Vol. 172, No. 12

Complete Nucleotide Sequence and Polypeptide Analysisof Multicomponent Phenol Hydroxylase from

Pseudomonas sp. Strain CF600INGRID NORDLUND, JUSTIN POWLOWSKI, AND VICTORIA SHINGLER*

The Unit for Applied Cell and Molecular Biology, The University of Umea, S-901 87 Umea, Sweden

Received 21 May 1990/Accepted 12 September 1990

Pseudomonas sp. strain CF600 metabolizes phenol and some of its methylated derivatives via a plasmid-encoded phenol hydroxylase and meta-cleavage pathway. The genes encoding the multicomponent phenolhydroxylase of this strain are located within a 5.5-kb SacI-NruI fragment. We report the nucleotide sequenceand the polypeptide products of this 5.5-kb region. A combination of deletion analysis, expression ofsubfragments in tac expression vectors, and identification of polypeptide products in maxicells was used todemonstrate that the polypeptides observed are produced from the six open reading frames identified in thesequence. Expression of phenol hydroxylase activity in a laboratory Pseudomonas strain allows growth onphenol, owing to expression of this enzyme and the chromosomally encoded ortho-cleavage pathway. Thissystem, in conjunction with six plasmids that each expressed all but one of the polypeptides, was used todemonstrate that all six polypeptides are required for growth on phenol.

Soil microorganisms, notably Pseudomonas spp., are ca-pable of degrading a wide variety of aromatic compounds(9). These metabolic capabilities are often encoded on cata-bolic plasmids (7). Pseudomonas sp. strain CF600 can catab-olize phenol and some of its methylated derivatives, o-, m-,and p-cresol and 3,4-dimethylphenol, as the sole carbon andenergy source. The phenol-dimethylphenol meta-cleavagepathway of this strain is encoded on a large Inc P2 plasmiddesignated pVI150 (22). All the structural genes of thispathway have been cloned and expressed from a 20-kbfragment, and the genes encoding the first four enzymaticsteps have been individually mapped and expressed andtheir nucleotide sequences have been determined (2, 3, 17;see below).Like other phenol-utilizing bacteria, Pseudomonas strain

CF600 metabolizes phenol by hydroxylation to catechol (22).This central intermediate is further dissimilated in the dif-ferent strains by either the meta- or ortho-fission pathway(4). The phenol hydroxylase of CF600 is unusual in that it isa multicomponent enzyme (22). All previously characterizedbacterial hydroxylases of aromatic compounds that alreadybear a hydroxyl group are single-component flavoproteins(1). In this communication we report the complete nucleo-tide sequence and polypeptide analysis of a 5.5-kb phenolhydroxylase-encoding region. We also demonstrate that sixdistinct polypeptides produced from this DNA region are allrequired to allow PB2701, a pseudomonad with a chromo-somally encoded ortho-cleavage pathway for catechol catab-olism, to grow on phenol.

MATERIALS AND METHODS

Bacterial strains and culture conditions. Escherichia coliK-12 strains DH5 (10) and XL1-blue (Stratagene), used forconstruction and maintenance of plasmids, and maxicellstrain CSR603 (20) were cultured at 37°C. Pseudomonasstrain PB2701 (22) was cultured at 30°C. Luria broth (16) wasused as the complete medium, M9-salts (16) supplemented

* Corresponding author.

with 2.5 mM phenol as the sole carbon source, and 0.5 mMisopropyl-p-D-thiogalactopyranoside (IPTG) was used as theminimal medium. Methionine assay medium (Difco) wasused in maxicell analysis of polypeptides. Plasmids wereintroduced into E. coli strains by the procedure of Kushner(14) and into Pseudomonas strain PB2701 by electroporationwith a Bio-Rad Gene Pulser. Ampicillin at 100 ,ug/ml andcarbenicillin at 1 to 2 mg/ml were used for selection ofplasmid-encoded ,-lactamase in E. coli and Pseudomonasstrains, respectively.

Plasmids expressing phenol hydroxylase genes. Plasmidswere constructed by using the broad-host-range tac expres-sion vectors pMMB66HE and pMMB66EH (8), and deriva-tives thereof, pMMB66HEA and pMMB66EHA (22), thatlack expression of the plasmid-encoded lacdq repressor gene.Subfragments of the DNA shown in Fig. 1 were cloned intothe polycloning site of pMMB66HE or pMMB66EH andsubsequently transferred, on HindIII-EcoRI fragments, topMMB66HEA or pMMB66EHA. Coordinates of fragmentscloned in each plasmid are given with respect to Fig. 1A. AA symbol distinguishes pMMB66A-based plasmids frompMMB66-based plasmids carrying the same DNA fragment.Plasmids pVI221, pVI257, pVI258, and pVI261 have beendescribed previously (3, 22) and carry SmaI-NruI (kb 4.4 to5.55), BamHI-NruI (kb 1.2 to 5.55), EcoRV-EcoRV (kb 0 to4.6) and BglII-NruI (kb 0.35 to 5.55) fragments, respectively.Plasmids pVI270 and pVI275 are Bal31 deletion derivativesof pVI261 whose endpoints have been determined by DNAsequencing. pVI276 was generated by PstI digestion andreligation of pVI261. pVI273 and pVI277-288 are subclonesof pVI261 generated by standard techniques. The fragmentscloned in these plasmids are as follows: pVI273, SmaI-NruI(kb 1.1 to 5.55); pVI277, SalI-NruI (kb 2.4 to 5.55); pVI278,XmnI-NruI (kb 2.75 to 5.55); pVI279, BglII-NruI (kb 3.8 to5.55); pVI281, PvuII-BamHI (kb 0.65 to 1.2); pVI283, BglII-XhoI (kb 0.35 to 2.45); pVI284, SmaI-XhoI (kb 1.1 to 2.45);pVI285, PstI-XmnI (kb 2.15 to 2.75); pVI287, XhoI-SauI (kb2.45 to 4); and pVI288, SauI-EcoRV (kb 4 to 4.6). pVI289contains the 0.86-kb HindIII-BamHI fragment of pVI261,with the recessive end of the BamHI site filled in with T4

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A

lane

1 pMMB66HEA2 pVI261A

3 pVI270A4 pVI273A

5 pVI275A

6 pVI257A

7 pVI276A

8 pVI277A

9 pVI278A

10 pVI279A

11 pVI221A

EScPBgPv SmB

0 1

PSaPSaX XmAs

I I I II I

2 3

BgSu XPvSm Pv E

A4 ) IICCN

5 kbP0 P1 P2 P3 P4 P5

+ + + + + +

+ +- + + + +

+ + + + +

+ + + + +

+ + + +

+ + + +

+ + +

+ +

+ +-

B 1 2 34 5 6 7 8 9101194-67-

43-_ _

_---

p3

--'DP1

20

14 -

6 -

P4_,-. low_ w! -11,-PO

-.", ^-.P2FIG. 1. Deletion analysis of the polypeptides produced from the phenol hydroxylase-encoding region. (A) The extent of the DNA in the

deletion series is shown with a summary of the polypeptides produced from each plasmid. Restriction recognition sites: As, Asp718; B,BamHI; Bg, BglII; E, EcoRV; N, NruI; P, PstI; Pv, PvuII; Sc, SacI; Sa, Sall; Su, Saul; Sm, SmaI; X, XhoI; Xm, XmnI. (B) Maxicell analysisof plasmid-encoded polypeptides. Polypeptides from CSR603 harboring the plasmids indicated in panel A were separated by 10 to 20%gradient SDS-PAGE with 32 mM NaCl in the analytical gel and 3 M urea in the sample buffer. Molecular mass standards are shown inkilodaltons. PO to P5 indicate polypeptides produced from the cloned DNA.

polymerase, cloned between the HindIII and the T4 poly-merase-treated PstI sites of pVI276. pVI290 was generatedby cloning a 1.93-kb HindIII-Bal31 fragment between theHindIII and T4 polymerase-treated Sall sites of pVI277.pVI291 contains the HindIII-XmnI fragment of pVI261cloned between the HindIII and T4 polymerase-treated SalIsites of pVI279. pVI292 was generated by partial XhoIdigestion of pVI261 followed by treatment with T4 polymer-ase and religation. Restriction enzymes and DNA-modifyingenzymes were purchased from Boehringer Mannheim orNew England BioLabs and used as recommended by thesuppliers.

Analysis of plasmid-encoded polypeptides. Plasmids wereintroduced into the maxicell CSR603 strain, and plasmid-encoded polypeptides were prepared, labeled with L-[35S]methionine (Amersham), and analyzed essentially as de-scribed previously (20). To aid the separation of smallpolypeptides, we performed sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) (15), whereindicated, with 32 mM NaCl added to the analytical gel and3 M urea added to the sample buffer. Size determinations ofpolypeptides were made by using the Pharmacia LMW

calibration kit (no. 17-044601) and Bethesda Research Lab-oratories prestained low-molecular-weight standards (no.6040SA).

Nucleotide sequence determinations. Nucleotide sequenceswere determined directly from plasmids by using the Strat-agene DNA sequencing kit (no. 70700). To determine thenucleotide sequence of the 5.5-kb SacI-NruI fragment, wecloned subfragments, in both orientations, into the polyclon-ing site of pBluescript SK(+) sequencing vector (Strata-gene). Ordered deletion libraries of the resulting plasmidswere generated by using exonuclease III and mung beannuclease essentially as described in the Stratagene Exo/Mung DNA sequencing manual. Each part of both strandswas sequenced at least twice.To sequence the novel junctions in plasmids pVI270,

pVI275, and pVI289-292, DNA fragments spanning the junc-tions were cloned into pBluescript SK(+) and the sequenceof one strand was determined.

Nucleotide sequence accession number. The sequence datain this paper have been submitted to the GenBank DataLibrary under accession number M37764.

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SacIfi bG(7~G GGGCGGACCTGACCAGACC CTTGIGTGAA TGCATCTGCG GCCCAGCGAG GAACATGCCG ACTTCGCTGG CATTCGGTCT 90CAGCTTCCTG GCCAGTTCGG CATCCTTCAG GCCGGACTGG TAACCATGGC GCAGGAACAA CGTTCGATGC CCAGGGTATT 180

PStI Bg1iIGACCATTTCC CGGCGAAAGC TGGCCATtGC=CAAAAC AOCAACAGCATGCGCTGTG CATGCTCTG 270GAAGTGGATC AGGTTGGTCA C ATCGGGTGC TGGATTTCAG GCTTGTACTT GATCGGCATG TAAG~GAG COCTATTTAT 360TTTTAGATGG GGAAAATCAG GGTCGCCGCT ATAGCGCAAG GCAGGCGGCG ATTCCA.GATG GGGTCATGGG AAAATCGGCA GrlTTCAC 450CTGGCGCAGC TCOCCATAGC CAGICTCAGT ATCTGGCAAA AGTCAACCAA ATGATCAATC GACGGCAGTG ATTTTAGTAT TAGAGATCAG 540

PvuIICGTTQLG= CGCCATAAGC ATTTGCTCAA GCGGCCTTGG GCAATTGATC AAATGCTTAA AAAGTCTGCG CAAGCGCGGC TTAATTTCGC 630

___> < -- ** ** ***

TCGCTCCGAT CATTCTAAAA ATTAGAAACA CATTGAAAAA CAATACCTTG AAGTCTGTTT TCAGACCTTG GCACAGCCGT TGCTTGATGT 720CCTGCGCAAG CCGCCAACCT GGAGATGACC GTGACCAATA CCCCACACC GACTTTCGAT CAGCTCACTC GTTACATCCG TGTGCGCAGC 810

M T V T N T P T P T F D Q L T R Y I R V R S

GAACCAGAAG CCAAGTTCGT CGAGTTCGAT TTCGCCATTG GTCATCCGGA GCTGTTOGTC GAGTTGGTGC TGCCGCAAGACGOCTTCGTG 900E P E A K F V E F D F A I G H P E L F V E L V L P Q D A F V

AAGTTTTGCC AGCACAACCG CGTGGTGGCA ATGGACGAAG CGATGGCCAA GGCGGTGGAC GACGACATGG TCAAGTGGCG CTXCGGCGAT 990K F C Q H N R V V A M D E A M A K A V D D D M V K W R F G D

SmaIGTOGGTCGCC GCTTGCCGA.A AGAQ:C TGAGAACCCT GCCGACAGGC AGATGGGCAT CCAACAACAA GAGGGTACGG TTGATATGAG 1080V G R R L P K D P G M S

BamHICGTAGAGATA AAGACCAATA CGGT90= GATCCGCCAG ACCTACGGCA ACCTGCAACG GCGCTTCGGC GACAAGCCGG CTAGCCGTTA 1170V E I K T N T V D P I R Q T Y G N L Q R R F G D K P A S R Y

TCAGGAAGCC AGCTACGACA TCGAAGCGGT CACCAACTTT CACTATOGCC CGCTGTGGGAOCCGCAGCAC GAGCTGCACG ATCCGACCCG 1260Q E A S Y D I E A V T N F H Y R P L W D P Q H E L H D P T R

CACGCGGATC CGCATGACCG ATTGGCACAA GGTCACCGAC CCCCGCCAAT TCTACTACGG CGCCTATGTG CAGACCCGCG CGCGGATGCA 1350T A I R M T D W H K V T D P R Q F Y Y G A Y V Q T R A R M Q

GGAAGCCACC GAACACGCCT ATGGCTTCTG CGAAAAGCGT GAGCTGCTGA GCCGTCTGCC GGCCGAGTTG CAGGCCAAGC TGCTGCGCTG 1440E A T E H A Y G F C E K R E L L S R L P A E L Q A K L L R C

CCTGGTGCCG CTGCGGCATG CCGAGCTGGG CGCCAACATG AATAACAGCA GCATCGCCGG CGACAGCATC GCCGCCACCG TGACCCAGAT 1530L V P L R H A E L G A N M N N S S I A G D S I A A T V T Q M

GCACATCTAC CAGGCGATGG ACCGCCTGGG CATGGGCCAG TACCTCTCGC GCATCGGCCT GCTGCTOGAT GGOGGCACOG GCGAGGCGTT 1620I Y Q A M D R L G M G Q Y L S R I G L L L D G G T G E A L

GGATCAAGCC AAGGCCTATT GGCTCGACGA CCCGATCTGG CAGGGCCTGC GTCGCTACGT CGAAGACAGC TTCGTGATCC GCGACTGGTT 1710D Q A K A Y W L D D P I W Q G L R R Y V E D S F V I R D W F

PstICGAGTTGGGC CTGGCGCAGA ACCTGGTGCT CGACGGCTTG =WAQCCGC TGATGTACCA GCGCTTCGAC CAATGGCTCA CAGAGAACGG 1800

E L G L A Q N L V L D G L L Q P L M Y Q R F D Q W L T E N GSalI

TGGCAGCGAT GTGGCCATGC TCACCGAGTT CATGCGCGAC TGGTACGGCG AAAGCACGCG CTGCG39G GCCATGTTCA AGACCGTGCT 1890G S D V A M L T E F M R D W Y G E S T R W V D A M F K T V L

TGCCGAAAAT GACGCTAACC GTGAGCAGGT GCAGGCCTGG CTGGAGGTCT GGGAGCCGCG TGCCTACGAG GCATTGTTGC CCCTGGCCGA 1980A E N D A N R E Q V Q A W L E V W E P R A Y E A L L P L A E

PstIGGAAGCCACC GGTATCGCCG CGCTGGATGA AGTCOGCAGC GCCTTCGCTA CTCGAZZLkAAATCGGC CTGAAAAGCC GCGAGGAATA 2070

E A T G I A A L D E V R S A F A T R L Q K I G L K S R E E

AAGCATGTCA TCACTCGTCT ACATCGCCTT CCAGGATAAC GACAACGCGC GTTACGTGGT GGAAGCGATC ATCCAGGACA ACOCCCACGC 2160M S S L V Y I A F Q D N D N A R Y V V E A I I Q D N P H A

OGTCGTCCAG CACCACCCGG CGATGATCCG TATCGAGGCC GAGAAGCGCC TGGAGATCCG CAGGGAAACC GTGGAAGAGA ACCTCGGCCG 2250V V Q H H P A M I R I E A E K R L E I R R E T V E E N L G R

SalI XhoICGCZCTGGGAC GTCCAGGAAA TGCTGGTGGA CGTAATCACC ATCGGCGGCA AC93rG&OGA GGACGATGAC C 2340A W D V Q E M L V D V I T I G G N V D E D D D R F V L E W K

GAACTAGGAG ACAAGCTCAT GGCTACCCAC AACAAGAAAC GCCTCAACCT GAAAGACAAA TACCGCTACC TGACCCGCGA TCTGGXXTGG 2430N M A T H N KK R L N L K D K Y R Y L T R D L A W

GAAACGACCT ACCAGAAGAA AGAAGACGTG TTCCCGCTGG AGCACTTCGA GGGCATCAAG ATCACCGACT GGGACAAGTG GGAAGACCCC 2520E T T Y Q K K E D V F P L E H F E G I K I T D W D K W E D P

TTcc~cCTGA CCATGGACAC CTACTGGAAA TAOCAGGCGG AGAAAGAGAA GAAGCTCTAC GCGATCTTCG ACGCCTTTGC CCAGAACAAT 2610F R L T M D T Y W K Y Q A E K E K K L Y A I F D A F A Q N N

XnnIGGTCATCAGA ACATTTCCGA TGCGCGCTAC GTCAACGCCC T C G C C GTICACCGC TGGAATACCA GGCCTTCCAG 2700G H Q N I S D A R Y V N A L K L F L T A V S P L E Y Q A F Q

GGCTCT~rGC GGGTIGGCG GCAGrTCAGT GGCGCCGGTG CGCGGGTCGC CTGrCAGATG CAGGCGATCG ACGAGCTGCG CCATGTGCAG 2790G F S R V G R Q F S G A G A R V A C Q M Q A I D E L R H V Q

Asp718ACGCAAGTCC ACGCCATGAG CCATTACAAC AAGCACTTCG ATGGTTTGCA TGACTTCGCC CACATGTACG ACCGGGTC ~rC 2880T Q V H A M S H Y N K H F D G L H D F A H M Y D R V W Y L S

FIG. 2. Nucleotide sequence of the 5,448-bp phenol hydroxylase-encoding region. The sense strand numbered from the first base of theSacI site is shown along with translation of six ORFs, that are preceded by ribosome-binding sites. The deduced amino acid sequences areshown in their one-letter code. The start of ORF 0 encoding P0 may be the GTG two codons downstream of the ATG shown. Arrows andasterisks above the untranslated nucleotide sequence indicate the location of putative regulatory signals that are further elaborated in Fig. 3.Restriction enzyme recognition sites shown in Fig. 1 are underlined.

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Asp7l8GTACCCAAGT CCTATATGGA CGATGCGCGG ACCGCCGGTC CGTTCGAGTT CCTCACCGCC G T CCTCGAGA CGTGCTGACC 2970V P K S Y M D D A R T A G P F E F L T A V S F S F E Y V L T

AACCTGTTGT TCGTACCCTT CATGTCCGGT GCCGCCTACA ACGGCGATAT GGCCACGGTC ACCTTCGGT TCTCCGCGCA GTVGGACGAG 3060N L L F V P F M S G A A Y N G D M A T V T F G F S A Q S D E

GCGCGGCACA TGACCCTGGG TCTGGAAGTG ATCAAGTTCA TGCTCGAACA GCATGAAGAC AACTGCCCA TCATCCAGCG CTGGATCGAT 3150A R H M T L G L E V I K F M L E Q H E D N V P I I Q R W I D

AAGTGGTTCT GGCGCGGTTA CCGCCTGCTG ACCCTGATCG GCATGATGAT GGACTACATG CTGCOGAACA AAGTGATGTC CTGGCTGAG 3240K W F W R G Y R L L T L I G M M M D Y M L P N K V M S W S E

GCCTGGGGGG TCTACTTCGA GCAGGCCGGT GGCGCGCTGT TCAAGGATCT GGAGCGCTAT GGCATCCGGC CGCCGAAATA CGTCGAGCAG 3330A W G V Y F E Q A G G A L F K D L E R Y G I R P P K Y V E Q

ACCACCATCG GCAAGGAGCA CATCACCCAC CAGGTGTGGG GGGCCTTATA TCAATACAGC AAGGCCACCA GCTTCCATAC CTGGATACCC 3420T T I G K E H I T H Q V W G A L Y Q Y S K A T S F H T W I P

GGCGACGAGG AACTGAACTG GCTGTCGGAG AAATACCCGG ACACCTTCGA CAAATACTAC CGCCOGOGCT TCGAGTTCTG GOGTGAGCAG 3510G D E E L N W L S E K Y P D T F D K Y Y R P R F E F W R E Q

CAGGCCAAGG GTGAGCGCTT CTACAACGAC ACCCTGCCGC ACCTCTGCCA GGTGTGCCAG TTACCGGTGA TTTTCACCGA GCCGGACGAT 3600Q A K G E R F Y N D T L P H L C Q V C Q L P V I F T E P D D

CCGACCAAGC TCAGCCTGCG CAGCCTGGTG CACGAGGGGG AGCGCTATCA ATTCTGCTCG GATGGCTGCT GCGACATCTT CAAGAACGAG 3690P T K L S L R S L V H E G E R Y Q F C S D G C C D I F K N E

BglIICCGGTGAAGT ACATCCAGGC CTGGCTGCCG GTGCACCAGQ&T-aACCAGGG CAACTGCGAA GGCGGGGATG TCGAAACGGT GGTGCAGAAG 3780P V K Y I Q A W L P V H Q I Y Q G N C E G G D V E T V V Q K

TACTACCACA TCAAAAGCGG CGTGGACAAT TTGGAGTACC TGGGCTCGCC CGAGCACCAG CGCTGGCTGG CCCTGAAAGG TCAGACCCCA 3870Y Y H I K S G V D N L E Y L G S P E H Q R W L A L K G Q T P

SauICCAACTGCCG CCCCGGCGGA CAAGAGCCTG GGCGCCICCTGA.GLGCAGCGC CAGCCGCTCA GGGGTGAAGC ACCGCCCCTG AGCCATTCCA 3960P T A A P A D K S L G A A

AGAACAAGAG GTTTCGATCA TGACTGTCAA CTCAATCGGC GAATACACCG CCACGCCACG GGATGTGCAG GCCAACTTCA ACGGCATGCA 4050M T V N S I G E Y T A T P R D V Q A N F N G M Q

ACTGCTCTAC CTCTACTGGG AAGAGCACCT GATGTACTGC TCCGCGCTCG CGTTCTTGGT AGCCCCCGGC ATGCCCTTI'G CCGAGTTCCS 4140L L Y L Y W E E H L M Y C S A L A F L V A P G M P F A E F L

XhoI PvuIICGh&rCAGGTG CTCAAGCCCG CGATCCACGC CCATCCGGAC AGCGCGAAGA TCGATTTCAG CCAGGCGCTC TGGCAGCTGA ACGACCAGCC 4230

E Q V L K P A I H A H P D S A K I D F S Q A L W Q L N D Q PSmaI

GTTCACCCCG GACTACGCCG CCAGCCTGGA AGCCAACGGC ATCGACCACA AAAGCATGCT GCGTCTGAAC ACCQCCGGGC TGAACGGCAT 4320F T P D Y A A S L E A N G I D H K S M L R L N T P G L N G I

PvuIICCAGGGTTCG TGCAGCTGAG AGGTGTGTCA TGAGTTACAA CGTCACCATT GAACCGACCG GCGAAGTGAT CGAAGTGGAG GAOGGCCAGA 4410Q G S C S M S Y N V T I E P T G E V I E V E D G Q

CCATCCTCCA GGCCGCTCTG CGCCAGGGCG TCTGGCTGCC GTTCGCCTGC GGCCACGGCA CCTGCSCCAC CTGCAAGGTG CAGGTGGTCG 4500T I L Q A A L R Q G V W L P F A C G H G T C A T C K V Q V V

EcoRVAGGGCGAAGT GfATaITGGC GAAGCCTCGC CGTTCGCCCT GATGGACATC GAGCGCGACG AGCGCAAGGT GCTGGCCTGC TGCGCCATTC 4590E G E V D I G E A S P F A L M D I E R D E R K V L A C C A I

CGCTGTCCGA CCTGGTGATC GAAGCCGACG TCGATGCCGA CCCGGACTTC CTCGGCCATC CGGTGGAGGA TTACCGGGGG GTGGTCAGCG 4680P L S D L V I E A D V D A D P D F L G H P V E D Y R G V V S

CCCTGGTTGA CCTGTCGCCG ACCATCAAGG GCCTGCACAT CAAGCTGGAT CGGCCCATGC OGlSCCAGGC CGGGCAGTAC GTCAACCTGG 4770A L V D L S P T I K G L H I K L D R P M P F Q A G Q Y V N L

CATTGCCGGG CATCGACGGC ACCCGCGCCT TCTCGCTGGC CAACCOGCCG AGCCGGAACG ACGAAGTCGA GTTGCACGTG CGCCTGGTCG 4860A L P G I D G T R A F S L A N P P S R N D E V E L H V R L V

AGGGCGGTGC GGCCACCGGC TTTATCCACA AGCAACTGAA AGTCGGCGAC GCGGTGGAGC TGTCCGGGCC TTATGGGCAG 1iTCGTGC 4950E G G A A T G F I R K Q L K V G D A V E L S G P Y G Q F F V

GCGATTCGCA GGCCGGCGAC CTGATCT1CA TCGCCGGCGG CTCGGGCTTA TCGAGCCCGC AGTCGATGAT CCTCGATCTG CTTGAACGCG 5040R D S Q A G D L I F I A G G S G L S S P Q S M I L D L L E R

GCGATACGCG GCGGATCACC CTGTTCCAGG GCGCGCGCAA CCGCGCCGAG CTGTACAACT GCGAACTGTT CGAGGAACTG GCCGCGCGCC 5130G D T R R I T L F Q G A R N R A E L Y N C E L F E E L A A R

ACCCCAACTT CAGTTACGTG OCGGCACTCA ACCAGGCCAA CGACGATCCC GAATGGCAGG GTTTCAAGGG CTlXGTCCAC GACGCCGCCA 5220H P N F S Y V P A L N Q A N D D P E W Q G F K G F V H D A A

AGGCGCATTT CGACGGCCGC TTCGGCGGGC AGAAAGCCTA CCTGTGCGGC CCACCGCCGA TGATCGACGC GGCCATCACC ACCCTGATGC 5230K A H F D G R F GG Q K A Y L C G P P P M I D A A I T T L M

AAGGTOGCTT GTTOGAGCGC GACATCTTTA TGGAGCGCTT CTACAcCGcC GtCGATGGGG CCGCOGAGAG CAGCCGTTCG GCCCTGTTCA 5400Q G R L F E R D I F M E R F Y T A A D G A G E S S R S A L F

NruIAGCGCATCTG AGGTGAACCA TGAAOCGTGC CGGTTATGAG ATjrGCL& 5448K R I

FIG. 2-Continued.

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Putative ribosome binding sites for ORF's

ORF 0 745-1020 GCGCAAGCCGCCAACCTGGAGATG____ Met

ORF 1 1076-2068 CAACAAGAGGGTACGGTTGATATGMet

ORF 2 2075-2344 AAAAGCCGCAAGGAATAAAGCATGMet

ORF 3 2359-3909 GAAGAACTAGGAGACAAGCTCATG_____ Met

ORF 4 3980-4336 CAAGAACAAGAGGTTTCGATCATGMet

ORF 5 4350-5408 CGTGCAGCTGAGAGGTGTGTCATGMet

Homology of putative promoter at 699-714

TGGCACAGCCGTTGCT PTGGCATGGCGGTTGCT PdmPTGGCGTTATTTTTGCT PXYlCs-24 -12 xlGG GC

Inverted repeat upstream of the putativepromoter: 640-660

TCATTCTAAAAATTAGAAACA

TNTNANxNTNANA

FIG. 3. Putative regulation signals of the phenol hydroxylase-encoding region. Over- and underlined bases indicate sequencescomplementary to the 3' end of the 16S rRNAs of E. coli and P.aeruginosa, respectively (21). Homologous regions of promotersequences are marked with asterisks. Arrows indicate the extent ofan inverted repeat.

RESULTSPolypeptide products of the phenol hydroxylase-encoding

region. The phenol hydroxylase of Pseudomonas strainCF600 is a multicomponent enzyme (22). The genes thatencode this enzyme are clustered and have previously beenlocated to a 5.2-kb BglII-NruI fragment (Fig. 1, coordinates0.35 to 5.55) (3). The polypeptide product of one of thecomponents, encoded by the SmaI-NruI fragment (Fig. 1,coordinates 4.4 to 5.55) has been shown to be an Mr 39,500polypeptide (22).To investigate the number and location of all the genes

involved in phenol hydroxylase activity, we generated adeletion series of the 5.5-kb SacI-NruI fragment spanningthis region by subcloning fragments into the broad-host-range tac expression vectors pMMB66 and pMMB66A, asdescribed in Materials and Methods. These vectors differ inthat pMMB66A contains a deletion of the lacF' repressorgene. The extent of the DNA in these plasmids is shown inFig. 1A, and a A symbol distinguishes plasmids based onpMMB66A from those carrying the same DNA in pMMB66.To obtain high expression of the polypeptide products, we

introduced the A deletion series into the maxicell strainCSR603 and analyzed the plasmid-encoded polypeptides bySDS-PAGE (Fig. 1B). Plasmids pVI261A and pVI270A(lanes 2 and 3) mediate the production of six novel polypep-tides, designated P0 (Mr 12,500), P1 (Mr 34,000), P2 (Mr10,000), P3 (Mr 58,000), P4 (M, 13,000), and P5 (Mr 39,000).Further deletion of the DNA carried by these plasmidsresults in the sequential loss of P0, P1, P2, P3, and P4 (lanes4 to 11). Thus, the polypeptides are encoded in the order P0to P5. P5 is the previously defined component of phenolhydroxylase (22).Pseudomonas strain PB2701 harboring pVI261 can grow

on phenol as the sole carbon and energy source by virtue of

TABLE 1. Correlation of ORFs 0 to 5 with PO to P5

.macd Predicted p I EstimatedORF residues molecular mass peptide molecular mass

0 92 10.6 PO 12.51 331 38.2 P1 342 90 10.5 P2 103 517 60.5 P3 584 119 13.2 P4 135 353 38.5 P5 39

the plasmid-encoded phenol hydroxylase and the chromo-somally encoded ortho-cleavage pathway (3). To furtherdefine the start of the phenol hydroxylase-encoding region,pVI261 and pVI270 to pVI275 were introduced into PB2701and tested for their ability to mediate growth on phenol.Plasmids pVI261 and pVI270, which encode P0 to P5, canmediate growth on phenol, whereas pVI273 and pVI275,which encode P1 to P5, cannot. Thus, the start of the phenolhydroxylase-encoding region lies between endpoints of theDNA in pVI270 and pVI273 (Fig. 1A).

Nucleotide sequence of the phenol hydroxylase-encodingregion. The nucleotide sequence, from the SacI site to theNruI site, of the phenol hydroxylase-encoding region wasdetermined as described in Materials and Methods. Thesense strand of this 5,448-bp region is shown in Fig. 2, alongwith translation of six open reading frames (ORFs 0 to 5).These ORFs are the only ones in the defined phenol hydrox-ylase-encoding region that are preceded by putative ribo-some-binding sites (Fig. 3). ORF 1 (Fig. 2, bp 1076 to 2068)is part of a larger ORF (bp 989 to 2068) that contains twoother possible ATG start sites 5' to the translational startindicated. However, these ATG codons are not preceded byputative translation signals, and amino acid sequencing of P1(see Discussion) has confirmed the translational start shown.As has been found for other Pseudomonas genes (see

reference 24 and references therein), the coding regions areG+C rich (59.4, 63.4, 60.7, 59.4, 61.8, and 65.6%) as a resultof preferential usage of codons with G or C at the thirdposition. The size and order of ORFs 0 to 5 correlate wellwith the size and order of the polypeptides observed (Table1).The nucleotide sequence was also analyzed for potential

regulatory signals. No regions with homology to the E. coliconsensus promoter sequence or translational terminators(18) were found. However, regions with strong homology tothe xylCAB and xylS promoters of TOL plasmid pWWO (11,12), and the proposed symmetrical recognition site for E. coliregulators (6), were identified and are shown in Fig. 3.

Independent expression of P0 to P5. To directly confirmthat ORFs 0 to 5 do indeed encode P0 to P5, constructs thatexpressed DNA spanning each of these ORFs were gener-ated. The extent of the DNA in the resulting plasmids(pVI281A, pVI284A, pVI285A, pVI287A, pVI288A, andpVI221A) is illustrated in Fig. 4A. Maxicell analysis of thepolypeptides produced by these plasmids (Fig. 4B) demon-strates that each plasmid independently expresses polypep-tides of the size of P0 to P5. For plasmids spanning the ORFsfor P1, P3, P4, and P5, only novel proteins of the corre-sponding Mr were observed (Fig. 4B, lanes 6, 8, 9, and 10).Plasmid pVI281A (lane 2) encodes a polypeptide of the sameMr as P0. However, the expression vector used, pMMB66HEA, also encodes a protein that comigrates with P5(compare lanes 1 and 2). The high levels of vector-encodedproteins observed in lane 2 account for the band at the same

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EScPBg Pv SmB

)l YV I IORF 1

:SaPSaX XmAs

i1 I 11 I i[ 2 =

pMMB66HEApVI261 A

2 pVI281A5 pVI283A6 pVI284A7 pVI285A8 pVI287A

BgSj XPvSmPvE N

3 N 1 _. 1 P0 P1 P2 P3 P4 P5

+ + + + + +

+ +

+

pVI288 ApVI221 A

+

+

B 1 2 394-67 -

43 -

4 5 6 7 8 9 10

- p3-- 5-..5_--- -P 1 -

30-- - . _

20-

14 - _ P4\6- -PO

FIG. 4. Independent expression of PO to P5. (A) The extent of the DNA in plasmid constructs is shown aligned with the location of ORFs0 to 5. A summary of the polypeptides that are produced from these plasmids is also shown. Abbreviations of restriction sites are as in Fig.1. (B) Maxicell analysis of plasmid-encoded polypeptides. Polypeptides of CSR603 harboring the plasmids indicated in panel A were separatedby 10-20% gradient SDS-PAGE. Molecular mass standards are shown in kilodaltons. PO to- P5 indicate polypeptides produced from the clonedDNA. Lanes 1 to 3 were run in the presence of 32 mM NaCl in the analytical gel and 3 M urea in the sample buffer. Consequently, P4 andPO are more clearly separated in these lanes than in lanes 4 to 10.

Mr as P5 (compare with Fig. 1B). Plasmid pVI285A carriesDNA spanning ORF 2, and a polypeptide of the same Mr asP2 is observed in lane 7. This construct also contains DNAencoding the N-terminal portion of P3, and a truncationproduct of the predicted size is observed in the Mr 12,600region of lane 7.

Polypeptide requirement for growth on phenol. PlasmidspVI261 and pVI270 express P0 to P5 and can mediate thegrowth of Pseudomonas strain PB2701 on phenol. pVI273and pVI275, which express only P1 to P5, cannot do so; thisdemonstrates the requirement of P0 for this property. Todirectly investigate the requirement of each of the polypep-tides P1 to PS for growth on phenol, we used a series ofplasmids lacking expression of each of the polypeptides.Plasmids pVI289 to pVI292 lack expression of each of thepolypeptides P1 to P4, respectively, and pVI258 (3) lacksexpression of P5. The DNA present in these plasmids isshown in Fig. SA. The nucleotide sequence of the noveljunctions in these plasmids was determined, and their effecton ORFs 1 to 5 could be deduced from the sequence shownin Fig. 2. Plasmid pVI289 has a large out-of-frame deletion inORF 1 that would result in fusion of the first 11 amino acidsof P1 with four unrelated amino acids. pVI290 has anin-frame deletion in ORF 2 that results in the fusion of thefirst 33 and last 13 amino acids of P2 with leucine inserted at

the junction. The out-of-frame deletion in ORF 3 of pVI291would result in a polypeptide containing the first 99 aminoacids of P3 fused to an unrelated sequence of 44 amino acids.pVI292 was generated by removing the protruding endsproduced by digestion with XhoI at the site indicated in Fig.5A. This treatment resulted in deletion of one more basethan expected and hence a frameshift in ORF 4, resulting infusion of the first 53 amino acids of P4 with an unrelated69-amino-acid sequence. The in-frame deletion of ORF 5 inpVI258 fuses the first 55 amino acids of P5 with a 19-residueunrelated amino acid sequence. The polypeptides producedfrom each of these plasmids were analyzed by expression inmaxicells. Each of these plasmids has lost expression of theexpected polypeptide (Fig. SB).

Plasmids pVI289-292 and pVI258 were introduced intoPseudomonas strain PB2701, and the resulting strains weretested for their ability to grow on phenol. None of theseplasmids were able to confer the ability to grow on phenol,clearly demonstrating the requirement of each of P1 to PS.Thus, we conclude that all six polypeptides, P0 to PS, arerequired for this property. The gene(s) for phenol hydroxy-lase has previously been designated dmpA. In view of thefinding that six polypeptides are produced from the dmpA-encoding region, we propose the names dmpKLMNOP forthe genes encoding P0 to P5, respectively.

Alane

1

3 &4

9

10

-94

-6;7

- 43

- 30

- 20

-14

- 6

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6832 NORDLUND ET AL.

Alane

1 pMMB66HEA

2 & 5 pVI261A3 pVI273A4 pVI289A6 pVI290A

7 pVI291 &

8 pVI292A

9 pVI258 A

EScPBg Pv

ORF

SmB PSaP SaX XmAs

~~~1 LBg SuX PvSm Pv E N

3 4-I"'

) 5 P0 P1 P2 P3 P4 P5

+ 4 + + + _

+ + +

+ + + + _

+ _ + + _

+ + + + -

+ + + _

+ - + + +

;-; 12 34

-7P 3

30i2 0 __ f. s . e ---

4~~~- P Z

r

20..__-t

5 6 7 8 9- 9 4- 67

_ 43

AQrn.. - 30

- 20

14 -- e-l-P46- 1P2 .

6 -----

- 1 4

-- 6

FIG. 5. Deletion analysis of individual ORFs. (A) The extent of the DNA in plasmid constructs is shown aligned with the location of ORFs0 to 5. V, Site of the small deletion in ORF 4 that results in a frameshift mutation of P4. A summary of the polypeptides that are producedfrom these plasmids is shown. Abbreviations of restriction sites are as in Fig. 1. (B) Maxicell analysis of plasmid-encoded polypeptides.Polypeptides of CSR603 harboring the plasmids indicated in panel A were separated by 10 to 20% gradient SDS-PAGE. Molecular massstandards are shown in kilodaltons. PO to P5 indicate polypeptides produced from the cloned DNA. These gels were run without NaCI in theanalytical gel and urea in the sample buffer. Consequently, P4 and PO are not completely resolved and run as a thick band, with P4 constitutingthe upper half and PO constituting the lower half.

DISCUSSIONThe first step in the biodegradation of phenol by Pseudo-

monas strain CF600 is catalyzed by the multicomponentenzyme phenol hydroxylase. Determination of the nucleo-tide sequence of the coding region of this enzyme reveals thepresence of six ORFs (ORFs 0 to 5) arrayed in an operonstructure (Fig. 2). Possible regulatory signals upstream of thecoding region have been located within the sequence (Fig.3). A putative promoter sequence 30 bp upstream of thetranslational start of ORF 0 shows strong homology to agroup of promoters that includes the xylCAB operon pro-moter and the xylS promoter of the toluene-xylene catabolicplasmid pWWO. These promoters have little in common withthe E. coli -35 TTGACA -10 TATAAT consensus se-quence. However, they do contain the invariant -24 GG-12 GC sequence recognized by the RpoN alternative sigmafactor of E. coli (5). Expression from these promoters hasrecently been shown to require the rpoN gene product ofPseudomonas putida (13). Another striking feature is thepresence of an inverted repeat, upstream of the promoterregion, that is similar to the symmetrical recognition se-quence proposed to be involved in the binding of E. colirepressors and activators (6). Expression of the genes of thephenol degradative pathway has previously been shown tobe tightly regulated (22), and this inverted repeat may be the

recognition site for a regulatory protein. An investigation ofthe role of these sequences in the regulation of expression ofthe catabolic genes of Pseudomonas strain CF600 is underway.

Six polypeptides, designated P0 to P5 and ranging in sizefrom 10 to 58 kDa, were produced from the phenol hydrox-ylase-encoding region (Fig. 1). The genes for these polypep-tides correlate with the ORFs identified as shown by threeindependent means. First, deletion of DNA in the directionof transcription resulted in sequential loss of P0, P1, P2, P3,and P4, demonstrating the coding order P0 to P5. Thus,expression of a polypeptide was lost when all or part of asuitably sized ORF was deleted. Second, independentexpression of DNA spanning each of the ORFs in turnresulted in the production of polypeptides indistinguishable,on the basis of size, from P0 to P5 (Fig. 4). Third, deletion ofpart of the ORFs resulted in six plasmids that each expressall but one of the polypeptides (Fig. 5). The correlation ofORF 1, ORF 2, and ORF 5 with P1, P2, and P5, respectively,has been confirmed by N-terminal amino acid sequencing ofthe purified polypeptides (19; data not shown). Thus, weconclude that ORFs 0 to 5, designated dmpKLMNOP, arethe genes encoding P0 to P5, respectively.By using the protein sequences of P0 to P5, computer-

assisted searches (Geneus version 2.0 and Wisconsin GCG

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version 6) only revealed significant homology of P5 to otherproteins. The N-terminal sequence of P5 has 33 to 42%identity with numerous ferredoxins. The cysteine residuesthat ligate the iron-sulfur center of these ferredoxins (23) are

conserved in P5. Analysis of purified PS, reported in theaccompanying paper (19), confirms the presence of an iron-sulfur center in this protein.Phenol hydroxylase catalyzes the conversion of phenol to

catechol, which can be further metabolized by the enzymesof the chromosomally encoded ortho-cleavage pathway ofPseudomonas strain PB2701. We have used growth on

phenol of PB2701 expressing cloned DNA to identify thegenes required to encode phenol hydroxylase. Plasmids thateach express all but one of PO to P5 were used to demon-strate that all six of these polypeptides are required tomediate growth on phenol. The in vitro requirements for thephenol hydroxylase enzyme activity are reported in theaccompanying paper (19). These studies demonstrate thatonly P1 to P5 are required for in vitro enzyme activity as

measured by oxygen uptake and product formation. Thisfinding suggests that PO is not absolutely required for hy-droxylase activity. However, the requirement of PO forgrowth but not in vitro activity could be explained if thisprotein had a function in binding and/or transporting ofphenol to the hydroxylase or in regulation of the hydroxylaseactivity. The role of P1 to PS in phenol hydroxylase activityis discussed in the accompanying paper (19).

ACKNOWLEDGMENTS

We thank Martin Gullberg for fruitful discussions and criticalreading of the manuscript.

This research was supported by grants STUF87-316 and STUF89-470 from the National Swedish Board for Technical Development.J.P. was supported by guest researcher stipends STU88-4347 andSTU89-4714.

LITERATURE CITED1. Ballou, D. P. 1982. Flavoprotein monooxygenases, p. 301-310.

In V. Massey and C. H. Williams (ed.), Flavins and flavopro-teins. Elsevier Science Publishing Co., Amsterdam.

2. Bartilson, M., and V. Shingler. 1989. Nucleotide sequence andexpression of the catechol 2,3-dioxygenase-encoding gene ofphenol catabolizing Pseudomonas CF600. Gene 85:233-238.

3. Bartilson, M., I. Nordlund, and V. Shingler. 1990. Location andgene organization of the dimethylphenol catabolic genes ofPseudomonas CF600. Mol. Gen. Genet. 220:294-300.

4. Dagley, S. 1986. Biochemistry of aromatic hydrocarbon degra-dation in pseudomonads. p. 527-556. In J. R. Sokatch (ed.), Thebacteria, vol. X. The biology ofPseudomonas. Academic Press,Inc. (London), Ltd., London.

5. Dixon, R. 1986. The xylABC promoter from Pseudomonasputida TOL plasmid is activated by nitrogen fixation regulatorygenes in Escherichia coli. Mol. Gen. Genet. 203:129-136.

6. Ebright, R. H. 1986. Proposed amino acid-base pair contact for13 sequence specific DNA binding proteins, p. 207-229. In D.Oxender (ed.), Protein structure, folding and design. Alan R.Liss, Inc., New York.

7. Frantz, B., and A. M. Chakrabarty. 1986. Degradative plasmidsin Pseudomonas, p. 295-317. In J. R. Sokatch (ed.), Thebacteria, vol. X. The biology ofPseudomonas. Academic Press,Inc. (London), Ltd., London.

8. Furste, J. P., W. Pansegrau, R. Frank, H. Blocker, P. Scholz, M.Bagdasarian, and E. Lanka. 1986. Molecular cloning of the RP4DNA primase region in a multirange tacP expression vector.Gene 48:119-131.

9. Gibson, D. T. (ed.). 1984. Microbial degradation of organiccompounds, vol. 13. Marcel Deckker, Inc., New York.

10. Hanahan, D. 1985. Techniques for transformation of E. coli, p.109-136. In D. M. Glover (ed.), DNA cloning, vol. 1. A practicalapproach. IRL Press Ltd., Oxford.

11. Inouye, S., A. Nakazowa, and T. Nakazowa. 1984. Nucleotidesequence surrounding the transcription initiation site ofxylABCoperon of TOL plasmid. Proc. Natl. Acad. Sci. USA 81:1688-1691.

12. Inouye, S., A. Nakazowa, and T. Nakazowa. 1987. Over produc-tion of the xylS gene product and activation of the xyIDLEGFoperon of the TOL plasmid. J. Bacteriol. 169:3587-3592.

13. Kohler, T., S. Harayarma, J.-L. Ramos, and K. T. Timmis.1989. Involvement of Pseudomonas putida RpoN a factor inregulation of various metabolic functions. J. Bacteriol. 171:4326-4333.

14. Kushner, S. R. 1978. An improved method for transformation ofEscherichia coli with ColEl derived plasmids, p. 17-23. InH. W. Boyer and S. Nicosia (ed.), Genetic engineering.Elsevier/North Holland Publishing Co., Amsterdam.

15. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

16. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

17. Nordlund, I., and V. Shingler. 1990. Nucleotide sequences ofthe meta-cleavage pathway enzymes 2-hydroxymuconic semi-aldehyde dehydrogenase and 2-hydroxymuconic semialdehydehydrolase from Pseudomnonas CF600. Biochim. Biophys. Acta1049:227-230.

18. Platt, T. 1986. Transcription termination and the regulation ofgene expression. Annu. Rev. Biochem. 55:339-372.

19. Powlowski, J., and V. Shingler. 1990. In vitro analysis ofpolypeptide requirements of multicomponent phenol hydroxy-lase from. Pseudomonas sp. strain CF600. J. Bacteriol. 172:6834-6840.

20. Sancar, A., A. M. Hack, and W. D. Rupp. 1978. Simple methodfor the identification of plasmid-coded proteins. J. Bacteriol.137:692-693.

21. Shine, J., and L. Dalgarno. 1975. Determination of cistronspecificity in bacterial ribosomes. Nature (London) 254:34-38.

22. Shingler, V., M. Tsuda, D. Holroyd, F. C. H. Franklin, and M.Bagdarsaian. 1989. Molecular analysis of the plasmid encodedphenol hydroxylase of Pseudomonas CF600. J. Gen. Microbiol.135:1083-1092.

23. Stout, C. D. 1982. Iron-sulfur protein crystallography, p. 97-146. In T. G. Spiro (ed.), Iron sulfur proteins. John Wiley &Sons, Inc., New York.

24. Zylstra, G. J., and D. T. Gibson. 1989. Toluene degradation ofPseudomonas putida Fl. Nucleotide sequence of thetodClC2BADE genes and their expression in Escherichia coli.J. Biol. Chem. 264:14940-14946.

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