOR,1 RONALD H. OLSEN,'* AND DAVID P. BALLOU2Departments of Microbiology and Immunology' and Biological Chemistry,2 University of Michigan Medical School,

Ann Arbor, Michigan 48109

Received 18 December 1987/Accepted 27 June 1988

A 9.9-kilobase (kb) BamHI restriction endonuclease fragment encoding the catA and catBC gene clusters wasselected from a gene bank of the Pseudomonas aeruginosa PAO1c chromosome. The catA, catB, and catC genes

encode enzymes that catalyze consecutive reactions in the catechol branch of the beta-ketoadipate pathway:catA, catechol-1,2-dioxygenase (EC 1.13.11.1); catB, muconate lactonizing enzyme (EC 5.5.1.1); and catC,muconolactone isomerase (EC 5.3.3.4). A recombinant plasmid, pRO1783, which contains the 9.9-kb BamHIrestriction fragment complemented P. aeruginosa mutants with lesions in the catA, catB, or catC gene;

however, this fragment of chromosomal DNA did not contain any other catabolic genes which had been placednear the catA or catBC cluster based on cotransducibility of the loci. Restriction mapping, deletion subcloning,and complementation analysis showed that the order of the genes on the cloned chromosomal DNA fragmentis catA, catB, catC. The catBC genes are tightly linked and are transcribed from a single promoter that is onthe 5' side of the catB gene. The catA gene is approximately 3 kb from the catBC genes. The cloned P.aeruginosa catA, catB, and catC genes were expressed at basal levels in blocked mutants of Pseudomonas putidaand did not exhibit an inducible response. These observations suggest positive regulation of the P. aeruginosacatA and catBC cluster, the absence of a positive regulatory element from pRO1783, and the inability of the P.putida regulatory gene product to induce expression of the P. aeruginosa catA, catB, and catC genes.

Degradation of numerous aromatic organic compounds byaerobic bacteria proceeds through protocatechuate and cat-echol via the two arms of the beta-ketoadipate pathway (39).The central reactions of the pathway, indicated in Fig. 1,involve two analogous routes by which oxygenative fissionof the aromatic nucleus leads to production of tricarboxylicacid cycle intermediates. In addition to protocatechuate andcatechol, which are illustrated in Fig. 1, many other com-pounds such as p-hydroxybenzoate, benzoate, quinate, shi-kimate, mandelate, tryptophan, and anthranilate are dissim-ilated through the beta-ketoadipate pathway (30, 41).

Considerable research has been done on the enzymologyand regulation of enzyme induction of the beta-ketoadipatepathway in Pseudomonas putida and Pseudomonas aerugi-nosa (20, 29-32). Three enzymes of the catechol branch ofthe pathway, catechol dioxygenase (CTD) (encoded bycatA), muconate lactonizing enzyme (encoded by catB), andmuconolactone isomerase (encoded by catC) share a com-mon inducer, cis, cis-muconate (3, 20, 29). In P. putida, catBand catC are coordinately controlled and have been found tobe closely linked on the chromosome (22, 40). In P. aerugi-nosa, transductional analysis has shown that catA, catB, andcatC are linked on the chromosome and that this cat clusteris grouped with several independently regulated genes thatcode for enzymes with related catabolic functions (38).Two of the enzymes of the catechol branch of the beta-

ketoadipate pathway have been purified and extensivelycharacterized from P. putida (21). cis, cis-Muconate lacto-nizing enzyme is an oligomeric protein composed of eighthomologous protomers, each with a molecular weight of40,000 (2, 12). The complete nucleotide sequence for thecatB gene, which encodes muconate lactonizing enzyme,has been published (1) for P. putida. Muconolactone isom-erase is a decamer consisting of 10 identical 11,000-molecu-

* Corresponding author.

lar-weight subunits (19). Catechol-1,2-dioxygenase (CTD)has been purified from several microbial sources (35). Ex-tensive characterization of the enzyme has been carried outin two strains of fluorescent pseudomonads (23, 25), in whichthe enzyme was found to consist of two nonidentical sub-units. The enzymes of the catechol branch of the beta-ketoadipate pathway have not previously been purified fromP. aeruginosa PAO1c.

In this report, we describe a molecular cloning analysis ofthe catA and catBC gene clusters from P. aeruginosa. Thesegenes were isolated on a 24.6-kilobase-pair fragment of theP. aeruginosa chromosome. Although this fragment is muchlarger than that required for these genes, none of thecatabolic genes that were previously placed near the catA,catB, and catC genes by transductional analysis (37, 38)were found. The cloned catA, catB, and catC genes were

expressed at elevated levels in blocked mutants of P. aeru-

ginosa; however, their low level of expression and theabsence of an inducible response in mutants of P. putidasuggest an incompatibility between the regulation andexpression of the cat genes in P. putida and P. aeruginosa.Furthermore, it can be concluded that the cloned fragmentdoes not code for all the regulatory elements of these genes.

MATERIALS AND METHODS

Bacterial strains and plasmids. The bacterial strains andplasmids used in this study, together with their relevantcharacteristics, are listed in Table 1.Media and growth conditions. Minimal medium (VBG or

MMO) and complex medium (TN) were prepared as de-scribed previously (7, 27). Tetracycline and carbenicillinwere used in selective media for P. aeruginosa at concen-

trations of 50 and 500 ,ug/ml, respectively. For P. putida,tetracycline was used at 25 ,ug/ml and carbenicillin at 2,000Lg/ml. When grown for enzyme assays, bacteria were cul-tured in 100 ml of minimal medium (MMO) with aeration,

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P. AERUGINOSA catABC CLUSTER 4459

,OH

protocatechuate

protocatechuatedioxygenase (pcaA)

beta-carboxy- -000cis,cis-muconate

beta-carboxy-muconatelactonizing enzyme(pcaB) -040

gamma-carboxy- Cmuconolactone

gamma-carboxy-muconolactonedecarboxylase (pcaC)

catechol

,COO-

-Qoc

OC

COO -

0>?2

COO-

OC/0

-0CC

'OH

catecholdioxygenase

COO- (catA)

cis,cis-muconate

mpconateCOO- lactonizing

enzyme (catB)

muconolactone

0 muconolactoneisomerase(catC)

beta-ketoadipate enollactone

beta-ketoadipate enollactone hydrolase(pcaD)

beta-ketoadipate

FIG. 1. Beta-ketoadipate pathway in P. aeruginosa showing the steps for conversion of protocatechuate and catechol to beta-ketoadipate.The gene designation is shown in parentheses for eaqh enzyme.

supplemented with 10 mM benzoate or 20 mM glucose. P.aeruginosa was grown at 37°C, and P. putida was grown at300C.

Preparation of mutants. Bacterial mutants were derivedfrom P. aeruginosa PAOlc (16) or P. putida PP0200, aderivative of strain mt-2 (42) cured of its TOL plasmid.Mutagenesis with nitrosoguanidine was performed as re-ported previously (7). Mutants were purified by single-colony isolation on complex medium and were tested onminimal benzoate medium. Mutants were further character-ized by analysis of compounds accumulated after growih on20 mM glucose plus 10 mM benzoate. Catechol was detectedby color formation (A605) after reaction with ferrous JtDTA(6), cis, cis-muconate was detected. by its' UV absorptionspectrum (32), muconolactone was detected by the hydro-xamate method (15), and beta-ketoadipate was detected bythe Rothera reaction (9), which involves reaction with 5%sodium nitroprusside in a saturated ammonium sulfate solu-tion overlaid with concentrated (14,8 M) ammonium hydrox-ide. The production of a dark purple band' at the interface ofthe two-layers is a positive reaction for beta-ketoadipate.

Preparation of DNA. The chromosomal DNA fragmentsused in this study were derived from a BamHI gene bank of

the P. aeruginosa chromosome described previously (26).Plasmid D,NA was prepired by a modification of the alka-line-sodium dodrcyl sulfate' (SDS) procedures of Birnboimand Doly (4) and Ish-Horowicz and Burke (18), using-cesiumchloride-ethidium bromide density gradient centrifugation.Recombinant' plasmids were surveyed for their, sizes withcells harvested from a patch of growth on selective mediumand lysis by a small-scale modification of the alkaline-SDSprocedure-(8, 13).DNA. restriction mapping, ligation, and transformation.

DNA procedures were done as'described previously'(8, 13,26).Measurement of enzyme activity. Cells were grown for

enzyme assays to a level that gave an apparent A425 of 0.8 to1.0 (Bausch & Lom,b Spectronic 21 spectrophotometer) andwere harvested by centrifugation at 10,000 x g for 15 min.The cell pellets were washed tWice in 20 mM Tris hydro-chloride (pH 8.0), and the cells were disrupted sonically byfour 15-s 200-W bursts-with a Braun-Sonic 1510 apparatus.Cellular debris was removed by centrifugation at 100,000 xg for 2 h, 'and the clear supernatant solution was: usedimmediately for enzyme assays. Activities' for enzymesencoded by the catA, catB, and catC gene.s were determined

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TABLE 1. Bacterial strains and plasmids

Strains and Reference orplasmids Phenotype or genotype' source

Pseudomonasaeruginosa

PAO1c Prototroph 16PAO2.1 catA ser-3 This studyPA02324 catA met-9020 tvu-9009 Matsumoto"

nar-9011 puuD6PAO1.93 catA This studyPA04032 catA met-9020 nar-9011 Matsumoto

mtu-9002 tvu-9030PAO1.94 catA This studyPAO1.169 catB This studyPAO1.92 catC This study

Pseudomonasputida

PPO200' Prototroph 42PPO200-17 catA This studyPPO200-2 catB This studyPPO200-16 catC This study

PlasmidspRO1614 Cbr Tcr IncPl 26pRO2317 Cbr Tcr IncW Zyistra and

Olsen'pRO1772 Cb' CatA+ CatB+ CatC+ 26pRO1783 Cbr CatA+ CatB' CatC+ 26pRO1944 Cbr CatA- CatB+ CatC+ This studypRO1875 Cb CatA- CatB+ CatC+ This studypRO1876 Cbr CatA1+ CatA2- CatB+ This study

CatC+pRO2337 Tcr CatA+ CatB- CatC- This studypRO2338 CatA1+ CatA2- CatB- This study

CatC-pRO2339 CatA- CatB- CatC- This studypRO1937 Cbr CatA- CatB+ CatC- This studypRO1938 Cbr CatA- CatB+ CatC+ This studypRO1941 Cbr CatA- CatB+ CatC+ This studypRO1942 Cb CatA CatB CatC This study

Marker abbreviations for bacterial strains are as in reference 17, exceptcatA, CTD; catB, muconate lactonizing enzyme; catC; muconolactone isom-erase; for plasmids, Cbr, carbenicillin resistance; Tcr, tetracycline resistance.

b Matsumoto collection, received from P. Phibbs.P. putida PPO200 is a derivative of strain mt-2 (ATCC 33015) that has

been cured by us of its TOL plasmid.d G. Zylstra and R. H. Olsen, manuscript in preparation. This vector has an

IncW replicon derived from R388, a Tcr determinant derived from pBR322with unique BamHI and Sall cloning sites, and a Cbr determinant derivedfrom Tnl with a unique PstI cloning site.

by established procedures (14, 28). Protein was determinedby the method of Bradford (5) with bovine serum albumin asthe standard. Specific activities are reported as micromolesof product formed per minute per milligram of protein.

Purification of CTD. CTD (EC 1.13.11.1) was obtainedfrom P. aeruginosa PAO1c by a modification of publishedprocedures (10, 36). Cells were grown in a 200-liter NewBrunswick F250 fermentor with benzoate as the sole carbonsource. Packed cells (450 g, wet weight) suspended in 1 literof 50 mM Tris hydrochloride (pH 8.0) containing 10 mMNaCI and 0.4 mM phenylmethylsulfonyl fluoride (referred tohereafter as Tris buffer) were disrupted in a Manton-Gaulinhomogenizer, and solid streptomycin sulfate was added to afinal concentration of 1.5% (wt/vol). The precipitated nucleicacids and cell debris were removed by centrifugation at100,000 x g for 1 h, and the resulting supernatant solutionwas applied to a DEAE-Sepharose column (5 by 24 cm). Thecolumn was washed with 1 liter of Tris buffer, and the

enzyme was eluted with 2 liters of a linear NaCl gradientfrom 100 to 300 mM in Tris buffer. Fractions with a specificactivity greater than 1 were pooled, and solid ammoniumsulfate was added to give 30% saturation. The precipitatewas removed by centrifugation, and the supernatant solutionwas brought to 55% saturation with ammonium sulfate. Theresulting precipitate, collected by centrifugation, was dis-solved in a minimum volume of Tris buffer and was dialyzedovernight against the same buffer. The enzyme solution wasthen applied to a column of Sephadex G-100 (5 by 90 cm)equilibrated with the Tris buffer and eluted with the samebuffer. Fractions containing CTD activity were pooled,concentrated in an Amicon stirred cell, and further fraction-ated by high-resolution anion-exchange chromatographywith a Pharmacia fast protein liquid chromatography system.Fast protein liquid chromatography separations were carriedout at a flow rate of 1.0 ml/min with an HR 5/5 Mono Qcolumn equilibrated with Tris buffer containing 5 mM NaCl.Proteins were eluted with a 5 to 500 mM linear NaClgradient. Active fractions were pooled, dialyzed against Trisbuffer, concentrated in an Amicon stirred cell, and stored at- 700C.SDS-polyacrylamide gel electrophoresis was performed

by the method of Neville as modified by Piccioni et al. (34)on 12.3% total acrylamide, 2.7% cross-linked, and 0.1% SDSdenaturing gels. Protein marker standards included bovinealbumin (66,000 molecular weight), ovalbumin (45,000), gly-ceraldehyde-3-phosphate dehydrogenase (36,000), carbonicanhydrase (29,000), trypsinogen (24,000), soybean trypsininhibitor (20,100), and alpha-lactalbumin (14,200). Samplesfor denaturing gel electrophoresis contained approximately20 jig of protein as well as 6 mM dithiothreitol, 0.1% SDS,and 0.044 mM phenylmethylsulfonyl fluoride. Protein bandswere visualized by being stained with Coomassie brilliantblue R-250 (34).

Chemicals and reagents. All the chemicals, enzymes, andreagents used in these studies were of the highest puritycommercially available. Enzymes and reagents used forDNA manipulations were purchased from International Bio-technologies, Inc. (New Haven, Conn.) or Bethesda Re-search Laboratories, Inc. (Gaithersburg, Md.) and wereused as suggested by the supplier. cis, cis-Muconate wasproduced enzymatically from catechol by using purifiedCTD. Muconolactone was produced enzymatically from cis,cis-muconate by established procedures (14, 32).

RESULTS

Mutant isolation and characterization. Enzyme levels ofthe catA, catB, and catC mutants isolated after exposure ofparent cells to nitrosoguanidine are listed in Table 2. Mu-tants with a catA phenotype could potentially have lesions inthe catA structural gene for either of the catA peptides or ina regulatory locus governing expression of catA. However,results described below with the P. aeruginosa catA mutantslisted in Table 2 indicate that these strains have mutations inthe structural gene for CTD.

Cloning of catABC cluster. Plasmid pRO1772, isolatedpreviously by us (26), was the starting point for the currentwork. This plasmid was used to transform a set of P.aeruginosa mutants to determine whether other genes of thebeta-ketoadipate pathway are carried on the cloned DNAfragment. Wild-type transformants were obtained whenpRO1772 was introduced into strains carrying mutations inthe catA, catB, or catC structural gene. However, thisplasmid did not complement strains with mutations for

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TABLE 2. Levels of enzymes in mutant strains of P. aeruginosaand P. putida after growth on 20 mM glucose with 10 mM

benzoate as a source of inducer

Enzyme levelStrain Mutation (pLmollmin/mg of protein)'

CTD MLE MI

P. aeruginosaPAO1c Prototroph 1.467 0.281 0.580PAO2.1 catA 0.001 0.004 0.005PA02324 catA 0.023 0.004 0.004PAO1.93 catA 0.005 0.004 0.003PA04032 catA 0.045 0.022 0.030PAO1.94 catA 0.005 0.007 0.007PAO1.169 catB 1.409 0.001 0.330PAO1.92 catC 1.023 0.200 0.001

P. putidaPP0200 Prototroph 1.237 0.338 0.304PP0200-17 catA 0.003 0.001 0.001PPO200-2 catB 1.620 0.002 0.300PP0200-16 catC 0.994 0.147 0.001

aAbbreviations: MLE, muconate lactonizing enzyme; MI, muconolactoneisomerase. Each value is the mean of three independent determinations.

benzoate, anthranilate, benzaldehyde, benzoylformate, ormandelate utilization (data not shown).The two BamHI fragments of chromosomal DNA in

plasmid pRO1772 were subcloned separately. The subclonewhich contained the 14.6-kilobase (kb) BamHI fragment wasdesignated pRO1784, and the other subclone with the 9.9-kbBamHI fragment was designated pRO1783. PlasmidpRO1783 complemented the catA, catB, and catC mutantswhen used to transform.P. aeruginosa cat mutants listed inTable 3. No complementation was obtained for any of thecat mutants when they were transformed with pRO1784.

Subcloning and characterization of cat4 region of pRO1783.Plasmid pRO1783 was digested with restriction endonu-cleases, and the resulting map is shown in Fig. 2. A seriesof EcoRI deletions were made to localize the catA, catB,and catC genes on pRO1783. Three plasmids, designatedpRO1944, pRO1875, and pRO1876, respectively (Fig. 2),were obtained from this EcoRI deletion-subcloning. Theseplasmids were used to transform the set of catA mutantslisted in Table 3. No complementation was observed forcatA mutants of P. aeruginosa by pRO1944 or pROi815.Complementation by PROi876 was observed for catA mu-tants PA04032 and PAO1.94, but not for catA mutantsPAO2.1, PA02324, and PAO1.93. These results suggest thatcatA spans the internal EcoRI site (at map coordinate 3.5 kb;Fig. 2) in pRO1783. To isolate the catA-containing portion ofpRO1783, we digested the plasmid with PstI and subcloned

it into the unique PstI cloning site which is located in thecarbenicillin resistance determinant of vector pRO2317(Table 1). This ligation mixture was used to transform eachof the catA mutants listed in Table 3, and each culture was

plated onto minimal benzoate medium to select for transfor-mants that grew on benzoate. Plasmid DNA purified fromeach of the benzoate-positive transformants contained a

4.25-kb Pstl fragment of pRO1783. Comparative restrictionanalysis of this PstI fragment demonstrated that it consistedof 1.25 kb of the internal EcoRI piece of pRO1783 as well as

3.0 kb of the adjacent BamHI-EcoRI fragment. The resultingPstI subclone containing the catA gene was designatedpRO2337.To further localize the catA gene on plasmid pRO2337, we

made a series of Sall deletions. Plasmid pRO2338 (Fig. 2),which contains a 2.2-kb PstI-SalI fragment of pRO2337DNA, complemented the P. aeruginosa catA mutantsPA04032 and PAO1.94, but none of the other catA mutants(Table 3). These results are similar to the complementationpattern found for pRO1876, described above. PlasmidpRO2339, which contains a 3.2-kb PstI-SalI fragment ofpRO2337 DNA (Fig. 2), did not complement any of the P.aeruginosa catA mutants (Table 3), which was similar to theresult for pRO1944 described above.

In vitro reconstruction of CTD activity. SDS-polyacryl-amide gel electrophoresis of denatured CTD in the presenceof dithiothreitol demonstrated two protein bands with esti-mated molecular weights of 34,000 and 33,000 (Fig. 3). Themolecular weight of the enzyme was estimated to be approx-imately 70,000 by gel filtration (data not shown). Thus, theenzyme appears to be a dimer composed of two nonidenticalsubunits which differ slightly in molecular weight.The catA mutants used in this study could be classified

into two groups based on complementation by pRO1876.This suggested that these two groups of mutants representedstrains with lesions in different subunits of CTD. Thispossibility was tested by partial in vitro reconstitution ofCTD activity by mixing cell extracts of the catA mutants thathad been grown on benzoate plus glucose. CTD activitycould be restored to approximately 10%. of that found inwild-type PAO1c when extracts of PA02324 or PAO1.93were combined with extracts of PA04032 or PAO1.94 (Table4). However, no restoration of enzyme activity was ob-served when extracts of PA02324 were combined withextracts of PAO1.93 or when PA04032 was combined withPAO1.94. These results, which are consistent with the dataobtained from subcloning of the catA region of pRO1783 andcomplemnentation of catA mutants of P. aeruginosa, indicatethat PAO2.1, PA02324, and PAO1.93 have mutations in onesubunit of CTD and that PA04032 and PAO1.94 havemutations in the other subunit of the enzyme.

TABLE 3. Complementation of P. aeruginosa mutants by pRO1783 and its subtlones'

Strain Genotypeb 1783 1944 1875 1876 2337 2338 2339 1937 1938 1941 1942

PA02.1 catA + - - - + - - - - - -PA02324 catA + - - - +PA01.93 catA + - - - +PA04032 catA + - - + + +PA01.94 catA + - - + + +PA01.169 catB + + + + - - - + + +PA01.92 catC + + + + - - - - + +

a Complementation is based on the ability of each mutant, when carrying the indicated recombinant plasmid, to grow on minimal media supplemented with theappropriate carbon source.

b Marker abbreviations are as in Table 1.

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coo(- ° o°-= 7;

UI 1) wif W1.1CL

I- I

cat A

CZ ia X5 'a c t co(X) cn X- XCJ

cat B cat C

pROl 944

pRO 1875

pRO1 876

pRO2337

pRO2338

pRO2339

pRO0 937

pROl 938

pRO0 941

pRO0 942

0 1 2 3 4 5 6 7 8 9 10 kbp

FIG. 2. Restriction map of plasmid pRO1783 and the deletion subclones derived from it. The positions of relevant restriction sites fromthe plasmid cloning vectors are shown flanking the cloned chromosomal DNA fragment. The approximate locations of the catA, catB, andcatC genes are shown.

Subc!oning and characterization of catBC region ofpRO1783. Plasmid pRO1875, an EcoRI deletant of pRO1783(Fig. 2), did not complement any of the catA mutants of P.aeruginosa, but did complement catB and catC mutants(Table 3). All attempts at separately subcloning catB andcatC proved futile,. which suggested that these two genes

are transcribed fromn a single promoter. To determine therelative positions of cattB and catC, plasmid pRO1875 was

cleaved at its BamHI site and then digested with the double-stranded exonuclease,Bal 31. Such digests were ligated andtransformed into catB and catC mutants of P. aeruginosa.One of the Bal 31 deletants, designated pRO1937, comple-mented PA01.169, a catB mutant, but not PA01.92, a catCmutant. Restriction analysis demonstrated that this plasmidhad approximately 1.4 kb of DNA deleted from the BamHIside of pRO1875. A second Bal 31 deletant, designatedpRO1938, which had approximately 1.0 kb of DNA deletedfrom the BamHI side of pRO1875, was able to complement

36

29

FIG. 3. SDS-polyacrylamide gel electrophoretic profile of thepartially purified CTD of P. aeruginosa PA01c. Estimated molecu-lar masses of the CTD subunits are 34,000 and 33,000 daltons. Thepositions of protein size standards (in kilodaltons) are indicated tothe left.

both the catB and catC mutants of P. aeruginosa. Fromthese results, catC can be placed at approximately 1.5 kbaway from the BamHI site of pRO1875 (map coordinate 8.5kb; Fig. 2).When pRO1875 was cleaved at its EcoRI site and digested

with Bal 31, two sizes of deletion plasmids were obtained.One, designated pRO1941, lacked approximately 0.4 kb ofDNA and was able to complement both PA01.169, a catBmutant, and PA01.92, a catC mutant. However, pRO1942,which had approximately 1.0 kb ofDNA deleted, was unableto complement either the catB or the catC mutant. Fromthese results, catB can be placed approximately 0.5 kb away

from the EcoRI site, at map coordinate 5.0 kb (Fig. 2) ofpRO1875. Furthermore, the deletion data support the infer-ence drawn from attempted subcloning of pRO1875 that catBand catC are tightly linked on this DNA fragment and are

transcribed from a single promoter adjacent to catB anddistal to catC.

TABLE 4. In vitro reconstruction of CTD activity for catAmutants of P. aeruginosa grown on benzoate plus glucose

Strain Enzymeactivity"

PA02324 ...................................... 0.023PAO1.93 ...................................... 0.009PA04032 ...................................... 0.045PAO1.94 ...................................... 0.004PA02324 + PAO1.93 ............................................... 0.025PA02324 + PA04032 .............................................. 0.217PA02324 + PAO1.94 .............................................. 0.102PAO1.93 + PA04032 ............................................... 0.215PAO1.93 + PAO1.94 ............................................... 0.130PA04032 + PAO1.94 ............................................... 0.039

a Values are units of CTD activity (micromoles of muconate produced perminute) per milligram of protein. The activity for fully induced (benzoate-grown) cells of PAO1c was 1.467.

O -DE =

Il I .m

pROl 783-0.W-;l....... ---L-"

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TABLE 5. Expression of the catABC genes carried on pRO1783 in P. aeruginosa

Enzyme activity" for complementing gene in:Strain Genotype of Enzymehost strain assayed Benzoate-grown Glucose-grown

(induced) cells (uninduced) cells

PAO2.1(pRO1783) catA CTD 1.050 0.009PA02324(pRO1783) catA CTD 1.741 0.022POA1.93(pRO1783) catA CTD 1.613 0.009PA04032(pRO1783) catA CTD 1.301 0.004PAO1.94(pRO1783) catA CTD 1.837 0.001PAO1.169(pRO1783) catB MLE 0.346 0.004PAO1.92(pRO1783) catC MI 0.531 0.001

aValues are units of enzyme activity (micromoles of product produced or substrate convered per minute) per milligram of protein. The activities for fullyinduced (benzoate-grown) cells of PAO1c were 1.467 (CTD), 0.281 (muconate lactonizing enzyme [MLE]), and 0.58 (muconolactone isomerase [MI]). Theactivities for uninduced (glucose-grown) cells of PAO1c were 0.005 (CTD), 0.002 (MLE), and 0.001 (MI).

Expression of catABC genes in P. aeruginosa. Enzymes ofthe beta-ketoadipate pathway, including those encoded bycatA, catB, and catC, are inducible in P. aeruginosa andrelated bacteria. When the bacteria are grown in the pres-

ence of an inducer, enzymes are produced at elevated levels(33). The catA, catB, and catC genes on pRO1783 exhibitedinducible expression when present in blocked mutants of P.aeruginosa (Table 5). The level of expression of the clonedgenes in the P. aeruginosa mutants was higher than foundfor fully induced cells of wild-type PAO1c, which may reflectthe copy number of the cloned fragments.

Expression of P. aeruginosa catABC genes in P. putida.

When carried on the recombinant plasmid pRO1783, thecatA, catB, and catC genes were expressed at very lowlevels in P. putida (Table 6), levels which were similar tothose found in uninduced cells of P. putida or P. aeruginosa(Table 6). The level of activity for each of the enzymes was1/10th to 1/50th of that found in fully induced cells of thewild-type PAO1c, and there was no significant elevation ofenzyme activity when the P. putida mutants were grownunder inducing conditions. Furthermore, the level of activityof the P. aeruginosa enzymes was 1/10th of that found infully induced cells of the wild-type P. putida PP0200 (Table6). These results suggest that a required positive regulatorylocus (catR) was not present in pRO1783.

DISCUSSION

On the basis of restriction digest analysis and subcloningof pRO1783, as well as complementation of blocked mutantsof P. aeruginosa, we place the gene order of the cat clusteras catA, catB, catC. This differs from the catA, catC, catBarrangement of genes given by Holloway and Morgan (17) inthe most recent map of the PAO chromosome. PlasmidpRO1783 contained the catA, catB, and catC gene cluster,but it did not contain any of the related catabolic genes of the

beta-ketoadipate and mandelate pathways that are known tobe clustered at 64 minutes on the P. aeruginosa chromosome(17, 37, 38).Our inability to subclone catB and catC separately to-

gether with the polar effects of Bal 31 deletions that inacti-vated both catB and catC indicate that these genes aretightly linked. This linkage is consistent with the cotransduc-tion data of Kemp and Hegeman (20) and Rosenberg andHegeman (37, 38) for P. aeruginosa and is similar to obser-vations for P. putida (22, 40). In addition, the fact that Bal 31deletions from the catC side of pRO1875 gave an active catBgene and an inactive catC gene, whereas deletions from thecatB side inactivated both genes, suggests that these genesare transcribed as a unit from a single promoter and thattranscription proceeds from catB through catC.

Subcloning of the catA-containing region of pRO1783revealed that the catA mutants of P. aeruginosa could bedifferentiated into two groups based on their ability to becomplemented by pRO1876, a deletant of pRO1783 thatlacks an internal 1.5-kb EcoRI DNA segment. Since theCTD purified from P. aeruginosa, was found to be a dimercomposed of nonidentical subunits, it appeared that the twogroups of catA mutants represented mutant P. aeruginosastrains that carried lesions in different subunits of the en-

zyme. This hypothesis was supported by the partial restora-tion of CTD activity when extracts of each of the two classesof catA mutants were combined.The 34- and 33-kilodalton subunits of P. aeruginosa CTD

would require approximately 1.7 kb of DNA for synthesis.Localization of the catA genes that encode these peptideswas derived from complementation experiments with over-

lapping deletion subclones of the catA+ plasmid, pRO2337.Plasmid pRO2339, which contains a 3.2-kb PstI-SalI frag-ment of pRO2337, did not complement any of the catAmutants; nor did plasmid pRO1944, which overlaps pRO2337

TABLE 6. Expression of P. aeruginosa catABC genes in P. putida

Enzyme activitya for complementing gene in:Strain Genotype of Enzyme

host strain assayed Benzoate-grown Glucose-grown(induced) cells (uninduced) cells

PP0200-17(pRO1783) catA CTD 0.125 0.112PP0200-2(pRO1783) catB MLE 0.042 0.039PP0200-16(pRO1783) catC MI 0.034 0.029

" Values are units of enzyme activity (micromoles of product produced or substrate converted per minute) per milligram of protein. The activities for fullyinduced (benzoate-grown) cells of P. aeruginosa PAO1c were 1.467 (CTD), 0.281 (muconate lactonizing enzyme [MLE]), and 0.589 (muconolactone isomerase[MI]), and for P. putida PP0200 they were 1.237 (CTD), 0.338 (MLE), and 0.304 (MI). The activities for uninduced (glucose-grown) cells of P. putida PP0200were 0.009 (CTD), 0.003 (MLE), and 0.002 (MI), and for P. aeruginosa PAO1c they were 0.005 (CTD), 0.002 (MLE), and 0.001 (MI).

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4464 KUKOR ET AL.

and pRO2339 by carrying the 1.5-kb internal EcoRI fragmentof pRO1783. On the other hand, plasmids pRO1876 andpRO2338, which contain DNA fragments from the smallerBamHI-EcoRI portion of pRO1783, complemented only twoof the catA mutants, PA04032 and PAO1.94. From theseresults, the catA genes can be positioned in the smallerBamHI-EcoRI fragment of pRO1783 (map coordinates 0.5 to3.5 kb; Fig. 2), and furthermore, it can be concluded thattranscription of the catA genes is initiated in the PstI-SalIregion of pRO2337 (map coordinates 0.5 to 1.75 kb; Fig. 2)and proceeds through the EcoRI site at map coordinate 3.5kb (Fig. 2) in pRO2337. This portion of the catA clone iscurrently being analyzed by DNA sequence analysis.When carried in mutants of P. putida, the cloned catA,

catB, and catC genes from P. aeruginosa were expressed atvery low levels, generally 10-fold lower than those found infully induced cells of the prototroph, P. aeruginosa PAO1c.Moreover, the cloned P. aeruginosa cat genes in P. putidashowed no significant elevation of activity under inducingconditions. One possible interpretation of these results isthat the P. aeruginosa promoters for catA and catBC are notrecognized in P. putida. However, this explanation seemsunlikely in the light of previous successful expression ofcloned P. aeruginosa catabolic genes in P. putida (8). Analternative explanation is that these results indicate differ-ences in the regulation of the cat genes between the twoPseudomonas species. An explanation for these observa-tions may be that in P. aeruginosa there is a requiredpositive regulatory gene product (an activator) that is notencoded by plasmid pRO1783. Thus, when placed in P.putida, the activator would not be present and the cloned P.aeruginosa genes would be expressed only at a basal level.Since our data show that catA and catBC are separatelytranscribed, it is possible that there are two positive regula-tory genes missing from pRO1783 that control transcriptionof the P. aeruginosa cat genes. However, results fromexperiments on the effects of catabolite repressors on syn-thesis of CTD, muconate lactonizing enzyme, and mucono-lactone isomerase in P. aeruginosa (20; Kukor and Olsen,unpublished data) indicate that catA and catBC are equallysensitive to catabolite repression. These results would sug-gest that a single regulatory gene (catR) controls transcrip-tion of both catA and catBC in P. aeruginosa.

If the P. aeruginosa catA, catB, and catC genes werenegatively controlled, their expression in P. putida catA,catB, or catC mutants should be constitutive if they are notregulated from the P. putida chromosome and if the negativeregulatory gene was missing from pRO1783. However, as-suming a model of negative transcriptional control, if the P.putida repressor was able to regulate expression of the P.aeruginosa genes, then the cloned P. aeruginosa catA, catB,and catC genes should exhibit an inducible response in a P.putida genetic background. Our findings on the lack ofconstitutive expression and the absence of an inducibleresponse of the cloned P. aeruginosa cat genes in a P. putidagenetic background suggest that the P. aeruginosa cat genesare under positive transcriptional control.

Analysis of gene regulation and expression in heterogene-tic backgrounds has proved to be a useful technique. Wehave used this approach previously in the analysis of clonedcarbohydrate catabolic genes from P. aeruginosa expressedin P. putida (8). In the work reported here, we extended thisapproach to an analysis of the regulation of the catecholbranch of the beta-ketoadipate pathway. Since the beta-ketoadipate pathway is widely distributed among bacteriaand its enzymology is well understood, analysis of hetero-

specific differences in expression of cloned DNA fragmentscan provide a useful approach to understanding differencesin modes of regulation.

In addition to the CTD cloned from P. aeruginosa in thisstudy, the gene for this enzyme has also been cloned fromthe chromosome of Acinetobacter calcoaceticus (24). Genesspecifying CTDs that accommodate halogenated catecholshave also been cloned from gram-negative soil bacteria (11;Kukor and Olsen, unpublished data). A suitable data set isnow available for comparison of the structure of these genesand also for an analysis of factors affecting enzyme substratespecificity in this group of dioxygenases.

ACKNOWLEDGMENTSWe thank Gerben Zyistra, Charles Mountjoy, and Chris Batie for

advice and technical assistance. We are also grateful to James Lutefor the derivation of the P. putida PPO200 mutants.

This work was supported in part by grants from the MichiganBiotechnology Institute (J.J.K.), Public Health Service grantGM20877 from the National Institutes of Health (D.P.B.), and U.S.Environmental Protection Agency Cooperative Agreement CR-812679 (R.H.O.).

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