Gene Cluster of Arthrobacter ilicis Rü61a Involved in the Degradation of Quinaldine to Anthranilate
2003; Elsevier BV; Volume: 278; Issue: 30 Linguagem: Inglês
10.1074/jbc.m301330200
ISSN1083-351X
AutoresKatja Parschat, Bernhard Hauer, Reinhard Kappl, Roswitha Kraft, Jürgen Hüttermann, Susanne Fetzner,
Tópico(s)Microbial metabolism and enzyme function
ResumoA genetic analysis of the anthranilate pathway of quinaldine degradation was performed. A 23-kb region of DNA from Arthrobacter ilicis Rü61a was cloned into the cosmid pVK100. Although Escherichia coli clones containing the recombinant cosmid did not transform quinaldine, cosmids harboring the 23-kb region, or a 10.8-kb stretch of this region, conferred to Pseudomonas putida KT2440 the ability to cometabolically convert quinaldine to anthranilate. The 10.8-kb fragment thus contains the genes coding for quinaldine 4-oxidase (Qox), 1H-4-oxoquinaldine 3-monooxygenase, 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, and N-acetylanthranilate amidase. The qoxLMS genes coding for the molybdopterin cytosine dinucleotide-(MCD-), FeSI-, FeSII-, and FAD-containing Qox were inserted into the expression vector pJB653, generating pKP1. Qox is the first MCD-containing enzyme to be synthesized in a catalytically fully competent form by a heterologous host, P. putida KT2440 pKP1; the catalytic properties and the UV-visible and EPR spectra of Qox purified from P. putida KT2440 pKP1 were essentially like those of wild-type Qox. This provides a starting point for the construction of protein variants of Qox by site-directed mutagenesis. Downstream of the qoxLMS genes, a putative gene whose deduced amino acid sequence showed 37% similarity to the cofactor-inserting chaperone XdhC was located. Additional open reading frames identified on the 23-kb segment may encode further enzymes (a glutamyl tRNA synthetase, an esterase, two short-chain dehydrogenases/reductases, an ATPase belonging to the AAA family, a 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase/5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase-like protein, and an enzyme of the mandelate racemase group) and hypothetical proteins involved in transcriptional regulation, and metabolite transport. A genetic analysis of the anthranilate pathway of quinaldine degradation was performed. A 23-kb region of DNA from Arthrobacter ilicis Rü61a was cloned into the cosmid pVK100. Although Escherichia coli clones containing the recombinant cosmid did not transform quinaldine, cosmids harboring the 23-kb region, or a 10.8-kb stretch of this region, conferred to Pseudomonas putida KT2440 the ability to cometabolically convert quinaldine to anthranilate. The 10.8-kb fragment thus contains the genes coding for quinaldine 4-oxidase (Qox), 1H-4-oxoquinaldine 3-monooxygenase, 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, and N-acetylanthranilate amidase. The qoxLMS genes coding for the molybdopterin cytosine dinucleotide-(MCD-), FeSI-, FeSII-, and FAD-containing Qox were inserted into the expression vector pJB653, generating pKP1. Qox is the first MCD-containing enzyme to be synthesized in a catalytically fully competent form by a heterologous host, P. putida KT2440 pKP1; the catalytic properties and the UV-visible and EPR spectra of Qox purified from P. putida KT2440 pKP1 were essentially like those of wild-type Qox. This provides a starting point for the construction of protein variants of Qox by site-directed mutagenesis. Downstream of the qoxLMS genes, a putative gene whose deduced amino acid sequence showed 37% similarity to the cofactor-inserting chaperone XdhC was located. Additional open reading frames identified on the 23-kb segment may encode further enzymes (a glutamyl tRNA synthetase, an esterase, two short-chain dehydrogenases/reductases, an ATPase belonging to the AAA family, a 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase/5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase-like protein, and an enzyme of the mandelate racemase group) and hypothetical proteins involved in transcriptional regulation, and metabolite transport. The genetic diversity and flexibility of prokaryotes has led to the evolution of an impressive variety of metabolic pathways to transform or degrade natural as well as numerous xenobiotic compounds. The genes coding for enzymes involved in degradative pathways are often organized as operons and supraoperonic clusters comprising “pathway modules” (1van der Meer J.R. de Vos W.M. Harayama S. Zehnder A.J. Microbiol. Rev. 1992; 56: 677-694Crossref PubMed Google Scholar, 2Wyndham R.C. Cashore A.E. Nakatsu C.H. Peel M.C. Biodegradation. 1994; 5: 323-342Crossref PubMed Scopus (91) Google Scholar, 3Romine M.F. Stillwell L.C. Wong K.K. Thurston S.J. Sisk E.C. Sensen C. Gaasterland T. Fredrickson J.K. Saffer J.D. J. Bacteriol. 1999; 181: 1585-1602Crossref PubMed Google Scholar, 4Parke D. D'Argenio D.A. Ornston L.N. J. Bacteriol. 2000; 182: 257-263Crossref PubMed Scopus (44) Google Scholar).N-Heteroaromatic compounds are metabolized and even mineralized by various bacteria (for a review, see Ref. 5Fetzner S. Appl. Microbiol. Biotechnol. 1998; 49: 237-250Crossref Scopus (258) Google Scholar and references therein). Quinaldine (2-methylquinoline) is utilized by Arthrobacter ilicis Rü61a as a source of carbon, nitrogen, and energy; its degradation proceeds via the “anthranilate pathway” (5Fetzner S. Appl. Microbiol. Biotechnol. 1998; 49: 237-250Crossref Scopus (258) Google Scholar, 6Hund H.-K. de Beyer A. Lingens F. Biol. Chem. Hoppe-Seyler. 1990; 371: 1005-1008Crossref PubMed Scopus (48) Google Scholar). The initial step, the hydroxylation of quinaldine in para position to the N-heteroatom, is catalyzed by the inducible enzyme quinaldine 4-oxidase (Qox). 1The abbreviations used are: Qox, quinaldine 4-oxidase; Qor, quinoline 2-oxidoreductase; Ior, isoquinoline 1-oxidoreductase; MCD, molybdopterin cytosine dinucleotide; Tc, tetracycline; Km, kanamycin; INT, iodonitrotetrazolium chloride; HPLC, high pressure liquid chromatography; aa, amino acid(s); MOP, aldehyde oxidoreductase from D. gigas; MOD, aldehyde oxidoreductase from D. desulfuricans; XDHRc, xanthine dehydrogenase from R. capsulatus; XOb, xanthine oxidase from cow's milk; ORF, open reading frame; Hod, 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase; TetR, tetracycline repressor; HTH, helix-turn-helix; SDR, short chain dehydrogenase/reductase; COG, cluster of orthologous groups; HHDD, 2-hydroxyhepta-2,4-diene-1,7-dioate; OPET, 5-oxopent-3-ene-1,2,5-tricarboxylate; MR, mandelate racemase; MLE, muconate lactonizing enzyme; MPT, molybdopterin cofactor; HxA, xanthine dehydrogenase from E. nidulans; Ap, ampicillin.1The abbreviations used are: Qox, quinaldine 4-oxidase; Qor, quinoline 2-oxidoreductase; Ior, isoquinoline 1-oxidoreductase; MCD, molybdopterin cytosine dinucleotide; Tc, tetracycline; Km, kanamycin; INT, iodonitrotetrazolium chloride; HPLC, high pressure liquid chromatography; aa, amino acid(s); MOP, aldehyde oxidoreductase from D. gigas; MOD, aldehyde oxidoreductase from D. desulfuricans; XDHRc, xanthine dehydrogenase from R. capsulatus; XOb, xanthine oxidase from cow's milk; ORF, open reading frame; Hod, 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase; TetR, tetracycline repressor; HTH, helix-turn-helix; SDR, short chain dehydrogenase/reductase; COG, cluster of orthologous groups; HHDD, 2-hydroxyhepta-2,4-diene-1,7-dioate; OPET, 5-oxopent-3-ene-1,2,5-tricarboxylate; MR, mandelate racemase; MLE, muconate lactonizing enzyme; MPT, molybdopterin cofactor; HxA, xanthine dehydrogenase from E. nidulans; Ap, ampicillin. 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Whereas genes coding for molybdenum hydroxylases containing molybdopterin or the molybdopterin guanine dinucleotide form of the cofactor have been successfully expressed in Escherichia coli (24Pollock V.V. Barber M.J. J. Biol. Chem. 1997; 272: 3355-3362Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 25Garrett R.M. Rajagopalan K.V. J. Biol. Chem. 1994; 269: 272-276Abstract Full Text PDF PubMed Google Scholar, 26Temple C.A. Rajagopalan K.V. J. Biol. Chem. 2000; 275: 40202-40210Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), attempts to produce MCD-containing enzymes in E. coli clones failed (27Black G.W. Lyons C.M. Williams E. Colby J. Kehoe M. O′Reilly C. FEMS Microbiol. Lett. 1990; 58: 249-254PubMed Google Scholar, 28Bläse M. Bruntner C. Tshisuaka B. Fetzner S. Lingens F. J. Biol. Chem. 1996; 271: 23068-23079Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). 2K. Parschat, U. Frerichs-Deeken, and S. Fetzner, unpublished results.2K. Parschat, U. Frerichs-Deeken, and S. Fetzner, unpublished results. We have recently been working at the construction of expression clones for the synthesis of Qor and Ior. Synthesis of catalytically fully competent Qor was only achieved when using a ΔqorMSL mutant of the donor strain P. putida 86 as recipient for the expression plasmid (29Frerichs-Deeken U. Goldenstedt B. Gahl-Janβen R. Kappl R. Hüttermann J. Fetzner S. Eur. J. Biochem. 2003; 270: 1567-1577Crossref PubMed Scopus (27) Google Scholar). Ior protein showing minor activity was synthesized from the respective expression plasmid when using the Qor-producing strain P. putida 86 as a host, suggesting that accessory gene(s) encoding product(s) essential for the synthesis or assembly of the enzyme is(are) part of the quinoline regulon in P. putida 86 (30Israel I. Sohni M. Fetzner S. FEMS Microbiol. Lett. 2002; 210: 123-127Crossref PubMed Google Scholar).Here we report on a gene cluster from A. ilicis Rü61a that comprises several genes coding for enzymes of the anthranilate pathway of quinaldine degradation. The amino acid sequences deduced from the qox genes are compared with those of other molybdenum hydroxylases. Due to the broad substrate specificity of Qox, which in addition to different N-heteroaromatic compounds oxidizes aromatic aldehydes (8Stephan I. Tshisuaka B. Fetzner S. Lingens F. Eur. J. Biochem. 1996; 236: 155-162Crossref PubMed Scopus (36) Google Scholar), sequence comparisons to the crystallized aldehyde oxidoreductases are of special interest. Moreover, we present the functional heterologous expression of the qoxLMS genes in P. putida KT2440 pKP1.MATERIALS AND METHODSBacterial Strains, Plasmids, and Growth Conditions—The bacterial strains and plasmids used are listed in Table I. A. ilicis Rü61a was grown in mineral salts medium (8Stephan I. Tshisuaka B. Fetzner S. Lingens F. Eur. J. Biochem. 1996; 236: 155-162Crossref PubMed Scopus (36) Google Scholar) at 30 °C with 0.5 ml/liter quinaldine. E. coli HB101 (31Boyer H.W. Roulland-Dussoix D. J. Mol. Biol. 1969; 41: 459-472Crossref PubMed Scopus (2566) Google Scholar), which served as host strain for recombinant cosmids, and both E. coli DH5α (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) and E. coli XL1-Blue MRF′ (Stratagene), used for cloning procedures with pUC18, were grown in Luria-Bertani (LB) broth (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) at 37 °C. If appropriate, E. coli and P. putida KT2440 cultures contained the following antibiotics: ampicillin (60 and 500 μg/ml for E. coli DH5α and P. putida KT2440 pKP1, respectively), tetracycline (15 μg/ml), and kanamycin (50 μg/ml). To investigate functional expression of the qox genes in the cosmid clone E. coli HB101 pVK55B/5, it was grown at 37 °C on mineral salts medium containing (per liter) 0.2 g of MgSO4 × 7H2O, 4 g of (NH4)2SO4, 5.25 g of K2HPO4, 2.25 g of KH2PO4, 2.7 mg of FeCl3 × 6H2O, and 8 g of glucose as carbon source. The medium was supplemented with 15 μg/ml of each proline and leucine, and 2 ml/liter vitamin solution (33Röger P. Erben A. Lingens F. Biol. Chem. Hoppe-Seyler. 1990; 371: 511-513Crossref PubMed Scopus (25) Google Scholar); 0.1 ml/liter quinaldine was added when the culture reached an optical density (600 nm) of about 0.8. P. putida KT2440 pKP1 was grown in the mineral salt medium described by Tshisuaka et al. (15Tshisuaka B. Kappl R. Hüttermann J. Lingens F. Biochemistry. 1993; 32: 12928-12934Crossref PubMed Scopus (28) Google Scholar) with 8 mm benzoate as carbon and energy source and as XylS effector, and with 1 g/liter (NH4)2SO4. As an additional XylS effector, 2 mm 2-methylbenzoate was added at an optical density (600 nm) of 0.8–1.2. To generate biomass for protein purification, cells were grown in two glass fermenters (4-liters each) to which benzoate was added repeatedly. At an optical density (600 nm) of about 3, cells were harvested by centrifugation at 14,000 × g for 15 min at 4 °C. For the preparation of electrocompetent cells, E. coli DH5α, E. coli XL1-Blue MRF′, and P. putida KT2440 were cultured in TB medium (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar).Table IBacterial strains and plasmidsBacterial strains and plasmidsDescriptionSource or referenceA. ilicis Rü61aWild-type strain utilizing quinaldine as sole source of carbon, nitrogen, and energy106Dembek G. Rommel T. Lingens F. Höke H. FEBS Lett. 1989; 246: 113-116Crossref Scopus (23) Google ScholarE. coli HB101F- leuB6 Δ(gpt-proA)62 glnV44 recA13 ara-14 lacY1 galK2 xyl5 mtl-1 rpsL20 (Strr) Δ(mcrC-mrr)31Boyer H.W. Roulland-Dussoix D. J. Mol. Biol. 1969; 41: 459-472Crossref PubMed Scopus (2566) Google ScholarE. coli DH5αendA1(rK-mK+) glnV44 thi-1 gyrA (Nalr) relA1 Δ(laclZYA r argF)U169 deoR (φ 80dlacΔ(lacZ)M15) hsdR17 recA132Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google ScholarE. coli XL1-Blue MRF′F′::Tn10 proA + B + lacl q Δ(lacZ)M15 recA1 endA1 gyrA96(Nalr) thi hsdR17 (rK-mK+) glnV44 relA1 lacStratageneP. putida KT2440r- derivative of P. putida mt-234Bagdasarian M. Lurz R. Rückert B. Franklin F.C.H. Bagdasarian M.M. Frey J. Timmis K.N. Gene (Amst.). 1981; 16: 237-247Crossref PubMed Scopus (769) Google Scholar, 51Nelson K.E. Weinel C. Paulsen I.T. Dodson R.J. Hilbert H. Martins dos Santos V.A. Fouts D.E. Gill S.R. Pop M. Holmes M. Brinkac L. Beanan M. DeBoy R.T. Daugherty S. Kolonay J. Madupu R. Nelson W. White O. Peterson J. Khouri H. Hance I. Chris Lee P. Holtzapple E. Scanlan D. Tran K. Moazzez A. Utterback T. Rizzo M. Lee K. Kosack D. Moestl D. Wedler H. Lauber J. Stjepandic D. Hoheisel J. Straetz M. Heim S. Kiewitz C. Eisen J. Timmis K.N. Dusterhoft A. Tummler B. Fraser C.M. Environ. Microbiol. 2002; 4: 799-808Crossref PubMed Scopus (1038) Google ScholarpVK100IncP Tcr Kmr39Knauf V.C. Nester E W. Plasmid. 1982; 8: 45-54Crossref PubMed Scopus (331) Google ScholarpUC18ColE1 lacZ Apr38Vieira J. Messing J. Gene (Amst.). 1982; 19: 259-268Crossref PubMed Scopus (3770) Google ScholarpJB653Broad-host-range cloning vector: RK2 replicon, Pm promoter, xylS for transcriptional regulation, Apr41Blatny J.M. Brautaset T. Winther-Larsen H.C. Haugan K. Valla S. Appl. Environ. Microbiol. 1997; 63: 370-379Crossref PubMed Google ScholarpVK55B/523-kb insert of A. ilicis Rü61a DNA in HindIII site of pVK100This workpVK55/1110.8-kb HindIII fragment of pVK55B/5 in HindIII site of pVK100This workpUC55/1110.8-kb HindIII fragment of pVK55B/5 in HindIII site of pUC18This workpUC55/3.23.2-kb SmaI fragment of pUC55/11 in SmaI site of pUC18This workpUC55/4.54.58-kb SmaI fragment of A. ilicis Rü61a genomic DNA in SmaI site of pUC18This workpUC55/32.9-kb SmaI/HindIII fragment of pUC55/11 in SmaI and HindIII sites of pUC18This workpKP1qoxLMS (PCR amplificate of the structural genes of quinaldine 4-oxidase) inserted in EcoRI and SmaI sites of pJB653This work Open table in a new tab For transfection of E. coli HB101, 20 ml of LB broth with 0.2 ml of 1 m MgSO4 and 0.2 ml 20% (w/v) maltose were inoculated with a single bacterial colony and grown to an optical density (600 nm) of 0.8. After harvesting the cells by centrifugation at 2000 × g and 4 °C for 10 min, cells were diluted to an optical density (600 nm) of 1 with ice-cold, sterile 10 mm MgSO4.Analysis of the Degradative Potential of P. putida KT2440 pVK55B/5 and P. putida KT2440 pVK55/11—To determine Qox activity and to identify metabolites of quinaldine catabolism, recombinant cosmids were transferred to P. putida KT2440 (34Bagdasarian M. Lurz R. Rückert B. Franklin F.C.H. Bagdasarian M.M. Frey J. Timmis K.N. Gene (Amst.). 1981; 16: 237-247Crossref PubMed Scopus (769) Google Scholar) by electroporation. The clones P. putida KT2440 pVK55B/5 and P. putida KT2440 pVK55/11 were grown in mineral salt medium (8Stephan I. Tshisuaka B. Fetzner S. Lingens F. Eur. J. Biochem. 1996; 236: 155-162Crossref PubMed Scopus (36) Google Scholar) with 30 mm succinate, 1 g/liter (NH4)2SO4, and 1 ml/liter vitamin solution (33Röger P. Erben A. Lingens F. Biol. Chem. Hoppe-Seyler. 1990; 371: 511-513Crossref PubMed Scopus (25) Google Scholar) at 30 °C. At an optical density (600 nm) of about 0.8, 0.1 ml/liter quinaldine was added. Quinaldine conversion was monitored spectrophotometrically in the culture supernatant. Spectra were compared with those of authentic references diluted in the same medium. Qox activity was measured in the cell free extracts, obtained after cell disruption by sonification, and centrifugation at 48,000 × g for 40 min at 4 °C.DNA Techniques—Genomic DNA of A. ilicis Rü61a was isolated according to Hopwood et al. (35Hopwood D.A. Bibb M.J. Chater K.F. Kieser T. Bruton C.J. Kieser H.M. Lydiate C.P. Ward J.M. Schrempf H. Genetic Manipulation of Streptomyces, a Laboratory Manual. The John Innes Foundation, Norwich, UK1985Google Scholar). Plasmid and cosmid DNA was isolated with the Qiagen Plasmid Mini- and Midi kits, respectively (Qiagen, Hilden, Germany). Gel extraction of DNA fragments for cloning was done with the Nucleo Spin® extraction kit of Macherey-Nagel (Düren, Germany); however, DNA fragments larger than 10 kb were size-fractionated in 0.5% low melting agarose gels and extracted by agarase treatment. DNA restriction, dephosphorylation, and ligation and agarose gel electrophoresis were carried out using standard procedures (32Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Electrocompetent cells were generated according to Dower et al. (36Dower W.J. Miller J.F. Ragsdale C.W. Nucleic Acids Res. 1988; 16: 6127-6145Crossref PubMed Scopus (2152) Google Scholar) and Iwasaki et al. (37Iwasaki K. Uchiyama H. Yagi O. Kurabayashi T. Ishizuku K. Takamura Y. Biosci. Biotechnol. Biochem. 1994; 58: 851-854Crossref PubMed Scopus (44) Google Scholar).Construction of Genomic Libraries—To generate an enriched gene library for A. ilicis Rü61a, genomic DNA, restricted with SmaI, was separated in agarose gels and vacuum-blotted to nylon membranes (parablot NY plus from Macherey-Nagel, Düren, Germany). Fragments in the size of 4–5 kb showing positive hybridization signals with the probe “b-DIG” (see below) were extracted from an agarose gel and ligated to the SmaI-digested, dephosphorylated vector pUC18 (38Vieira J. Messing J. Gene (Amst.). 1982; 19: 259-268Crossref PubMed Scopus (3770) Google Scholar). E. coli XL1-Blue MRF′ transformants were screened by colony blotting and identified by Southern hybridization of SmaI-restricted plasmids, using the probe b-DIG.For construction of a cosmid library, genomic DNA of A. ilicis Rü61a was partially restricted with HindIII. DNA fragments ranging in size from 15 to 25 kb were extracted from a 0.5% low melting agarose gel and ligated to the HindIII-digested, dephosphorylated cosmid vector pVK100 (39Knauf V.C. Nester E W. Plasmid. 1982; 8: 45-54Crossref PubMed Scopus (331) Google Scholar). The cosmids were packaged in vitro into lambda phage particles using a commercial extract (DNA Packaging kit from Roche Applied Science, Mannheim, Germany). The preparation was used to infect E. coli HB101, which was selected for tetracycline resistance (Tcr) and kanamycin sensitivity (Kms). The clone library was screened by colony blotting and hybridization with a probe described below as “1.1 DpnI.”DNA Probes and Hybridization—The oligonucleotide probe b-DIG, which was 5′-end-labeled with a digoxigenin derivative, was a degenerated 29-mer: 5′-TTY ATG CAY CCN TTY CAR TTY ATH ACN CC-3′ (following the IUPAC ambiguity code), deduced from the N-terminal amino acid sequence of the medium-sized subunit of Qox (FMH-PFQFITP) (7de Beyer A. Lingens F. Biol. Chem. Hoppe-Seyler. 1993; 374: 101-110Crossref PubMed Scopus (19) Google Scholar). Prehybridization for 2 h and hybridization overnight with b-DIG was carried out at 54.5 °C. The membranes were stringently washed for 2 × 15 min in 2× SSC, 0.1% SDS at room temperature, 2 × 15 min in 0.5× SSC, 0.1% SDS at 54.5 °C, and 2 × 15 min in 0.2× SSC, 0.1% SDS at 54.5 °C. Screening an enriched gene library of A. ilicis Rü61a established in the vector pUC18 with b-DIG revealed a clone containing an insert of 4580 bp (pUC55/4.5). Isolation of a 1052-bp DpnI fragment from this insert led to the specific probe 1.1 DpnI, which was used to screen the cosmid clone library. 1.1 DpnI hybridizes with the 5′-terminal half of qoxL (Fig. 1). After prehybridization for 2 h and hybridization overnight with the probe 1.1 DpnI at 68 °C, the membranes were washed twice for 15 min in 2× SSC, 0.1% SDS at room temperature and twice for 15 min in 0.5× SSC, 0.1% SDS at 68 °C. Random primed labeling of the probe 1.1 DpnI using the DIG-High Prime DNA labeling kit (Roche Applied Science, Mannheim, Germany), Southern and colony blotting, hybridization, and colorimetric detection with nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate were performed following the DIG System User's Guide for Filter Hybridization (40Roche molecular biochemicals The DIG System User′s Guide for Filter Hybridization. Boehringer Mannheim GmbH, Mannheim, Germany1995Google Scholar).Subcloning Procedures—Restriction of the recombinant cosmid pVK55B/5 with HindIII produced two fragments of 12,203 and 10,812 bp, respectively, beside the 23-kb vector. These two fragments were separately inserted into the HindIII cleavage site of pUC18, generating pUC55/12 and pUC55/11. The 10.8-kb fragment was also inserted into the cosmid vector pVK100 (forming pVK55/11) to propagate it in P. putida KT2440. When pUC55/11 was restricted with SmaI, three fragments were generated, the internal 4.58-kb fragment showing the positive signal with probe b-DIG, and two fragments of 2,978 and 3,254 bp, respectively (Fig. 1). The 4.58-kb fragment was cloned into the SmaI site of pUC18 (forming pUC55/4.5). The 2.97-kb fragment and the 3.25-kb fragment were removed from pUC55/11 by SmaI-HindIII and SmaI digestion, respectively. Both fragments were separately cloned into the multiple cloning site of pUC18, yielding pUC55/3 and pUC55/3.2. All pUC derivatives were transferred to E. coli DH5α by electroporation.Expression Cloning of qoxLMS Genes—Using the cosmid pVK55B/5 as template, the qoxLMS genes were amplified with Pfu polymerase. The forward primer was chosen to contain the assumed Shine-Dalgarno sequence (in italics) preceding the qox genes, and an EcoRI recognition site (underlined): 5′-ACGCGAATTC GTGACGAAGTTAAGGAGACC-3′; the nucleotides set in boldface are complementary to nucleotides 17,492–17,511 of EMBL accession number AJ537472. The reverse primer was completely complementary to nucleotides 21,401–21,375 of EMBL accession number AJ537472: 5′-TTTGGAATGCGCAGTGAGGAGATTTGC-3′. After EcoRI restriction the PCR product was ligated into the EcoRI- and SmaI-restricted plasmid pJB653 (41Blatny J.M. Brautaset T. Winther-Larsen H.C. Haugan K. Valla S. Appl. Environ. Microbiol. 1997; 63: 370-379Crossref PubMed Google Scholar), generating pKP1. The recipient P. putida KT2440 was transformed by electroporation (36Dower W.J. Miller J.F. Ragsdale C.W. Nucleic Acids Res. 1988; 16: 6127-6145Crossref PubMed Scopus (2152) Google Scholar).DNA Sequencing and Comparative Sequence Analysis—The genes and open reading frames shown in Fig. 1 were deduced from computer-assisted analysis of sequences obtained from single strand sequencing of the inserts of pUC55/12, pUC55/3, pUC55/4.5, and pUC55/3.2; sequences of the qox genes were verified by sequencing both strands. Sequencing was carried out according to the method of Sanger et al. (42Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52361) Google Scholar) on a sequencer 377 from Applied Biosystems.Analysis of the DNA sequences were performed with the HUSAR 4.0 program package (EMBL, GENIUSnet, DKFZ Heidelberg, Germany) using the BLAST family of programs (43Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59167) Google Scholar) for data base searches, GAP for calculating similarities and identities, and ClustalW (44Higgins D.G. Thompson J.D. Gibson T.J. Methods Enzymol. 1996; 266: 383-402Crossref PubMed Scopus (1288) Google Scholar) for calculating multiple alignments. Conserved protein domain sequences and fingerprint motifs were found at Pfam (Sanger Institute, Hinxton, Cambridge, UK) and PRINTS (45Attwood T.K. Beck M.E. Flower D.R. Scordis P. Selley J.N. Nucleic Acids Res. 1998; 26: 304-308Crossref PubMed Scopus (88) Google Scholar). Gene-coding
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