JOURNAL OF BACTERIOLOGY, Sept. 1991, p. 5260-5265 Vol. 173, No. 170021-9193/91/175260-06$02.00/0Copyright ©) 1991, American Society for Microbiology

Isolation, Sequence, and Expression in Escherichia coli of thePseudomonas sp. Strain ACP Gene Encoding 1-Aminocyclopropane-

1-Carboxylate DeaminaseRAYMOND E. SHEEHY,' MAMORU HONMA,2 MASATAKA YAMADA,2 TORU SASAKI,2 BELINDA

MARTINEAU,' AND WILLIAM R. HIATTl*Calgene, Inc., 1920 Fifth Street, Davis, California 95616,1 and Department of Agricultural Chemistry, Hokkaido

University, Sapporo 060, Japan2Received 27 March 1991/Accepted 17 June 1991

Pseudomonas sp. strain ACP is capable of growth on l-aminocyclopropane-l-carboxylate (ACC) as anitrogen source owing to induction of the enzyme ACC deaminase and the subsequent conversion of ACC toa-ketobutyrate and ammonia (M. Honma, Agric. Biol. Chem. 49:567-571, 1985). The complete amino acidsequence of purified ACC deaminase was determined, and the sequence information was used to clone the ACCdeaminase gene from a 6-kb EcoRI fragment of Pseudomonas sp. strain ACP DNA. DNA sequence analysis ofan EcoRI-PstI subclone demonstrated an open reading frame (ORF) encoding a polypeptide with a deducedamino acid sequence identical to the protein sequence determined chemically and a predicted molecular massof 36,674 Da. The ORF also contained an additional 72 bp of upstream sequence not predicted by the aminoacid sequence. Escherichia coli minicells containing the 6-kb clone expressed a major polypeptide of the sizeexpected for ACC deaminase which was reactive with ACC deaminase antiserum. Furthermore, a lacZ fusionwith the ACC deaminase ORF resulted in the expression of active enzyme in E. coli. ACC is a key intermediatein the biosynthesis of ethylene in plants, and the use of the ACC deaminase gene to manipulate this pathwayis discussed.

Pseudomonas sp. strain ACP and the yeast Hansenulasaturnus are capable of utilizing the cyclopropanoid aminoacid 1-aminocyclopropane-1-carboxylate (ACC) as a nitro-gen source owing to induction of the enzyme ACC deami-nase (EC 4.1.99.4) in these organisms (8). PseudomonasACC deaminase has an estimated molecular mass of 110 kDaand is composed of three identical subunits, each with amolecular mass estimated to be 36.5 kDa (7, 8). ACCdeaminase utilizes pyridoxal 5'-phosphate as a cofactor incatalyzing the cleavage of ACC to a-ketobutyrate and am-monia (7, 22).ACC was originally identified as a natural product in the

juices of several fruits (2) and is now regarded as a keyintermediate in the biosynthesis of the plant hormone ethyl-ene (23). Ethylene is biosynthesized in higher plants frommethionine via S-adenosylmethionine and ACC (1, 24). Therate-limiting step in ethylene biosynthesis is the productionof ACC by a ring-closing displacement of methylthioadenos-ine from S-adenosylmethionine in a reaction catalyzed byACC synthase (1). The reverse of this reaction can beviewed as analogous to the reaction catalyzed by ACCdeaminase (22).

Ethylene influences many aspects of plant development,including leaf and flower senescence and fruit ripening (23).Techniques for the integration and expression of bacterialgenes in plants utilizing Agrobacterium tumefaciens areavailable (14), and the expression of an ACC deaminase genein plants may provide a means to perturb ACC levels andethylene biosynthesis, thus leading to a better understandingof the role of this plant hormone. As an initial step in thisprocess, the ACC deaminase gene from Pseudomonas sp.strain ACP was isolated and characterized. The amino acid

* Corresponding author.

sequence of purified ACC deaminase was determined andused to design oligonucleotide primers. The primers wereused in polymerase chain reactions (PCR) to isolate a portionof the gene, which was used to screen a Pseudomonasgenomic library. The DNA sequence of the ACC deaminasegene is presented, and its authenticity is demonstrated byexpression of the gene in Escherichia coli.

MATERIALS AND METHODS

Bacterial strains and growth media. Lambda Zap II and E.coli XL1-Blue and SURE were obtained from Stratagene (LaJolla, Calif.). Bluescript plasmids pBCSK+, pBSK-, andpBCKS+ (Stratagene) in E. coli SURE or DH5a (5) wereused for DNA sequencing. pUC19 (25) was used for sub-cloning in E. coli SURE. E. coli XL1-Blue and SURE strainswere maintained on Luria-Bertani (LB) agar plates supple-mented with 20 jig of tetracycline per ml and routinely grownat 37°C in LB medium or Terrific broth (T-broth) (13).Pseudomonas sp. strain ACP was grown and maintained inT-broth or 2% glucose4.5% Bacto-Peptone (Difco)-0.3%yeast extract (PYG medium) and transferred to M9 salts (17)containing 2% glucose and 0.1% ACC for induction of ACCdeaminase. The minicell-producing E. coli YS1 (thr leu thistr minA endI; obtained from D. Stalker) was grown andmaintained as previously described (3). E. coli containingplasmid pCGN2332, pCGN1479, or pCGN1472 or pBSK-subclones was grown on LB agar plates or LB mediumsupplemented with 300 jig of penicillin per ml. E. colicontaining pBCSK+ or pBCKS+ plasmids was grown onthe same medium but supplemented with 20 p,g of chloram-phenicol per ml.Chemical modification for amino acid sequencing. ACC

deaminase was purified from cultures of Pseudomonas sp.strain ACP as described previously (8) and stored in 3 M

5260

ACC DEAMINASE GENE FROM A PSEUDOMONAS SP. 5261

ammonium sulfate. The enzyme preparation was dialyzedagainst 0.1 M potassium phosphate (pH 7.5), reduced withsodium borohydride, and dialyzed against the same buffer.Reduction of the enzyme leads to irreversible binding of thepyridoxal phosphate coenzyme, which can be monitored bythe disappearance of an absorption band at 416 nm. ReducedACC deaminase or reduced enzyme digested with trypsin(see below) was pyridylethylated by the following proce-dure. Enzyme or peptides were denatured with 2-mercapto-ethanol (1 [lI/mg of enzyme) in 6 M guanidine hydrochlorideunder nitrogen for 15 h at room temperature. Pyridylethyla-tion of cysteine residues was done by addition of 4-vinylpy-ridine (1.5 [lI/mg of protein) and further incubation for 1 h.The reaction was stopped by acidifying to pH 3.0 with glacialacetic acid.

Proteolytic digestions and purification of peptides. Reducedenzyme was digested with trypsin before denaturation, anddenatured enzyme or peptides were digested with fourdifferent proteolytic enzymes. One milligram of dialyzed andreduced enzyme in 0.5 ml of 0.1 M potassium phosphate (pH7.5) was incubated with 20 ,ug of tolylsulfonyl phenylalanylchloromethyl ketone (TPCK)-treated trypsin (Sigma Chem-ical Co.) at 30°C for 12 to 24 h. Tryptic peptides weremodified with vinylpyridine and fractionated with a FinepakSILC8-P column (4.6 by-50 mm) run at 70°C and a gradientof 20 to 30% propyl alcohol in 0.1% trifluoroacetic acid.Peptides and pyridylethylated ACC deaminase were purifiedby using A'sahipak C4P-50 (4.6 by 250 mm) or Microbonda-sphere C4-300 A (3.9 by 150 mm) columns run at 50°C and agradient of 0 to 40% acetonitrile in 0.1% trifluoroacetic acid.One peptide (T3) was purified by using a gradient of 25 to35% acetonitrile.Lysyl endopeptidase digestions were performed by dis-

solving purified pyridylethylated ACC deaminase or trypticpeptides in 8 M urea (pH 9.0). Peptide and urea concentra-tions were adjusted to 0.5 mg/nil and below 4 M, respec-tively, by the addition of 0.01 M Tris (pH 9.0). One micro-gram of lysyl endopeptidase (WAKO Pure Chemical, Inc.)was added per 50 pug of peptide and incubated at 30°C for 24h. Staphylococcal protease digestions were performed bydissolving peptides in a small amount of 8 M urea (pH 7.8).Four or more volumes of 0.05 M ammonium bicarbonate (pH7.8)-S5 mM EDTA were added to adjust peptide concentra-tions to 0.25 mg/ml. After the addition of 1 ,ug of proteasefrom Staphylococcus aureus V8 (Sigma) per 50 ,ug of pep-tide, the mixture was incubated at 30°C for 24 h. Peptideswere digested with endoproteinase Asp-N or chymotrypsinby dissolving samples in 8 M urea and then adding 0.05 Msodium phosphate (pH 8.0) to adjust peptide concentrationsto 0.1 mg/ml and urea concentrations below 1 M for endopro-teinase Asp-N (Boehringer Mannheim) or below 2 M fortolysulfonyl lysyl chloromethyl ketone (TLCK)-treated chy-motrypsin (Sigma) digestions. The appropriate enzyme (2pug) was added to 1 ml of the mixture and incubated at 37°Cfor 12 to 24 h. Peptides were separated by using AsahipakC4P-50 or C8P-50 columns or Microbondasphere C4 col-umns and a gradient of 0 to 40% acetonitrile as describedabove. Chromatograms were monitored at 216 nm for pep-tides, 280 nm for tryptophan, and 320 nm for the coenzyme.

Peptide sequencing and other methods. Fractions contain-ing purified peptides were evaporated and applied to aprotein sequencer (Applied Biosystems model 477A-120Asystem) for analysis. The C-terminal amino acid was deter-mined by the method of Hayashi (6) by carboxypeptidase Yand amino acid analysis (Applied Biosystems). Phosphateion was detected by the method described by Kempers (10).

Peptide molecular mass was determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)(12).DNA manipulations. Chromosomal DNA was isolated

from cultures of Pseudomonas sp. strain ACP grown over-night in PYG medium. Cells were harvested by centrifuga-tion, suspended in 3 ml of 10 mM Tris-HCl (pH 7.5)-l mMEDTA (TE buffer), and disrupted by the addition of 1 ml of10% SDS-1 ml of pronase (5 mg/ml) and incubation for 30min at 37°C. DNA was precipitated with ethanol, dissolvedin TE buffer, and extracted with phenol-chloroform (1:1,vol/vol) and then again precipitated with ethanol and resus-pended in TE buffer. PCR, using oligonucleotide primers andPseudomonas sp. strain ACP DNA as a template, wereconducted with a DNA Thermal Cycler (Perkin-Elmer Ce-tus) under standard reaction conditions suggested by themanufacturer. The PCR products were separated by electro-phoresis through a 1% agarose gel.For Southern analysis, Pseudomonas DNA was digested

with various restriction endonucleases (Boehringer Mann-heim), and the resulting fragments were separated by elec-trophoresis through 1.5% agarose gels and blotted ontonylon membranes (Nytran; Schleicher & Schuell). DNAblots were probed with DNA fragments radioactively labeledwith 32P by the nick translation method (nick translation kit;Boehringer Mannheim). Prehybridization and hybridizationsolutions were as previously described (17) except that theconcentration of Denhardt's solution was reduced to 2 x anddextran sulfate was added to a final concentration of 10% forhybridization. Prehybridizations and hybridizations weredone at 42°C. After hybridization, the membranes werewashed with 0.1 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 Msodium citrate)-0.1% SDS at 55 to 65°C and exposed toKodak X-AR film.Pseudomonas sp. strain ACP genomic library construction

and screening. Pseudomonas DNA was digested to comple-tion with EcoRI, and fragments of 3 to 7 kb were isolatedfrom an agarose gel by electroelution onto dialysis mem-brane. The size-fractionated DNA was ligated into EcoRI-digested and alkaline phosphatase-treated lambda ZAP IIbacteriophage arms and packaged to produce viable phageparticles following the manufacturer's instructions (Strata-gene). The phage (75% recombinant) were plated on a lawnof E. coli XL1-Blue or SURE and screened for ACCdeaminase clones by hybridization with a PCR-generatedfragment (9) and immunologically (9, 26) with antibodyraised against purified ACC deaminase in rabbits. Recombi-nant plaques identified as positive were purified by sequen-tial isolation and replating. Plasmid pBSK- containing thePseudomonas DNA insert was then excised from the lambdaZAP II phage arms in E. coli SURE as described by thesupplier (Stratagene).

Restriction mapping, DNA sequencing, and subcloning.Restriction enzyme sites were mapped by standard proce-dures (13, 17). Deletion mutants for sequencing were createdwith a deletion kit (Bluescript Exo/Mung DNA SequencingSystem; Stratagene) and by religating plasmids after diges-tion with various restriction enzymes. Novel oligonucleotideprimers were also synthesized and used in sequencing reac-tions. The sequence of overlapping DNA fragments wasdetermined by the dideoxy chain termination method (18).Both DNA strands were sequenced. DNA sequence infor-mation was analyzed by using Intelligenetics Gel and Seqprograms.

Protein manipulations. Minicells obtained from 500-mlcultures of E. coli YS1 were purified as described previously

VOL. 173, 1991

5262 SHEEHY ET AL.

1 MNLQRFPRYPLTFGPTPIQPLARLSKHLGGKVHLYAKREDCNSGLAFGGNi T2 | T~~~~~~~2ESnET2K rE4-

51 XTRKLEYLIPEALAQGCDTLVSIGGIQSNQTRQVAAVAAHLGMKCVLVQEI?I-2D6

101 NWVNYSDAVYDRVGNIQMSRILGADVRLVPDGFDIGFRRSWEDALESVRAK10I I-K1OD4 T3-

151 AGGKPYAIPAGCSDHPLGGLGFVGFAEEVRAQEAELGFKFDYVVVCSVTGi Kg 1 1 K4-

201 STQAGMVVGFAADGRADRVIGVDASAKPAQTREQITRIARQTAEKVGLERK4 11 -K3 I K

251 DIMRADVVLDERFAGPEYGLPNEGTLEAIRLCARTEGMLTDPVYEGKSMHK7 Ti I T1C7-

301 GMIEMVRNGEFPEGSRVLYAHLGGVPALNGYSFIFRDGK9 1

FIG. 1. Peptide sequence of Pseudomonas sp. strain ACP ACC deaminase. Peptide sequences were assigned as a result of Edmandegradation by an automatic sequencer of tryptic digests (T) or lysyl endopeptidase digests (K) or by staphylococcal protease (TE),endoproteinase Asp-N (TD), chymotrypsin (TC), or lysyl endopeptidase (TK) digests of the various tryptic peptides. Vertical bars indicatethe ends of individual peptides.

(3, 16), suspended in 2 ml of Bacto-Methionine AssayMedium (13.1 mg/ml) (Difco) in M9 salts with 0.5% glucose,labeled with 40 ,uCi of [35S]methionine (specific activity, 800Ci/mmol; Amersham) for 45 min, pelleted, and frozen.Pellets were resuspended in 400 ,ul of sample buffer (12) andheated to 90°C for 2 min. Aliquots of 20 ,ul were thenseparated by electrophoresis on SDS 12.5% polyacrylamidegels (12). 35S-labeled polypeptides were electroblotted tonitrocellulose membranes for autoradiography. The sameblots were then probed with antibody produced againstdenatured ACC deaminase and developed by using theProtoblot system as described by the vendor (PromegaBiotec, Madison, Wis.).

Extracts from E. coli and Pseudomonas sp. strain ACPwere prepared and assayed for ACC deaminase activity bymeasuring the production of a-ketobutyrate with 2,4-dinitro-phenylhydrazine as a colorimetric indicator (8). One unit ofACC deaminase activity was defined as a change in A540 of0.001/min. Protein concentrations were determined withBradford reagent (Bio-Rad Laboratories), using bovine se-rum albumin as a standard.

Nucleotide sequence accession number. The nucleotidesequence of the Pseudomonas sp. strain ACP ACC deami-nase gene has been given GenBank accession numberM73488.

RESULTS AND DISCUSSION

Amino acid sequence analysis. The amino acid sequence ofpurified ACC deaminase is shown in Fig. 1. ACC deaminasewas incubated with trypsin and separated into four fractionsby using a C8 column and a gradient of propanol. Furtherpurification of two fractions with a C4 column gave peptidesTi (9 kDa) and T2 (15 kDa). The third fraction was a mixtureof three peptides (14, 20, and 28 kDa); the smallest peptidewas designated T3. The other peptides were thought to becombinations of tryptic peptides, T3-T1 and T2-T3. Thefourth fraction comprised a peptide with a molecular massapproximately the same as intact ACC deaminase (36.5kDa). N-terminal and C-terminal sequence analysis indi-cated that the T2 peptide was the N-terminal peptide andthat the C-terminal amino acid, glycine, of the Ti peptidecorresponded to the C-terminal residue of the intact subunit.

A reasonable order of the tryptic peptides was thus estab-lished as T2-T3-T1.The ratio ofA32JA216 was 0.0013 for Ti, 0.014 for T2, and

0.0038 for T3, indicating that the T2 peptide bound thecofactor pyridoxal phosphate. The T2 peptide was furtherfragmented by lysyl endopeptidase (T2K peptides) or sta-phylococcal protease (T2E peptides), and the resulting pep-tides were separated on a C4 or C8 column and sequenced.Digestion with either protease generated a peptide of highA320, and the sequences of these two peptides overlapped(T2K1 and T2E4, Fig. 1). Phosphate ion corresponding tophosphopyridoxyllysine was detected in the Edman degra-dation product at position 51, indicating that the residuedesignated X in Fig. 1 is lysine. Analyses of five peptidesfrom lysyl endopeptidase digestion, six peptides from sta-phylococcal protease digestion, and a tryptophan-containingpeptide from endoproteinase Asp-N (T2D6), as indicated byA280, provided the amino acid sequence for an additional 135residues of the T2 peptide.Reduced and pyridylethylated ACC deaminase was then

digested with lysyl endopeptidase and separated into 10peptides (KI to K10) on C4 and C8 columns. The sequencesof these peptides covered the T3 and Ti regions, except forthe sequence provided by peptide T1C7, which was gener-ated by chymotrypsin digestion of Ti. One of the K peptidesshowed markedly higher A280. This peptide, K10, producedtwo tryptophan-containing peptides by endoproteinaseAsp-N digestion. One of them (K1OD4) was analyzed to givea sequence connecting T2 and T3.As mentioned above, the conversion of S-adenosylmethio-

nine to ACC is catalyzed by the enzyme ACC synthase inplants, and the reverse of this reaction is analogous to thereaction performed by ACC deaminase (22). Comparison ofthe amino acid sequence of ACC deaminase with the de-duced amino acid sequence for ACC synthase (20) fromtomato demonstrated two regions (residues 163 to 167 and169 to 174 ofACC deaminase) of greater than 70% similarity.One of these regions (169 to 174, with 75% similarity)coincided with the active site of ACC synthase. This regionof the ACC deaminase enzyme is distant from the predictedcofactor-binding lysine at position 51, and the significance ofthis homology is unknown.

J. BACTERIOL.

ACC DEAMINASE GENE FROM A PSEUDOMONAS SP. 5263

Xba IACCD1 5'-CGTAICTAGATGAAQQTNCAPJGNTTQCCNJG-3'

Eco RI P

ACCD3 5'-TCACGAA3ICKNMLCATQTGKATPTrNCCNAC-3'

Bam HI Nde IACCD55 5'-TATCTAGGATCCCCATATGAACCrGCAACGATTCCCTC-3'

Asp 718ACCD53 5'-TATTGGIACCGTTTCGCATACAGATGCAC-3'

Asp718 ClaIACCD35 5'-TTATAGGTACCGGCAAATCGATGCACGGCATG-3'

Sst IACCD33 5'-ATTAAGAGTAGCCGTCTCGGAAGATAAAGC-3'

FIG. 2. Oligonucleotide primers used for PCR reactions. Thenucleotide sequences of the six oligonucleotide primers are shownwith their respective restriction endonuclease recognition site(s)underlined. Symbols used to designate degeneracy in ACCD1 andACCD3: P, A or G; Q, C or T; J, C or A; K, T or G; L, T or A; M,C or G.

Isolation and sequence analysis of ACC deaminase gene.The amino acid sequence of the N-terminal region of theACC deaminase protein, for which reverse translation indi-cated a relatively unambiguous nucleotide sequence, wasused to design oligonucleotide fragments for use as primersin PCR (Fig. 2). Recognition sites for various restrictionendonucleases were also added to the primers to facilitatecloning of Pseudomonas DNA sequences. Primers ACCD1

and ACCD3, representing amino acid sequences 1 to 7 and113 to 120, respectively, were used in PCR with Pseudomo-nas sp. strain ACP DNA as the template to produce a 370-bpfragment of the ACC deaminase gene. This fragment wasisolated and ligated into pBSK- to produce pCGN2332.

Southern analysis of Pseudomonas sp. strain ACP DNAwas conducted with the insert from pCGN2332 as a probe.The results of this analysis (data not shown) indicated thatthe ACC deaminase gene was contained on an EcoRI DNAfragment of approximately 6 kb. A Pseudomonas sp. strainACP genomic library constructed in lambda ZAP II andenriched for the 6-kb EcoRI fragment was prepared, andapproximately 6,000 PFUs were screened with the 370-bpinsert from pCGN2332 as a hybridization probe. Six positiveplaques were identified, and two of these were rescreenedwith antibody raised against ACC deaminase. One plaquereacted with antibody and was purified. The plasmid form ofthis clone, pCGN1479, was used as a source of DNA formapping common restriction sites (Fig. 3A) and for DNAsequence analysis.The nucleotide sequence of a 2.2-kb EcoRI-PstI fragment

that hybridized to the 370-bp ACC deaminase fragment frompCGN2332 is shown in Fig. 3B. The clone contains an openreading frame (ORF) which includes the entire coding regionfor the mature protein subunit with a predicted molecularmass of 36,674 Da. The deduced amino acid sequence frompCGN1479 is identical to the ACC deaminase protein se-quence shown in Fig. 1. An in-frame ATG codon is located

A

l l

I..- L-

_- oz X~

I lkb IB

1 gaattctccgatctcgcatgtcgcgaggagggttatgggttcgcccaaggcccgcaaatggataaaaacggccccgggcggccgccgctctgtcaggtgc101 ggcatgtaactttttcagcgaacggcacgcgctgtccggcaaaacacacgacttgcacgcggtcgatggtccgtcagacggacatttgtctaaccagacc201 ggtgcgaaagatttgccgatggatgcatctttgcaaaatgcgggcccgagcagtggagcgtaggttcggattgcgccggcgcggccggcttgaccgtggc301 cgcgagcgagacatgtgcaactgacgcgccggctttcaaccgcctcgagatcgcccgatcgaggcgctcgatcgattgcagcggacctgccagcactgca401 acggcaagggtaagggcaaccgcagtccgacttgggtcgtcacgaatttttgttgcatccataaccatctttaccaatcctttgttaattcaaattgcca501 tacctattttcaaaattatgtctagttaaattttttcgcaagcacatttccaatgcatttgcctaccatttctataccgcgaacggatgctgttcgcgtc601 tcttttaccgatcgct cgcATGAACCTGCAACGATTCCCTCGTTACCCGCTGACTTTCGGGCCGACGCCAATCCAACCGCTAGCGCGTCTGAGCA701 AGCACCTCGGCGGCAAAGTGCATCTGTATGCGAAACGCGAAGACTGCAACAGCGGCCTGGCGTTCGGTGGCAACAAGACACGCAACTCGAATATCTGAT801 CCCTGAAGCGCTTGCTCAGGGTTGCGAACGCTCGTGTCGATCGGCGGCATTCAGTCGAACCAGACGCGCCAGGTGGCGGCCGTGGCGGCTCATCTGGGC901 ATGAAGTGCGTGCTGGTGCAGGAGAACTGGGTCAACTATTCGGACGCAGTCTACGACCGCGTCGGCAACATCCAGATGTCGCGCATTCTCGGCGCCGATG1001 TTCGCCTCGTGCCCGACGGCTTCGAQTCGGTTTTCGCAGGAGCTGGGAGGATGCGCTGGAAAGCGTGCGGGCGGCCGGCGGCAAGCCGTATGCGATTCC1101 GGCAGGCTGCTCGGATCACCCGCTCGGCGGCCTGGGTTTCGTCGGCTTCGCGGAGGAGGTGCGGGCGQGGACGAATTGGGCTTCAAATTCGACTAT1201 GTCGTCGTGTGTTCCGTGACCCAGC CGGCATGGTGGTGGGCTTCGCCGCTGACGGCCGCGCCGATCGCGTGATCGGCGTCGACGCTTCGG1301 CCAAACCCGCGCAGACGCGCGAGCAGATQCCCGCATCGCGAGAQGACCGCGGAGAAAGTCGGCCTGGAGCGCGATATCATGCGGGCCGACGTGGTGCT1401 CGACGAGCGCTTCGCGGGTCCGGAATACGGATTGCCGAACGAAGGCACGCTGGAAGCGATCCGCTTGTGCGCGCGCACGGAGGGCATGCTGACCGATCCC1501 GTCTACGAAGGCAAATCGATGCACGGCATGATCGAAATGGTGCGCAACGGCGAATTTCCGGAAGGCTCGCGCGTGCTGTATGCGCACCTCGGCGGGGTGC1601 CGGCGTTGAACGGCTACAGCTTTATCTTCCGAGACGGCTGAacgctccgaccggcggccagcaccgg_gcc_ tgtccaqccq_tc_c1701 tqctttactcgtgcgcttt_ctcgtgc_ctttact_gtgcgct_ctcgtgcg_ctttac_attgcaccggatcaccgcccccggctgacacgc1801 acgcggtcaaacaaggctttacgcgctcaagcgcttcctcaaatgctctctcaagaactccgcgaacgcccggatcgtgagggagctttgccggtgctgc1901 ggataaacggcgtagacgctcgtcgcagtaggcagaaactcttcgagcaccggtacgagctgaccggtcttcagcgcgtccgcgacgataaaatcaggaa2001 gccgaacaatcccgaggcccgccacggccgcatcgcgcaccagttcgccattgttggcgcgcaatggaccgtgtacctcgacgcccttcgtcacgccatc2101 cacgacgaactcccagttcaccgccgccatggccatacagcagacacgaatgacgcgccagatcgccggngaccgccggtncgccgcggcggcgcaga2201 tagcccggactgcag

FIG. 3. Restriction map and nucleotide sequence of Pseudomonas sp. strain ACP ACC deaminase genomic clone. (A) Restriction map ofthe 6-kb genomic clone. Restriction endonuclease recognition sites are indicated. The location of the ACC deaminase ORF is indicated byarrows. (B) Nucleotide sequence of the 2.2-kb EcoRI-PstI fragment containing the ACC deaminase gene. The ORF corresponding to theamino acid sequence of purified ACC deaminase is shown in uppercase letters. A consensus Shine-Dalgarno sequence in the 5' upstreamregion is underlined. A series of repeated sequences are found in the 3' nontranslated region, including a possible stem-loop motif (doubleunderline) and a series of direct repeats (single underline).

VOL. 173, 1991

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5264 SHEEHY ET AL.

A1 2 3 B1 2 3

-69

-46

-30

FIG. 4. Expression of ACC deaminase in minicells containingpCGN1479. (A) Autoradiogram of 35S-labeled polypeptides from E.coli YS1(pCGN1479) lysates after separation by SDS-PAGE andtransfer to nitrocellulose. (B) Immunoblot of the same nitrocelluloseblot used in panel A but reacted with ACC deaminase antibodies anddeveloped as described in Materials and Methods. Lanes 1, E.coli(pBSK-); 2, E. coli(pCGN1479); 3, E. coli YS1. The molecularmasses of protein standards are indicated to the right (kilodaltons).

72 bp upstream of the ATG encoding the N-terminal methi-onine of the purified protein. The significance of this se-quence is unknown. A hydropathic plot of the predicted24-amino-acid peptide (data not shown) is not typical ofother prokaryotic signal sequences (21). In addition, a Shineand Dalgarno consensus ribosome-binding sequence (19) islocated at position -11 (Fig. 3B) in relation to the ATGcorresponding to the first methionine found in the proteinsequence. A TGA termination codon is preceded in the ORFby a glycine codon; glycine is also the C-terminal residue ofthe ACC deaminase protein (Fig. 1). A number of unusualrepeated sequences found in the ACC deaminase gene 3'noncoding region are indicated in Fig. 3B. The sequenceTACTCGTGCGCTT is present as multiple direct repeats(single underline), and the sequence AAGCAGCGACCGGappears as an inverted repeat (double underline) potentiallycapable of forming a stem-loop structure. The significance ofthese sequences, as for repeated sequences found in otherPseudomonas genes, such as algP (11), is unclear but mayrepresent regions which interact with transcriptional factors.

Expression of ACC deaminase in E. coli. pCGN1479, whichcontains the ACC deaminase gene on a 6-kb EcoRI frag-ment, was used to transform E. coli YS1, a minicell-produc-ing strain. 35S-labeled proteins encoded by the plasmid wereseparated by SDS-PAGE, blotted to nitrocellulose, andvisualized by autoradiography (Fig. 4A). YS1 transformedwith the vector pBSK- without insert (lane 1) produced theprecursor (31.5 kDa) and mature (26 kDa) forms of P-lacta-mase (15). YS1 transformed with pCGN1479 (lane 2), whichis pBSK- containing the 6-kb insert, synthesized additionalpolypeptides including a major protein of approximately 36kDa, the expected size for ACC deaminase. The identity ofthe 36-kDa polypeptide as ACC deaminase was confirmed byreaction of the same nitrocellulose filter shown in Fig. 4Awith antibodies raised against ACC deaminase. As shown inFig. 4B, lane 2, the 36-kDa protein reacted with ACCdeaminase antibody, resulting in a band visible by secondantibody-dependent color development.The plasmid pCGN1479 contains over 600 bp of Pseudo-

monas DNA sequences upstream from the ATG codonrepresenting the first amino acid in the ACC deaminaseprotein sequence. Expression of the ACC deaminase gene in

E. coli did not require induction by ACC. It is possible thatthe E. coli lacZ promoter is responsible for the observedexpression of the ACC deaminase gene in E. coli YS1minicells as well as during immunological screening of thePseudomonas genomic library in E. coli SURE. Conversely,sequences resembling the -10 and -35 promoter regions ofother Pseudomonas genes which are recognized by E. coliRNA polymerase (4) can be found upstream from the ACCdeaminase gene coding region. Defining the regulatory re-gions responsible for ACC deaminase gene expression willrequire further studies, including establishing the transcrip-tional start site.

Activity of ACC deaminase in E. coli. The ORF of the ACCdeaminase gene corresponding to the amino acid sequence ofthe purified protein was subcloned from pCGN1479 intopUC19 to form a translational fusion with the lacZ geneproduct. This was accomplished by using PCR primersdesigned to introduce restriction sites to facilitate precisecloning of the ORF and ensure proper frame alignment withthe lacZ gene product. Primers ACCD55 and ACCD53, andACCD35 and ACCD33 (Fig. 2) were used in PCR withpCGN1479 as the template to construct the 5' and 3' regionsof the ORF, respectively. The sequences of these 5' and 3'PCR products were confirmed by DNA sequencing. Theinternal portion of the ACC deaminase gene from pCGN1479was then isolated as an NheI-to-ClaI fragment and ligatedinto a plasmid containing the 5' and 3' PCR fragments. Thefinal plasmid was designated pCGN1472. Levels of ACCdeaminase activity were determined in extracts of E. coliSURE transformed with pCGN1472 and grown in T-brothand compared with levels found in E. coli without the ACCdeaminase gene construct grown in T-broth and in Pseudo-monas sp. strain ACP grown on M9 salts with 2% glucosecontaining 0.1% ACC as the sole nitrogen source. Thespecific activity of ACC deaminase in E. coli(pCGN1472)(56.4 U/mg of protein) was comparable to levels measured inPseudomonas sp. strain ACP induced for ACC deaminaseactivity (29.5 U/mg of protein) and was undetectable innontransformed E. coli. We were, however, unable to obtaingrowth of E. coli(pCGN1472) on medium containing ACC asthe sole nitrogen source, despite the relatively high levels ofACC deaminase activity expressed in this strain.

In summary, ACC deaminase purified from Pseudomonassp. strain ACP was sequenced, and the amino acid sequenceinformation was utilized to clone the gene encoding thisenzyme. DNA sequence analysis of the ACC deaminasegene demonstrated the deduced amino acid sequence to beidentical to the chemically determined amino acid sequence.The authenticity of the ACC deaminase clone was alsodemonstrated through expression of the gene product in E.coli minicells and the demonstration of active enzyme inextracts from E. coli cells transformed with a lacZ-ACCdeaminase gene fusion.

Levels of ACC limit ethylene biosynthesis in plants.Ethylene evolution, in turn, affects numerous characteristicsof plant physiology. In particular, the induction of ethylenebiosynthesis triggers the initiation of several stages of plantdevelopment including fruit ripening and leaf and flowersenescence. Introduction of the ACC deaminase gene intoplants by A. tumefaciens-mediated transformation may pro-vide a mechanism by which to shunt ACC away fromethylene production and a means to control the onset ofthese developmental processes. Such experiments are cur-rently under way.

J. BACTERIOL.

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ACC DEAMINASE GENE FROM A PSEUDOMONAS SP.

ACKNOWLEDGMENTS

We thank Mindy Wilke-Douglas for technical assistance, BurtRose for DNA sequencing, Elaine Best and Gregory Thompson forcritical reading of the manuscript, and Shang Fa Yang for advice andhelpful discussions.

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