Vol. 176, No. 24JOURNAL OF BACrERIOLOGY, Dec. 1994, p. 7757-77620021-9193/94/$04.00+0Copyright © 1994, American Society for Microbiology

Organization and Evolution of Naphthalene Catabolic Pathways:Sequence of the DNA Encoding 2-Hydroxychromene-2-Carboxylate

Isomerase and trans-o-HydroxybenzylidenepyruvateHydratase-Aldolase from the NAH7 Plasmidt

RICHARD W. EATON*Environmental Research Laboratory, U.S. Environmental Protection Agency, Gulf Breeze, Florida 32561

Received 1 August 1994/Accepted 7 October 1994

The sequence of a 2,437-bp DNA segment from the naphthalene upper catabolic pathway operon of plasmidNAH7 was determined. This segment contains three large open reading frames designated nahQ', nahE, andnahD. The first of these is the 3' end of an open reading frame that has no known function, the second (993bp) encodes trans-o-hydroxybenzylidenepyruvate hydratase-aldolase (deduced molecular weight, 36,640), andthe third (609 bp) encodes 2-hydroxychromene-2-carboxylate isomerase (deduced molecular weight, 23,031).This DNA has a high degree of sequence homology (greater than 91% for the first 2161 bp) with a DNA segmentfrom the dox (dibenzothiophene oxidation) operon of Pseudomonas sp. strain C18, which encodes a pathwayanalogous to that encoded by NAH7. However, 84 bp downstream from nahD, the last gene in the nah operon,this homology ends. This 84-bp sequence at the downstream end of nah and dox homology has 76% homologyto a sequence that occurs just upstream of the nah promoter in NAH7. These directly repeated 84-bp sequencesthus encompass the upper-pathway nah operon and constitute the ends of a highly conserved region.

The naphthalene catabolic pathway in Pseudomonas putidaG7 can be divided into two parts, an upper pathway and alower pathway. Enzymes of the upper pathway convert naph-thalene to salicylate, which is subsequently transformed bylower-pathway enzymes to amphibolic intermediates. The up-per and lower pathways are encoded by separate operonsunder the control of a common regulatory protein encoded bynahR (32). The two nah operons and nahR are located on aDNA segment of about 25 kb within a 37.5-kb defectivetransposon, Tn4655 (29), on the 83-kb plasmid NAH7 (6, 32).

Naphthalene is metabolized to salicylate by a six-step path-way (Fig. 1) (7). The enzyme-catalyzed reactions of the firstthree steps in this pathway (Fig. 1, enzymes A, B, and C) aresimilar to those involved in the metabolism of single-ringaromatic hydrocarbons such as toluene (13, 23). Subsequentreactions differ because the instability of the 1,2-dihy-droxynaphthalene ring cleavage product (Fig. 1, chemical IV)leads to a rearrangement followed by the formation of ahemiketal product, 2-hydroxychromene-2-carboxylate (Fig. 1,chemical VI), having no analogs among intermediates in thedegradation of simple aromatic hydrocarbons. Isomerase (Fig.1, enzyme D) and hydratase-aldolase (Fig. 1, enzyme E)enzymes such as those of the naphthalene catabolic pathwaythus represent classes of enzymes that are unique to thebiodegradation of aromatic compounds having two or morerings. As a result, the genes that encode these enzymes shouldbe valuable as relatively specific probes for the identification orenumeration of bacteria that degrade polycyclic aromaticcompounds. In addition, the isomerase and hydratase-aldolaseenzymes may have interesting, novel reaction mechanismswhich merit further study (7); the amino acid sequences ofthese enzymes would be useful to have when initiating these

* Phone: (904) 934-9345. Fax: (904) 934-9201. Electronic mailaddress: reaton@gulfbr.gbr.epa.gov.

t Contribution 892 from the Environmental Research Laboratory,U.S. Environmental Protection Agency, Gulf Breeze, Fla.

studies. For these reasons, the nucleotide sequence of a DNAsegment from the NAH7 plasmid that carries nahD and nahE,the genes that encode 2-hydroxychromene-2-carboxylate isomer-ase and trans-o-hydroxybenzylidenepyruvate hydratase-aldo-lase, respectively, was determined.The sources of DNA for sequencing were pRE714 and

pRE701 (7), which carry DNA fragments on which nahD andnahE are located and which were cloned from the naphthalenecatabolic plasmid NAH7, originally from P. putida G7 (ATCC17485) (6, 32) but obtained from I. C. Gunsalus in the P. putidaG1 (ATCC 17453) derivative PpG1064 (31). For sequencing,DNA fragments were subcloned from pRE714 and pRE701into the vectors pBluescriptll SK and pBluescriptII KS (1)(Stratagene Cloning Systems, La Jolla, Calif.). DNA sequencedeterminations were then carried out by the DNA SequencingCore Laboratory of the Interdisciplinary Center for Biotech-nology Research at the University of Florida, Gainesville.Sequencing reactions were performed by the dideoxy chaintermination method (24) with a fluorescent dye-labelled T3 orT7 primer complementary to vector sequences adjacent tocloned fragments or with the Applied Biosystems DyeDeoxyfluorescent terminators and the following custom primers: A,5'-CACGCGGAAACAATGTCG-3'; B, 5'-CGGAGTTCTCGAAGTATA-3'; and C, 5'-CCCCGCGCTGCATGTTGG-3'.Custom oligonucleotide primers were prepared by the DNASynthesis Laboratory, Interdisciplinary Center for Biotechnol-ogy Research, University of Florida, Gainesville. Sequencedata were obtained with Applied Biosystems, Inc., model 373aDNA sequencers and were subsequently provided as computerfiles and paper copies. Sequence data were aligned and editedby using DNASTAR (DNASTAR, Inc., Madison, Wis.). Pub-lished sequences were obtained from the GenBank data base(3); searches for specific sequences were carried out by usingthe BLAST program (2).A restriction map of the sequenced DNA segment is shown

in Fig. 2 along with the strategy used in sequencing. Theapproximate locations of nahE and nahD were known prior to

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H pH OH

Q Q 7A- B 00 H

NADH NAD+ 11 NAD+ NADH III

02IV V

<z~4~400-- cHONADH NADx H+ H20O

+ H3CyCOO 0O- -

Vl0l VH2°Vill VII

00CD 1;OH

VI

FIG. 1. Reactions of the NAH7-encoded naphthalene catabolic pathway. Chemicals: I, naphthalene; II, cis-1,2-dihydroxy-1,2-dihydronaphtha-lene; III, 1,2-dihydroxynaphthalene; IV, 2-hydroxy-4-(2'-oxo-3,5-cyclohexadienyl)-buta-2,4-dienoate; V, cis-o-hydroxybenzylidenepyruvate; VI,2-hydroxychromene-2-carboxylate; VII, trans-o-hydroxybenzylidenepyruvate; VIII, pyruvate; IX, salicylaldehyde; X, salicylate. Enzymes: A,naphthalene dioxygenase; B, cis-1,2-dihydroxy-1,2-dihydronaphthalene dehydrogenase; C, 1,2-dihydroxynaphthalene dioxygenase; D, 2-hydroxy-chromene-2-carboxylate isomerase; E, trans-o-hydroxybenzylidenepyruvate hydratase-aldolase; F, salicylaldehyde dehydrogenase.

sequence determination: recombinant bacteria carrying theMluI-StuI fragment (bp 233 to 1225) produced trans-o-hy-droxybenzylidenepyruvate hydratase-aldolase, while bacteriacarrying the KpnI-BglII fragment (bp 646 to about 2460)produced 2-hydroxychromene-2-carboxylate isomerase (7).The segment (Fig. 3) contains three large open reading frames(ORFs); the first extends from bp 1 to 157 and is thedownstream end of an ORF, here designated nahQ, thatspecifies an unknown product and possibly fills most of thespace in the operon between nahC, the gene that encodes1,2-dihydroxynaphthalene dioxygenase, and nahE (see below);the second ORF, of 993 bp, extends from bp 209 to 1201 andcorresponds to the location of nahE; and the third ORF, of 609bp, corresponds to nahD and extends from bp 1469 to 2077.The trans-o-hydroxybenzylidenepyruvate hydratase-aldolase

gene, nahE, has a G+C content of 53.87% and encodes a

protein with a molecular weight of 36,640 (331 amino acids)and an isoelectric point of 5.43. The 2-hydroxychromene-2-carboxylate isomerase gene, nahD, has a G+C content of49.42% and encodes a protein with a molecular weight of23,031 (203 amino acids) and an isoelectric point of 5.40.By searching the GenBank Bacteria data base (1, 3), one

DNA sequence (accession number M60405) having a highlevel of homology was identified. This 9,841-bp sequence isthat of the dox (dibenzothiophene oxidation) operon of a

Pseudomonas sp. strain C18 (5). Like the NAH7 upper-

pathway nah operon (7), this operon specifies the conversion

nahQ' nahE nahDhydratase-aldolase isomerase

. ~̂ ~~~~~

0 400 800 1200 1600 2000 2400base pairs

FIG. 2. Restriction enzyme cleavage map of the segment of NAH7sequenced in this study and the sequencing strategy. A, B, and Cindicate the sites where sequencing reactions with custom primerswere initiated.

of dibenzothiophene to 3-hydroxybenzothiophene-2-carboxy-late as well as the conversion of naphthalene to salicylate. Thedox sequence that was compared with the nah sequence (Fig.4) is that extending from dox bp 6459 to 8736 and containsthree ORFs. It begins with the last 157 bp of a 636-bp ORFhaving no known function and called doxH and includes thegenes doxI and dox, putatively encoding a hydratase-aldolaseand an isomerase, respectively. Homologies are as follows(numbering is that for the nah sequence): (i) the ORFextending from bp 1 to 157, nahQ', had 90% homology todoxH; (ii) the 50 bp between nahQ/doxH and nahE/doxI had78% homology; (iii) nahE, extending from bp 209 to 1201, had94% homology with doxl; (iv) the 267 bp between the hy-dratase-aldolase and isomerase genes had 85% homology; (v)nahD, extending from bp 1469 to 2077, had 92% homologywith doxJ (a 35-bp gap in the nah isomerase gene after bp 2043was counted as one difference); and (vi) the first 84 bpdownstream from nahD had 92% homology with the corre-sponding DNA downstream from dox!, after which there wasno apparent homology. The deduced amino acid sequences ofthe hydratase-aldolases are 96% identical, while the deducedamino acid sequences of isomerases are 91% identical up tothe deletion. That the deletion from nahD occurred in NAH7and was not generated during cloning was confirmed bysequencing the same region of DNA derived from two addi-tional independently isolated clones. The 35-bp nahD deletioneliminated a stop codon present in doxJ, resulting in theproduction of an active enzyme that is 4 amino acid residueslonger than that encoded by dox.The high degree of sequence conservation between the

ORFs doxH and nahQ suggests that they may be genesencoding active and useful proteins which provide a selectiveadvantage to naphthalene-degrading bacteria that producethem. However, the function of these proteins remains un-known.Kuhm et al. (19) recently purified a trans-o-hydroxybenzy-

lidenepyruvate hydratase-aldolase from the naphthalenesul-fonate-degrading strain BN6 (Pseudomonas vesicularis DSM6383). This enzyme appears to be a trimer with a molecularweight of about 120,000 containing identical 38,000-molecular-weight subunits. The NH2-terminal amino acid sequence wasdetermined (19) and is compared here with the deducedNH2-terminal amino acid sequences for the hydratase-aldola-ses from nah and dox (Fig. 5). While the nah and dox enzymesare identical at 25 of 26 amino acid residues (96%), the enzyme

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TGCCGATATTGACGTCGAGTCGCAACTGGTTCGTCAGCATTGATATCCGGAAGTTATACCTGAAAA CCGACGCATCAGGTTA6CTTAGGGCCA CAGGAGGCTAAGCA AGTCTCTTGAC 121AlaAspIleA~spValSerAxgAsnTrpPheValrerIleAspIleArgLyaLeuTyrLeuLyeThrA~pAlaSerGlyTyrLeuGlyProGlnGluAlaLysAlaLysValThrLeuAsp

End nahQ RBS Start nahE KluICCATTGATAACATCGATCGCGATCGGACGCCAATTCTGATGACCCCTTTTAAGGCTCTCTATCTATCTAACTGCAAAGGTATTTTTATGTTr = =AAAGTTATTAAAAQCACGCGTCTT 241ProLeuIleThrSerIleAlaIleGlyArgGlnPheTer MetLeuAAnLysValIleLysThrThrAxgLeu

SailACCGCTGAAGATATCAACGGTGCCTGGACTATAATGCCCACACCGTCGACGCCTGATGCTTCTGATTGGCGCAGQACTAACACTGTGGACTTAGACGAGACTGCCCGCATAGTTG~A~AG 361ThrAlaGluAspIleAnnGlyAlaTrpThrIletletProThrProSerThrProA pAlaSerAspTrpArgS-rThrA nTh~rVaLA~pLeuAxpGluThrAlaArgIleValGluGlu

CTGATTGCAGCTGGTGTCAACGGTATTTTAAGTATGGGTACTTTTGGTGAGTGCGCCACCTTGACCTCCC(aTGATTATGTTTCGACGGTTGTCGAGA1CCTTCGTGGTCGT 481LeulleklaAlaGlyValAsnr~lylleLeuSerMetGlyThrPheGlyGluCyaAlaThrLeuTh~rTrpGluGluLy&ArgA pTyrValS-rThrValVal~luThrIleArqGlyArg

Cla3GTGCCTTATTTCTGTGGCACGACGGCCCTGAATACCCGAAATCTCCGCCAGACCCCaAGC4TTATCGATATTGGCGCTAACGGCACCATGCTAGGCGTGCCGATGTGGGTGAAGATG 601ValProTyrPheCynGlyTh~rThrAla~veuAsnThrA~rgGluVal~leArgGlnTh~rArgGluIeuIle~pIleGlyA3laAanGlyTh~rMetIeuGlyVal~lroMetTrpValLysMet

K4nIGACCTGCCCACAGCGGTCCAGTTCTATCGTGATGTTGCAGGCGCGGTACCGCAGGCTGCCATTGCGATTTACGCCAACCCCGAAGCATTCAAATTCGACTTCCCTCGCCCATTTTGGGCA 721A~spLeuProThrAlaValGlnPheTyrArgAspValAlaGlyAlaValProGluA]laAlaIlehlaIleTyrAlaAanProGluAlaPheLyaPheA~spE'heProArgProPheTrpAla

NoI. . . . . . . . .

GAGATGTCCAAAATTCCTCAGGTAGTGACTGCCAAGTATCTAGGCATCGGAATGCTTGACTTGGACCTGAAATTGGCGCCTAAQATCCGCTTCCTTCQQCCGAGGACGACTATTACGCG 841GluMetSerLysIleProGlnValValThrAlaLysTyrLeuGlyIleGlyMetLeuAspLeuAspLeuLysLeuAlaProAsnIleArgPheLeuProHiaGluAspA~spTyrTyrAla

NotIGCCGCACGCATCAATCCCGAGCGCATAACCGCGTTCTGGTCAAGCGGGGCCATGTGCGG;CCCGGCTACCGCTATCATGTTGCGTGATGAAGTGGAGCGGGCCAtACATACCGGTGACTGG 961AlaAlaArgIleAsnProGluArgIleTh~rAlaPheTrpSerSer~lyAlaMetCys~lyProAlaThrAlaIleMetLeuArgAspGluValGluArgAlaLysSerThrGlyAxpTrp

ATCAAGCCAAAGCCQTCTCCGATGATATGCGTGCAGCCGATTCGACATTGTTTCCGCGTGGCGACTTTTCGGAGTTCTCGAAGTATAACATCGGGCTTGAAzAGGCACGGATGGACGCG 1081IleLyaAlaLyzAlaIle~erAspAspM-tA~rgAlaAlalspS-rThr~euPh ProArgGlyAspPhe8-rGluPheS-rLysTyrA nIleGlyLouGluLyzAlaArgM*tA~spAla

End nahEGCTGGTTGGCTCAAGGCTGGTCCCTGCCGTCCTCCCTACAATCTTGTTCCAGAAGA TTACCTCGTTGGTGCQCGAACGCAGGCGTGGGGCCGCGCTGCACGCTAATCGTA 1201AlaGlyTrp~euLynAlaGlyProCyaArgProProTyrAsnLeuValProGluAzpTyrLeuValGlyA]laGlnLys~erGlyLyaAl~aTrplilaAlaLeu~isilaLysTyrSerLys

StuITAATTAAAGTAGTTCACCTCCGCAGGCCTGAGCGCGAGGGTTACGAACACGCTGAGCGGTGCGCGAAGTAAGTGAGTTAAAGCTCATCTCTTGCGCCAGGCACTGCTAGATC Q=UCAAAGT 1321Ter

TAGCTGATCTGGCAGTCTCGAAAATTTTGGCGAA3TGATCTTAGGAATGCGGGATAAAGGCGTACACCGTAACGATGGGGGTGTGCCGTTCATGTTGAACGACGCCGCTATTGCGCC 1441

RBS . Start nahD HindIIIGACTTCTCTTCTTCGGAGTGTTTGATTGTGATTGTCGATTTTTATTTCGATTTTTTGAGTCCGTTCTCTTACTTGGCCAATQCAGCCTTGTCAAGCTTGCGCAAATTATGGCCTTACC 1561

)EtIleValAapPheTyrPheAspPheLeuSerProPheSerTyrLsuAlaAanGlnArgLeuSerLyaLouAlaGlnAspTyrGlyLeuThr

* . . .........HindIII

GTCGGTTATGGGGAGAA CATGGCATGTCGATGGGTAAAGCTTGCCTGCCCTGGTCATCTGAACTAGGCTGGATCGTAGTGTTCGAGCACTTTCTCAGTAGCAATGGCAACAGGAGG 1921AI~lerTyrp~nlyau~yleAsLealrvArgpaLeu~luerLa~euolyasVal~e~uy~uly~o~r~nrpAp~eurg~e~al~heIlu~iss~hLeuySeraeAspelnAralThrplu

. . . . . . . . . .Xho

CAGGTATGACGCAGACACATGCGTTCCACGAGCGACTCAAGGTGCGGTGTGCCAACATTGTTTTATGGCACTGAAGTGGTCGGGGC~GGACCGCTGCTATTCTGCATCGAGTAGCGAAT 18041AglneTyr~Glu~lnehroi~l~a~eeurgValPhe~r~as~rsS~Glyalro~Thsn~etPhe~eurlyrspGluye~rlaGlyAsa~nAspArg~Leu~he~eLeun~lu~eAlahet

*End nahD III

GGCGCTTGTGCC=GCAATGCCGATTT~AzAGTATTGTCTGATC$GTTATTCTCGAT^GCAGTGCTTGGTCGAA TGTTCGAGCGGATACTGAA CAGCTA AATGCGGACTACAQGAG 1216

GlyArgLeuCyaArgGlnksrAlaAspLeuSerSerTerXhoI ~ ~ 28CAGGCTCATGACGGCAMGGCGGCQCGAGCGTGCAAGGTAGTTTCGCTGTGCATCGATGTTTTTAAGCTGCTGATTTCGTGCCTGGGaCTGACTCTCTGGTTTGGTCGTGACGCGCCAG 2281

* . . . . . . . . . . .NhI

ATAGGTTTCCAATCTCGCGTTGTACCTGACQAGAGATACGGGTGTTTATGACGGAATTGCTGATAQAGCGCATGACGCCATGCTGCTTQACTTCGAGTGTACTQACGACCTCGACGCTAG 2401

CAATCTCAACGAGCCAGATGACCGCCAGGGCGGAAC 2437

FIG. 3. Sequence (5' to 3') of the DNA segment carrying nahQ', nahE, and nahD and the deduced amino acid sequences of the proteinsencoded by the ORFs. Potential ribosome binding sites (RBS) are underlined.

from strain BN6 is identical to the other two enzymes at only isopropyl-3-D-thiogalactopyranoside. From the sequence dem-11 of 26 residues (42%). The BN6 enzyme also appears to have onstrated here (Fig. 3), it can be seen that the nahE ORFundergone the removal of six amino acids from the NH2 end. begins 24 bp upstream of the MluI cleavage site. In pRE701Determination of whether the nah- and dox-encoded enzymes these 24 bp have been removed and replaced by 24 bp of theare similarly processed will await their purification and NH2- P-galactosidase gene and polylinker (ATG ATT ACG AATterminal sequencing. In a previous study (7), the MluI-StuI TCC CGG GGA TCC), which encode the eight amino acidsfragment (bp 233 to 1225; Fig. 3) was inserted in the pUC19 Met Ile Thr Asn Ser Arg Gly Ser. Apparently the missing(30) derivative, pUCBM20, to produce pRE701. Escherichia NH2-terminal amino acids are not required for enzyme activ-coli JM1O9(pRE701) produced a high level of hydratase- ity.aldolase activity (1.55 pLmol min- mg-') when induced with The high degree of homology between nah and dox operons

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TGCCGATATTGAC GTGAGTCcCAaCTGGTTCTA ccsTATACCGGaAGATACCTcccGAcsAACCGCAAGGTTACTTAGGGCCACG GTAACAGT TCTTQ&CT AC TGGT T G G CG

End nahQ/doxff . Start nahz/doxlCCQTTGATAACATCGATCGCGATCGGC^ACCCAATTCTG&iTGACCCCTTTTAAGGCTCTCTATCTATCTAACTGCAAAGGGTATTTTTATCTTGAATAAAlTTATTAAAACCACGCGTCTT

AC T TT TG A T T C G CC C A G GT

ACCGCTGAGTTCAGGTG CCTGGACTATAATG CCCACACCGTCGACGCCTGATG CTTCTGATTGGCGCAaGACTAA CACTGTGGACTTAGACGGA"TCTcGCCGATAGTTGAAGAGC C C GC C A

CTGATTGCAGCTGGTGTCAACGGTATTTCCTAGTACTCGG~GTTACTTT=GRGCGCCAGTGATTGTTCACTGGTTGTCGAIGACCQTTCGTGGTCGTC T A C A C

GTGCCTTATTTCTGTGGCACGACGGCCCTGAATACCCCGAGACTQTCCGCCAGACCCCWAACTTATCGATATTGGCGCTAALCGGCACQATGCTACGCGTGCCGATGTGCCTGUAGTGA T A C T C G

GILCCT(;CCACAGCGGTCCAGTTCTATCGTGATGTTGCAGGCGCGGTACCGGa^GCTGCCATTGCGATTTACGCCAACCsCGAACTTCAAATTCGACTTCsCTCGCCCQTTTTGGGCAT T AT A G C

GAGATGTCCAAAATTCCTCAGGTAGTGACTGCCAAGTATCTAGGCATCGGAATGCTTGACTTGGACCTGAAATTGGCGCCTAACATCCGCTTCCTTCQCGCCAGGACGALCTATTACGCGG G G C A C C A T

GCCGCACGCATCAATCeCGAGC~CATAACCGCGTTCTGTCAAGCGGGGCCATGTGCGGCsCGGCTACCGCTATQATGTTGCGTGATrzAGTGGAGCGGGCC~aGAACCGGTGACTGGC C T C

ATCAAGGCCLAACAGTCTCCGATGALTATGCGTGCAGCCGATTCGALCATTGTTTCCGCGTGGCGACTTTTCGGAGTTCTCGAAATAAACATCGGGCTT=QAAGCiCGATGGACGCGGC C T T

End nahEGCTGGTTGGCTCAAGGCTGGTCCCTGCCGTCCTCCCTACAATCTTGTTCCAQAGTTACCTCGTTGGTGCACAQA =AGCQGGGTGGGCCGCGCTGCACGCTAAATA QG;TAAA

G G C C C T TEnd doxI

T ATTAAAGTAGTTCACCTCCGCAGGCCTGAGCGCCAG^GGTTACGAACACGCTGAGCGGTGCGGGAAGTAAGTGAGTTAAAGCTCATCTCTTGCGCCAGGCACTGCTAGATQCcAGCAGTG G A T AC GG C G - A T C A T T C

TAGCTGATCTGGC AGTCTCGAAAATTTTGCAACG TCTAGAATGCGGAAGCGACGTAACGATGGGGGTCTGCCC TTCATCTTGAACCACGCCCGCTATTGCGCCT T A G A C A G A C C- A G A TTTT T

Start nahD/doxJ.GACTTCTCTTCTTCGGAGTGTTTGATTGTGATTGTCGIAT TTATTTCGILTTTTTTfGATCCGTTCTCTTACTTGGCCAATCAGCCGCTTGTCAAAGCTTGCGCAAGATTATGGCCTTACC

G - C T T C T T

ATTC2TTATAAoCGCATCGATTTGGCGCGGGTCAAAATAGCCQTTGGAAACGTTGGTCCATCCAATCGCGACTTGAAAGTCAAATTGGACTATTTGAAAGTAGALTTTGCAACGGTGGGCCT A A T C T C T GC

CAGCTTTACGGAATACCGCTGGTATTCCCAGCTAACTACAACAGCCGALCGGAkTGAATATTGGGTTTTATTACTCGGGGGCCGAGGGCAGGCCGCTGCCTATGTGAATGTAGTATTTAATG A T C C A AT A AT G

CCC220CCAOAACTCGTTGAGTGCTGCCCTGGTATCTGAAAAGCTAGGCTGGGATCGTAGTGCTTTCGAGCACTTTCTCQGCAGCAATCCCGQAACAGAGT T A C C G A G

AGGTATGACGAGCAGAoCACATCCGCQLTCGAGCGCAAGGTGTTCGGTGTGCCAASCA~TGTTTTTGGGCGATGAAALGGTGGGGGGn~A~ACACCGTCTATTCQTGCTCGAGAGCCCAALTGG A A A T A G

End doxJ, End nahD . .GG-----------------------------------GCGCTTTGCCCCCAAAATGCCGCTTTAAGTAGTTGNTCTGATCGTTATTTGCTCGATGAGTCCCTTTCAAAATCAGCGCATAAGGTGCGCCTGTAAATCCACAATAGTCCCTACGGA T A G C ACAG C

1216579

2416699

3616819

4816939

6017059

7217179

8417299

9617419

10817539

12017659

13217778

14417897

15618016

16818136

18018256

19218376

20418496

21268616

CTGAAGTCAGCTAAAAATGCCGCACTACTTCAGGCCQTGCTTCAGGGACGGTQCAGCGCGACCGGCGTGTGCAAGGTAGTTGTCGCGTGAGTCATGTTTTTAAGCTGCTATTTTCAGTGC 2246A ATCCTG GC AC AAAGTTTTACCTGTAATTGTCCACCTA TCCGAGTTTGGA TGGTA C GACTC ATGCGACCAGCGAT 8736

FIG. 4. Comparison of the sequence of nah DNA with the sequence of dox DNA. The complete nah sequence (5' to 3') from bp I to 2246 isshown. Below the sequence are indicated only those bases for which dox differs from nah. Start and stop codons are underlined.

followed by a complete lack of homology downstream from theisomerase genes suggests that the genes that encode thenaphthalene upper pathway may have moved as a unit fromone replicon to another. Such movable genetic elements oftenhave features such as repeated DNA sequences that occur attheir ends and that are involved in their mobilization. To lookfor such repeated DNA sequences, a search for homologiesbetween the nah sequence obtained here, the dox sequence (5),and the recently published sequences of DNAs encodingnaphthalene dioxygenase (26) from the NAH7 plasmid (Gen-Bank accession number M83949) and from pDTG1 (27) of P.putida 9816-4 (4) (GenBank accession number M83950) and

encoding polycyclic aromatic hydrocarbon dioxygenase fromthe chromosome of P. putida OUS82 (18, 28) (GenBankaccession number D16629) was carried out (Fig. 6). Thesecomparisons (Fig. 7) demonstrated that there is a segment of84 bp located just upstream of the nah promoter (25) andupstream of nahAa in NAH7 that has 76% homology withDNA downstream from nahD and dox. This segment, havingsuffered a 9-bp deletion, is also present in DNA upstream ofthe pah operon, but it is not present in DNA from pDTG1. AsSimon et al. (27) showed, homology between DNA fromNAH7 and pDTG1 begins just upstream of the nah promotersequence; this is immediately downstream from the 84-bp

NAH e alIleyhrrhrArgLeuThrAlaGluAspIleAsnGlyAlaTrpDOX kfee leMetLy.sSer hr sp leAsnGlyAlaTrpBN6 AlaArg LeuValLysProAspp alLys IyAIITrp

NAH hrleMetProThrProSerThrProAspAiaSerAspTrpArgSerThr sn rVaDOX le roThrPro erThrProAspAlaaerAspTrpAraSerThr laBN6 Ala lleiaLysAspksgAlafhr

FIG. 5. Comparison of the NH2-terminal amino acid sequences of the hydratase-aldolases from the naphthalenesulfonate-degrading strain BN6and the deduced sequences of those encoded by nahE and by dox.

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pDTG1 nah I EEl

OUS82 pah Aa b Ac Ad B I F

C18 dox &]I B I D I E I F I G I l H I I I E

NAH7 nah I| Aa c Ad B F I C Q l E I

Cuo u - 3 cr

l I l l l l l l l l l Il I0 1 2 3 4 5 6 7 8 9 10 11 12

kilobase pairs

FIG. 6. Alignment of genes of naphthalene upper-pathway operons from NAH7 (nah), pDTG1 (nah), P. putida OUS82 (pah), andPseudomonas strain C18 (dox). The restriction map and relative locations of genes from NAH7 are from reference 7. The genes nahA to nahFencode the enzymes that catalyze the corresponding reactions A to F shown in Fig. 1. Genes indicated by boxes have been sequenced (5, 16, 27,28). The solid boxes indicate the locations of the short homologous sequences compared in Fig. 7.

repeated element in the NAH7 DNA. Thepah DNA has a highdegree of homology with nah DNA from NAH7 (93%), andthis extends upstream of the sequence shown in Fig. 7 for over300 bp. At the other end of the operon, the repeated sequenceextends downstream from the termination codon, TGA, ofnahD, to the border of nah and dox homology. These datasuggest that genes encoding the naphthalene upper pathwaymay have moved as a unit between different replicons by amechanism involving these 84-bp sequences. This mobilizationmechanism would have differed from the mobilization of thesegenes by transposition of Tn4655, the 37.5-kb (now defective)transposon of NAH7 that extends from about 2 kb to the leftof nahAa and encompasses the upper-pathway genes and thegenes that encode the enzymes of the lower pathway (29).

Naphthalene dioxygenase (Fig. 1, enzyme A) is a four-component enzyme consisting of a two-subunit terminal oxy-genase, a ferredoxin, and a ferredoxin reductase (9, 10, 14, 15);these components are encoded by nahAc, nahAd, nahAb, andnahAa, respectively (Fig. 6). It is likely that the naphthalene/dibenzothiophene dioxygenase encoded by the dox operon hasa similar composition, yet from the sequences, it can be seen

that a dox gene corresponding to nahAa is missing (Fig. 7).Despite the fact that it lacks the dox ferredoxin reductase gene,the cloned dox operon still specifies activity toward dibenzo-thiophene and naphthalene (5). This is in agreement withprevious studies showing that the cloned naphthalene dioxyge-nase (11, 20, 27) and isopropylbenzene dioxygenase (8) haveactivities in the absence of a cloned ferredoxin reductase; it hasbeen suggested that ferredoxin reductase activities may beprovided by host cells (8, 11). This means, however, that thecomplete dox operon was not cloned and sequenced and that itis not yet possible to search for upstream homologous regionsin the dox operon.The two 84-bp directly repeated sequences are located about

10 kb apart in NAH7 and encompass the entire upper-pathwayoperon; homologous recombination between the two se-quences may be responsible for some of the differences(deletions and rearrangements) observed between SAL (salic-ylate catabolism) plasmids that lack the upper-pathway genesthat encode the metabolism of naphthalene to salicylate andNAH7 (17, 21, 32).

Several papers (5, 12, 22) have implicated plasmids in the

5 # -70GGGTTTGGTGC-CTTGCCGCCTTTCAC -T---ATCGCCACCTCGCTATCACCAACATTCCTTCAGGGCTGAGTGCGTAATTTTCTGAAGGGGAGCCAGGTTATG AGTATTCACATTGGT

AGAAATGAGCGCGC Gkc~ccGATcGcc-TTTG&TcQATTcTccGcTTTCAAA GTGCcAGAGCCTGAAGTcAccGGAA&TAC-CCACA "'TC~AACATTCATGCTGGTAGAAATGAGCG- CCGATCGCC-TTTGTATcTCTccCCTTTCAAAATGGGCGGGGCTGAAGTcAG ccA cTaGTAcTTc&G ATCG TCACGCTGGT

CGATTTAAGTA- GATCTGATCGTTATTTGCTCGATG&GTCGCTTTCAAAATCAGCGGATACTGAAGTCA.GCTA&AAATGCGGGACTACTTC&GG CATGCTTCAGGGACGGTCAGGG

CGATATAAGTG-G CTGATCTTATTTGCT CGCTTTCAAAATCACCCTACTCAMTCAGATAAAAATGCGGGACTACTTCAGG&TCCTGTGCGACACAA&GTTTT

pDTG1 upstream of nahAa

OUS82 upstream of pahAa

NAH7 upstream of nahAa

downstream from nahD

downstream from doxJ

GATAAACAACCTC&CTTATGCGTTATTG&CATATAACGTCGTATTC&CGATTATTTACCATATAAGTCTTATAATAACGAAGCCATATT- ATGCAACTCCTCATACAACCGAACAATCGC pDTG1 nahAs

GATmTAAAATTCAACTATGcTTTATTGACAATAAAAGcACACTCACCATcATcACGAATACAA&TcTTATAA-AATTAAGCcGGATTI ATGAAACTTCTCATACAGCCAAACAATCGC OUS82 pahAa

GATAAACAAATTCAACTATGCTTTATTGACAAATAAAAGCACGCTCACCATCATCGCGAATACAAATCTTATAAAAATTAAGCCGGATTS CGC NAH7 nahAa_oc en an

transcription begin nshAa & pahAa

FIG. 7. Comparison of naphthalene upper-pathway operon DNA sequences from the beginning of the nah operons from pDTG1 and NAH7,the beginning of the pah operon of P. putida OUS82, the end of the nah operon from NAH7, and the end of the dox operon from Pseudomonasstrain C18. The box indicates the approximately 84-bp homologous sequences present in all DNAs except that from pDTG1. The solid arrowsindicate 5-bp inverted repeats in dox. Partial boxes indicate the beginnings of promoter sequences and ferredoxin reductase genes. NAH7 promotersequences were identified by Schell (25).

NOTES 7761

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7762 NOTES

transformation of dibenzothiophene by a pathway analogousto the naphthalene catabolic pathway. The plasmids alsoencode the degradation of naphthalene, and it is likely, on thebasis of sequence data presented here and elsewhere (5, 27,28), that many if not all of these dibenzothiophene catabolicplasmids are actually naphthalene catabolic plasmids, in manycases closely related to NAH7.

Nucleotide sequence accession number. The DNA se-quences obtained in this study are available from GenBank(accession number U09057).

I am grateful to the DNA Sequencing Core Laboratory, Interdisci-plinary Center for Biotechnology Research (ICBR), University ofFlorida, Gainesville (Ernesto Almira, Director) and the DNA Synthe-sis Laboratory, ICBR, University of Florida, for providing high-qualitysequence data and custom DNA primers, respectively. I thank P. J.Chapman for his encouragement and I. C. Gunsalus and T. J. Lessiefor reading the manuscript.

REFERENCES1. Alting-Mees, M. A., and J. M. Short. 1989. pBluescript II: gene

mapping vectors. Nucleic Acids Res. 17:9494.2. Altschul, S. F., W. Gish, W. Miller, E. W. Myers, and D. J. Lipman.

1990. Basic local alignment search tool. J. Mol. Biol. 215:403-410.3. Benson, D., D. J. Lipman, and J. Ostell. 1993. GenBank. Nucleic

Acids Res. 21:2963-2965.4. Davies, J. L., and W. C. Evans. 1964. Oxidative metabolism of

naphthalene by soil pseudomonads. The ring-fission mechanism.Biochem. J. 91:251-261.

5. Denome, S. A., D. C. Stanley, E. S. Olson, and K. D. Young. 1993.Metabolism of dibenzothiophene and naphthalene in Pseudomo-nas strains: complete DNA sequence of an upper naphthalenecatabolic pathway. J. Bacteriol. 175:6890-6901.

6. Dunn, N., and I. C. Gunsalus. 1973. Transmissible plasmids codingearly enzymes of naphthalene oxidation in Pseudomonasputida. J.Bacteriol. 114:974-979.

7. Eaton, R. W., and P. J. Chapman. 1992. Bacterial metabolism ofnaphthalene: construction and use of recombinant bacteria tostudy ring cleavage of 1,2-dihydroxynaphthalene and subsequentreactions. J. Bacteriol. 174:7542-7554.

8. Eaton, R. W., and K. N. Timmis. 1986. Characterization of aplasmid-specified pathway for catabolism of isopropylbenzene inPseudomonas putida RE204. J. Bacteriol. 168:123-131.

9. Ensley, B. D., and D. T. Gibson. 1983. Naphthalene dioxygenase:purification and properties of a terminal oxygenase component. J.Bacteriol. 155:505-511.

10. Ensley, B. D., D. T. Gibson, and A. L Laborde. 1982. Oxidation ofnaphthalene by a multicomponent enzyme system from Pseudo-monas sp. strain NCIB 9816. J. Bacteriol. 149:948-954.

11. Ensley, B. D., T. D. Osslund, M. Joyce, and M. J. Simon. 1987.Expression and complementation of naphthalene dioxygenaseactivity in Eschenichia coli, p. 437-455. In S. R. Hagedorn, R. S.Hansen, and D. A. Kunz (ed.), Microbial metabolism and thecarbon cycle. Harwood Academic Publishers, New York.

12. Focht, J. M., and D. W. S. Westlake. 1990. Expression of diben-zothiophene-degradative genes in two Pseudomonas species. Can.J. Microbiol. 36:718-724.

13. Gibson, D. T., and V. Subramanian. 1984. Microbial degradation ofaromatic hydrocarbons, p. 181-252. In D. T. Gibson (ed.), Microbialdegradation of organic compounds. Marcel Dekker, Inc., New York.

14. Haigler, B. E., and D. T. Gibson. 1990. Purification and propertiesof NADH-ferredoxinNAp reductase, a component of naphthalenedioxygenase from Pseudomonas sp. strain NCIB 9816. J. Bacteriol.172:457-464.

15. Haigler, B. E., and D. T. Gibson. 1990. Purification and propertiesof ferredoxinNAP, a component of naphthalene dioxygenase fromPseudomonas sp. strain NCIB 9816. J. Bacteriol. 172:465-468.

16. Harayama, S., and M. Rekik. 1989. Bacterial aromatic ring-cleavage enzymes are classified into two different gene families. J.Biol. Chem. 264:15328-15333.

17. Heinaru, A. L., C. J. Duggleby, and P. Broda. 1978. Molecularrelationships of degradative plasmids determined by in situ hybrid-ization of their endonuclease-generated fragments. Mol. Gen.Genet. 160:347-351.

18. Kiyohara, H., S. Torigoe, N. Kaida, T. Asaki, T. lida, H. Hayashi,and N. Takizawa. 1994. Cloning and characterization of a chro-mosomal gene cluster, pah, that encodes the upper pathway forphenanthrene and naphthalene utilization by Pseudomonas putidaOUS82. J. Bacteriol. 176:2439-2443.

19. Kuhm, A. E., H.-J. Knackmuss, and A. Stolz. 1993. Purificationand properties of 2'-hydroxybenzalpyruvate aldolase from a bac-terium that degrades naphthalenesulfonates. J. Biol. Chem. 268:9484-9489.

20. Kurkela, S., H. Lehvaslaiho, E. T. Plava, and T. H. Teeri. 1991.Cloning, nucleotide sequence and characterization of genes en-coding naphthalene dioxygenase of Pseudomonas putida strainNCIB9816. Gene 73:355-362.

21. Lehrbach, P. R., I. McGregor, J. M. Ward, and P. Broda. 1983.Molecular relationships between Pseudomonas INC P-9 degrada-tive plasmids TOL, NAH, and SAL. Plasmid 10:164-174.

22. Monticello, D. J., D. Bakker, and W. R. Finnerty. 1985. Plasmid-mediated degradation of dibenzothiophene by Pseudomonas spe-cies. Appl. Environ. Microbiol. 49:756-760.

23. Ribbons, D. W., and R. W. Eaton. 1982. Chemical transformationsof aromatic hydrocarbons that support the growth of microorgan-isms, p. 59-84. In A. M. Chakrabarty (ed.), Biodegradation anddetoxification of environmental pollutants. CRC Press, Boca Ra-ton, Fla.

24. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencingwith chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA74:5463-5467.

25. Schell, M. A. 1986. Homology between nucleotide sequences ofpromoter regions of nah and sal operons of NAH7 plasmid ofPseudomonas putida. Proc. Natl. Acad. Sci. USA 83:369-373.

26. Serdar, C. M., and D. T. Gibson. 1989. Isolation and character-ization of altered plasmids in mutant strains of Pseudomonasputida NCIB 9816. Biochem. Biophys. Res. Commun. 164:764-771.

27. Simon, M. J., T. D. Osslund, R. Saunders, B. D. Ensley, S. Suggs,A. Harcourt, W.-C. Suen, D. T. Gibson, and G. J. Zylstra. 1993.Sequences of genes encoding naphthalene dioxygenase in Pseudo-monas putida strains G7 and NCIB 9816-4. Gene 127:31-37.

28. Takizawa, N., N. Kaida, S. Torigoe, T. Moritani, T. Sawada, S.Satoh, and H. Kiyohara. 1994. Identification and characterizationof genes encoding polycyclic aromatic hydrocarbon dioxygenaseand polycyclic aromatic hydrocarbon dihydrodiol dehydrogenasein Pseudomonas putida OUS82. J. Bacteriol. 176:2444-2449.

29. Tsuda, M., and T. lino. 1990. Naphthalene degrading genes onplasmid NAH7 are on a defective transposon. Mol. Gen. Genet.223:33-39.

30. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. ImprovedM13 phage cloning vectors and host strains: nucleotide sequencesof the M13mpl8 and pUC19 vectors. Gene 33:103-119.

31. Yen, K.-M., and I. C. Gunsalus. 1982. Plasmid gene organization:naphthalene/salicylate oxidation. Proc. Natl. Acad. Sci. USA 79:874-878.

32. Yen, K.-M., and C. M. Serdar. 1988. Genetics of naphthalenecatabolism in pseudomonads. Crit. Rev. Microbiol. 15:247-268.

J. BACTERIOL.

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