Dijkhuizen, L.

Hessels, G.I.

Hylckama Vlieg, J. van

Put, N.M.J. van der

Vrijbloed, J.W.

English

An efficient restriction barrier for methylated DNA in the actinomycete Amycolatopsis metanolica could be avoided by using a nonmethylating Escherichia coli strain for DNA isolations. The A. methanolica prephenate dehydratase gene was cloned from a gene bank in a pMEA300-derived shuttle vector in E. coli and characterized.</p

Proceedings - University of Groningen

   University of GroningenMolecular Cloning with a pMEA300-Derived Shuttle Vector and Characterization of theAmycolatopsis methanolica Prephenate Dehydratase GeneVrijbloed, J.W.; Hylckama Vlieg, J. van; Put, N.M.J. van der; Hessels, G.I.; Dijkhuizen, L.Published in:Journal of BacteriologyIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1995Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Vrijbloed, J. W., Hylckama Vlieg, J. V., Put, N. M. J. V. D., Hessels, G. I., & Dijkhuizen, L. (1995). MolecularCloning with a pMEA300-Derived Shuttle Vector and Characterization of the Amycolatopsis methanolicaPrephenate Dehydratase Gene. Journal of Bacteriology, 177(22), 6666-6669.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.JOURNAL OF BACTERIOLOGY, Nov. 1995, p. 6666–6669 Vol. 177, No. 220021-9193/95/$04.0010Copyright q 1995, American Society for MicrobiologyNOTESMolecular Cloning with a pMEA300-Derived Shuttle Vectorand Characterization of the Amycolatopsis methanolicaPrephenate Dehydratase GeneJ. W. VRIJBLOED, J. VAN HYLCKAMA VLIEG, N. M. J. VAN DER PUT, G. I. HESSELS,AND L. DIJKHUIZEN*Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute,University of Groningen, 9751 NN Haren, The NetherlandsReceived 26 June 1995/Accepted 10 September 1995An efficient restriction barrier for methylated DNA in the actinomycete Amycolatopsis methanolica could beavoided by using a nonmethylating Escherichia coli strain for DNA isolations. The A. methanolica prephenatedehydratase gene was cloned from a gene bank in a pMEA300-derived shuttle vector in E. coli and character-ized.Transformation of Amycolatopsis methanolica (6, 15) withvarious actinomycete vectors was unsuccessful (24). An indig-enous, integrative, A. methanolica plasmid (pMEA300; 13.3kb) (23) was subsequently selected for the construction ofcloning vectors. A. methanolica WV1, devoid of both inte-grated and free pMEA300 sequences, was used to establish anefficient transformation protocol (24).We are interested in the enzymology and regulation of aro-matic amino acid biosynthesis in A. methanolica (1, 8, 10).Prephenate dehydratase (PDT) is a key regulatory enzyme inL-phenylalanine (L-Phe) biosynthesis in A. methanolica (6).The PDT protein has been purified to homogeneity and char-acterized as a homotetrameric enzyme with 34-kDa subunits(10).Here we report the construction of a gene library of A.methanolica total DNA in Escherichia coli with a pMEA300-derived shuttle vector and the characterization of the pdt gene.Growth, DNA manipulations, and transformation. A. meth-anolica and E. coli strains and plasmids used are shown inTable 1. The complex (23) and mineral (7) media and trans-formation protocols used have been described elsewhere (22,24). DNA sequencing was done as previously described (23).Open reading frames were identified with a Streptomycescodon usage table (26).Transformation with (non)methylated DNA. Attempts totransform strain WV1 with plasmids pWV102 and pWV110,isolated from strain HB101, were unsuccessful. The possiblepresence of a restriction barrier between E. coli and A. meth-anolica, as reported for several actinomycetes (2, 3, 5, 16, 17),was investigated. The Streptomyces avermitilis restriction bar-rier can be avoided by performing DNA isolations from non-methylating strains, e.g., E. coli JM110 (dam, dcm) and Strep-tomyces lividans (16). Reisolation of pWV102 and pWV110from strain JM110 indeed resulted in successful transforma-tion of strain WV1 (approximately 105 pocks per mg of DNA).The effect of pMEA300 methylation on the transformationfrequency was further studied by treating pMEA300 DNA withBamHI (GGATCmC) and SssI (CmG) methylases. The meth-ylation efficiency was checked by the (in)ability of the appro-priate restriction enzymes, BamHI and SalI, respectively, todigest the in vitro-methylated DNA. Treatment of pMEA300with BamHI methylase caused a 90% reduction in transforma-tion frequency with strain WV1. The SssI methylase recognizesmany sites on pMEA300 with its high G1C content (68%;GenBank accession no. L36679), reducing the transformationfrequency even more severely (by 99.8%).Construction of a gene library in pWV138. Attempts toconstruct a gene bank of A. methanolica DNA in pWV137failed; apparently, this vector could not stably maintain DNAfragments of A. methanolica. Cloning of DNA fragments maybe greatly facilitated by the use of transcriptional terminators(4). Therefore, the tetracycline resistance marker flanked byomega (V) fragments from pHP45V-Tc (11) was cloned intothe BamHI site of pWV136, yielding pWV138 (Fig. 1). Thisplasmid contains a unique BamHI site that can be used forcloning, resulting in insertional inactivation of the Tcr gene inE. coli. The ligation mixture of pWV138 DNA digested withBamHI and a partial Sau3A digest (.3 kb) of total DNA of A.methanolica was used to transform strain MC1061, yieldingapproximately 15,000 independent transformants in six differ-ent batches. The average insert frequency was 84%; plasmidDNA of 34 Tcs colonies was analyzed by restriction analysis,showing an average insert size of 4 kb.Cloning of the A. methanolica pdt gene. The oligonucleotidepdt3 (38 nucleotides, 256-fold degenerate) (TTCATGGC[CG]GC[CG]GG[CG]GACGA[AG]CT[CG]GT[CG]GC[CG]GC[GC]AGAC), based on the N-terminal amino acid sequence ofthe PDT protein of A. methanolica (10), was used as a probe toscreen total DNA of A. methanolica and the pWV138 genelibrary for the pdt gene. pdt3 was labelled with the D.I.G.oligonucleotide tailing kit (Boehringer, Mannheim, Germany).Positive signals were observed with total DNA, digested withBamHI (9.0 kb), BclI (5.5 kb), and PvuII (3.4 kb), and in two* Corresponding author. Mailing address: Department of Microbi-ology, Groningen Biomolecular Sciences and Biotechnology Institute,University of Groningen, Kerklaan 30, 9751 NN Haren, The Nether-lands. Phone: 31.50.3632150. Fax: 31.50.3632154. Electronic mail ad-dress: L.Dijkhuizen@biol.rug.nl.6666 at university library on May 24, 2007 jb.asm.orgDownloaded from batches of the gene library, digested with PvuII (3.4 kb). Theresults indicated that an internal 3.4-kb chromosomal fragmenthad been cloned entirely. Batch 1 (approximately 2,500 plas-mids) was transformed to strain DH5a. After replica plating ofthe transformants, plasmid DNA from pooled colonies wasisolated, digested with PvuII, and hybridized with pdt3. A pos-itive signal was obtained in two subsets of batch 1. In threeconsecutive steps, a colony that contained a single plasmid(pNAT115, 27 kb [Fig. 1]) that hybridized strongly to pdt3 andcontained a chromosomal DNA insert of 15 kb with a 3.5-kbPvuII fragment was isolated. Unmethylated plasmid DNA iso-lated from strain JM110 (pNAT115) subsequently was used totransform A. methanolica GH71, a PDT-deficient mutant (10).Strain GH71 (pNAT115) grew on glucose mineral media with-out requiring L-Phe as supplement. The specific activities ofPDT assayed (10) in crude extracts of A. methanolica wild typeand GH71 (pNAT115) were 17 and 16 nmol/mg/min, respec-FIG. 1. Restriction maps of the plasmids pWV138 and pNAT115. Numbersbetween brackets indicate various restriction sites on the plasmids. Other num-bers indicate distances from the unique BamHI site on pWV138 and pNAT115.Arrows on pNAT115 and pWV138 indicate open reading frames encoding func-tions required for PDT activity (pdt), maintenance and autonomous replication(korA, orfA, orfB) (22), conjugational transfer (traA) (25), stimulation of trans-formation frequency (stf) (24), and high mutation frequency (mut) (21).FIG. 2. Nucleotide sequence of the pdt gene of A. methanolica. The deducedamino acid sequence (single-letter code) is shown below the DNA sequence. Thenucleotide sequence data have been deposited in the GenBank database underthe accession number L47666.TABLE 1. A. methanolica and E. coli strains and pMEA300-derived plasmids usedStrain or plasmid Description ReferenceStrainsA. methanolica NCIB 11946 wild-type strain 6WV1 pMEA300-free derivative strain of A. methanolica 24WV2 Spectinomycin-resistant (150 mg/ml) derivative of strain WV1 24GH71 PDT-negative mutant, derivative of WV2 9E. coliMC1061 hsdR mcrB araD139 D(araABC-leu) 7679 DlacX74 galU galK rspL thiDH5a supE44 dlacU169 (V80lacZdM15) hsdR17 recA1 gyrA96 thi-1 relA1JM110 dam dcm supE44 hsdR thi leu rpsL lacY galK galT ara tonA thr tsx D(lac-proAB) F9(traD36proAB1 lacZ DM15)HB101 supE44 hsdS20 recA 13 ara-14 proA2 lacY1 galK 2 rpsL20 xyl-5 mtl-1 19PlasmidspMEA300 Wild-type plasmid of A. methanolica 23pWV102 pUC18 (18) containing BamHI-digested pMEA300 in the BamHI site This workpWV110 pUC18 containing BglII-digested pMEA300 in the BamHI site This workpWV136 Integration- and conjugation-deficient derivative of pMEA300 with the pHSS6 ColE1 replicon 22pWV137 pWV136 containing the lacZ region of pUC18 as a HaeII fragment in the EcoRI site This workpWV138 pWV136 containing the tetracycline resistance gene flanked by transcription terminators ofpHP45V-Tc (11) as a HindIII fragment in the EcoRI siteThis workpNAT115 pWV138 containing a 15-kb chromosomal insert encoding the pdt gene This workVOL. 177, 1995 NOTES 6667 at university library on May 24, 2007 jb.asm.orgDownloaded from tively. These results confirmed that the entire A. methanolicapdt gene had been cloned.Nucleotide sequence of the pdt gene. An 0.9-kb DNA frag-ment was sequenced (GC content, 73%), revealing the pres-ence of a single large open reading frame that would encode aprotein of 304 amino acids (Fig. 2). Its deduced amino acidsequence was nearly identical (33 of 35 residues correct) withthe previously determined N-terminal amino acid sequence ofthe A. methanolica PDT protein (10). Also, the calculatedmolecular mass of 32,288 Da is very close to the estimatedmolecular mass (34 kDa) of the purified PDT protein (10). Thededuced amino acid sequence aligned well with known se-quences of other PDT enzymes (Fig. 3) and showed highestsimilarity (61%) with PheA of Corynebacterium glutamicum(13).PDT of A. methanolica is allosterically inhibited by L-Pheand activated by L-tyrosine (L-Tyr) (10). Interestingly, the shortPTGXD amino acid sequence (starting at position 186, A.methanolica PDT protein) is only present in all PDT proteinsthat are activated by tyrosine (Fig. 3). Analysis of the aminoacid sequences of deregulated mutant PDT proteins (10) willprovide further information about factors determining L-Pheinhibition and L-Tyr stimulation.This study was supported by grant no. GBI81.1510 from the Neth-erlands Technology Foundation (STW), which is subsidized by theNetherlands Organization for the Advancement of Pure Research(NWO).We are indebted to Peter Terpstra for assistance in the sequenceanalysis.REFERENCES1. Abou-Zeid, A., G. J. W. Euverink, G. I. Hessels, R. A. Jensen, and L.Dijkhuizen. 1995. Biosynthesis of L-phenylalanine and L-tyrosine in the ac-tinomycete Amycolatopsis methanolica. Appl. Environ. Microbiol. 61:1298–1302.2. Bailey, C. R., and D. J. Winstanley. 1986. Inhibition of restriction inStreptomyces clavuligerus by heat treatment. J. Gen. 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Evol. 36:107–120.VOL. 177, 1995 NOTES 6669 at university library on May 24, 2007 jb.asm.orgDownloaded from 

Molecular Cloning with a pMEA300-Derived Shuttle Vector and Characterization of the Amycolatopsis methanolica Prephenate Dehydratase Gene

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Molecular Cloning with a pMEA300-Derived Shuttle Vector and Characterization of the Amycolatopsis methanolica Prephenate Dehydratase Gene

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