Article | Published:

Bipartite interactions, antibiotic production and biosynthetic potential of the Arabidopsis leaf microbiome

Nature Microbiologyvolume 3pages909919 (2018) | Download Citation


Plants are colonized by phylogenetically diverse microorganisms that affect plant growth and health. Representative genome-sequenced culture collections of bacterial isolates from model plants, including Arabidopsis thaliana, have recently been established. These resources provide opportunities for systematic interaction screens combined with genome mining to discover uncharacterized natural products. Here, we report on the biosynthetic potential of 224 strains isolated from the A. thaliana phyllosphere. Genome mining identified more than 1,000 predicted natural product biosynthetic gene clusters (BGCs), hundreds of which are unknown compared to the MIBiG database of characterized BGCs. For functional validation, we used a high-throughput screening approach to monitor over 50,000 binary strain combinations. We observed 725 inhibitory interactions, with 26 strains contributing to the majority of these. A combination of imaging mass spectrometry and bioactivity-guided fractionation of the most potent inhibitor, the BGC-rich Brevibacillus sp. Leaf182, revealed three distinct natural product scaffolds that contribute to the observed antibiotic activity. Moreover, a genome mining-based strategy led to the isolation of a trans-acyltransferase polyketide synthase-derived antibiotic, macrobrevin, which displays an unprecedented natural product structure. Our findings demonstrate that the phyllosphere is a valuable environment for the identification of antibiotics and natural products with unusual scaffolds.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Dewick, P. M. Medicinal Natural Products: A Biosynthetic Approach 3rd edn (Wiley, Chichester, 2009).

  2. 2.

    Newman, D. J. & Cragg, G. M. Natural products as sources of new drugs from 1981 to 2014. J. Nat. Prod. 79, 629–661 (2016).

  3. 3.

    Mlot, C. Antibiotics in nature: beyond biological warfare. Science 324, 1637–1639 (2009).

  4. 4.

    Meiser, P., Bode, H. B. & Müller, R. The unique DKxanthene secondary metabolite family from the myxobacterium Myxococcus xanthus is required for developmental sporulation. Proc. Natl Acad. Sci. USA 103, 19128–19133 (2006).

  5. 5.

    Hawver, L. A., Jung, S. A. & Ng, W. L. Specificity and complexity in bacterial quorum-sensing systems. FEMS Microbiol. Rev. 40, 738–752 (2016).

  6. 6.

    Höfer, I. et al. Insights into the biosynthesis of hormaomycin, an exceptionally complex bacterial signaling metabolite. Chem. Biol. 18, 381–391 (2011).

  7. 7.

    Phelan, V. V., Liu, W. T., Pogliano, K. & Dorrestein, P. C. Microbial metabolic exchange—the chemotype-to-phenotype link. Nat. Chem. Biol. 8, 26–35 (2012).

  8. 8.

    Saha, R., Saha, N., Donofrio, R. S. & Bestervelt, L. L. Microbial siderophores: a mini review. J. Basic Microbiol. 53, 303–317 (2013).

  9. 9.

    Wilson, M. C. et al. An environmental bacterial taxon with a large and distinct metabolic repertoire. Nature 506, 58–62 (2014).

  10. 10.

    Lincke, T., Behnken, S., Ishida, K., Roth, M. & Hertweck, C. Closthioamide: an unprecedented polythioamide antibiotic from the strictly anaerobic bacterium Clostridium cellulolyticum. Angew. Chem. 49, 2011–2013 (2010).

  11. 11.

    Pidot, S. J., Coyne, S., Kloss, F. & Hertweck, C. Antibiotics from neglected bacterial sources. Int. J. Med. Microbiol. 304, 14–22 (2014).

  12. 12.

    Wilson, M. C. & Piel, J. Metagenomic approaches for exploiting uncultivated bacteria as a resource for novel biosynthetic enzymology. Chem. Biol. 20, 636–647 (2013).

  13. 13.

    Rondon, M. R. et al. Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms. Appl. Environ. Microbiol. 66, 2541–2547 (2000).

  14. 14.

    Banik, J. J. & Brady, S. F. Recent application of metagenomic approaches toward the discovery of antimicrobials and other bioactive small molecules. Curr. Opin. Microbiol. 13, 603–609 (2010).

  15. 15.

    Vorholt, J. A., Vogel, C., Carlström, C. I. & Müller, D. B. Establishing causality: opportunities of synthetic communities for plant microbiome research. Cell Host Microbe 22, 142–155 (2017).

  16. 16.

    Bai, Y. et al. Functional overlap of the Arabidopsis leaf and root microbiota. Nature 528, 364–369 (2015).

  17. 17.

    Vorholt, J. A. Microbial life in the phyllosphere. Nat. Rev. Microbiol. 10, 828–840 (2012).

  18. 18.

    Ryffel, F. et al. Metabolic footprint of epiphytic bacteria on Arabidopsis thaliana leaves. ISME J. 10, 632–643 (2016).

  19. 19.

    Blin, K. et al. antiSMASH 4.0-improvements in chemistry prediction and gene cluster boundary identification. Nucleic Acids Res. 45, W36–W41 (2017).

  20. 20.

    Medema, M. H. et al. Minimum information about a biosynthetic gene cluster. Nat. Chem. Biol. 11, 625–631 (2015).

  21. 21.

    Yang, S. C., Lin, C. H., Sung, C. T. & Fang, J. Y. Antibacterial activities of bacteriocins: application in foods and pharmaceuticals. Front. Microbiol. 5, 241 (2014).

  22. 22.

    Helfrich, E. J. & Piel, J. Biosynthesis of polyketides by trans-AT polyketide synthases. Nat. Prod. Rep. 33, 231–316 (2016).

  23. 23.

    Blin, K., Medema, M. H., Kottmann, R., Lee, S. Y. & Weber, T. The antiSMASH database, a comprehensive database of microbial secondary metabolite biosynthetic gene clusters. Nucleic Acids Res. 45, D555–D559 (2017).

  24. 24.

    Hillenmeyer, M. E., Vandova, G. A., Berlew, E. E. & Charkoudian, L. K. Evolution of chemical diversity by coordinated gene swaps in type II polyketide gene clusters. Proc. Natl Acad. Sci. USA 112, 13952–13957 (2015).

  25. 25.

    Lazos, O. et al. Biosynthesis of the putative siderophore erythrochelin requires unprecedented crosstalk between separate nonribosomal peptide gene clusters. Chem. Biol. 17, 160–173 (2010).

  26. 26.

    Lombó, F. et al. Deciphering the biosynthesis pathway of the antitumor thiocoraline from a marine actinomycete and its expression in two Streptomyces species. ChemBioChem 7, 366–376 (2006).

  27. 27.

    Arrebola, E. et al. Mangotoxin: a novel antimetabolite toxin produced by Pseudomonas syringae inhibiting ornithine/arginine biosynthesis. Physiol. Mol. Plant Pathol. 63, 117–127 (2003).

  28. 28.

    Bassler, B. L. & Losick, R. Bacterially speaking. Cell 125, 237–246 (2006).

  29. 29.

    Pandey, S. S., Patnana, P. K., Rai, R. & Chatterjee, S. Xanthoferrin, the α-hydroxycarboxylate-type siderophore of Xanthomonas campestris pv. campestris, is required for optimum virulence and growth inside cabbage. Mol. Plant Pathol. 18, 949–962 (2017).

  30. 30.

    Barona-Gomez, F., Wong, U., Giannakopulos, A. E., Derrick, P. J. & Challis, G. L. Identification of a cluster of genes that directs desferrioxamine biosynthesis in Streptomyces coelicolor M145. J. Am. Chem. Soc. 126, 16282–16283 (2004).

  31. 31.

    Lee, J. Y. et al. Biosynthetic analysis of the petrobactin siderophore pathway from Bacillus anthracis. J. Bacteriol. 189, 1698–1710 (2007).

  32. 32.

    Barry, S. M. & Challis, G. L. Recent advances in siderophore biosynthesis. Curr. Opin. Chem. Biol. 13, 205–215 (2009).

  33. 33.

    Lindow, S. E. & Brandl, M. T. Microbiology of the phyllosphere. Appl. Environ. Microbiol. 69, 1875–1883 (2003).

  34. 34.

    Lindow, S. E. & Leveau, J. H. Phyllosphere microbiology. Curr. Opin. Biotechnol. 13, 238–243 (2002).

  35. 35.

    Schöner, T. A. et al. Aryl polyenes, a highly abundant class of bacterial natural products, are functionally related to antioxidative carotenoids. ChemBioChem 17, 247–253 (2016).

  36. 36.

    Wang, M. et al. Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking. Nat. Biotechnol. 34, 828–837 (2016).

  37. 37.

    Mootz, H. D. & Marahiel, M. A. The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains. J. Bacteriol. 179, 6843–6850 (1997).

  38. 38.

    Gebhardt, K., Pukall, R. & Fiedler, H. P. Streptocidins A-D, novel cyclic decapeptide antibiotics produced by Streptomyces sp. Tu 6071. I. Taxonomy, fermentation, isolation and biological activities. J. Antibiot. 54, 428–433 (2001).

  39. 39.

    Zgurskaya, H. I., Löpez, C. A. & Gnanakaran, S. Permeability barrier of Gram-negative cell envelopes and approaches to bypass it. ACS Infect. Dis. 1, 512–522 (2015).

  40. 40.

    Zhou, X. et al. Marthiapeptide A, an anti-infective and cytotoxic polythiazole cyclopeptide from a 60 L scale fermentation of the deep sea-derived Marinactinospora thermotolerans SCSIO 00652. J. Nat. Prod. 75, 2251–2255 (2012).

  41. 41.

    Grubbs, K. J. et al. Large-scale bioinformatics analysis of Bacillus genomes uncovers conserved roles of natural products in bacterial physiology. mSystems 2, e00040-17 (2017).

  42. 42.

    Donia, M. S. et al. A systematic analysis of biosynthetic gene clusters in the human microbiome reveals a common family of antibiotics. Cell 158, 1402–1414 (2014).

  43. 43.

    Russel, J., Røder, H. L., Madsen, J. S., Burmølle, M. & Sørensen, S. J. Antagonism correlates with metabolic similarity in diverse bacteria. Proc. Natl Acad. Sci. USA 114, 10684–10688 (2017).

  44. 44.

    Maida, I. et al. Antagonistic interactions between endophytic cultivable bacterial communities isolated from the medicinal plant Echinacea purpurea. Environ. Microbiol. 18, 2357–2365 (2016).

  45. 45.

    Hassani, M. A., Durán, P. & Hacquard, S. Microbial interactions within the plant holobiont. Microbiome 6, 58 (2018).

  46. 46.

    Venturi, V. & Keel, C. Signaling in the rhizosphere. Trends Plant Sci. 21, 187–198 (2016).

  47. 47.

    Chodkowski, J. L. & Shade, A. A synthetic community system for probing microbial interactions driven by exometabolites. mSystems 2, e00129-17 (2017).

  48. 48.

    Stringlis, I. A., Zhang, H., Pieterse, C. M. J., Bolton, M. D. & de Jonge, R. Microbial small molecules—weapons of plant subversion. Nat. Prod. Rep. 35, 410–433 (2018).

  49. 49.

    Raaijmakers, J. M. & Mazzola, M. Diversity and natural functions of antibiotics produced by beneficial and plant pathogenic bacteria. Annu. Rev. Phytopathol. 50, 403–424 (2012).

  50. 50.

    Peyraud, R. et al. Demonstration of the ethylmalonyl-CoA pathway by using 13C metabolomics. Proc. Natl Acad. Sci. USA 106, 4846–4851 (2009).

  51. 51.

    Asnicar, F., Weingart, G., Tickle, T. L., Huttenhower, C. & Segata, N. Compact graphical representation of phylogenetic data and metadata with GraPhlAn. PeerJ 3, e1029 (2015).

  52. 52.

    Müller, D. B., Schubert, O. T., Rost, H., Aebersold, R. & Vorholt, J. A. Systems-level proteomics of two ubiquitous leaf commensals reveals complementary adaptive traits for phyllosphere colonization. Mol. Cell. Proteom. 15, 3256–3269 (2016).

  53. 53.

    Yamanaka, K. et al. Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A. Proc. Natl Acad. Sci. USA 111, 1957–1962 (2014).

  54. 54.

    Finn, R. D. et al. The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res. 44, 279–285 (2016).

  55. 55.

    Ceniceros, A., Dijkhuizen, L., Petrusma, M. & Medema, M. H. Genome-based exploration of the specialized metabolic capacities of the genus Rhodococcus. BMC Genom. 18, 593 (2017).

  56. 56.

    Yang, J. Y. et al. Primer on agar-based microbial imaging mass spectrometry. J. Bacteriol. 194, 6023–6028 (2012).

  57. 57.

    Ueoka, R. et al. Metabolic and evolutionary origin of actin-binding polyketides from diverse organisms. Nat. Chem. Biol. 11, 705–712 (2015).

  58. 58.

    Durante-Rodríguez, G., de Lorenzo, V. & Martínez-García, E. The Standard European Vector Architecture (SEVA) plasmid toolkit. Methods Mol. Biol. 1149, 469–478 (2014).

  59. 59.

    Radeck, J. et al. The Bacillus BioBrick Box: generation and evaluation of essential genetic building blocks for standardized work with Bacillus subtilis. J. Biol. Eng. 7, 29 (2013).

Download references


This work was financially supported by SNF grant NRP72 to J.P. and J.A.V. and by European Research Council Advanced Grants (PhyMo to J.A.V. and SynPlex to J.P.).

Author information

Author notes

  1. These authors contributed equally: Eric J. N. Helfrich, Christine M. Vogel.


  1. Institute of Microbiology, ETH Zurich, Zurich, Switzerland

    • Eric J. N. Helfrich
    • , Christine M. Vogel
    • , Reiko Ueoka
    • , Martin Schäfer
    • , Florian Ryffel
    • , Daniel B. Müller
    • , Silke Probst
    • , Markus Kreuzer
    • , Jörn Piel
    •  & Julia A. Vorholt


  1. Search for Eric J. N. Helfrich in:

  2. Search for Christine M. Vogel in:

  3. Search for Reiko Ueoka in:

  4. Search for Martin Schäfer in:

  5. Search for Florian Ryffel in:

  6. Search for Daniel B. Müller in:

  7. Search for Silke Probst in:

  8. Search for Markus Kreuzer in:

  9. Search for Jörn Piel in:

  10. Search for Julia A. Vorholt in:


E.J.N.H., C.M.V., R.U., M.S., F.R., D.B.M., J.P. and J.A.V. designed the research. C.M.V., M.S., F.R., D.B.M. and M.K. performed binary interaction screens. E.J.N.H., C.M.V., F.R. and S.P. performed genome mining studies. C.M.V. and D.B.M. conducted statistical analyses. E.J.N.H., C.M.V. and M.S. conducted MALDI imaging experiments. E.J.N.H., C.M.V., M.S., F.R. and S.P. conducted bioassays. E.J.N.H., C.M.V., R.U., F.R. and S.P. isolated and structure-elucidated metabolites. M.S. generated Brevibacillus knockout mutants. E.J.N.H., C.M.V., D.B.M., J.P. and J.A.V. wrote the manuscript with contributions from all authors.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Jörn Piel or Julia A. Vorholt.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–48, Supplementary Results 1, Supplementary Methods, Supplementary References.

  2. Reporting Summary

  3. Supplementary Table

    Supplementary Tables 1–26.

About this article

Publication history




Issue Date