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.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Scientific Reports Open Access 04 November 2023
Integrated omics approach to unveil antifungal bacterial polyynes as acetyl-CoA acetyltransferase inhibitors
Communications Biology Open Access 12 May 2022
Nature Communications Open Access 23 June 2021
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
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.
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.
Depoorter, E. et al. Burkholderia: an update on taxonomy and biotechnological potential as antibiotic producers. Appl. Microbiol. Biotechnol. 100, 5215–5229 (2016).
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).
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).
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).
Seyedsayamdost, M. R. et al. Quorum-sensing-regulated bactobolin production by Burkholderia thailandensis E264. Org. Lett. 12, 716–719 (2010).
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).
Flórez, L. V. et al. Antibiotic-producing symbionts dynamically transition between plant pathogenicity and insect-defensive mutualism. Nat. Commun. 8, 15172 (2017).
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).
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).
Mansfield, J. et al. Top 10 plant pathogenic bacteria in molecular plant pathology. Mol. Plant Pathol. 13, 614–629 (2012).
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).
Agnoli, K. et al. Exposing the third chromosome of Burkholderia cepacia complex strains as a virulence plasmid. Mol. Microbiol. 83, 362–378 (2012).
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).
Tawfik, K. A. et al. Burkholdines 1097 and 1229, potent antifungal peptides from Burkholderia ambifaria 2.2N. Org. Lett. 12, 664–666 (2010).
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).
Parker, W. L. et al. Cepacin A and cepacin B, two new antibiotics produced by Pseudomonas cepacia. J. Antibiot. (Tokyo) 37, 431–440 (1984).
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).
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).
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).
Bricio-Moreno, L. et al. Evolutionary trade-offs associated with loss of PmrB function in host-adapted Pseudomonas aeruginosa. Nat. Commun. 9, 2635 (2018).
Duerkop, B. A. et al. Quorum-sensing control of antibiotic synthesis in Burkholderia thailandensis. J. Bacteriol. 191, 3909–3918 (2009).
Lee, J. & Zhang, L. The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein Cell 6, 26–41 (2015).
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).
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).
Fritsche, K. et al. Biosynthetic genes and activity spectrum of antifungal polyynes from Collimonas fungivorans Ter331. Environ. Microbiol. 16, 1334–1345 (2014).
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).
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).
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).
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).
Eberl, L. & Vandamme, P. Members of the genus Burkholderia: good and bad guys. F1000Res. 5, 1007 (2016).
LiPuma, J. J. The changing microbial epidemiology in cystic fibrosis. Clin. Microbiol. Rev. 23, 299–323 (2010).
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).
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).
Hunt, M. et al. Circlator: automated circularization of genome assemblies using long sequencing reads. Genome Biol. 16, 294 (2015).
Pritchard, L. et al. Genomics and taxonomy in diagnostics for food security: soft-rotting enterobacterial plant pathogens. Anal. Methods 8, 12–24 (2016).
Richter, M. & Rossello-Mora, R. Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl Acad. Sci. USA 106, 19126–19131 (2009).
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).
Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068–2069 (2014).
Morgulis, A. et al. BLAST+: architecture and applications. Bioinformatics 24, 1757–1764 (2008).
Ondov, B. D. et al. Mash: fast genome and metagenome distance estimation using MinHash. Genome Biol. 17, 132 (2016).
Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003).
Page, A. J. et al. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics 31, 3691–3693 (2015).
Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2—approximately maximum-likelihood trees for large alignments. PLoS ONE 5, e9490 (2010).
Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014).
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).
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).
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).
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).
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).
Hadfield, J. et al. Phandango: an interactive viewer for bacterial population genomics. Bioinformatics 34, 292–293 (2018).
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.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
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
This article is cited by
Scientific Reports (2023)
Microbial Ecology (2023)
Nature Reviews Drug Discovery (2022)
Integrated omics approach to unveil antifungal bacterial polyynes as acetyl-CoA acetyltransferase inhibitors
Communications Biology (2022)
Genome mining of Burkholderia ambifaria strain T16, a rhizobacterium able to produce antimicrobial compounds and degrade the mycotoxin fusaric acid
World Journal of Microbiology and Biotechnology (2022)