1887

Abstract

The polyketide metabolite 2,4-diacetylphloroglucinol (2,4-DAPG) plays a major role in the biological control of soil-borne plant diseases by 2P24. Two mutants (PM810 and PM820) with increased extracellular accumulation of 2,4-DAPG were isolated using transposon mutagenesis. The disrupted genes in these two mutants shared >80 % identity with the genes of the EmhR–EmhABC resistance-nodulation-division (RND) efflux system of cLP6a. The deletion of (PM802), (PM803) or (PM804) genes in strain 2P24 increased the extracellular accumulation of 2,4-DAPG, whereas the deletion of the (PM801) gene decreased the biosynthesis of 2,4-DAPG. The promoter assay confirmed the elevated transcription of in the EmhR disrupted strain (PM801) and an indirect negative regulation of 2,4-DAPG biosynthetic locus transcription by the EmhABC efflux pump. Induction by exogenous 2,4-DAPG led to remarkable differences in transcription of chromosome-borne  : :  fusion in PM901 and PM811 ( ) strains. Additionally, the EmhABC system in strain 2P24 was involved in the resistance to a group of toxic compounds, including ampicillin, chloramphenicol, tetracycline, ethidium bromide and crystal violet. In conclusion, our results suggest that the EmhABC system is an important element that influences the production of antibiotic 2,4-DAPG and enhances resistance to toxic compounds in 2P24.

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2010-01-01
2020-08-03
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References

  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 Bacteriol182:3913–3919
    [Google Scholar]
  2. Abbas A., Morrissey J. P., Marquez P. C., Sheehan M. M., Delany I. R., 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 Bacteriol184:3008–3016
    [Google Scholar]
  3. Abbas A., McGuire J. E., Crowley D., Baysse C., Dow M., O'Gara F.. 2004; The putative permease PhlE of Pseudomonas fluorescens F113 has a role in 2,4-diacetylphloroglucinol resistance and in general stress tolerance. Microbiology150:2443–2450
    [Google Scholar]
  4. Aendekerk S., Ghysels B., Cornelis P., Baysse C.. 2002; Characterization of a new efflux pump, MexGHI-OpmD, from Pseudomonas aeruginosa that confers resistance to vanadium. Microbiology148:2371–2381
    [Google Scholar]
  5. Baehler E., de Werra P., Wick L. Y., Péchy-Tarr M., Mathys S., Maurhofer M., Keel C.. 2006; Two novel MvaT-like global regulators control exoproduct formation and biocontrol activity in root-associated Pseudomonas fluorescens CHA0. Mol Plant Microbe Interact19:313–329
    [Google Scholar]
  6. Bangera M. G., Thomashow L. S.. 1999; Identification and characterization of a gene cluster for synthesis of the polyketide antibiotic 2,4-diacetylphloroglucinol from Pseudomonas fluorescens Q2–87. J Bacteriol181:3155–3163
    [Google Scholar]
  7. Chan Y. Y., Chua K. L.. 2005; The Burkholderia pseudomallei BpeAB–OprB efflux pump: expression and impact on quorum sensing and virulence. J Bacteriol187:4707–4719
    [Google Scholar]
  8. Chilton M. D., Currier T. C., Farrand S. K., Bendich A. J., Gordon M. P., Nester E. W.. 1974; Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. Proc Natl Acad Sci U S A71:3672–3676
    [Google Scholar]
  9. Delany I., Sheehan M. M., Fenton A., Bardin S., Aarons S., O'Gara F.. 2000; Regulation of production of the antifungal metabolite 2,4-diacetylphloroglucinol in Pseudomonas fluorescens F113: genetic analysis of PhlF as a transcriptional repressor. Microbiology146:537–546
    [Google Scholar]
  10. Duffy B. K., Defago G.. 1999; Environmental factors modulating antibiotic and siderophore biosynthesis by Pseudomonas fluorescens biocontrol strains. Appl Environ Microbiol65:2429–2438
    [Google Scholar]
  11. Dwivedi D., Johri B. N.. 2003; Antifungals from fluorescent pseudomonads: biosynthesis and regulation. Curr Sci85:1693–1703
    [Google Scholar]
  12. Fenton A. M., Stephens P. M., Crowley J., O'Callaghan M., O'Gara F.. 1992; Exploitation of gene(s) involved in 2,4-diacetylphloroglucinol biosynthesis to confer a new biocontrol capability to a Pseudomonas strain. Appl Environ Microbiol58:3873–3878
    [Google Scholar]
  13. Goldberg J. B., Hancock R., Parales R. E., Loper J., Cornelis P.. 2008; Pseudomonas 2007. J Bacteriol190:2649–2662
    [Google Scholar]
  14. Haas D., Défago G.. 2005; Biological control of soil-borne pathogens by fluorescent pseudomonads. Nat Rev Microbiol3:307–319
    [Google Scholar]
  15. Haas D., Blumer C., Keel C.. 2000; Biocontrol ability of fluorescent pseudomonads genetically dissected: importance of positive feedback regulation. Curr Opin Biotechnol11:290–297
    [Google Scholar]
  16. Hearn E. M., Dennis J. J., Gray M. R., Foght J. M.. 2003; Identification and characterization of the emhABC efflux system for polycyclic aromatic hydrocarbons in Pseudomonas fluorescens cLP6a. J Bacteriol185:6233–6240
    [Google Scholar]
  17. Hearn E. M., Gray M. R., Foght J. M.. 2006; Mutations in the central cavity and periplasmic domain affect efflux activity of the resistance-nodulation-division pump EmhB from Pseudomonas fluorescens cLP6a. J Bacteriol188:115–123
    [Google Scholar]
  18. Herrero M., De Lorenzo V., Timmis K. N.. 1990; Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in Gram-negative bacteria. J Bacteriol172:6557–6567
    [Google Scholar]
  19. Keen N. T., Tamaki S., Kobayashi D., Trollinger D.. 1988; Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria. Gene70:191–197
    [Google Scholar]
  20. Kieboom J., de Bont J.. 2001; Identification and molecular characterization of an efflux system involved in Pseudomonas putida S12 multidrug resistance. Microbiology147:43–51
    [Google Scholar]
  21. King E. O., Ward M. K., Raney D. E.. 1954; Two simple media for the demonstration of pyocyanin and fluorescein. J Lab Clin Med44:301–307
    [Google Scholar]
  22. Laville J., Voisard C., Keel C., Maurhofer M., Defago G., Haas D.. 1992; Global control in Pseudomonas fluorescens mediating antibiotic synthesis and suppression of black root rot of tobacco. Proc Natl Acad Sci U S A89:1562–1566
    [Google Scholar]
  23. Li X. Z., Nikaido H., Poole K.. 1995; Role of MexA-MexB-OprM in antibiotic efflux in Pseudomonas aeruginosa. Antimicrob Agents Chemother39:1948–1953
    [Google Scholar]
  24. Linares J. F., Lopez J. A., Camafeita E., Albar J. P., Rojo F., Martinez J. L.. 2005; Overexpression of the multidrug efflux pumps MexCD-OprJ and MexEF-OprN is associated with a reduction of type III secretion in Pseudomonas aeruginosa. J Bacteriol187:1384–1391
    [Google Scholar]
  25. Mavrodi O. V., Mavrodi D. V., Park A. A., Weller D. M., Thomashow L. S.. 2006; The role of dsbA in colonization of the wheat rhizosphere by Pseudomonas fluorescens Q8r1–96. Microbiology152:863–872
    [Google Scholar]
  26. Miller J. H.. 1972; Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  27. Nikaido H.. 1996; Multidrug efflux pumps of Gram-negative bacteria. J Bacteriol178:5853–5859
    [Google Scholar]
  28. Pearson J. P., Van Delden C., Iglewski B. H.. 1999; Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals. J Bacteriol181:1203–1210
    [Google Scholar]
  29. Poole K.. 2001; Multidrug resistance in Gram-negative bacteria. Curr Opin Microbiol4:500–508
    [Google Scholar]
  30. Poole K.. 2004; Efflux-mediated multiresistance in Gram-negative bacteria. Clin Microbiol Infect10:12–26
    [Google Scholar]
  31. Poole K.. 2008; Bacterial multidrug efflux pumps serve other functions. Microbe3:179–185
    [Google Scholar]
  32. Raaijmakers J. M., Weller D. M., Thomashow L. S.. 1997; Frequency of antibiotic-producing Pseudomonas spp. in natural environments. Appl Environ Microbiol63:881–887
    [Google Scholar]
  33. Rahmati S., Yang S., Davidson A. L., Zechiedrich E. L.. 2002; Control of the AcrAB multidrug efflux pump by quorum-sensing regulator SdiA. Mol Microbiol43:677–685
    [Google Scholar]
  34. Ramos J. L., Duque E., Godoy P., Segura A.. 1998; Efflux pumps involved in toluene tolerance in Pseudomonas putida DOT-T1E. J Bacteriol180:3323–3329
    [Google Scholar]
  35. Sambrook J., Russell D. W.. 2001; Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
  36. Schnider-Keel U., Seematter A., Maurhofer M., Blumer C., Duffy B., Gigot-Bonnefoy C., Reimmann C., Notz R., Defago G., Haas D.. 2000; Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the biocontrol agent Pseudomonas fluorescens CHA0 and repression by the bacterial metabolites salicylate and pyoluteorin. J Bacteriol182:1215–1225
    [Google Scholar]
  37. Schwalbe R. S., Steele-Moore L., Goodwin A. C.. 2007; Antimicrobial Susceptibility Testing Protocols Boca Raton, FL: CRC Press;
  38. Shanahan P., O'Sullivan D. J., Simpson P., Glennon J. D., O'Gara F.. 1992; Isolation of 2,4-diacetylphloroglucinol from a fluorescent pseudomonad and investigation of physiological parameters influencing its production. Appl Environ Microbiol58:353–358
    [Google Scholar]
  39. Tseng T. T., Gratwick K. S., Kolman J., Park D., Nies D. H., Goffeau A., Saier M. H.. 1999; The RND permease: an ancient, ubiquitous and diverse family that includes human disease and development proteins. J Mol Microbiol Biotechnol1:107–125
    [Google Scholar]
  40. Tusnady G. E., Simon I.. 2001; The hmmtop transmembrane topology prediction server. Bioinformatics17:849–850
    [Google Scholar]
  41. Valverde C., Heeb S., Keel C., Haas D.. 2003; RsmY, a small regulatory RNA, is required in concert with RsmZ for GacA-dependent expression of biocontrol traits in Pseudomonas fluorescens CHA0. Mol Microbiol50:1361–1379
    [Google Scholar]
  42. Van den Eede G., Deblaere R., Goethals K., Van Montagu M., Holsters M.. 1992; Broad host range and promoter selection vectors for bacteria that interact with plants. Mol Plant Microbe Interact5:228–234
    [Google Scholar]
  43. Vincent M. N., Harrison L. A., Brackin J. M., Kovacevich P. A., Mukerji P., Weller D. M., Pierson E. A.. 1991; Genetic analysis of the antifungal activity of a soilborne Pseudomonas aureofaciens strain. Appl Environ Microbiol57:2928–2934
    [Google Scholar]
  44. Wei H. L., Zhang L. Q.. 2005; Cloning and functional characterization of the gacS gene of the biocontrol strain Pseudomonas fluorescens 2P24. Wei Sheng Wu Xue Bao45:368–372 in Chinese
    [Google Scholar]
  45. Wei H. L., Zhang L. Q.. 2006; Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24. Antonie Van Leeuwenhoek89:267–280
    [Google Scholar]
  46. Yan Q., Wu X. G., Wei H. L., Wang H. M., Zhang L. Q.. 2009; Differential control of the PcoI/PcoR quorum-sensing system in Pseudomonas fluorescens 2P24 by sigma factor RpoS and the GacS/GacA two-component regulatory system. Microbiol Res164:18–26
    [Google Scholar]
  47. Yang S., Lopez C. R., Zechiedrich E. L.. 2006; Quorum sensing and multidrug transporters in Escherichia coli. Proc Natl Acad Sci U S A103:2386–2391
    [Google Scholar]
  48. Zgurskaya H. I., Krishnamoorthy G., Tikhonova E. B., Lau S. Y., Stratton K. L.. 2003; Mechanism of antibiotic efflux in Gram-negative bacteria. Front Biosci8:s862–s873
    [Google Scholar]
  49. Zhou H. Y., Wei H. L., Liu X. L., Wang Y., Zhang L. Q., Tang W. H.. 2005; Improving biocontrol activity of Pseudomonas fluorescens through chromosomal integration of 2,4-diacetylphloroglucinol biosynthesis genes. Chin Sci Bull50:775–781
    [Google Scholar]
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