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New PCR Primers for the Screening of NRPS and PKS-I Systems in Actinomycetes: Detection and Distribution of These Biosynthetic Gene Sequences in Major Taxonomic Groups

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Abstract

Nonribosomal peptide synthetases (NRPS) and type I polyketide synthases (PKS-I) are biosynthetic systems involved in the synthesis of a large number of important biologically active compounds produced by microorganisms, among others by actinomycetes. In order to assess the occurrence of these biosynthetic systems in this metabolically active bacterial group, we designed new PCR primers targeted to specifically amplify NRPS and PKS-I gene sequences from actinomycetes. The sequence analysis of amplified products cloned from two model systems and used to validate these molecular tools has shown the extreme richness of NRPS or PKS-I-like sequences in the actinomycete genome. When these PCR primers were tested on a large collection of 210 reference strains encompassing all major families and genera in actinomycetes, we observed that the wide distribution of these genes in the well-known productive Streptomyces species is also extended to other minor lineages where in some cases very few bioactive compounds have been identified to date.

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References

  1. AS Anderson Z An WR Strohl (2000) Polyketide antibiotics. Lederberg (Eds) Encyclopedia for Microbiology, vol. 3 Academic Press San Diego 241–254

    Google Scholar 

  2. AS Anderson D Clark P Gibbons J Sigmund (2002) ArticleTitleThe detection of diverse aminoglycoside phosphotransferases within natural populations of actinomycetes. J Ind Microbiol Biotechnol 29 60–69 Occurrence Handle1:CAS:528:DC%2BD38XlslymtLY%3D Occurrence Handle12161772

    CAS  PubMed  Google Scholar 

  3. JF Aparicio I Molnar T Schwecke A König SF Haydock LE Khaw J Staunton PF Leadlay (1996) ArticleTitleOrganization of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of the enzymatic domains in the modular polyketide synthase. Gene 169 9–16 Occurrence Handle1:CAS:528:DyaK28XhsVaktr0%3D Occurrence Handle8635756

    CAS  PubMed  Google Scholar 

  4. JF Aparicio R Fouces VM Mendes N Oliver JF Martín (2000) ArticleTitleA complex multienzyme system encoded by five polyketide synthase genes is involved in the biosynthesis of the 26-membered polyene macrolide pimaricin in Streptomyces natalensis. Chem Biol 7 895–905 Occurrence Handle1:CAS:528:DC%2BD3cXosVWitLo%3D Occurrence Handle11094342

    CAS  PubMed  Google Scholar 

  5. RL Arslanian L Tang S Blough W Ma RG Qiu L Katz JR Carney (2002) ArticleTitleA new cytotoxic epothilone from modified polyketide synthases heterologously expressed in Myxococcus xanthus. J Nat Prod 65 1061–1064 Occurrence Handle1:CAS:528:DC%2BD38Xkt1Clu7k%3D Occurrence Handle12141877

    CAS  PubMed  Google Scholar 

  6. SD Bentley KF Chater AM Cerdeño-Tárraga GL Challis NR Thomson KD James DE Harris MA Quail H Kieser D Harper A Bateman S Brown G Chandra CW Chen M Collins A Cronin A Fraser A Goble J Hidalgo T Hornsby S Howarth CH Huang T Kieser L Larke L Murphy K Oliver S O’Neil E Rabbinowitsch MA Rajandream K Rutherford S Rutter K Seeger D Saunders S Sharp R Squares S Squares K Taylor T Warren A Wietzorrek J Woodward BG Barrell J Parkhill DA Hopwood (2002) ArticleTitleComplete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417 141–147 Occurrence Handle12000953

    PubMed  Google Scholar 

  7. S Beyer B Kunze B Silakowski R Müller (1999) ArticleTitleMetabolic diversity in myxobacteria: identification of the myxalamid and the stigmatellin biosynthetic gene cluster of Stigmatella aurantiaca Sg a15 and a combined polyketide–(poly)peptide gene cluster from the epothilone producing strain Sorangium cellulosum So ce90. Biochim Biophys Acta 1445 185–195 Occurrence Handle1:CAS:528:DyaK1MXjs1ehtr4%3D Occurrence Handle10320771

    CAS  PubMed  Google Scholar 

  8. LE Bingle CM Lazarus (1999) ArticleTitleKetosynthase domain probes identify two subclasses of fungal polyketide synthase genes. Fungal Genet Biol 26 209–223 Occurrence Handle1:CAS:528:DyaK1MXjsFKnsLo%3D Occurrence Handle10361035

    CAS  PubMed  Google Scholar 

  9. T Brautaset ON Sekurova H Sletta TE Ellingsen AR Stronm S Valla S Zotchev (2000) ArticleTitleBiosynthesis of the polyene antifungal antibiotic nystatin in Streptomyces noursei ATCC 11455: analysis of the gene cluster and deduction of biosynthetic pathway. Chem Biol 7 395–403 Occurrence Handle1:CAS:528:DC%2BD3cXktl2nsLk%3D Occurrence Handle10873841

    CAS  PubMed  Google Scholar 

  10. GL Challis J Ravel CA Townsend (2000) ArticleTitlePredictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains. Chem Biol 7 211–224 Occurrence Handle1:CAS:528:DC%2BD3cXitVOkt7k%3D Occurrence Handle10712928

    CAS  PubMed  Google Scholar 

  11. YQ Cheng GL Tang B Shen (2003) ArticleTitleType I polyketide synthase requiring a discrete acyltransferase for polyketide biosynthesis. Proc Natl Acad Sci USA 100 3149–3154 Occurrence Handle1:CAS:528:DC%2BD3sXisVCnsbk%3D Occurrence Handle12598647

    CAS  PubMed  Google Scholar 

  12. G Christiansen E Dittmann LV Ordorika R Rippka M Herdman T Börner (2001) ArticleTitleNonribosomal peptide synthase genes occur in most cyanobacterial genera as evidenced by their distribution in axenic strains of the PCC. Arch Microbiol 176 452–458 Occurrence Handle1:CAS:528:DC%2BD38XhtFyitQ%3D%3D Occurrence Handle11734889

    CAS  PubMed  Google Scholar 

  13. JJ Coque JF Martin JG Calzada P Liras (1991) ArticleTitleThe cephamycin biosynthetic genes pcbAB, encoding a large multidomain peptide synthetase, and pcbC of Nocardia lactamdurans are clustered together in an organization different from the same genes in Acremonium chrysogenum and Penicillium chrysogenum. Mol Microbiol 5 1125–1133 Occurrence Handle1:CAS:528:DyaK3MXksVaqsLc%3D Occurrence Handle1956290

    CAS  PubMed  Google Scholar 

  14. V de Crecy-Lagard W Saurin D Thibaut P Gil L Naudin J Crouzet V Blanc (1997) ArticleTitleStreptogramin B biosynthesis in Streptomyces pristinaespiralis and Streptomyces virginiae: molecular characterization of the last structural peptide synthetase gene. Antimicrob Agents Chemother 41 1904–1909 Occurrence Handle1:CAS:528:DyaK2sXlvFOgtLY%3D Occurrence Handle9303382

    CAS  PubMed  Google Scholar 

  15. S Donadio L Katz (1992) ArticleTitleOrganization of the enzymatic domains in the multifunctional polyketide synthase involved in erythromycin formation in Saccharopolyspora erythraea. Gene 111 51–60 Occurrence Handle1:CAS:528:DyaK38XltVOitLw%3D Occurrence Handle1547954

    CAS  PubMed  Google Scholar 

  16. SF Haydock JF Aparicio I Molnar T Schwecke LE Khaw A König AF Marsden IS Galloway J Staunton PF Leadlay (1995) ArticleTitleDivergent sequence motifs correlated with the substrate specificity of (methyl)malonyl-CoA:acyl carrier protein transacylase domains in modular polyketide synthases. FEBS Lett 374 246–248 Occurrence Handle1:CAS:528:DyaK2MXptlCmtrk%3D Occurrence Handle7589545

    CAS  PubMed  Google Scholar 

  17. MP Heaton FC Neuhaus (1992) ArticleTitleBiosynthesis of D-alanyl-lipoteichoic acid: cloning, nucleotide sequence, and expression of the Lactobacillus casei gene for D-alanine-activating enzyme. J Bacteriol 174 4707–4717 Occurrence Handle1:CAS:528:DyaK3sXkt1emsL8%3D Occurrence Handle1385594

    CAS  PubMed  Google Scholar 

  18. GL Igloi (1998) ArticleTitleVariability in the stability of DNA-peptide nucleic acid (PNA) single-base mismatched duplexes: real-time hybridization during affinity electrophoresis in PNA-containing gels. Proc Natl Acad Sci USA 95 8562–8567 Occurrence Handle1:CAS:528:DyaK1cXkvFamu7Y%3D Occurrence Handle9671717

    CAS  PubMed  Google Scholar 

  19. H Ikeda T Nonomiya M Usami T Ohta S Omura (1999) ArticleTitleOrganization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis. Proc Natl Acad Sci USA 96 9509–9514 Occurrence Handle1:CAS:528:DyaK1MXlsVyms74%3D Occurrence Handle10449723

    CAS  PubMed  Google Scholar 

  20. MA Innis DH Gelfand JJ Sninsky TJ White (1990) PCR Protocols. A Guide to Methods and Applications Academic Press San Diego

    Google Scholar 

  21. SJ Kakavas L Katz D Stassi (1997) ArticleTitleIdentification and characterization of the niddamycin polyketide synthase genes from Streptomyces caelestis. J Bacteriol 179 7515–7522 Occurrence Handle1:CAS:528:DyaK2sXnvFeksbw%3D Occurrence Handle9393718

    CAS  PubMed  Google Scholar 

  22. C Khosla RS Gokhale JR Jacobsen DE Cane (1999) ArticleTitleTolerance and specificity of polyketide synthases. Annu Rev Biochem 68 219–253 Occurrence Handle1:CAS:528:DyaK1MXlvFajsL0%3D Occurrence Handle10872449

    CAS  PubMed  Google Scholar 

  23. D Konz MA Marahiel (1999) ArticleTitleHow do peptide synthases generate structural diversity? Chem Biol 6 39–48

    Google Scholar 

  24. SY Lee SK Rhee CH Kim JW Suh (1998) ArticleTitleRapid and efficient isolation of genes for biosynthesis of peptide antibiotics from gram-positive bacterial strain. J Microbiol Biotechnol 8 310–317 Occurrence Handle1:CAS:528:DyaK1cXmsVeksr8%3D

    CAS  Google Scholar 

  25. MA Marahiel (1997) ArticleTitleProtein templates for the biosynthesis of antibiotics. Chem Biol 4 561–567 Occurrence Handle1:CAS:528:DyaK2sXmt1ansrk%3D Occurrence Handle9281530

    CAS  PubMed  Google Scholar 

  26. HD Mootz D Schwarzer A Marahiel (2002) ArticleTitleBiosynthetic strategies of nonribosomal peptide synthetases. Chem Bio Chem 3 490–504 Occurrence Handle1:CAS:528:DC%2BD38XksFemsr8%3D Occurrence Handle12325005

    CAS  PubMed  Google Scholar 

  27. H Motamedi A Shafiee (1998) ArticleTitleThe biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506. Eur J Biochem 256 528–534 Occurrence Handle1:CAS:528:DyaK1cXmtlCgtbg%3D Occurrence Handle9780228

    CAS  PubMed  Google Scholar 

  28. TP Nicholson M Dawson CM Lazarus TJ Simpson RJ Cox (2001) ArticleTitleDesign and utility of oligonucleotide gene probes for fungal polyketide synthases. Chem Biol 8 157–178 Occurrence Handle1:CAS:528:DC%2BD3MXjtV2kurk%3D Occurrence Handle11251290

    CAS  PubMed  Google Scholar 

  29. S Omura H Ikeda J Ishikawa A Hanamato C Takahashi M Shinose et al. (2001) ArticleTitleGenome sequence of an industrial microorganism Streptomyces avermitilis: deducing the ability of producing secondary metabolites. Proc Natl Acad Sci USA 98 12215–12220 Occurrence Handle1:CAS:528:DC%2BD3MXns1Cjtrk%3D Occurrence Handle11572948

    CAS  PubMed  Google Scholar 

  30. S Pelzer W Reichert M Huppert D Heckmann W Wohlleben (1997) ArticleTitleCloning and analysis of a peptide synthetase gene of the balhimycin producer Amycolatopsis mediterranei DSM 5908 and development of a gene disruption/replacement system. J Biotechnol 56 115–128 Occurrence Handle1:CAS:528:DyaK2sXltl2ktLk%3D Occurrence Handle9304873

    CAS  PubMed  Google Scholar 

  31. M Perego P Glaser A Minutello MA Strauch K Leopold W Fisher (1995) ArticleTitleIncorporation of D-alanine into lipoteichoic acid and wall teichoic acid in Bacillus subtilis. J Biol Chem 26 15598–15606

    Google Scholar 

  32. J Piel (2002) ArticleTitleA polyketide synthase–peptide synthetase gene cluster from an uncultured bacterial symbiont of Paederus beetles. Proc Natl Acad Sci USA 99 14002–14007 Occurrence Handle1:CAS:528:DC%2BD38XosF2isbw%3D Occurrence Handle12381784

    CAS  PubMed  Google Scholar 

  33. N Rajendran (1999) ArticleTitleIdentification and cloning of a gene locus encoding peptide synthase of Pseudomonas fluorescens by two sets of PCR primers. Z Naturforsch 54 105–109 Occurrence Handle1:CAS:528:DyaK1MXhsFOgtLo%3D

    CAS  Google Scholar 

  34. J Recktenwald R Shawky O Puk F Pfennig U Keller W Wohlleben S Pelzer (2002) ArticleTitleNonribosomal biosynthesis of vancomycin-type antibiotics: a heptapeptide backbone and eight peptide synthetase modules. Microbiology 148 1105–1118 Occurrence Handle1:CAS:528:DC%2BD38Xjt1ems7Y%3D Occurrence Handle11932455

    CAS  PubMed  Google Scholar 

  35. X Ruan D Stassi SA Lax L Katz (1997) ArticleTitleA second type-I PKS gene cluster isolated from Streptomyces hygroscopicus ATCC 29253, a rapamycin-producing strain. Gene 203 1–9 Occurrence Handle1:CAS:528:DyaK2sXotFSqtrc%3D Occurrence Handle9426000

    CAS  PubMed  Google Scholar 

  36. J Sambrook DW Russel (2001) Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY

    Google Scholar 

  37. M Sauser P Lu R Sangari S Kennedy J Polishook G Bills Z An (2002) ArticleTitleEstimating polyketide metabolic potential among nonsporulating fungal endophytes of Vaccinium macrocarpon. Mycol Res 106 460–470

    Google Scholar 

  38. F Schauwecker F Pfennig W Schroder U Keller (1998) ArticleTitleMolecular cloning of the actinomycin synthetase gene cluster from Streptomyces chrysomallus and functional heterologous expression of the gene encoding actinomycin synthetase II. J Bacteriol 180 2468–2474 Occurrence Handle1:CAS:528:DyaK1cXivFOlsL4%3D Occurrence Handle9573200

    CAS  PubMed  Google Scholar 

  39. T Schupp C Toupet E Nathalie S Goff (1998) ArticleTitleCloning and sequence analysis of the putative rifamycin polyketide synthase gene cluster from Amycolatopsis mediterranei. FEMS Microbiol Lett 159 201–207 Occurrence Handle1:CAS:528:DyaK1cXht1Wgt7c%3D Occurrence Handle9503613

    CAS  PubMed  Google Scholar 

  40. L Serre EC Verbree A Dauter AR Stuitje ZS Derewenda (1995) ArticleTitleThe Escherichia coli malonyl-CoA:acyl carrier protein transacylase at 1.5 Å resolution. J Biol Chem 270 12961–12964 Occurrence Handle1:CAS:528:DyaK2MXmtV2itrs%3D Occurrence Handle7768883

    CAS  PubMed  Google Scholar 

  41. S Shah Q Xue L Tang JR Carney M Betlach R McDaniel (2000) ArticleTitleCloning, characterization and heterologous expression of a polyketide synthase and P-450 oxidase involved in the biosynthesis of the antibiotic oleandomycin. J Antibiot 53 502–508 Occurrence Handle1:CAS:528:DC%2BD3cXjslejurg%3D Occurrence Handle10908114

    CAS  PubMed  Google Scholar 

  42. JM Sigmund DJ Clark FA Rainey AS Anderson (2003) ArticleTitleDetection of eubacterial 3-hydroxy-3-methylglutaryl coenzyme A reductases from natural populations of actinomycetes. Microb Ecol 46 106–112 Occurrence Handle1:CAS:528:DC%2BD3sXnt1Git78%3D Occurrence Handle12754661

    CAS  PubMed  Google Scholar 

  43. M Sosio M Bossi A Bianchi S Donadio (2000) ArticleTitleMultiple peptide synthase gene clusters in actinomycetes. Mol Gen Genet 264 213–221 Occurrence Handle1:CAS:528:DC%2BD3cXot1Gkt7g%3D Occurrence Handle11085259

    CAS  PubMed  Google Scholar 

  44. T Stachelhaus MA Marahiel (1995) ArticleTitleModular structure of genes encoding multifunctional peptide synthetases required for non-ribosomal peptide synthesis. FEMS Microbiol Lett 125 3–14 Occurrence Handle1:CAS:528:DyaK2MXivVKlsbo%3D Occurrence Handle7867917

    CAS  PubMed  Google Scholar 

  45. DA Stahl R Amann (1991) Development and application of nucleic acid probes. E Stackebrandt M Goodfellow (Eds) Nucleic Acid Techniques in Bacterial Systematics Wiley Chichester, UK 205–248

    Google Scholar 

  46. JD Thompson DG Higgins TJ Gibson (1994) ArticleTitleCLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22 4673–4680 Occurrence Handle1:CAS:528:DyaK2MXitlSgu74%3D Occurrence Handle7984417

    CAS  PubMed  Google Scholar 

  47. JW Trauger CT Walsh (2000) ArticleTitleHeterologous expression in Escherichia coli of the first module of the nonribosomal peptide synthetase for chloroeremomycin, a vancomycin-type glycopeptide antibiotic. Prac Natl Acad Sci USA 97 3112–3117 Occurrence Handle1:CAS:528:DC%2BD3cXitlWru7Y%3D

    CAS  Google Scholar 

  48. K Turgay MA Marahiel (1994) ArticleTitleA general approach for identifying and cloning peptide synthetase genes. Pept Res 7 238–241 Occurrence Handle1:CAS:528:DyaK2MXht1aksr8%3D Occurrence Handle7849417

    CAS  PubMed  Google Scholar 

  49. KJ Weissman M Bycroft J Staunton F Leadlay (1998) ArticleTitleOrigin of starter units for erythromycin biosynthesis. Biochemistry 37 11012–11017 Occurrence Handle1:CAS:528:DyaK1cXkvVylsL4%3D Occurrence Handle9692995

    CAS  PubMed  Google Scholar 

  50. K Wu L Chung WP Revill L Katz CD Reeves (2000) ArticleTitleThe FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units. Gene 251 81–90 Occurrence Handle1:CAS:528:DC%2BD3cXktV2itLk%3D Occurrence Handle10863099

    CAS  PubMed  Google Scholar 

  51. Y Xue D Wilson DH Sherman (2000) ArticleTitleGenetic architecture of the polyketide synthases for methylmycin and pikromycin series macrolides. Gene 245 203–211 Occurrence Handle1:CAS:528:DC%2BD3cXhsFanurs%3D Occurrence Handle10713461

    CAS  PubMed  Google Scholar 

  52. G Zhu MJ LaGier F Stejskal JJ Millership X Cai JS Keithly (2002) ArticleTitleCryptosporidium parvum: the first protist known to encode a putative polyketide synthase. Gene 298 79–89 Occurrence Handle1:CAS:528:DC%2BD38XotFOmur8%3D Occurrence Handle12406578

    CAS  PubMed  Google Scholar 

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Acknowledgments

A. Ayuso was recipient of a pre-doctoral fellowship from the MIT program, Ministerio de Ciencia y Tecnología, Spain. We are grateful to O. Salazar for his help on sequence data analysis and for providing many of the genome DNAs tested from the reference strain collection. We also thank A. Anderson and Z. An for sharing with us their results prior to publication, and especially A. Anderson for her helpful comments and discussions during the preparation of the manuscript.

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  1. Centro de Investigatión Básica, Merck Research Laboratories, Merck Sharp and Dohme de España S.A., Josefa Valcárcel 38, E-28027, Madrid, Spain

    A. Ayuso-Sacido & O. Genilloud

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Ayuso-Sacido, A., Genilloud, O. New PCR Primers for the Screening of NRPS and PKS-I Systems in Actinomycetes: Detection and Distribution of These Biosynthetic Gene Sequences in Major Taxonomic Groups. Microb Ecol 49, 10–24 (2005). https://doi.org/10.1007/s00248-004-0249-6

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