Indexing the Pseudomonas specialized metabolome enabled the discovery of poaeamide B and the bananamides

  • Nature Microbiology 2, Article number: 16197 (2016)
  • doi:10.1038/nmicrobiol.2016.197
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Pseudomonads are cosmopolitan microorganisms able to produce a wide array of specialized metabolites. These molecules allow Pseudomonas to scavenge nutrients, sense population density and enhance or inhibit growth of competing microorganisms. However, these valuable metabolites are typically characterized one-molecule–one-microbe at a time, instead of being inventoried in large numbers. To index and map the diversity of molecules detected from these organisms, 260 strains of ecologically diverse origins were subjected to mass-spectrometry-based molecular networking. Molecular networking not only enables dereplication of molecules, but also sheds light on their structural relationships. Moreover, it accelerates the discovery of new molecules. Here, by indexing the Pseudomonas specialized metabolome, we report the molecular-networking-based discovery of four molecules and their evolutionary relationships: a poaeamide analogue and a molecular subfamily of cyclic lipopeptides, bananamides 1, 2 and 3. Analysis of their biosynthetic gene cluster shows that it constitutes a distinct evolutionary branch of the Pseudomonas cyclic lipopeptides. Through analysis of an additional 370 extracts of wheat-associated Pseudomonas, we demonstrate how the detailed knowledge from our reference index can be efficiently propagated to annotate complex metabolomic data from other studies, akin to the way in which newly generated genomic information can be compared to data from public databases.

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Financial support was provided by the National Institutes of Health (NIH) grant GM097509 (to B.S.M. and P.C.D.). M.H.M. was supported by Rubicon (825.13.001) and Veni (863.15.002) grants from the Netherlands Organization for Scientific Research (NWO). J.R. and V.J.C. were supported by a grant from the Netherlands BEBasic Foundation (project F07.003.01) R.D.M. was supported by KU Leuven grant GOA/011/2008. M.G.K.G. is the recipient of a post-doctoral fellowship from FWO Vlaanderen (12M4615N). A.M.K. and T.L.C. were supported by NSF grants DEB-1115895 and DEB-1336290 and US FWS grant F12AP01081. L.M.S. was supported by a National Institutes of Health IRACDA K12 GM068524 grant award. J.G.M. was supported by BBSRC Institute Strategic Program (ISPG) grant BB/J004553/1 and University of East Anglia start-up funding. T.H.M. was supported by the BBSRC Institute Strategic Program (ISPG) ‘Optimization of nutrients in soil-plant systems’ (BBS/E/C/00005196). The authors acknowledge Bruker and NIH grants GMS10RR029121 and P41-GM103484 for support in the form of shared instrumentation and the computational infrastructure. NMR data were acquired at the University of California, San Diego Skaggs School of Pharmacy and Pharmaceutical Sciences NMR Facility. The authors thank V.V. Phelan for a review of this manuscript and M. Wang, A.T. Nelson and L.-F. Nothias-Scaglia for contributions.

Author information

Author notes

    • Don D. Nguyen
    •  & Alexey V. Melnik

    These authors contributed equally to this work.


  1. Department of Chemistry and Biochemistry, University of California San Diego, California 92093, USA

    • Don D. Nguyen
    •  & Jinshu Fang
  2. Collaborative Mass Spectrometry Innovation Center, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, California 92093, USA

    • Alexey V. Melnik
    • , Nobuhiro Koyama
    •  & Pieter C. Dorrestein
  3. Graduate School of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641, Japan

    • Nobuhiro Koyama
  4. Bioinformatics Group, Wageningen University, Droevendaalsesteeg 1, 6708PB Wageningen, The Netherlands

    • Xiaowen Lu
    •  & Marnix H. Medema
  5. Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography, University of California San Diego, California 92093, USA

    • Michelle Schorn
    • , Bradley S. Moore
    •  & Pieter C. Dorrestein
  6. Ion Torrent by Thermo Fisher, 5781 Van Allen Way, Carlsbad, California 92008, USA

    • Kristen Aguinaldo
    •  & Tommie L. Lincecum Jr
  7. Centre of Microbial and Plant Genetics, KU Leuven, 3001 Heverlee, Belgium

    • Maarten G. K. Ghequire
    •  & René De Mot
  8. Department of Microbial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), 6708PB Wageningen, The Netherlands

    • Victor J. Carrion
    •  & Jos M. Raaijmakers
  9. Department of Ecology and Evolutionary Biology, University of California, 1156 High Street, Santa Cruz, California 95064, USA

    • Tina L. Cheng
    •  & A. Marm Kilpatrick
  10. Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, California 92093, USA

    • Brendan M. Duggan
    • , Laura M. Sanchez
    • , Bradley S. Moore
    •  & Pieter C. Dorrestein
  11. Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK

    • Jacob G. Malone
  12. School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK

    • Jacob G. Malone
  13. Department of AgroEcology, Rothamsted Research, West Common, Harpenden AL5 2JQ, UK

    • Tim H. Mauchline


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D.D.N., A.V.M., X.L., M.H.M. and P.C.D. designed the research. D.D.N., A.V.M., N.K., X.L., M.S., J.F., K.A., T.L.L., B.M.D., B.S.M., M.H.M. and P.C.D. performed research. D.D.N., A.V.M., N.K., X.L., M.S., M.G.K.G., J.F., B.M.D., R.D.M., M.H.M. and P.C.D. analysed data. M.G.K.G., V.J.C., T.L.C., J.G.M., T.H.M., L.M.S., A.M.K., J.M.R. and R.D.M. contributed microbial strains or extracts. D.D.N., A.V.M. and P.C.D. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Marnix H. Medema or Pieter C. Dorrestein.

Supplementary information

PDF files

  1. 1.

    Supplementary information

    Legends for Supplementary Tables 1–8, Supplementary Figures 1–16

Excel files

  1. 1.

    Supplementary Table 1

    Known Pseudomonas specialized metabolites versus strain collection. A list of the 260 Pseudomonas isolates and the additional 370 wheat-associated samples from the UK, their collection information (if available), and the known compounds they contribute to.

  2. 2.

    Supplementary Table 2

    Known Pseudomonas specialized metabolites molecular families versus strain collection. A list of the 260 Pseudomonas isolates and the additional 370 wheat- associated samples from the UK, their collection information (if available), and the molecular families they contribute to.

  3. 3.

    Supplementary Table 3

    A summary table of known Pseudomonas specialized metabolites observed and the number of strains that contribute to them.

  4. 4.

    Supplementary Table 4

    A summary table of known Pseudomonas specialized metabolite molecular families that were observed and the number of strains that contribute to them.

  5. 5.

    Supplementary Table 5

    Supplementary Table 5. Poaeamide B (m/z 1,253) NMR table. MS/MS provided the amino acid sequence tag. Amino acid, 3-hydroxy acyl chain chemical shifts and measured coupling constants obtained from 2D 1H-13C heteronuclear single quantum coherence (HSQC), 2D 1H-13C heteronuclear multiple bond correlation (HMBC), 2D 1H-13C double quantum filtered correlation spectroscopy (COSY), and 1H-NMR spectra were used to confirm the identity of the amino acid residues and 3-hydroxy acyl chain specification.

  6. 6.

    Supplementary Table 6

    Bananamide 1, 2, and 3 NMR table. MS/MS provided the amino acid sequence tag. Amino acid, 3-hydroxy acyl chain chemical shifts and measured coupling constants obtained from 2D 1H-13C heteronuclear single quantum coherence (HSQC), 2D 1H-13C heteronuclear multiple bond correlation (HMBC), 2D 1H-13C double quantum filtered correlation spectroscopy (DQF-COSY), and 1H-NMR spectra were used to confirm the identity of the amino acid residues and 3-hydroxy acyl chains specification.

  7. 7.

    Supplementary Table 7

    Poaeamide B gene cluster table. Genes in the poaeamide B biosynthetic gene cluster from P. synxantha CR32 based on homology and protein domain analysis.

  8. 8.

    Supplementary Table 8

    Bananamide gene cluster table. Genes in the bananamide biosynthetic gene cluster from P. fluorescens BW11P2 based on homology and protein domain analysis.