Abstract:
An Enterobacter cloacae-derived gene encoding an enzyme involved in the biosynthetic pathway from tryptophan to indoleacetic acid via indolepyruvic acid and indoleacetaldehyde as intermediates.

Description:
FIELD OF THE INVENTION 
     This invention relates to a gene encoding an enzyme involved in the biosynthetic pathway from tryptophan to indoleacetic acid via indolepyruvic acid and indoleacetaldehyde as intermediates. 
     BACKGROUND OF THE INVENTION 
     Indoleacetic acid (IAA) is a typical plant hormone. Its actions that have been demonstrated include, among others, promoting plant rooting, inducing stem and leaf elongation, inducing parthenocarpy and preventing aging. IAA is therefore a substance of considerable significance from the plant physiology viewpoint. It has been established that IAA is produced not only by plants but also by microorganisms and animals. As regards microorganisms, in particular, various investigations are in progress concerning the meaning of the production of plant hormones by bacteria occurring on or in the plant stem, leaf and root regions. Thus, for instance, it has been shown that Pseudomonas syringae pv. savastanoi infects olives and produces IAA, leading to tumor formation (Physiol. Plant Pathol., 13, 203-214, 1978). Furthermore, Agrobacterium tumefaciens infects dicotyledons and the T-DNA region of the Ti plasmid carried by this microorganism is integrated into the nucleus of said plants causing formation of tumors called crown galls. It has been revealed that the T-DNA contains a gene involved in the biosynthesis of IAA (Eur. J. Biochem., 138, 387-391, 1984). 
     It has been established that IAA is generally produced from tryptophan as a precursor via the following three biosynthetic pathways (Biol. Rev. , 48, 510-515, 1973). 
     1) Tryptophan→indoleacetamide→IAA; 
     2) Tryptophan→tryptamine→indoleacetaldehyde→IAA; 
     3) Tryptophan→indolepyruvic acid→indoleacetaldehyde→IAA. 
     The Pseudomonas and Agrobacterium species mentioned above have been shown to produce IAA by the biosynthetic pathway 1), and the enzymes involved therein and the genes coding therefor have been isolated (Proc. Natl. Acid. Sci. USA, 81, 1728-1732, 1984; ibid., 82, 6522-6526, 1985). 
     On the other hand, as regards the biosynthetic pathway 3), which is considered to be the main one in plants, neither the enzymes involved therein nor the genes coding therefor have been isolated because of instability of the intermediates involved. Accordingly, the mechanisms of control thereof have not been made clear as yet. As the above facts suggest, the plant physiology of IAA, a representative plant hormone, remains unknown in many respects and its functions have been known only insufficiently. 
     SUMMARY OF THE INVENTION 
     An object of the present invention is to isolate a gene encoding the biosynthetic route to IAA, to reveal the structure thereof, to introduce IAA gene of an enzyme or enzymes involved in the biosynthetic route to IAA into a plant or microorganism, to create a novel plant or microorganism of industrial value, and to utilize the same commercially. 
     As a result of intensive investigation, the present inventors found that Enterobacter cloacae isolated from the root region of a plant has the ability to produce IAA as one of the mechanisms of its plant growth promoting action. They also found, by identifying the reactions involved in conversion to IAA from its precursor, that the biosynthetic pathway to IAA involves indolepyruvic acid and indoleacetaldehyde as intermediates in the same manner as in plants. Furthermore, they isolated a DNA corresponding to the full-length IAA synthetase gene, introduced said DNA into Escherichia coli, confirmed the function of said gene by verifying the ability of an Escherichia coil transformant obtained by introducing said gene thereinto to produce IAA and determined the base sequence of said gene. 
     Thus the gist of the invention consists of a gene borne by Enterobacter cloacae and involved in the biosynthesis of IAA from tryptophan via indolepyruvic acid and indoleacetaldehyde as intermediates. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIGS. 1A-1C show a gene encoding indoleacetic acid synthetase. Amino acids are indicated according to the &#34;one-letter notation&#34; [&#34;Seikagaku Jiten (Dictionary of Biochemistry)&#34;. published by Tokyo Kagaku Dojin, page 1392, 1984]. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Enterobacter cloacae isolated from the root region of a plant has been deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology of Japan, of 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki 305 Japan, since Sep. 12, 1986 under the deposit number FERM BP-1529 in accordance with the Budapest Treaty. 
     The IAA synthetase gene according to the present invention is isolated in the following manner. 
     Enterobacter cloacae is suspension-cultured in LB medium (1% Bactotryptone (Difco), 0.5% yeast extract (Difco) and 0.5% NaCl, pH 7.0) at 37° C. for 24 hours with shaking. The nuclear DNA is extracted from the cells obtained by the alkaline method. This nuclear DNA is partially digested with Sau3AI and the resulting fragments are inserted into the plasmid pUC119 (Takara Shuzo) at the BamHI site. Usable plasmids are pUC118 (Takara Shuzo), PTZ19 and PTZ18 (Toyobo). The resulting recombinant plasmid DNA mixture is used to transform host cells such as Escherichia coli DH5α, JM109, JM105, DH1 and HB101 to give a genomic library. Preferred as a host is E. coli DH5α (BRL) which is incapable of producing IAA. Colonies are each suspension-cultured in LB medium at 37° C. for 24 hours. A clone having the IAA biosynthesis gene can be obtained by selecting an IAA producer. The base sequence of the thus-obtained IAA biosynthesis gene is determined by the dideoxy method and the corresponding amino acid sequence is as shown in FIGS. 1A-1C. 
     The following examples are further illustrative of the present invention but are by no means limitative of the scope thereof. 
     The techniques, reactions and analysis for use in the practice of the invention are well known in the art, as described in Molecular Cloning, a laboratory mannual, Cold Spring Harbor Laboratory Press, New York. 
     EXAMPLE 1 
     Nuclear DNA isolation from Enterobacter cloacae by the alkaline method 
     Enterobacter cloacae (FERM BP-1529) was shake-cultured in 100 ml of LB medium at 37° C. for 24 hours. Cells were recovered by centrifuging the culture at 10,000 revolutions per minute for 20 minutes. The cells were suspended in 16 ml of a TESH solution (0.2M Tris-HCl, 0.02M EDTA, 0.05M NaCl, pH 8.0). To the suspension were added 4 ml of 0.5M EDTA, 0.2 ml of 0.5% RNase A (Sigma) and 0.2 ml of egg white lysozyme (Sigma). The mixture was incubated at 37° C. for 2 hours. Then 1 ml of 5% SDS solution was added and the resultant mixture was incubated overnight at 37° C. with gentle stirring. To this reaction mixture was added an equal volume of phenol-saturated TESH solution. The mixture was shaken at room temperature for 10 minutes and then centrifuged at 3,500 revolutions per minute at room temperature for 10 minutes. The aqueous layer was taken up with care to exclude the intermediate layer and subjected to three repetitions of the above-mentioned phenol treatment. Two volumes of ethanol were added to the aqueous layer thus obtained and the nuclear DNA was rolled around a glass rod with stirring therewith and resolved in a TE solution (0.01M Tris-HCl, 0.001M EDTA, pH 8.0) to give a nuclear DNA solution. 
     EXAMPLE 2 
     Preparation of an Enterobacter cloacae-derived genomic library 
     (1) Sau3AI (Toyobo) was added, in amounts of 0.25, 0.5, 1, 2, 4 , 8 and 16 units, to respective 100-μl portions of the nuclear DNA solution (450 μg/ml) prepared from Enterobacter cloacae as described in Example 1 and each mixture was incubated at 37° C. for 30 minutes. The reaction mixtures were examined by agarose gel electrophoresis. Those reaction mixtures that contained DNAs of 1 to 20 kbp in length were pooled and subjected to three repetitions of phenol extraction (phenol-chloroform-isoamyl alcohol=50:49:1 by volume). To the solution thus obtained was added 1/10 volume of 3 N sodium acetate and 2 volumes of ethanol, and the mixture was cooled at -20° C. for 20 minutes and then centrifuged. The DNA thus recovered was washed with cold 90% ethanol, dried in vacuo and dissolved in 100 μl of the TE solution. 
     (2) To 500 μl of the plasmid vector pUC119 (40 μg/ml) was added 160 units of BamHl (Toyobo), and the digestion reaction was allowed to proceed at 37° C. for 4 hours. After 2 phenol extractions, DNA was recovered by ethanol precipitation and dried in vacuo. Sterile water (400 μl), 50 μl of 0.5M Tris-HCl, pH 8.0 and 50 μl of alkaline phosphatase (Boehringer) (1 unit/μl) were added to the DNA and, after mixing, followed by 3 phenol extractions and ethanol precipitation. The DNA thus recovered was dissolved in 50 μl of the TE solution. 
     (3) To 10 μl of the DNA-containing TE solution obtained as described above in (1) were added 10 μl of the DNA-containing TE solution obtained as described above in (2), 7 μl of T4 ligase (Toyobo) (5 units/μl) and 3 μl of a buffer solution (660 mM Tris-HCl, pH 7.6, 66 mM MgCl 2 , 100 mM dithiothreitol, 660 μM ATP). After mixing, the ligation reaction was conducted at 16° C. for 16 hours. 
     EXAMPLE 3 
     Identification of an IAA synthetase-encoding clone 
     (1 ) The ligation reaction mixture obtained as described in Example 2-(3) was added to competent DH5α cells prepared by the method of Hanahan (J. Mol. Biol., 166, 557, 1983) for transformation of said cells. The cells were sown onto L agar medium containing 100 ppm of ampicillin and 40 ppm of Xgal and cultured at 37° C. for 24 hours. White colonies that had appeared were picked up and respectively cultured in a liquid LB medium containing 100 ppm of ampicillin and a strain producing IAA in said medium was selected as a clone holding an IAA synthetase gene. The ability to produce IAA was checked by growing each transformant in the conventional manner and judging, by high-performance liquid chromatography, whether IAA was produced in the culture. 
     (2) The above-mentioned IAA-producing strain was suspension-cultured in L medium. Cells were collected and the plasmid DNA was extracted therefrom by the alkaline minipreparation method (Nuclear Acids Res., 7 (6), 1513-1523, 1979). This DNA was cleaved with BamHI and subjected to 0.8% agarose gel electrophoresis, which confirmed the insertion of a foreign DNA of about 4 kbp. A pUC119 transformant carrying this insert of about 4 kbp was named pIA119. 
     EXAMPLE 4 
     Sequencing of the IAA synthetase gene 
     (1) The Escherichia coli strain DH5α transformed with the plasmid pIA119 by a conventional method was cultured in 100 ml of LB medium overnight at 37° C. and cells were collected by centrifuging the culture at 10,000 revolutions per minute for 20 minutes. To the cells were added 200 ml of a TES solution (0.1M Tris-HCl, 0.02M EDTA, 25% sucrose, pH 8.0), 0.4 ml of RNase A (5 mg/ml) and 0.4 ml of egg white lysozyme (30 mg/ml) to give a cell suspension, which was maintained at 37° C. for 2 hours to allow the reaction to proceed. Then 8 ml of 20% SDS solution was added and the resultant mixture was stirred gently at room temperature. After the mixture became clear and transparent, 1.6 ml of 3 N NaOH was added and the resultant mixture was shaken gently at room temperature for 1 hour. Thereto was added 6 ml of 2M Tris-HCl (pH 7.0), followed by 2  minutes of gentle stirring. Thereafter, 10 ml of 5M NaCl was added and the mixture was stirred again gently and then allowed to stand overnight at 0° C. The mixture was then centrifuged at 5° C. and 10,000 revolutions per minute for 15 minutes. The supernatant was separated and one-tenth volume of polyethylene glycol #1000 (Nakalai Tesque) was added thereto. The mixture was stirred gently and then allowed to stand overnight at 0° C. The resultant precipitate was recovered by centrifugation (10,000 revolutions per minute, 15 minutes, 5° C.) and dissolved in 3.5 ml of the TESH solution by adding the solution thereto. To the resultant solution was added 4.4 g of CsCl (Nakalai Tesque) and 0.5 ml of an ethidium bromide solution (5 mg/ml). After mixing, the resultant mixture was centrifuged at 3,500 revolutions per minute for 10 minutes to eliminate the precipitate occurring on the surface of the aqueous layer. The solution obtained was centrifuged at 50,000 revolutions per minute at 20° C. for 16 hours and the second (from the top) band so identified under ultraviolet irradiation was recovered as plasmid DNA. This solution was subjected to 4 repetitions of butanol extraction for elimination of the ethidium bromide and then dialyzed against the TE solution for purification of the plasmid pIA119. 
     (2) Both ends of the insert DNA in the purified plasmid pIA119 were deleted using the EXO/MUNG system (Stratagene) and Escherichia coli DH5α transformants carrying the respective deletion product DNAs were checked as to whether they had IAA producing activity. As a result, it was found that a DNA of 1.65 kbp is the minimum DNA fragment necessary for the production of IAA. Then, the 1.65 kbp DNA was curtailed from the 5&#39; terminus using the EXO/MUNG system to give 10 deletion product DNAs differing in length by about 200 bp. Furthermore, the 1.65 kbp insert DNA was subcloned into the plasmid pUC118 (Takara Shuzo) between the sites for recognition by EcoRI and HindIII (Toyobo) and curtailed in the same manner from the 5&#39; terminus, whereby 10 deletion products DNAs resulting from reverse direction curtailment were obtained. 
     (3) The 20 deletion product DNAs obtained as mentioned above were introduced into Escherichia coli JM109 by a conventional method. Single-stranded DNAs were recovered therefrom by the method of Messing (Vieira, J. and Messing, J., Methods in Enzymology, 153, 3-11 (1987)) and the base sequence of the 1.65 kbp insert DNA was determined by the dideoxy method of Sanger et al. The base sequence of the IAA biosynthesis gene as revealed by analyzing the base sequence of the 1.65 kbp insert DNA and the corresponding amino acid sequence are shown in FIGS. 1A-1C. 
     While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof. 
     
         __________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 2(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 1659 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: N(iv) ANTI-SENSE: N(vi) ORIGINAL SOURCE:(A) ORGANISM: Enterobacter cloacae(vii) IMMEDIATE SOURCE:(A) LIBRARY: genomic(B) CLONE: PIA119(ix) FEATURE:(A) NAME/KEY: matpeptide(B) LOCATION: 1..1659(D) OTHER INFORMATION:/product=&#34;indoleacetic acid synthetase&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:ATGCGAACCCCATACT GCGTCGCCGATTACCTGCTGGACCGTCTTACAGATTGTGGTGCC60GATCATCTGTTTGGCGTGCCGGGCGACTATAACCTGCAGTTTCTCGACCACGTAATAGAC120AGCCCGGATATTTGTTGGGTGGGCTGTGCCAATGAGCTGAACGCATCCTATGCCGCTGAC 180GGATACGCCCGATGTAAGGGCTTTGCCGCGCTACTGACCACATTCGGCGTTGGGGAGTTA240AGTGCCATGAACGGCATTGCCGGCAGCTATGCCGAGCATGTCCCGGTTTTACATATTGTG300GGGGCGCCGGGTACGGCGGCACAGCAAAGGGGAGAGTTG CTGCATCATACGTTGGGGGAT360GGGGAGTTCCGTCACTTTTATCATATGAGCGAGCCGATCACCGTCGCACAGGCGGTCCTT420ACCGAACAAAATGCCTGTTATGAAATCGACCGTGTGTTGACAACCATGCTTCGGGAACGC480CGCCCCGGTTATCTGA TGTTACCCGCCGATGTGGCAAAAAAAGCCGCCACGCCGCCTGTA540AACGCTCTCACTCATAAGCAGGCTCATGCCGATAGCGCCTGCCTGAAAGCGTTCCGGGAT600GCTGCTGAGAACAAACTGGCGATGAGCAAACGTACCGCGCTGCTGGCCGACTTCCTTGTT 660CTGCGCCATGGCCTGAAACATGCGCTACAGAAGTGGGTGAAAGAGGTACCGATGGCCCAT720GCCACCATGCTGATGGGGAAAGGGATATTCGACGAGCGTCAGGCGGGTTTTTACGGCACA780TACAGTGGTTCAGCCAGCACTGGCGCGGTAAAAGAGGCG ATTGAAGGGGCTGACACGGTA840TTGTGTGTTGGCACGCGTTTTACCGATACCCTGACGGCCGGGTTCACGCACCAGCTTACC900CCGGCGCAGACCATTGAAGTTCAGCCGCATGCCGCACGCGTCGGGGATGTCTGGTTTACC960GGCATCCCAATGAACC AGGCGATTGAGACGCTGGTCGAACTCTGCAAACAGCACGTGCAT1020GCTGGCCTTATGTCGTCATCATCCGGCGCAATACCGTTCCCGCAGCCGGACGGTTCGCTT1080ACCCAGGAGAATTTCTGGAGAACGTTGCAAACCTTTATTCGCCCGGGGGACATTATCCTT 1140GCCGACCAGGGAACATCGGCCTTCGGCGCGATTGATCTGCGTTTACCGGCTGATGTGAAC1200TTTATCGTCCAGCCGCTGTGGGGCTCGATTGGTTACACGCTGGCGGCGGCGTTTGGTGCA1260CAAACCGCATGCCCGAACCGGCGCGTGATTGTGCTGACG GGGGATGGCGCTGCCCAGCTC1320ACTATTCAGGAACTAGGCTCGATGCTGCGTGATAAACAGCACCCCATTATTCTGGTGCTC1380AACAACGAAGGTTACACGGTTGAGAGGGCTATCCACGGGGCGGAGCAGCGGTATAACGAC1440ATTGCTTTGTGGAACT GGACGCACATTCCGCAGGCGTTGAGCCTCGATCCTCAGTCTGAG1500TGCTGGCGGGTCAGTGAAGCGGAACAGCTGGCGGACGTACTTGAAAAAGTGGCGCACCAC1560GAGCGGCTCTCGTTGATTGAGGTGATGCTCCCGAAAGCGGATATCCCGCCGCTGCTCGGG 1620GCGCTTACCAAGGCTCTGGAAGCGTGTAATAACGCCTGA1659(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 552 amino acids(B) TYPE: amino acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(iii ) HYPOTHETICAL: Y(vi) ORIGINAL SOURCE:(A) ORGANISM: Enterobacter cloacae(ix) FEATURE:(A) NAME/KEY: Protein(B) LOCATION: 1..552(D) OTHER INFORMATION:/note=&#34;indoleacetic acid synthetase&#34;(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:MetArgThrProTyrCysValAlaAspTyrLeuLeuAspArg LeuThr151015AspCysGlyAlaAspHisLeuPheGlyValProGlyAspTyrAsnLeu2025 30GlnPheLeuAspHisValIleAspSerProAspIleCysTrpValGly354045CysAlaAsnGluLeuAsnAlaSerTyrAlaAlaAspGlyTyrAla Arg505560CysLysGlyPheAlaAlaLeuLeuThrThrPheGlyValGlyGluLeu65707580 SerAlaMetAsnGlyIleAlaGlySerTyrAlaGluHisValProVal859095LeuHisIleValGlyAlaProGlyThrAlaAlaGlnGlnArgGly Glu100105110LeuLeuHisHisThrLeuGlyAspGlyGluPheArgHisPheTyrHis115120125 MetSerGluProIleThrValAlaGlnAlaValLeuThrGluGlnAsn130135140AlaCysTyrGluIleAspArgValLeuThrThrMetLeuArgGluArg145 150155160ArgProGlyTyrLeuMetLeuProAlaAspValAlaLysLysAlaAla165170175 ThrProProValAsnAlaLeuThrHisLysGlnAlaHisAlaAspSer180185190AlaCysLeuLysAlaPheArgAspAlaAlaGluAsnLysLeuAlaMe t195200205SerLysArgThrAlaLeuLeuAlaAspPheLeuValLeuArgHisGly210215220LeuLys HisAlaLeuGlnLysTrpValLysGluValProMetAlaHis225230235240AlaThrMetLeuMetGlyLysGlyIlePheAspGluArgGlnAlaGly 245250255PheTyrGlyThrTyrSerGlySerAlaSerThrGlyAlaValLysGlu260265270 AlaIleGluGlyAlaAspThrValLeuCysValGlyThrArgPheThr275280285AspThrLeuThrAlaGlyPheThrHisGlnLeuThrProAlaGlnThr 290295300IleGluValGlnProHisAlaAlaArgValGlyAspValTrpPheThr305310315320Gl yIleProMetAsnGlnAlaIleGluThrLeuValGluLeuCysLys325330335GlnHisValHisAlaGlyLeuMetSerSerSerSerGlyAlaIlePro 340345350PheProGlnProAspGlySerLeuThrGlnGluAsnPheTrpArgThr355360365Leu GlnThrPheIleArgProGlyAspIleIleLeuAlaAspGlnGly370375380ThrSerAlaPheGlyAlaIleAspLeuArgLeuProAlaAspValAsn385 390395400PheIleValGlnProLeuTrpGlySerIleGlyTyrThrLeuAlaAla405410415 AlaPheGlyAlaGlnThrAlaCysProAsnArgArgValIleValLeu420425430ThrGlyAspGlyAlaAlaGlnLeuThrIleGlnGluLeuGlySerMet 435440445LeuArgAspLysGlnHisProIleIleLeuValLeuAsnAsnGluGly450455460TyrThrValG luArgAlaIleHisGlyAlaGluGlnArgTyrAsnAsp465470475480IleAlaLeuTrpAsnTrpThrHisIleProGlnAlaLeuSerLeuAsp 485490495ProGlnSerGluCysTrpArgValSerGluAlaGluGlnLeuAlaAsp500505510Va lLeuGluLysValAlaHisHisGluArgLeuSerLeuIleGluVal515520525MetLeuProLysAlaAspIleProProLeuLeuGlyAlaLeuThrLys 530535540AlaLeuGluAlaCysAsnAsnAla545550