Pseudomonas sp. Strain ACP Gene Encoding 1-Aminocyclopropane ...

Vol. 173, No. 17

JOURNAL OF BACTERIOLOGY, Sept. 1991, p. 5260-5265 0021-9193/91/175260-06$02.00/0 Copyright ©) 1991, American Society for Microbiology

Isolation, Sequence, and Expression in Escherichia coli of the Pseudomonas sp. Strain ACP Gene Encoding 1-Aminocyclopropane1-Carboxylate Deaminase RAYMOND 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, Japan2 Received 27 March 1991/Accepted 17 June 1991

Pseudomonas sp. strain ACP is capable of growth on l-aminocyclopropane-l-carboxylate (ACC) as a nitrogen source owing to induction of the enzyme ACC deaminase and the subsequent conversion of ACC to a-ketobutyrate and ammonia (M. Honma, Agric. Biol. Chem. 49:567-571, 1985). The complete amino acid sequence of purified ACC deaminase was determined, and the sequence information was used to clone the ACC deaminase gene from a 6-kb EcoRI fragment of Pseudomonas sp. strain ACP DNA. DNA sequence analysis of an EcoRI-PstI subclone demonstrated an open reading frame (ORF) encoding a polypeptide with a deduced amino acid sequence identical to the protein sequence determined chemically and a predicted molecular mass of 36,674 Da. The ORF also contained an additional 72 bp of upstream sequence not predicted by the amino acid sequence. Escherichia coli minicells containing the 6-kb clone expressed a major polypeptide of the size expected for ACC deaminase which was reactive with ACC deaminase antiserum. Furthermore, a lacZ fusion with the ACC deaminase ORF resulted in the expression of active enzyme in E. coli. ACC is a key intermediate in the biosynthesis of ethylene in plants, and the use of the ACC deaminase gene to manipulate this pathway is discussed. sequence of purified ACC deaminase was determined and used to design oligonucleotide primers. The primers were used in polymerase chain reactions (PCR) to isolate a portion of the gene, which was used to screen a Pseudomonas genomic library. The DNA sequence of the ACC deaminase gene is presented, and its authenticity is demonstrated by expression of the gene in Escherichia coli.

Pseudomonas sp. strain ACP and the yeast Hansenula saturnus are capable of utilizing the cyclopropanoid amino acid 1-aminocyclopropane-1-carboxylate (ACC) as a nitrogen source owing to induction of the enzyme ACC deaminase (EC 4.1.99.4) in these organisms (8). Pseudomonas ACC deaminase has an estimated molecular mass of 110 kDa and is composed of three identical subunits, each with a molecular mass estimated to be 36.5 kDa (7, 8). ACC deaminase utilizes pyridoxal 5'-phosphate as a cofactor in catalyzing the cleavage of ACC to a-ketobutyrate and ammonia (7, 22). ACC was originally identified as a natural product in the juices of several fruits (2) and is now regarded as a key intermediate in the biosynthesis of the plant hormone ethylene (23). Ethylene is biosynthesized in higher plants from methionine via S-adenosylmethionine and ACC (1, 24). The rate-limiting step in ethylene biosynthesis is the production of ACC by a ring-closing displacement of methylthioadenosine from S-adenosylmethionine in a reaction catalyzed by ACC synthase (1). The reverse of this reaction can be viewed as analogous to the reaction catalyzed by ACC deaminase (22). Ethylene influences many aspects of plant development, including leaf and flower senescence and fruit ripening (23). Techniques for the integration and expression of bacterial genes in plants utilizing Agrobacterium tumefaciens are available (14), and the expression of an ACC deaminase gene in plants may provide a means to perturb ACC levels and ethylene biosynthesis, thus leading to a better understanding of the role of this plant hormone. As an initial step in this process, the ACC deaminase gene from Pseudomonas sp. strain ACP was isolated and characterized. The amino acid *

MATERIALS AND METHODS Bacterial strains and growth media. Lambda Zap II and E. coli XL1-Blue and SURE were obtained from Stratagene (La Jolla, Calif.). Bluescript plasmids pBCSK+, pBSK-, and pBCKS+ (Stratagene) in E. coli SURE or DH5a (5) were used for DNA sequencing. pUC19 (25) was used for subcloning in E. coli SURE. E. coli XL1-Blue and SURE strains were maintained on Luria-Bertani (LB) agar plates supplemented with 20 jig of tetracycline per ml and routinely grown at 37°C in LB medium or Terrific broth (T-broth) (13). Pseudomonas sp. strain ACP was grown and maintained in T-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 ACC deaminase. The minicell-producing E. coli YS1 (thr leu thi str minA endI; obtained from D. Stalker) was grown and maintained as previously described (3). E. coli containing plasmid pCGN2332, pCGN1479, or pCGN1472 or pBSKsubclones was grown on LB agar plates or LB medium supplemented with 300 jig of penicillin per ml. E. coli containing pBCSK+ or pBCKS+ plasmids was grown on the same medium but supplemented with 20 p,g of chloramphenicol 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

Corresponding author. 5260

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ACC DEAMINASE GENE FROM A PSEUDOMONAS SP.

ammonium sulfate. The enzyme preparation was dialyzed against 0.1 M potassium phosphate (pH 7.5), reduced with sodium borohydride, and dialyzed against the same buffer. Reduction of the enzyme leads to irreversible binding of the pyridoxal phosphate coenzyme, which can be monitored by the disappearance of an absorption band at 416 nm. Reduced ACC deaminase or reduced enzyme digested with trypsin (see below) was pyridylethylated by the following procedure. Enzyme or peptides were denatured with 2-mercaptoethanol (1 [lI/mg of enzyme) in 6 M guanidine hydrochloride under nitrogen for 15 h at room temperature. Pyridylethylation of cysteine residues was done by addition of 4-vinylpyridine (1.5 [lI/mg of protein) and further incubation for 1 h. The reaction was stopped by acidifying to pH 3.0 with glacial acetic acid. Proteolytic digestions and purification of peptides. Reduced enzyme was digested with trypsin before denaturation, and denatured enzyme or peptides were digested with four different proteolytic enzymes. One milligram of dialyzed and reduced enzyme in 0.5 ml of 0.1 M potassium phosphate (pH 7.5) was incubated with 20 ,ug of tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma Chemical Co.) at 30°C for 12 to 24 h. Tryptic peptides were modified with vinylpyridine and fractionated with a Finepak SILC8-P column (4.6 by-50 mm) run at 70°C and a gradient of 20 to 30% propyl alcohol in 0.1% trifluoroacetic acid. Peptides and pyridylethylated ACC deaminase were purified by using A'sahipak C4P-50 (4.6 by 250 mm) or Microbondasphere C4-300 A (3.9 by 150 mm) columns run at 50°C and a gradient of 0 to 40% acetonitrile in 0.1% trifluoroacetic acid. One peptide (T3) was purified by using a gradient of 25 to 35% acetonitrile. Lysyl endopeptidase digestions were performed by dissolving purified pyridylethylated ACC deaminase or tryptic peptides in 8 M urea (pH 9.0). Peptide and urea concentrations were adjusted to 0.5 mg/nil and below 4 M, respectively, by the addition of 0.01 M Tris (pH 9.0). One microgram of lysyl endopeptidase (WAKO Pure Chemical, Inc.) was added per 50 pug of peptide and incubated at 30°C for 24 h. Staphylococcal protease digestions were performed by dissolving peptides in a small amount of 8 M urea (pH 7.8). Four or more volumes of 0.05 M ammonium bicarbonate (pH 7.8)-S5 mM EDTA were added to adjust peptide concentrations to 0.25 mg/ml. After the addition of 1 ,ug of protease from Staphylococcus aureus V8 (Sigma) per 50 ,ug of peptide, the mixture was incubated at 30°C for 24 h. Peptides were digested with endoproteinase Asp-N or chymotrypsin by dissolving samples in 8 M urea and then adding 0.05 M sodium phosphate (pH 8.0) to adjust peptide concentrations to 0.1 mg/ml and urea concentrations below 1 M for endoproteinase Asp-N (Boehringer Mannheim) or below 2 M for tolysulfonyl lysyl chloromethyl ketone (TLCK)-treated chymotrypsin (Sigma) digestions. The appropriate enzyme (2 pug) was added to 1 ml of the mixture and incubated at 37°C for 12 to 24 h. Peptides were separated by using Asahipak C4P-50 or C8P-50 columns or Microbondasphere C4 columns and a gradient of 0 to 40% acetonitrile as described above. Chromatograms were monitored at 216 nm for peptides, 280 nm for tryptophan, and 320 nm for the coenzyme. Peptide sequencing and other methods. Fractions containing purified peptides were evaporated and applied to a protein sequencer (Applied Biosystems model 477A-120A system) for analysis. The C-terminal amino acid was determined by the method of Hayashi (6) by carboxypeptidase Y and amino acid analysis (Applied Biosystems). Phosphate ion was detected by the method described by Kempers (10).

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Peptide molecular mass was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12). DNA manipulations. Chromosomal DNA was isolated from cultures of Pseudomonas sp. strain ACP grown overnight in PYG medium. Cells were harvested by centrifugation, suspended in 3 ml of 10 mM Tris-HCl (pH 7.5)-l mM EDTA (TE buffer), and disrupted by the addition of 1 ml of 10% SDS-1 ml of pronase (5 mg/ml) and incubation for 30 min at 37°C. DNA was precipitated with ethanol, dissolved in TE buffer, and extracted with phenol-chloroform (1:1, vol/vol) and then again precipitated with ethanol and resuspended in TE buffer. PCR, using oligonucleotide primers and Pseudomonas sp. strain ACP DNA as a template, were conducted with a DNA Thermal Cycler (Perkin-Elmer Cetus) under standard reaction conditions suggested by the manufacturer. The PCR products were separated by electrophoresis through a 1% agarose gel. For Southern analysis, Pseudomonas DNA was digested with various restriction endonucleases (Boehringer Mannheim), and the resulting fragments were separated by electrophoresis through 1.5% agarose gels and blotted onto nylon membranes (Nytran; Schleicher & Schuell). DNA blots were probed with DNA fragments radioactively labeled with 32P by the nick translation method (nick translation kit; Boehringer Mannheim). Prehybridization and hybridization solutions were as previously described (17) except that the concentration of Denhardt's solution was reduced to 2 x and dextran sulfate was added to a final concentration of 10% for hybridization. Prehybridizations and hybridizations were done at 42°C. After hybridization, the membranes were washed with 0.1 x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS at 55 to 65°C and exposed to Kodak X-AR film. Pseudomonas sp. strain ACP genomic library construction and screening. Pseudomonas DNA was digested to completion with EcoRI, and fragments of 3 to 7 kb were isolated from an agarose gel by electroelution onto dialysis membrane. The size-fractionated DNA was ligated into EcoRIdigested and alkaline phosphatase-treated lambda ZAP II bacteriophage arms and packaged to produce viable phage particles following the manufacturer's instructions (Stratagene). The phage (75% recombinant) were plated on a lawn of E. coli XL1-Blue or SURE and screened for ACC deaminase clones by hybridization with a PCR-generated fragment (9) and immunologically (9, 26) with antibody raised against purified ACC deaminase in rabbits. Recombinant plaques identified as positive were purified by sequential isolation and replating. Plasmid pBSK- containing the Pseudomonas DNA insert was then excised from the lambda ZAP II phage arms in E. coli SURE as described by the supplier (Stratagene). Restriction mapping, DNA sequencing, and subcloning. Restriction enzyme sites were mapped by standard procedures (13, 17). Deletion mutants for sequencing were created with a deletion kit (Bluescript Exo/Mung DNA Sequencing System; Stratagene) and by religating plasmids after digestion with various restriction enzymes. Novel oligonucleotide primers were also synthesized and used in sequencing reactions. The sequence of overlapping DNA fragments was determined by the dideoxy chain termination method (18). Both DNA strands were sequenced. DNA sequence information was analyzed by using Intelligenetics Gel and Seq programs. Protein manipulations. Minicells obtained from 500-ml cultures of E. coli YS1 were purified as described previously

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1 MNLQRFPRYPLTFGPTPIQPLARLSKHLGGKVHLYAKREDCNSGLAFGGN

i T2 | nET2K rE4T~~~~~~~2ES 51 XTRKLEYLIPEALAQGCDTLVSIGGIQSNQTRQVAAVAAHLGMKCVLVQE

I?I-2D6 101 NWVNYSDAVYDRVGNIQMSRILGADVRLVPDGFDIGFRRSWEDALESVRA

I -K1OD4 T3151 AGGKPYAIPAGCSDHPLGGLGFVGFAEEVRAQEAELGFKFDYVVVCSVTG i Kg 11 K4-

K10I

201 STQAGMVVGFAADGRADRVIGVDASAKPAQTREQITRIARQTAEKVGLER

K4

11

-K3

I

K

251 DIMRADVVLDERFAGPEYGLPNEGTLEAIRLCARTEGMLTDPVYEGKSMH T1C7K7 I Ti 301 GMIEMVRNGEFPEGSRVLYAHLGGVPALNGYSFIFRDG K9

1

FIG. 1. Peptide sequence of Pseudomonas sp. strain ACP ACC deaminase. Peptide sequences were assigned as a result of Edman degradation 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 indicate the ends of individual peptides.

(3, 16), suspended in 2 ml of Bacto-Methionine Assay Medium (13.1 mg/ml) (Difco) in M9 salts with 0.5% glucose, labeled with 40 ,uCi of [35S]methionine (specific activity, 800 Ci/mmol; Amersham) for 45 min, pelleted, and frozen. Pellets were resuspended in 400 ,ul of sample buffer (12) and heated to 90°C for 2 min. Aliquots of 20 ,ul were then separated by electrophoresis on SDS 12.5% polyacrylamide gels (12). 35S-labeled polypeptides were electroblotted to nitrocellulose membranes for autoradiography. The same blots were then probed with antibody produced against denatured ACC deaminase and developed by using the Protoblot system as described by the vendor (Promega Biotec, Madison, Wis.). Extracts from E. coli and Pseudomonas sp. strain ACP were prepared and assayed for ACC deaminase activity by measuring the production of a-ketobutyrate with 2,4-dinitrophenylhydrazine as a colorimetric indicator (8). One unit of ACC deaminase activity was defined as a change in A540 of 0.001/min. Protein concentrations were determined with Bradford reagent (Bio-Rad Laboratories), using bovine serum albumin as a standard. Nucleotide sequence accession number. The nucleotide sequence of the Pseudomonas sp. strain ACP ACC deaminase gene has been given GenBank accession number M73488. RESULTS AND DISCUSSION Amino acid sequence analysis. The amino acid sequence of purified ACC deaminase is shown in Fig. 1. ACC deaminase was incubated with trypsin and separated into four fractions by using a C8 column and a gradient of propanol. Further purification of two fractions with a C4 column gave peptides Ti (9 kDa) and T2 (15 kDa). The third fraction was a mixture of three peptides (14, 20, and 28 kDa); the smallest peptide was designated T3. The other peptides were thought to be combinations of tryptic peptides, T3-T1 and T2-T3. The fourth fraction comprised a peptide with a molecular mass approximately the same as intact ACC deaminase (36.5 kDa). N-terminal and C-terminal sequence analysis indicated that the T2 peptide was the N-terminal peptide and that the C-terminal amino acid, glycine, of the Ti peptide corresponded to the C-terminal residue of the intact subunit.

A reasonable order of the tryptic peptides was thus established as T2-T3-T1. The ratio of A32JA216 was 0.0013 for Ti, 0.014 for T2, and 0.0038 for T3, indicating that the T2 peptide bound the cofactor pyridoxal phosphate. The T2 peptide was further fragmented by lysyl endopeptidase (T2K peptides) or staphylococcal protease (T2E peptides), and the resulting peptides were separated on a C4 or C8 column and sequenced. Digestion with either protease generated a peptide of high A320, and the sequences of these two peptides overlapped (T2K1 and T2E4, Fig. 1). Phosphate ion corresponding to phosphopyridoxyllysine was detected in the Edman degradation product at position 51, indicating that the residue designated X in Fig. 1 is lysine. Analyses of five peptides from lysyl endopeptidase digestion, six peptides from staphylococcal protease digestion, and a tryptophan-containing peptide from endoproteinase Asp-N (T2D6), as indicated by A280, provided the amino acid sequence for an additional 135 residues of the T2 peptide. Reduced and pyridylethylated ACC deaminase was then digested with lysyl endopeptidase and separated into 10 peptides (KI to K10) on C4 and C8 columns. The sequences of these peptides covered the T3 and Ti regions, except for the sequence provided by peptide T1C7, which was generated by chymotrypsin digestion of Ti. One of the K peptides showed markedly higher A280. This peptide, K10, produced two tryptophan-containing peptides by endoproteinase Asp-N digestion. One of them (K1OD4) was analyzed to give a sequence connecting T2 and T3. As mentioned above, the conversion of S-adenosylmethionine to ACC is catalyzed by the enzyme ACC synthase in plants, and the reverse of this reaction is analogous to the reaction performed by ACC deaminase (22). Comparison of the amino acid sequence of ACC deaminase with the deduced amino acid sequence for ACC synthase (20) from tomato demonstrated two regions (residues 163 to 167 and 169 to 174 of ACC 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 region of the ACC deaminase enzyme is distant from the predicted cofactor-binding lysine at position 51, and the significance of this homology is unknown.

VOL. 173, 1991

ACC DEAMINASE GENE FROM A PSEUDOMONAS SP. Xba I

ACCD1

5'-CGTAICTAGATGAAQQTNCAPJGNTTQCCNJG-3'

ACCD3

Eco RI P 5'-TCACGAA3ICKNMLCATQTGKATPTrNCCNAC-3'

ACCD55

5'-TATCTAGGATCCCCATATGAACCrGCAACGATTCCCTC-3'

ACCD53

5'-TATTGGIACCGTTTCGCATACAGATGCAC-3'

ACCD35

5'-TTATAGGTACCGGCAAATCGATGCACGGCATG-3'

Bam HI

Asp

Nde I

718

Asp718

ClaI

Sst I

ACCD33

5'-ATTAAGAGTAGCCGTCTCGGAAGATAAAGC-3' FIG. 2. Oligonucleotide primers used for PCR reactions. The nucleotide sequences of the six oligonucleotide primers are shown with their respective restriction endonuclease recognition site(s) underlined. Symbols used to designate degeneracy in ACCD1 and ACCD3: 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 the ACC deaminase protein, for which reverse translation indicated a relatively unambiguous nucleotide sequence, was used to design oligonucleotide fragments for use as primers in PCR (Fig. 2). Recognition sites for various restriction endonucleases were also added to the primers to facilitate cloning of Pseudomonas DNA sequences. Primers ACCD1

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and ACCD3, representing amino acid sequences 1 to 7 and 113 to 120, respectively, were used in PCR with Pseudomonas sp. strain ACP DNA as the template to produce a 370-bp fragment of the ACC deaminase gene. This fragment was isolated and ligated into pBSK- to produce pCGN2332. Southern analysis of Pseudomonas sp. strain ACP DNA was conducted with the insert from pCGN2332 as a probe. The results of this analysis (data not shown) indicated that the ACC deaminase gene was contained on an EcoRI DNA fragment of approximately 6 kb. A Pseudomonas sp. strain ACP genomic library constructed in lambda ZAP II and enriched for the 6-kb EcoRI fragment was prepared, and approximately 6,000 PFUs were screened with the 370-bp insert from pCGN2332 as a hybridization probe. Six positive plaques were identified, and two of these were rescreened with antibody raised against ACC deaminase. One plaque reacted with antibody and was purified. The plasmid form of this clone, pCGN1479, was used as a source of DNA for mapping common restriction sites (Fig. 3A) and for DNA sequence analysis. The nucleotide sequence of a 2.2-kb EcoRI-PstI fragment that hybridized to the 370-bp ACC deaminase fragment from pCGN2332 is shown in Fig. 3B. The clone contains an open reading frame (ORF) which includes the entire coding region for the mature protein subunit with a predicted molecular mass of 36,674 Da. The deduced amino acid sequence from pCGN1479 is identical to the ACC deaminase protein sequence shown in Fig. 1. An in-frame ATG codon is located

A -

L-

_- oz X~ l

l

I.. I I

lkb

I

B 1 101 201 301 401

gaattctccgatctcgcatgtcgcgaggagggttatgggttcgcccaaggcccgcaaatggataaaaacggccccgggcggccgccgctctgtcaggtgc ggcatgtaactttttcagcgaacggcacgcgctgtccggcaaaacacacgacttgcacgcggtcgatggtccgtcagacggacatttgtctaaccagacc ggtgcgaaagatttgccgatggatgcatctttgcaaaatgcgggcccgagcagtggagcgtaggttcggattgcgccggcgcggccggcttgaccgtggc cgcgagcgagacatgtgcaactgacgcgccggctttcaaccgcctcgagatcgcccgatcgaggcgctcgatcgattgcagcggacctgccagcactgca acggcaagggtaagggcaaccgcagtccgacttgggtcgtcacgaatttttgttgcatccataaccatctttaccaatcctttgttaattcaaattgcca 501 tacctattttcaaaattatgtctagttaaattttttcgcaagcacatttccaatgcatttgcctaccatttctataccgcgaacggatgctgttcgcgtc 601 tcttttaccgatcgct cgcATGAACCTGCAACGATTCCCTCGTTACCCGCTGACTTTCGGGCCGACGCCAATCCAACCGCTAGCGCGTCTGAGCA 701 801 901 1001 1101 1201 1301 1401 1501

AGCACCTCGGCGGCAAAGTGCATCTGTATGCGAAACGCGAAGACTGCAACAGCGGCCTGGCGTTCGGTGGCAACAAGACACGCAACTCGAATATCTGAT

CCCTGAAGCGCTTGCTCAGGGTTGCGAACGCTCGTGTCGATCGGCGGCATTCAGTCGAACCAGACGCGCCAGGTGGCGGCCGTGGCGGCTCATCTGGGC ATGAAGTGCGTGCTGGTGCAGGAGAACTGGGTCAACTATTCGGACGCAGTCTACGACCGCGTCGGCAACATCCAGATGTCGCGCATTCTCGGCGCCGATG TTCGCCTCGTGCCCGACGGCTTCGAQTCGGTTTTCGCAGGAGCTGGGAGGATGCGCTGGAAAGCGTGCGGGCGGCCGGCGGCAAGCCGTATGCGATTCC

GGCAGGCTGCTCGGATCACCCGCTCGGCGGCCTGGGTTTCGTCGGCTTCGCGGAGGAGGTGCGGGCGQGGACGAATTGGGCTTCAAATTCGACTAT GTCGTCGTGTGTTCCGTGACCCAGC

CGGCATGGTGGTGGGCTTCGCCGCTGACGGCCGCGCCGATCGCGTGATCGGCGTCGACGCTTCGG CCAAACCCGCGCAGACGCGCGAGCAGATQCCCGCATCGCGAGAQGACCGCGGAGAAAGTCGGCCTGGAGCGCGATATCATGCGGGCCGACGTGGTGCT CGACGAGCGCTTCGCGGGTCCGGAATACGGATTGCCGAACGAAGGCACGCTGGAAGCGATCCGCTTGTGCGCGCGCACGGAGGGCATGCTGACCGATCCC GTCTACGAAGGCAAATCGATGCACGGCATGATCGAAATGGTGCGCAACGGCGAATTTCCGGAAGGCTCGCGCGTGCTGTATGCGCACCTCGGCGGGGTGC 1601 CGGCGTTGAACGGCTACAGCTTTATCTTCCGAGACGGCTGAacgctccgaccggcggccagcaccgg_gcc_ tgtccaqccq_tc_c 1701

tqctttactcgtgcgcttt_ctcgtgc_ctttact_gtgcgct_ctcgtgcg_ctttac_attgcaccggatcaccgcccccggctgacacgc

1801

1901 2001 2101 2201

acgcggtcaaacaaggctttacgcgctcaagcgcttcctcaaatgctctctcaagaactccgcgaacgcccggatcgtgagggagctttgccggtgctgc ggataaacggcgtagacgctcgtcgcagtaggcagaaactcttcgagcaccggtacgagctgaccggtcttcagcgcgtccgcgacgataaaatcaggaa gccgaacaatcccgaggcccgccacggccgcatcgcgcaccagttcgccattgttggcgcgcaatggaccgtgtacctcgacgcccttcgtcacgccatc cacgacgaactcccagttcaccgccgccatggccatacagcagacacgaatgacgcgccagatcgccggngaccgccggtncgccgcggcggcgcaga tagcccggactgcag

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

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2

J. BACTERIOL. 3

B1

2

3

-69

-46

-30 ...

.... ...... .. .. ... .... ....

.....

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..........

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

72 bp upstream of the ATG encoding the N-terminal methionine of the purified protein. The significance of this sequence is unknown. A hydropathic plot of the predicted 24-amino-acid peptide (data not shown) is not typical of other prokaryotic signal sequences (21). In addition, a Shine and Dalgarno consensus ribosome-binding sequence (19) is located at position -11 (Fig. 3B) in relation to the ATG corresponding to the first methionine found in the protein sequence. A TGA termination codon is preceded in the ORF by a glycine codon; glycine is also the C-terminal residue of the ACC deaminase protein (Fig. 1). A number of unusual repeated sequences found in the ACC deaminase gene 3' noncoding region are indicated in Fig. 3B. The sequence TACTCGTGCGCTT is present as multiple direct repeats (single underline), and the sequence AAGCAGCGACCGG appears as an inverted repeat (double underline) potentially capable of forming a stem-loop structure. The significance of these sequences, as for repeated sequences found in other Pseudomonas genes, such as algP (11), is unclear but may represent regions which interact with transcriptional factors. Expression of ACC deaminase in E. coli. pCGN1479, which contains the ACC deaminase gene on a 6-kb EcoRI fragment, was used to transform E. coli YS1, a minicell-producing strain. 35S-labeled proteins encoded by the plasmid were separated by SDS-PAGE, blotted to nitrocellulose, and visualized by autoradiography (Fig. 4A). YS1 transformed with the vector pBSK- without insert (lane 1) produced the precursor (31.5 kDa) and mature (26 kDa) forms of P-lactamase (15). YS1 transformed with pCGN1479 (lane 2), which is pBSK- containing the 6-kb insert, synthesized additional polypeptides including a major protein of approximately 36 kDa, the expected size for ACC deaminase. The identity of the 36-kDa polypeptide as ACC deaminase was confirmed by reaction of the same nitrocellulose filter shown in Fig. 4A with antibodies raised against ACC deaminase. As shown in Fig. 4B, lane 2, the 36-kDa protein reacted with ACC deaminase antibody, resulting in a band visible by second antibody-dependent color development. The plasmid pCGN1479 contains over 600 bp of Pseudomonas DNA sequences upstream from the ATG codon representing the first amino acid in the ACC deaminase protein sequence. Expression of the ACC deaminase gene in

E. coli did not require induction by ACC. It is possible that the E. coli lacZ promoter is responsible for the observed expression of the ACC deaminase gene in E. coli YS1 minicells as well as during immunological screening of the Pseudomonas genomic library in E. coli SURE. Conversely, sequences resembling the -10 and -35 promoter regions of other Pseudomonas genes which are recognized by E. coli RNA polymerase (4) can be found upstream from the ACC deaminase gene coding region. Defining the regulatory regions responsible for ACC deaminase gene expression will require further studies, including establishing the transcriptional start site. Activity of ACC deaminase in E. coli. The ORF of the ACC deaminase gene corresponding to the amino acid sequence of the purified protein was subcloned from pCGN1479 into pUC19 to form a translational fusion with the lacZ gene product. This was accomplished by using PCR primers designed to introduce restriction sites to facilitate precise cloning of the ORF and ensure proper frame alignment with the lacZ gene product. Primers ACCD55 and ACCD53, and ACCD35 and ACCD33 (Fig. 2) were used in PCR with pCGN1479 as the template to construct the 5' and 3' regions of the ORF, respectively. The sequences of these 5' and 3' PCR products were confirmed by DNA sequencing. The internal portion of the ACC deaminase gene from pCGN1479 was then isolated as an NheI-to-ClaI fragment and ligated into a plasmid containing the 5' and 3' PCR fragments. The final plasmid was designated pCGN1472. Levels of ACC deaminase activity were determined in extracts of E. coli SURE transformed with pCGN1472 and grown in T-broth and compared with levels found in E. coli without the ACC deaminase gene construct grown in T-broth and in Pseudomonas sp. strain ACP grown on M9 salts with 2% glucose containing 0.1% ACC as the sole nitrogen source. The specific activity of ACC deaminase in E. coli(pCGN1472) (56.4 U/mg of protein) was comparable to levels measured in Pseudomonas sp. strain ACP induced for ACC deaminase activity (29.5 U/mg of protein) and was undetectable in nontransformed E. coli. We were, however, unable to obtain growth of E. coli(pCGN1472) on medium containing ACC as the sole nitrogen source, despite the relatively high levels of ACC deaminase activity expressed in this strain. In summary, ACC deaminase purified from Pseudomonas sp. strain ACP was sequenced, and the amino acid sequence information was utilized to clone the gene encoding this enzyme. DNA sequence analysis of the ACC deaminase gene demonstrated the deduced amino acid sequence to be identical to the chemically determined amino acid sequence. The authenticity of the ACC deaminase clone was also demonstrated through expression of the gene product in E. coli minicells and the demonstration of active enzyme in extracts from E. coli cells transformed with a lacZ-ACC deaminase gene fusion. Levels of ACC limit ethylene biosynthesis in plants. Ethylene evolution, in turn, affects numerous characteristics of plant physiology. In particular, the induction of ethylene biosynthesis triggers the initiation of several stages of plant development including fruit ripening and leaf and flower senescence. Introduction of the ACC deaminase gene into plants by A. tumefaciens-mediated transformation may provide a mechanism by which to shunt ACC away from ethylene production and a means to control the onset of these developmental processes. Such experiments are currently under way.

VOL. 173, 1991

ACC DEAMINASE GENE FROM A PSEUDOMONAS SP.

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