Antonie van Leeuwenhoek

, Volume 97, Issue 3, pp 261–273 | Cite as

The Pseudomonas fluorescens secondary metabolite 2,4 diacetylphloroglucinol impairs mitochondrial function in Saccharomyces cerevisiae

  • Olive Gleeson
  • Fergal O’Gara
  • John P. Morrissey
Original Paper


Pseudomonas fluorescens strains are known to produce a wide range of secondary metabolites including phenazines, siderophores, pyoluteorin, and 2,4 diacetylphloroglucinol (DAPG). DAPG is of particular interest because of its antifungal properties and because its production is associated with inhibition of phytopathogenic fungi in natural disease-suppressive soils. This trait has been exploited to develop strains of P. fluorescens that have potential application as biocontrol agents. Although the biochemistry, genetics and regulation of DAPG production have been well-studied, relatively little is known about how DAPG inhibits fungal growth and how fungi respond to DAPG. Employing a yeast model and a combination of phenotypic assays, molecular genetics and molecular physiological probes, we established that inhibition of fungal growth is caused by impairment of mitochondrial function. The effect of DAPG on yeast is largely fungistatic but DAPG also induces the formation of petite cells. Expression of the multidrug export proteins Pdr5p and Snq2p is increased by DAPG-treatment but this appears to be a secondary effect of mitochondrial damage as no role in enhancing DAPG-tolerance was identified for either Pdr5p or Snq2p.


2,4 Diacetylphloroglucinol Yeast Pseudomonas Biocontrol Bacteria Yeast interactions 



We thank K. Kuchler for generous provisions of strains and constructs, Maurice O’Donoghue for help with flow cytometry, Pat Higgins for excellent technical support, and Lucy Holcombe for critical reading of the manuscript. Work in F. O’Gara’s and J. Morrissey’s laboratories is supported by grants awarded by the European Union (TRAMWAYS and MICROMAIZE: FP6#O36314), Science Foundation of Ireland (04/BR/B0597; 07/IN.1/B948; 08-RFP-GEN1319; 08/RFP/GEN1295), the Marine SSTI programme, and the Department of Agriculture (FIRM grants: 06RDC459; 06RDC506 and RSF grants: 06-321; 06-377).


  1. Aarons S, Abbas A, Adams C, Fenton A, O’Gara F (2000) A regulatory RNA (PrrB RNA) modulates expression of secondary metabolite genes in Pseudomonas fluorescens F113. J Bacteriol 182:3913–3919CrossRefPubMedGoogle Scholar
  2. Abbas A, Morrissey JP, Marquez PC, Sheehan MM, Delany IR, O’Gara F (2002) Characterization of interactions between the transcriptional repressor PhlF and its binding site at the phlA promoter in Pseudomonas fluorescens F113. J Bacteriol 184:3008–3016CrossRefPubMedGoogle Scholar
  3. Agarwal AK, Rogers PD, Baerson SR, Jacob MR, Barker KS, Cleary JD, Walker LA, Nagle DG, Clark AM (2003) Genome-wide expression profiling of the response to polyene, pyrimidine, azole, and echinocandin antifungal agents in Saccharomyces cerevisiae. J Biol Chem 278:34998–35015CrossRefPubMedGoogle Scholar
  4. Balba H (2007) Review of strobilurin fungicide chemicals. J Environ Sci Health B 42:441–451CrossRefPubMedGoogle Scholar
  5. Bartlett DW, Clough JM, Godwin JR, Hall AA, Hamer M, Parr-Dobrzanski B (2002) The strobilurin fungicides. Pest Manag Sci 58:649–662CrossRefPubMedGoogle Scholar
  6. Bauer BE, Wolfger H, Kuchler K (1999) Inventory and function of yeast ABC proteins: about sex, stress, pleiotropic drug and heavy metal resistance. Biochim Biophys Acta 1461:217–236CrossRefPubMedGoogle Scholar
  7. Bissinger PH, Kuchler K (1994) Molecular cloning and expression of the Saccharomyces cerevisiae STS1 gene product. A yeast ABC transporter conferring mycotoxin resistance. J Biol Chem 269:4180–4186PubMedGoogle Scholar
  8. Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, Boeke JD (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14:115–132CrossRefPubMedGoogle Scholar
  9. Brazelton JN, Pfeufer EE, Sweat TA, Gardener BB, Coenen C (2008) 2, 4-Diacetylphloroglucinol alters plant root development. Mol Plant Microbe Interact 21:1349–1358CrossRefPubMedGoogle Scholar
  10. Cabiscol E, Belli G, Tamarit J, Echave P, Herrero E, Ros J (2002) Mitochondrial Hsp60, resistance to oxidative stress, and the labile iron pool are closely connected in Saccharomyces cerevisiae. J Biol Chem 277:44531–44538CrossRefPubMedGoogle Scholar
  11. Charton C, Ulaszewski S, da Silva Vieira MR, Henoux V, Claisse ML (2004) Effects of oligomycins on adenosine triphosphatase activity of mitochondria isolated from the yeasts Saccharomyces cerevisiae and Schwanniomyces castellii. Biochem Biophys Res Commun 318:67–72CrossRefPubMedGoogle Scholar
  12. De Souza JT, Arnould C, Deulvot C, Lemanceau P, Gianinazzi-Pearson V, Raaijmakers JM (2003) Effect of 2, 4-diacetylphloroglucinol on pythium: cellular responses and variation in sensitivity among propagules and species. Phytopathology 93:966–975CrossRefPubMedGoogle Scholar
  13. de Waard MA, Andrade AC, Hayashi K, Schoonbeek HJ, Stergiopoulos I, Zwiers LH (2006) Impact of fungal drug transporters on fungicide sensitivity, multidrug resistance and virulence. Pest Manag Sci 62:195–207CrossRefPubMedGoogle Scholar
  14. di Rago JP, Coppee JY, Colson AM (1989) Molecular basis for resistance to myxothiazol, mucidin (strobilurin A), and stigmatellin. Cytochrome b inhibitors acting at the center o of the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae. J Biol Chem 264:14543–14548PubMedGoogle Scholar
  15. Ding MG, di Rago JP, Trumpower BL (2006) Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor. J Biol Chem 281:36036–36043CrossRefPubMedGoogle Scholar
  16. Duffy B, Keel C, Defago G (2004) Potential role of pathogen signaling in multitrophic plant-microbe interactions involved in disease protection. Appl Environ Microbiol 70:1836–1842CrossRefPubMedGoogle Scholar
  17. Edlind T, Smith L, Henry K, Katiyar S, Nickels J (2002) Antifungal activity in Saccharomyces cerevisiae is modulated by calcium signalling. Mol Microbiol 46:257–268CrossRefPubMedGoogle Scholar
  18. Gietz RD, Woods RA (2006) Yeast transformation by the LiAc/SS Carrier DNA/PEG method. Methods Mol Biol 313:107–120PubMedGoogle Scholar
  19. Goldstein AL, McCusker JH (2001) Development of Saccharomyces cerevisiae as a model pathogen. A system for the genetic identification of gene products required for survival in the mammalian host environment. Genetics 159:499–513PubMedGoogle Scholar
  20. Goossens A, Hakkinen ST, Laakso I, Oksman-Caldentey KM, Inze D (2003) Secretion of secondary metabolites by ATP-binding cassette transporters in plant cell suspension cultures. Plant Physiol 131:1161–1164CrossRefPubMedGoogle Scholar
  21. Haas D, Defago G (2005) Biological control of soil-borne pathogens by fluorescent pseudomonads. Nat Rev Microbiol 3:307–319CrossRefPubMedGoogle Scholar
  22. Hayashi K, Schoonbeek HJ, De Waard MA (2002) Bcmfs1, a novel major facilitator superfamily transporter from Botrytis cinerea, provides tolerance towards the natural toxic compounds camptothecin and cercosporin and towards fungicides. Appl Environ Microbiol 68:4996–5004CrossRefPubMedGoogle Scholar
  23. Hayashi K, Schoonbeek HJ, De Waard MA (2003) Modulators of membrane drug transporters potentiate the activity of the DMI fungicide oxpoconazole against Botrytis cinerea. Pest Manag Sci 59:294–302CrossRefPubMedGoogle Scholar
  24. Iavicoli A, Boutet E, Buchala A, Metraux JP (2003) Induced systemic resistance in Arabidopsis thaliana in response to root inoculation with Pseudomonas fluorescens CHA0. Mol Plant Microbe Interact 16:851–858CrossRefPubMedGoogle Scholar
  25. Jousset A, Lara E, Wall LG, Valverde C (2006) Secondary metabolites help biocontrol strain Pseudomonas fluorescens CHA0 to escape protozoan grazing. Appl Environ Microbiol 72:7083–7090CrossRefPubMedGoogle Scholar
  26. Jungwirth H, Kuchler K (2006) Yeast ABC transporters—a tale of sex, stress, drugs and aging. FEBS Lett 580:1131–1138CrossRefPubMedGoogle Scholar
  27. Kaur R, Castano I, Cormack BP (2004) Functional genomic analysis of fluconazole susceptibility in the pathogenic yeast Candida glabrata: roles of calcium signaling and mitochondria. Antimicrob Agents Chemother 48:1600–1613CrossRefPubMedGoogle Scholar
  28. Kessl JJ, Lange BB, Merbitz-Zahradnik T, Zwicker K, Hill P, Meunier B, Palsdottir H, Hunte C, Meshnick S, Trumpower BL (2003) Molecular basis for atovaquone binding to the cytochrome bc1 complex. J Biol Chem 278:31312–31318CrossRefPubMedGoogle Scholar
  29. Kim JH, Mahoney N, Chan KL, Molyneux RJ, Campbell BC (2004) Secondary metabolites of the grapevine pathogen Eutypa lata inhibit mitochondrial respiration, based on a model bioassay using the yeast Saccharomyces cerevisiae. Curr Microbiol 49:282–287CrossRefPubMedGoogle Scholar
  30. Kolaczkowski M, van der Rest M, Cybularz-Kolaczkowska A, Soumillion JP, Konings WN, Goffeau A (1996) Anticancer drugs, ionophoric peptides, and steroids as substrates of the yeast multidrug transporter Pdr5p. J Biol Chem 271:31543–31548CrossRefPubMedGoogle Scholar
  31. Kolaczkowski M, Kolaczowska A, Luczynski J, Witek S, Goffeau A (1998) In vivo characterization of the drug resistance profile of the major ABC transporters and other components of the yeast pleiotropic drug resistance network. Microb Drug Resist 4:143–158CrossRefPubMedGoogle Scholar
  32. Leppert G, McDevitt R, Falco SC, Van Dyk TK, Ficke MB, Golin J (1990) Cloning by gene amplification of two loci conferring multiple drug resistance in Saccharomyces. Genetics 125:13–20PubMedGoogle Scholar
  33. Li W, Mo W, Shen D, Sun L, Wang J, Lu S, Gitschier JM, Zhou B (2005) Yeast model uncovers dual roles of mitochondria in action of artemisinin. PLoS Genet 1:e36CrossRefPubMedGoogle Scholar
  34. Ludovico P, Sansonetty F, Corte-Real M (2001) Assessment of mitochondrial membrane potential in yeast cell populations by flow cytometry. Microbiology 147:3335–3343PubMedGoogle Scholar
  35. Mazzola M, Fujimoto DK, Thomashow LS, Cook RJ (1995) Variation in sensitivity of Gaeumannomyces graminis to antibiotics produced by fluorescent Pseudomonas spp. and effect on biological control of take-all of wheat. Appl Environ Microbiol 61:2554–2559PubMedGoogle Scholar
  36. McSpadden Gardener BB, Weller DM (2001) Changes in populations of rhizosphere bacteria associated with take-all disease of wheat. Appl Environ Microbiol 67:4414–4425CrossRefPubMedGoogle Scholar
  37. Morrissey JP, Walsh UF, O’Donnell A, Moenne-Loccoz Y, O’Gara F (2002) Exploitation of genetically modified inoculants for industrial ecology applications. Antonie Van Leeuwenhoek 81:599–606CrossRefPubMedGoogle Scholar
  38. Moynihan JA, Morrissey JP, Coppoolse ER, Stiekema WJ, O’Gara F, Boyd EF (2009) Evolutionary history of the phl gene cluster in the plant-associated bacterium Pseudomonas fluorescens. Appl Environ Microbiol 75:2122–2131CrossRefPubMedGoogle Scholar
  39. Paulsen IT, Press CM, Ravel J, Kobayashi DY, Myers GS, Mavrodi DV, DeBoy RT, Seshadri R, Ren Q, Madupu R, Dodson RJ, Durkin AS, Brinkac LM, Daugherty SC, Sullivan SA, Rosovitz MJ, Gwinn ML, Zhou L, Schneider DJ, Cartinhour SW, Nelson WC, Weidman J, Watkins K, Tran K, Khouri H, Pierson EA, Pierson LS 3rd, Thomashow LS, Loper JE (2005) Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5. Nat Biotechnol 23:873–878CrossRefPubMedGoogle Scholar
  40. Phillips DA, Fox TC, King MD, Bhuvaneswari TV, Teuber LR (2004) Microbial products trigger amino acid exudation from plant roots. Plant Physiol 136:2887–2894CrossRefPubMedGoogle Scholar
  41. Picard C, Bosco M (2006) Heterozygosis drives maize hybrids to select elite 2, 4-diacethylphloroglucinol-producing Pseudomonas strains among resident soil populations. FEMS Microbiol Ecol 58:193–204CrossRefPubMedGoogle Scholar
  42. Raaijmakers JM, Vlami M, de Souza JT (2002) Antibiotic production by bacterial biocontrol agents. Antonie Van Leeuwenhoek 81:537–547CrossRefPubMedGoogle Scholar
  43. Rezzonico F, Zala M, Keel C, Duffy B, Moenne-Loccoz Y, Defago G (2007) Is the ability of biocontrol fluorescent pseudomonads to produce the antifungal metabolite 2, 4-diacetylphloroglucinol really synonymous with higher plant protection? New Phytol 173:861–872CrossRefPubMedGoogle Scholar
  44. Rogers B, Decottignies A, Kolaczkowski M, Carvajal E, Balzi E, Goffeau A (2001) The pleitropic drug ABC transporters from Saccharomyces cerevisiae. J Mol Microbiol Biotechnol 3:207–214PubMedGoogle Scholar
  45. Schnider-Keel U, Seematter A, Maurhofer M, Blumer C, Duffy B, Gigot-Bonnefoy C, Reimmann C, Notz R, Defago G, Haas D, Keel C (2000) Autoinduction of 2, 4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin. J Bacteriol 182:1215–1225CrossRefPubMedGoogle Scholar
  46. Schoonbeek H, Del Sorbo G, De Waard MA (2001) The ABC transporter BcatrB affects the sensitivity of Botrytis cinerea to the phytoalexin resveratrol and the fungicide fenpiclonil. Mol Plant Microbe Interact 14:562–571CrossRefPubMedGoogle Scholar
  47. Schoonbeek HJ, Raaijmakers JM, De Waard MA (2002) Fungal ABC transporters and microbial interactions in natural environments. Mol Plant Microbe Interact 15:1165–1172CrossRefPubMedGoogle Scholar
  48. Schouten A, van den Berg G, Edel-Hermann V, Steinberg C, Gautheron N, Alabouvette C, de Vos CH, Lemanceau P, Raaijmakers JM (2004) Defense responses of Fusarium oxysporum to 2, 4-diacetylphloroglucinol, a broad-spectrum antibiotic produced by Pseudomonas fluorescens. Mol Plant Microbe Interact 17:1201–1211CrossRefPubMedGoogle Scholar
  49. Schouten A, Maksimova O, Cuesta-Arenas Y, van den Berg G, Raaijmakers JM (2008) Involvement of the ABC transporter BcAtrB and the laccase BcLCC2 in defence of Botrytis cinerea against the broad-spectrum antibiotic 2, 4-diacetylphloroglucinol. Environ Microbiol 10:1145–1157CrossRefPubMedGoogle Scholar
  50. Shanahan P, O’Sullivan DJ, Simpson P, Glennon JD, O’Gara F (1992) Isolation of 2, 4-diacetylphloroglucinol from a fluorescent Pseudomonad and investigation of physiological parameters influencing its production. Appl Environ Microbiol 58:353–358PubMedGoogle Scholar
  51. Sherman F (2002) Getting started with yeast. Methods Enzymol 350:3–41CrossRefPubMedGoogle Scholar
  52. Simons V, Morrissey JP, Latijnhouwers M, Csukai M, Cleaver A, Yarrow C, Osbourn A (2006) Dual effects of plant steroidal alkaloids on Saccharomyces cerevisiae. Antimicrob Agents Chemother 50:2732–2740CrossRefPubMedGoogle Scholar
  53. Stefanato FL, Abou-Mansour E, Buchala A, Kretschmer M, Mosbach A, Hahn M, Bochet CG, Metraux JP, Schoonbeek HJ (2009) The ABC transporter BcatrB from Botrytis cinerea exports camalexin and is a virulence factor on Arabidopsis thaliana. Plant J 58:499–510CrossRefPubMedGoogle Scholar
  54. Stergiopoulos I, Zwiers LH, De Waard MA (2003) The ABC transporter MgAtr4 is a virulence factor of Mycosphaerella graminicola that affects colonization of substomatal cavities in wheat leaves. Mol Plant Microbe Interact 16:689–698CrossRefPubMedGoogle Scholar
  55. Teixeira MC, Duque P, Sa-Correia I (2007) Environmental genomics: mechanistic insights into toxicity of and resistance to the herbicide 2, 4-D. Trends Biotechnol 25:363–370CrossRefPubMedGoogle Scholar
  56. Thevissen K, Cammue BP, Lemaire K, Winderickx J, Dickson RC, Lester RL, Ferket KK, Van Even F, Parret AH, Broekaert WF (2000) A gene encoding a sphingolipid biosynthesis enzyme determines the sensitivity of Saccharomyces cerevisiae to an antifungal plant defensin from dahlia (Dahlia merckii). Proc Natl Acad Sci USA 97:9531–9536CrossRefPubMedGoogle Scholar
  57. Thevissen K, Ferket KK, Francois IE, Cammue BP (2003) Interactions of antifungal plant defensins with fungal membrane components. Peptides 24:1705–1712CrossRefPubMedGoogle Scholar
  58. Ulrich JT, Mathre DE (1972) Mode of action of oxathiin systemic fungicides. V. Effect on electron transport system of Ustilago maydis and Saccharomyces cerevisiae. J Bacteriol 110:628–632PubMedGoogle Scholar
  59. Vermeulen T, Schoonbeek H, De Waard MA (2001) The ABC transporter BcatrB from Botrytis cinerea is a determinant of the activity of the phenylpyrrole fungicide fludioxonil. Pest Manag Sci 57:393–402CrossRefPubMedGoogle Scholar
  60. Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, Bangham R, Benito R, Boeke JD, Bussey H, Chu AM, Connelly C, Davis K, Dietrich F, Dow SW, El Bakkoury M, Foury F, Friend SH, Gentalen E, Giaever G, Hegemann JH, Jones T, Laub M, Liao H, Liebundguth N, Lockhart DJ, Lucau-Danila A, Lussier M, M’Rabet N, Menard P, Mittmann M, Pai C, Rebischung C, Revuelta JL, Riles L, Roberts CJ, Ross-MacDonald P, Scherens B, Snyder M, Sookhai-Mahadeo S, Storms RK, Veronneau S, Voet M, Volckaert G, Ward TR, Wysocki R, Yen GS, Yu K, Zimmermann K, Philippsen P, Johnston M, Davis RW (1999) Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285:901–906CrossRefPubMedGoogle Scholar
  61. Xie MW, Jin F, Hwang H, Hwang S, Anand V, Duncan MC, Huang J (2005) Insights into TOR function and rapamycin response: chemical genomic profiling by using a high-density cell array method. Proc Natl Acad Sci USA 102:7215–7220CrossRefPubMedGoogle Scholar
  62. Zwiers LH, Stergiopoulos I, Van Nistelrooy JG, De Waard MA (2002) ABC transporters and azole susceptibility in laboratory strains of the wheat pathogen Mycosphaerella graminicola. Antimicrob Agents Chemother 46:3900–3906CrossRefPubMedGoogle Scholar
  63. Zwiers LH, Stergiopoulos I, Gielkens MM, Goodall SD, De Waard MA (2003) ABC transporters of the wheat pathogen Mycosphaerella graminicola function as protectants against biotic and xenobiotic toxic compounds. Mol Genet Genomics 269:499–507CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Olive Gleeson
    • 1
    • 3
  • Fergal O’Gara
    • 1
    • 2
  • John P. Morrissey
    • 1
  1. 1.Department of MicrobiologyUniversity College CorkCorkIreland
  2. 2.BIOMERIT Research Centre, BioSciences InstituteUniversity College CorkCorkIreland
  3. 3.Biochemistry DepartmentNational University of Ireland GalwayGalwayIreland

Personalised recommendations