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Genome mining identifies cepacin as a plant-protective metabolite of the biopesticidal bacterium Burkholderia ambifaria

Nature Microbiology volume 4, pages 996–1005 (2019)Cite this article

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Abstract

Beneficial microorganisms are widely used in agriculture for control of plant pathogens, but a lack of efficacy and safety information has limited the exploitation of multiple promising biopesticides. We applied phylogeny-led genome mining, metabolite analyses and biological control assays to define the efficacy of Burkholderia ambifaria, a naturally beneficial bacterium with proven biocontrol properties but potential pathogenic risk. A panel of 64 B. ambifaria strains demonstrated significant antimicrobial activity against priority plant pathogens. Genome sequencing, specialized metabolite biosynthetic gene cluster mining and metabolite analysis revealed an armoury of known and unknown pathways within B. ambifaria. The biosynthetic gene cluster responsible for the production of the metabolite cepacin was identified and directly shown to mediate protection of germinating crops against Pythium damping-off disease. B. ambifaria maintained biopesticidal protection and overall fitness in the soil after deletion of its third replicon, a non-essential plasmid associated with virulence in Burkholderia cepacia complex bacteria. Removal of the third replicon reduced B. ambifaria persistence in a murine respiratory infection model. Here, we show that by using interdisciplinary phylogenomic, metabolomic and functional approaches, the mode of action of natural biological control agents related to pathogens can be systematically established to facilitate their future exploitation.

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Fig. 1: Core gene phylogeny of 64 B. ambifaria strains.
Fig. 2: Specialized metabolite BGC network analysis of 64 B. ambifaria strains.
Fig. 3: Unrooted phylogeny of LuxR protein homologues extracted from 64 B. ambifaria strains.
Fig. 4: Organization of the cepacin A BGC, LC–MS analysis of cepacin A production and antimicrobial screening of B. ambifaria BCC0191 WT and cepacin A-deficient derivative (::ccnJ).
Fig. 5: Biological control of Pythium damping-off disease is mediated by B. ambifaria cepacin.

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Code availability

The publicly available software and codes used for genome sequence determination, phylogenomics, mass spectrometry and general statistical analysis are described in the appropriate Methods sections.

Data availability

Sequence data that support the genomic findings of this study have been deposited in the European Nucleotide Archive with the accession/bioproject codes listed in Supplementary Table 1. The data that support the antimicrobial production, P. sativum and G. mellonella survival and murine infection model findings of this study are available from the corresponding authors on request. Bacterial strains and constructs will be made available on written request to the corresponding authors and after signing a Material Transfer Agreement. We are restricted in redistributing certain bacterial strains, such as those from recognized culture collections, but such requests will be redirected to the appropriate source.

References

  1. Depoorter, E. et al. Burkholderia: an update on taxonomy and biotechnological potential as antibiotic producers. Appl. Microbiol. Biotechnol. 100, 5215–5229 (2016).

    Article  CAS  Google Scholar 

  2. Parke, J. L. & Gurian-Sherman, D. Diversity of the Burkholderia cepacia complex and implications for risk assessment of biological control strains. Annu. Rev. Phytopathol. 39, 225–258 (2001).

    Article  CAS  Google Scholar 

  3. Mahenthiralingam, E. et al. Enacyloxins are products of an unusual hybrid modular polyketide synthase encoded by a cryptic Burkholderia ambifaria genomic island. Chem. Biol. 18, 665–677 (2011).

    Article  CAS  Google Scholar 

  4. Masschelein, J., Jenner, M. & Challis, G. L. Antibiotics from Gram-negative bacteria: a comprehensive overview and selected biosynthetic highlights. Nat. Prod. Rep. 34, 712–783 (2017).

    Article  CAS  Google Scholar 

  5. Seyedsayamdost, M. R. et al. Quorum-sensing-regulated bactobolin production by Burkholderia thailandensis E264. Org. Lett. 12, 716–719 (2010).

    Article  CAS  Google Scholar 

  6. Song, L. et al. Discovery and biosynthesis of gladiolin: a Burkholderia gladioli antibiotic with promising activity against Mycobacterium tuberculosis. J. Am. Chem. Soc. 139, 7974–7981 (2017).

    Article  CAS  Google Scholar 

  7. Flórez, L. V. et al. Antibiotic-producing symbionts dynamically transition between plant pathogenicity and insect-defensive mutualism. Nat. Commun. 8, 15172 (2017).

    Article  Google Scholar 

  8. Kim, J. et al. Quorum sensing and the LysR-type transcriptional activator ToxR regulate toxoflavin biosynthesis and transport in Burkholderia glumae. Mol. Microbiol. 54, 921–934 (2004).

    Article  CAS  Google Scholar 

  9. Ross, C., Scherlach, K., Kloss, F. & Hertweck, C. The molecular basis of conjugated polyyne biosynthesis in phytopathogenic bacteria. Angew. Chem. Int. Ed. Engl. 53, 7794–7798 (2014).

    Article  CAS  Google Scholar 

  10. Mansfield, J. et al. Top 10 plant pathogenic bacteria in molecular plant pathology. Mol. Plant Pathol. 13, 614–629 (2012).

    Article  Google Scholar 

  11. Howden, A. J. M., Rico, A., Mentlak, T., Miguet, L. & Preston, G. M. Pseudomonas syringae pv. syringae B728a hydrolyses indole-3-acetonitrile to the plant hormone indole-3-acetic acid. Mol. Plant Pathol. 10, 857–865 (2009).

    Article  CAS  Google Scholar 

  12. Agnoli, K. et al. Exposing the third chromosome of Burkholderia cepacia complex strains as a virulence plasmid. Mol. Microbiol. 83, 362–378 (2012).

    Article  CAS  Google Scholar 

  13. Schmidt, S. et al. Production of the antifungal compound pyrrolnitrin is quorum sensing-regulated in members of the Burkholderia cepacia complex. Environ. Microbiol. 11, 1422–1437 (2009).

    Article  Google Scholar 

  14. Tawfik, K. A. et al. Burkholdines 1097 and 1229, potent antifungal peptides from Burkholderia ambifaria 2.2N. Org. Lett. 12, 664–666 (2010).

    Article  CAS  Google Scholar 

  15. Hareland, W. A., Crawford, R. L., Chapman, P. J. & Dagley, S. Metabolic function and properties of 4-hydroxyphenylacetic acid 1-hydroxylase from Pseudomonas acidovorans. J. Bacteriol. 121, 272–285 (1975).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Parker, W. L. et al. Cepacin A and cepacin B, two new antibiotics produced by Pseudomonas cepacia. J. Antibiot. (Tokyo) 37, 431–440 (1984).

    Article  CAS  Google Scholar 

  17. Weber, T. et al. antiSMASH 3.0—a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res. 43, W237–W243 (2015).

    Article  CAS  Google Scholar 

  18. Ishida, K., Lincke, T., Behnken, S. & Hertweck, C. Induced biosynthesis of cryptic polyketide metabolites in a Burkholderia thailandensis quorum sensing mutant. J. Am. Chem. Soc. 132, 13966–13968 (2010).

    Article  CAS  Google Scholar 

  19. Fothergill, J. L., Neill, D. R., Loman, N., Winstanley, C. & Kadioglu, A. Pseudomonas aeruginosa adaptation in the nasopharyngeal reservoir leads to migration and persistence in the lungs. Nat. Commun. 5, 4780 (2014).

    Article  CAS  Google Scholar 

  20. Bricio-Moreno, L. et al. Evolutionary trade-offs associated with loss of PmrB function in host-adapted Pseudomonas aeruginosa. Nat. Commun. 9, 2635 (2018).

    Article  Google Scholar 

  21. Duerkop, B. A. et al. Quorum-sensing control of antibiotic synthesis in Burkholderia thailandensis. J. Bacteriol. 191, 3909–3918 (2009).

    Article  CAS  Google Scholar 

  22. Lee, J. & Zhang, L. The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein Cell 6, 26–41 (2015).

    Article  CAS  Google Scholar 

  23. Yabuuchi, E. et al. Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiol. Immunol. 36, 1251–1275 (1992).

    Article  CAS  Google Scholar 

  24. Vanlaere, E. et al. Burkholderia latens sp. nov., Burkholderia diffusa sp. nov., Burkholderia arboris sp. nov., Burkholderia seminalis sp. nov. and Burkholderia metallica sp. nov., novel species within the Burkholderia cepacia comple. Int. J. Syst. Evol. Microbiol. 58, 1580–1590 (2008).

    Article  CAS  Google Scholar 

  25. Fritsche, K. et al. Biosynthetic genes and activity spectrum of antifungal polyynes from Collimonas fungivorans Ter331. Environ. Microbiol. 16, 1334–1345 (2014).

    Article  CAS  Google Scholar 

  26. Kai, K., Sogame, M., Sakurai, F., Nasu, N. & Fujita, M. Collimonins A–D, unstable polyynes with antifungal or pigmentation activities from the fungus-feeding bacterium Collimonas fungivorans Ter331. Org. Lett. 20, 3536–3540 (2018).

    Article  CAS  Google Scholar 

  27. Haas, D. & Keel, C. Regulation of antibiotic production in root-colonizing Pseudomonas spp. and relevance for biological control of plant disease. Annu. Rev. Phytopathol. 41, 117–153 (2003).

    Article  CAS  Google Scholar 

  28. Palazzini, J. M., Dunlap, C. A., Bowman, M. J. & Chulze, S. N. Bacillus velezensis RC 218 as a biocontrol agent to reduce Fusarium head blight and deoxynivalenol accumulation: genome sequencing and secondary metabolite cluster profiles. Microbiol. Res. 192, 30–36 (2016).

    Article  CAS  Google Scholar 

  29. Law, J. W.-F. et al. The potential of Streptomyces as biocontrol agents against the rice blast fungus, Magnaporthe oryzae (Pyricularia oryzae). Front. Microbiol. 8, 3 (2017).

    PubMed  PubMed Central  Google Scholar 

  30. Eberl, L. & Vandamme, P. Members of the genus Burkholderia: good and bad guys. F1000Res. 5, 1007 (2016).

    Article  Google Scholar 

  31. LiPuma, J. J. The changing microbial epidemiology in cystic fibrosis. Clin. Microbiol. Rev. 23, 299–323 (2010).

    Article  Google Scholar 

  32. Kenna, D. T. D. et al. Prevalence of Burkholderia species, including members of Burkholderia cepacia complex, among UK cystic and non-cystic fibrosis patients. J. Med. Microbiol. 66, 490–501 (2017).

    Article  Google Scholar 

  33. Galardini, M., Biondi, E. G., Bazzicalupo, M. & Mengoni, A. CONTIGuator: a bacterial genomes finishing tool for structural insights on draft genomes. Source Code Biol. Med. 6, 11 (2011).

    Article  Google Scholar 

  34. Hunt, M. et al. Circlator: automated circularization of genome assemblies using long sequencing reads. Genome Biol. 16, 294 (2015).

    Article  Google Scholar 

  35. Pritchard, L. et al. Genomics and taxonomy in diagnostics for food security: soft-rotting enterobacterial plant pathogens. Anal. Methods 8, 12–24 (2016).

    Article  Google Scholar 

  36. Richter, M. & Rossello-Mora, R. Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl Acad. Sci. USA 106, 19126–19131 (2009).

    Article  CAS  Google Scholar 

  37. Connor, T. R. et al. CLIMB (the Cloud Infrastructure for Microbial Bioinformatics): an online resource for the medical microbiology community. Microb. Genomics 2, e000086 (2016).

    Google Scholar 

  38. Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068–2069 (2014).

    Article  CAS  Google Scholar 

  39. Morgulis, A. et al. BLAST+: architecture and applications. Bioinformatics 24, 1757–1764 (2008).

    Article  CAS  Google Scholar 

  40. Ondov, B. D. et al. Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol. 17, 132 (2016).

    Article  Google Scholar 

  41. Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).

    Article  CAS  Google Scholar 

  42. Page, A. J. et al. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics 31, 3691–3693 (2015).

    Article  CAS  Google Scholar 

  43. Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS ONE 5, e9490 (2010).

    Article  Google Scholar 

  44. Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).

    Article  CAS  Google Scholar 

  45. Flannagan, R. S., Aubert, D., Kooi, C., Sokol, P. A. & Valvano, M. A. Burkholderia cenocepacia requires a periplasmic HtrA protease for growth under thermal and osmotic stress and for survival in vivo. Infect. Immun. 75, 1679–1689 (2007).

    Article  CAS  Google Scholar 

  46. Gan, H. M. et al. Whole genome sequencing and analysis reveal insights into the genetic structure, diversity and evolutionary relatedness of luxI and luxR homologs in bacteria belonging to the Sphingomonadaceae family. Front. Cell. Infect. Microbiol. 4, 188 (2014).

    PubMed  Google Scholar 

  47. Katoh, K., Misawa, K., Kuma, K. & Miyata, T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30, 3059–3066 (2002).

    Article  CAS  Google Scholar 

  48. Toda, T., Iwasa, A., Fuji, S. & Furuya, H. Widespread occurrence of Pythium arrhenomanes pathogenic to rice seedlings around Japanese rice fields. Plant Dis. 99, 1823–1831 (2015).

    Article  Google Scholar 

  49. Vidal-Quist, J. C. et al. Arabidopsis thaliana and Pisum sativum models demonstrate that root colonization is an intrinsic trait of Burkholderia cepacia complex bacteria. Microbiology 160, 373–384 (2014).

    Article  CAS  Google Scholar 

  50. Hadfield, J. et al. Phandango: an interactive viewer for bacterial population genomics. Bioinformatics 34, 292–293 (2018).

    Article  CAS  Google Scholar 

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Acknowledgements

A.J.M. is funded by a Biotechnology and Biological Sciences Research Council (BBSRC) South West doctoral training partnership award (BY1910 7007). E.M., G.L.C., T.R.C. and J.P. acknowledge additional support for genome mining from BBSRC award BB/L021692/1; C.J. and M.J. were funded by this award. M.J. is currently the recipient of a BBSRC Future Leader Fellowship (BB/R01212/1). The Bruker maXis II UHPLC-ESI-Q-TOF-MS system used in this research was funded by the BBSRC (BB/M017982/1). G.W. was supported by awards to E.M. from the Life Sciences Bridging Fund and Wellcome Trust Institutional Strategic Support Fund held at Cardiff University. T.R.C. and M.J.B. acknowledge funding support from the Medical Research Council’s Cloud Infrastructure for Microbial Bioinformatics (MR/L015080/1), which provided the computational resources to undertake the analyses for this work. D.R.N. and A.E.G. acknowledge funding from a Wellcome Trust and Royal Society Sir Henry Dale Fellowship awarded to D.R.N. (grant number 204457/Z/16/Z). G.L.C. is the recipient of a Wolfson Research Merit Award from the Royal Society (WM130033). We thank L. Eberl and K. Agnoli for provision of the mini-c3 used for the third replicon deletion.

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Author notes

  1. Cerith Jones

    Present address: Faculty of Computing, Engineering and Science, University of South Wales, Pontypridd, UK

Authors and Affiliations

  1. Microbiomes, Microbes and Informatics Group, Organisms and Environment Division, School of Biosciences, Cardiff University, Cardiff, UK

    Alex J. Mullins, James A. H. Murray, Matthew J. Bull, Cerith Jones, Gordon Webster, Thomas R. Connor & Eshwar Mahenthiralingam

  2. Department of Chemistry and Warwick Integrative Synthetic Biology Centre, University of Warwick, Coventry, UK

    Matthew Jenner & Gregory L. Challis

  3. Institute of Infection and Global Health, University of Liverpool, Liverpool, UK

    Angharad E. Green & Daniel R. Neill

  4. Wellcome Sanger Institute, Wellcome Genome Campus, Cambridge, UK

    Julian Parkhill

  5. Department of Biochemistry and Molecular Biology,Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia

    Gregory L. Challis

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Contributions

The initial study to characterize the genomes of B. ambifaria as a biopesticide was conceived by E.M., with additional aspects of the study design added by A.J.M., G.L.C. and J.A.H.M. A.J.M. performed all aspects of the study with the exception of the LC–MS profiling, and was assisted by specific contributions from the following: data sets and input for genome sequencing and mining: E.M., G.L.C., J.P. and T.R.C.; genome assembly, phylogenomics, cluster mining and de-replication: M.J.B.; LuxR mining: E.M.; generation of a cepacin insertional mutant and antimicrobial activity screening: C.J.; extraction, identification and fractionation of Burkholderia metabolites by HPLC and enacyloxin minimum inhibitory concentration analysis: G.W.; LC–MS identification and confirmation of B. ambifaria antimicrobial metabolites: M.J. and G.L.C.; biocontrol modelling: E.M., G.W. and J.A.H.M.; evaluation and analysis of plant models: J.A.H.M.; Galleria virulence assays: G.W. and C.J.; and murine infection modelling and analysis: A.E.G. and D.R.N. A.J.M. and E.M. developed the first draft of the manuscript, and all authors read and contributed towards finalization of the study.

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Correspondence to Alex J. Mullins or Eshwar Mahenthiralingam.

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Mullins, A.J., Murray, J.A.H., Bull, M.J. et al. Genome mining identifies cepacin as a plant-protective metabolite of the biopesticidal bacterium Burkholderia ambifaria. Nat Microbiol 4, 996–1005 (2019). https://doi.org/10.1038/s41564-019-0383-z

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