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PCR Detection of Novel Non-ribosomal Peptide Synthetase Genes in Lipopeptide-Producing Pseudomonas

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Abstract

Bacterial lipopeptides (LPs) are a diverse group of secondary metabolites synthesized through one or more non-ribosomal peptide synthetases (NRPSs). In certain genera, such as Pseudomonas and Bacillus, these enzyme systems are often involved in synthesizing biosurfactants or antimicrobial compounds. Several different types of LPs have been reported for non-pathogenic plant-associated Pseudomonas. Focusing on this group of bacteria, we devised and validated a PCR method to detect novel LP-synthesizing NRPS genes by targeting their lipoinitiation and tandem thioesterase domains, thus avoiding amplification of genes for non-LP metabolites, such as the pyoverdine siderophores present in all fluorescent Pseudomonas. This approach enabled detection of as yet unknown NRPS genes in strains producing viscosin, viscosinamide, WLIP, or lokisin. Furthermore, it proved valuable to identify novel candidate LP producers among Pseudomonas rhizosphere isolates. By phylogenetic analysis of these amplicons, several of the corresponding NRPS genes can be tentatively assigned to the viscosin, amphisin, or entolysin biosynthetic groups, while some others may represent novel NRPS systems.

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References

  1. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Ansari MZ, Yadav G, Gokhale RS, Mohanty D (2004) NRPS-PKS: a knowledge-based resource for analysis of NRPS/PKS megasynthases. Nucleic Acids Res 32:W405–W413

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Ayuso-Sacido A, Genilloud O (2005) 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

    Article  CAS  PubMed  Google Scholar 

  4. Berti AD, Greve NJ, Christensen QH, Thomas MG (2007) Identification of a biosynthetic gene cluster and the six associated lipopeptides involved in swarming motility of Pseudomonas syringae pv. tomato DC3000. J Bacteriol 189:6312–6323

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Bumpus SB, Evans BS, Thomas PM, Ntai I, Kelleher NL (2009) A proteomics approach to discovering natural products and their biosynthetic pathways. Nat Biotechnol 27:951–956

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Cornelis P (2010) Iron uptake and metabolism in pseudomonads. Appl Microbiol Biotechnol 86:1637–1645

    Article  CAS  PubMed  Google Scholar 

  7. de Bruijn I, de Kock MJ, de Waard P, van Beek TA, Raaijmakers JM (2008) Massetolide A biosynthesis in Pseudomonas fluorescens. J Bacteriol 190:2777–2789

    Article  PubMed  Google Scholar 

  8. de Bruijn I, de Kock MJ, Yang M, de Waard P, van Beek TA, Raaijmakers JM (2007) Genome-based discovery, structure prediction and functional analysis of cyclic lipopeptide antibiotics in Pseudomonas species. Mol Microbiol 63:417–428

    Article  PubMed  Google Scholar 

  9. Drummond A, Ashton B, Cheung M, Heled J, Kearse M, Moir R, Stones-Havas S, Thierer T, Wilson A (2011) Geneious v5.3.6. Available from: http://www.geneious.com

  10. Fiore A, Mannina L, Sobolev AP, Salzano AM, Scaloni A, Grgurina I, Fullone MR, Gallo M, Swasey C, Fogliano V, Takemoto JY (2008) Bioactive lipopeptides of ice-nucleating snow bacterium Pseudomonas syringae strain 31R1. FEMS Microbiol Lett 286:158–165

    Article  CAS  PubMed  Google Scholar 

  11. Gross H, Loper JE (2009) Genomics of secondary metabolite production by Pseudomonas spp. Nat Prod Rep 26:1408–1446

    Article  CAS  PubMed  Google Scholar 

  12. Gross H, Stockwell VO, Henkels MD, Nowak-Thompson B, Loper JE, Gerwick WH (2007) The genomisotopic approach: a systematic method to isolate products of orphan biosynthetic gene clusters. Chem Biol 14:53–63

    Article  CAS  PubMed  Google Scholar 

  13. Grünewald J, Marahiel MA (2006) Chemoenzymatic and template-directed synthesis of bioactive macrocyclic peptides. Microbiol Mol Biol Rev 70:121–146

    Article  PubMed  PubMed Central  Google Scholar 

  14. Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696–704

    Article  PubMed  Google Scholar 

  15. Imker HJ, Krahn D, Clerc J, Kaiser M, Walsh CT (2010) N-acylation during glidobactin biosynthesis by the tridomain nonribosomal peptide synthetase module GlbF. Chem Biol 17:1077–1083

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ivanova EP, Gorshkova NM, Sawabe T, Hayashi K, Kalinovskaya NI, Lysenko AM, Zhukova NV, Nicolau DV, Kuznetsova TA, Mikhailov VV, Christen R (2002) Pseudomonas extremorientalis sp. nov., isolated from a drinking water reservoir. Int J Syst Evol Microbiol 52:2113–2120

    CAS  PubMed  Google Scholar 

  17. Kalaitzis JA, Lauro FM, Neilan BA (2009) Mining cyanobacterial genomes for genes encoding complex biosynthetic pathways. Nat Prod Rep 26:1447–1465

    Article  CAS  PubMed  Google Scholar 

  18. Koglin A, Walsh CT (2009) Structural insights into nonribosomal peptide enzymatic assembly lines. Nat Prod Rep 26:987–1000

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Kraas FI, Helmetag V, Wittmann M, Strieker M, Marahiel MA (2010) Functional dissection of surfactin synthetase initiation module reveals insights into the mechanism of lipoinitiation. Chem Biol 17:872–880

    Article  CAS  PubMed  Google Scholar 

  20. Kuiper I, Lagendijk EL, Pickford R, Derrick JP, Lamers GE, Thomas-Oates JE, Lugtenberg BJ, Bloemberg GV (2004) Characterization of two Pseudomonas putida lipopeptide biosurfactants, putisolvin I and II, which inhibit biofilm formation and break down existing biofilms. Mol Microbiol 51:97–113

    Article  CAS  PubMed  Google Scholar 

  21. Laycock MV, Hildebrand PD, Thibault P, Walter JA, Wright JLC (1991) Viscosin, a potent peptidolipid biosurfactant and phytopathogenic mediator produced by a pectolytic strain of Pseudomonas fluorescens. J Agric Food Chem 39:483–489

    Article  CAS  Google Scholar 

  22. Mortishire-Smith RJ, Nutkins JC, Packman LC, Brodey CL, Rainey PB, Johnstone K, Williams DH (1991) Determination of the structure of an extracellular peptide produced by the mushroom saprotroph Pseudomonas reactans. Tetrahedron 47:3645–3654

    Article  CAS  Google Scholar 

  23. Nett M, Ikeda H, Moore BS (2009) Genomic basis for natural product biosynthetic diversity in the actinomycetes. Nat Prod Rep 26:1362–1384

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Nybroe O, Sørensen J (2004) Production of cyclic lipopeptides by fluorescent pseudomonads. In: Ramos J-L (ed) Pseudomonas, biosynthesis of macromolecules and molecular metabolism, vol 3. Kluwer/Plenum, New York, pp 147–172

    Google Scholar 

  25. Raaijmakers JM, de Bruijn I, de Kock MJ (2006) Cyclic lipopeptide production by plant-associated Pseudomonas spp.: diversity, activity, biosynthesis, and regulation. Mol Plant Microbe Interact 19:699–710

    Article  CAS  PubMed  Google Scholar 

  26. Raaijmakers JM, De Bruijn I, Nybroe O, Ongena M (2010) Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol Rev 34:1037–1062

    Article  CAS  PubMed  Google Scholar 

  27. Roongsawang N, Hase K, Haruki M, Imanaka T, Morikawa M, Kanaya S (2003) Cloning and characterization of the gene cluster encoding arthrofactin synthetase from Pseudomonas sp. MIS38. Chem Biol 10:869–880

    Article  CAS  PubMed  Google Scholar 

  28. Roongsawang N, Lim SP, Washio K, Takano K, Kanaya S, Morikawa M (2005) Phylogenetic analysis of condensation domains in the nonribosomal peptide synthetases. FEMS Microbiol Lett 252:143–151

    Article  CAS  PubMed  Google Scholar 

  29. Roongsawang N, Washio K, Morikawa M (2007) In vivo characterization of tandem C-terminal thioesterase domains in arthrofactin synthetase. ChemBioChem 8:501–512

    Article  CAS  PubMed  Google Scholar 

  30. Roongsawang N, Washio K, Morikawa M (2011) Diversity of nonribosomal peptide synthetases involved in the biosynthesis of lipopeptide biosurfactants. Int J Mol Sci 12:141–172

    Article  CAS  Google Scholar 

  31. Scholz-Schroeder BK, Soule JD, Gross DC (2003) The sypA, sypS, and sypC synthetase genes encode twenty-two modules involved in the nonribosomal peptide synthesis of syringopeptin by Pseudomonas syringae pv. syringae B301D. Mol Plant Microbe Interact 16:271–280

    Article  CAS  PubMed  Google Scholar 

  32. Tapi A, Chollet-Imbert M, Scherens B, Jacques P (2010) New approach for the detection of non-ribosomal peptide synthetase genes in Bacillus strains by polymerase chain reaction. Appl Microbiol Biotechnol 85:1521–1531

    Article  CAS  PubMed  Google Scholar 

  33. Vallet-Gely I, Novikov A, Augusto L, Liehl P, Bolbach G, Pechy-Tarr M, Cosson P, Keel C, Caroff M, Lemaitre B (2010) Association of hemolytic activity of Pseudomonas entomophila, a versatile soil bacterium, with cyclic lipopeptide production. Appl Environ Microbiol 76:910–921

    Article  CAS  PubMed  Google Scholar 

  34. Vallet-Gely I, Opota O, Boniface A, Novikov A, Lemaitre B (2010) A secondary metabolite acting as a signalling molecule controls Pseudomonas entomophila virulence. Cell Microbiol 12:1666–1679

    Article  CAS  PubMed  Google Scholar 

  35. Wenzel SC, Müller R (2009) The impact of genomics on the exploitation of the myxobacterial secondary metabolome. Nat Prod Rep 26:1385–1407

    Article  CAS  PubMed  Google Scholar 

  36. Wong WC, Preece TF (1979) Identification of Pseudomonas tolaasii: the white line in agar and mushroom tissue block rapid pitting tests. Appl Bacteriol 47:401–407

    Article  Google Scholar 

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Acknowledgments

J. Sørensen kindly provided strains DR54 and CTS41. The authors acknowledge P. Hildebrand for the use of strain SH10-3B. AML was supported by an Erasmus scholarship through the Lifelong Learning Programme of the EU.

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  1. Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, 3001, Heverlee, Belgium

    Hassan Rokni-Zadeh, Alba Mangas-Losada & René De Mot

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  1. Hassan Rokni-Zadeh
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  2. Alba Mangas-Losada
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  3. René De Mot
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Correspondence to René De Mot.

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Rokni-Zadeh, H., Mangas-Losada, A. & De Mot, R. PCR Detection of Novel Non-ribosomal Peptide Synthetase Genes in Lipopeptide-Producing Pseudomonas . Microb Ecol 62, 941–947 (2011). https://doi.org/10.1007/s00248-011-9885-9

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