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Volume 162, Issue 12

The global regulator ANR is essential for strain PA23 biocontrol Free

Abstract

PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungus . The focus of the current study was to elucidate the role of the transcriptional regulator ANR in the biocontrol capabilities of this bacterium. An mutant was created, PA23, that was devoid antifungal activity. In other pseudomonads, ANR is essential for regulating HCN production. Characterization of PA23 revealed that, in addition to HCN, ANR controls phenazine (PHZ), pyrrolnitrin (PRN), protease and autoinducer (AHL) signal molecule production. In gene expression studies, , , and were found to be downregulated, consistent with our endproduct analysis. Because the phenotype of PA23 closely resembles that of quorum sensing (QS)-deficient strains, we explored whether there is a connection between ANR and the PhzRI QS system. Both and are positively regulated by ANR, whereas PhzR represses transcription. Complementation of PA23 with pUCP-, C-HSL or both yielded no change in phenotype. Conversely, PA23 harbouring pUCP23- exhibited partial-to-full restoration of antifungal activity, HCN, PRN and AHL production together with , , and expression. PHZ and protease production remained unchanged indicating that ANR can complement the QS-deficient phenotype with respect to some but not all traits. Our experiments were conducted at atmospheric O levels underscoring the fact that ANR has a profound effect on PA23 physiology under aerobic conditions.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): ANR , antibiotics , biocontrol , degradative enzymes , Pseudomonas and regulator
© 2016 The Authors
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2016-12-21
2024-04-19
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References

  1. Alexeyev M. F. 1999; The pKNOCK series of broad-host-range mobilizable suicide vectors for gene knockout and targeted DNA insertion into the chromosome of Gram-negative bacteria. Biotechniques 26:824–826[PubMed]
     [Google Scholar]
  2. Alvarez-Ortega C., Harwood C. S. 2007; Responses of Pseudomonas aeruginosa to low oxygen indicate that growth in the cystic fibrosis lung is by aerobic respiration. Mol Microbiol 65:153–165 [View Article][PubMed]
     [Google Scholar]
  3. Bates D. M., Popescu C. V., Khoroshilova N., Vogt K., Beinert H., Münck E., Kiley P. J. 2000; Substitution of leucine 28 with histidine in the Escherichia coli transcription factor FNR results in increased stability of the [4Fe-4S](2+) cluster to oxygen. J Biol Chem 275:6234–6240 [View Article][PubMed]
     [Google Scholar]
  4. Blumer C., Haas D. 2000; Mechanism, regulation, and ecological role of bacterial cyanide biosynthesis. Arch Microbiol 173:170–177 [View Article][PubMed]
     [Google Scholar]
  5. Castric P. A. 1983; Hydrogen cyanide production by Pseudomonas aeruginosa at reduced oxygen levels. Can J Microbiol 29:1344–1349 [View Article][PubMed]
     [Google Scholar]
  6. Castric P. 1994; Influence of oxygen on the Pseudomonas aeruginosa hydrogen cyanide synthase. Curr Microbiol 29:19–21 [View Article]
     [Google Scholar]
  7. Dietrich L. E., Price-Whelan A., Petersen A., Whiteley M., Newman D. K. 2006; The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa . Mol Microbiol 61:1308–1321 [View Article][PubMed]
     [Google Scholar]
  8. Fernando W. G. D., Zhang Y., Nakkeeran S., Savchuk S. 2007; Biological control of Sclerotinia sclerotiorum (Lib.) de bary by Pseudomonas and Bacillus species on canola petals. Crop Protection 26:100–107 [CrossRef]
     [Google Scholar]
  9. Finan T. M., Kunkel B., De Vos G. F., Signer E. R. 1986; Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J Bacteriol 167:66–72 [View Article][PubMed]
     [Google Scholar]
  10. Galimand M., Gamper M., Zimmermann A., Haas D. 1991; Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa . J Bacteriol 173:1598–1606 [View Article][PubMed]
     [Google Scholar]
  11. Hammond J. H., Dolben E. F., Smith T. J., Bhuju S., Hogan D. A. 2015; Links between anr and quorum sensing in Pseudomonas aeruginosa Biofilms. J Bacteriol 197:2810–2820 [View Article][PubMed]
     [Google Scholar]
  12. Hernandez M. E., Newman D. K. 2001; Extracellular electron transfer. Cell Mol Life Sci 58:1562–1571 [View Article][PubMed]
     [Google Scholar]
  13. Hernandez M. E., Kappler A., Newman D. K. 2004; Phenazines and other redox-active antibiotics promote microbial mineral reduction. Appl Environ Microbiol 70:921–928 [View Article][PubMed]
     [Google Scholar]
  14. House B. L., Mortimer M. W., Kahn M. L. 2004; New recombination methods for Sinorhizobium meliloti genetics. Appl Environ Microbiol 70:2806–2815 [View Article][PubMed]
     [Google Scholar]
  15. Ibrahim S. A., Crack J. C., Rolfe M. D., Borrero-de Acuña J. M., Thomson A. J., Le Brun N. E., Schobert M., Stapleton M. R., Green J. 2015; Three P seudomonas putida FNR family proteins with different sensitivities to O2 . J Biol Chem 290:16812–16823 [View Article][PubMed]
     [Google Scholar]
  16. Jervis A. J., Crack J. C., White G., Artymiuk P. J., Cheesman M. R., Thomson A. J., Le Brun N. E., Green J. 2009; The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion. Proc Natl Acad Sci USA 106:4659–4664 [View Article][PubMed]
     [Google Scholar]
  17. Jordan P. A., Thomson A. J., Ralph E. T., Guest J. R., Green J. 1997; FNR is a direct oxygen sensor having a biphasic response curve. FEBS Lett 416:349–352 [View Article][PubMed]
     [Google Scholar]
  18. Körner H., Sofia H. J., Zumft W. G. 2003; Phylogeny of the bacterial superfamily of Crp-Fnr transcription regulators: exploiting the metabolic spectrum by controlling alternative gene programs. FEMS Microbiol Rev 27:559–592 [View Article]
     [Google Scholar]
  19. Lambden P. R., Guest J. R. 1976; Mutants of Escherichia coli K12 unable to use fumarate as an anaerobic electron acceptor. J Gen Microbiol 97:145–160 [View Article][PubMed]
     [Google Scholar]
  20. Latifi A., Foglino M., Tanaka K., Williams P., Lazdunski A. 1996; A hierarchical quorum-sensing cascade in Pseudomonas aeruginosa links the transcriptional activators LasR and RhIR (VsmR) to expression of the stationary-phase sigma factor RpoS. Mol Microbiol 21:1137–1146 [View Article][PubMed]
     [Google Scholar]
  21. Laville J., Blumer C., Von Schroetter C., Gaia V., Défago G., Keel C., Haas D. 1998; Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0. J Bacteriol 180:3187–3196[PubMed]
     [Google Scholar]
  22. Livak K. J., Schmittgen T. D. 2001; Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408 [View Article][PubMed]
     [Google Scholar]
  23. Lu C. D., Winteler H., Abdelal A., Haas D. 1999; The ArgR regulatory protein, a helper to the anaerobic regulator ANR during transcriptional activation of the arcD promoter in Pseudomonas aeruginosa . J Bacteriol 181:2459–2464[PubMed]
     [Google Scholar]
  24. Manuel J., Selin C., Fernando W. G., de Kievit T. 2012; Stringent response mutants of Pseudomonas chlororaphis PA23 exhibit enhanced antifungal activity against Sclerotinia sclerotiorum in vitro . Microbiology 158:207–216 [View Article][PubMed]
     [Google Scholar]
  25. Mazzola M., Cook R. J., Thomashow L. S., Weller D. M., Pierson L. S. 1992; Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats. Appl Environ Microbiol 58:2616–2624[PubMed]
     [Google Scholar]
  26. Pessi G., Haas D. 2000; Transcriptional control of the hydrogen cyanide biosynthetic genes hcnABC by the anaerobic regulator ANR and the quorum-sensing regulators LasR and RhlR in Pseudomonas aeruginosa . J Bacteriol 182:6940–6949 [View Article][PubMed]
     [Google Scholar]
  27. Poritsanos N., Selin C., Fernando W. G., Nakkeeran S., de Kievit T. R. 2006; A GacS deficiency does not affect Pseudomonas chlororaphis PA23 fitness when growing on canola, in aged batch culture or as a biofilm. Can J Microbiol 52:1177–1188 [View Article][PubMed]
     [Google Scholar]
  28. Price-Whelan A., Dietrich L. E., Newman D. K. 2006; Rethinking ‘secondary’ metabolism: physiological roles for phenazine antibiotics. Nat Chem Biol 2:71–78 [View Article][PubMed]
     [Google Scholar]
  29. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning, a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
     [Google Scholar]
  30. Savchuk S., Dilantha Fernando W. G. 2004; Effect of timing of application and population dynamics on the degree of biological control of Sclerotinia sclerotiorum by bacterial antagonists. FEMS Microbiol Ecol 49:379–388 [View Article][PubMed]
     [Google Scholar]
  31. Sawers R. G. 1991; Identification and molecular characterization of a transcriptional regulator from Pseudomonas aeruginosa PAO1 exhibiting structural and functional similarity to the FNR protein of Escherichia coli . Mol Microbiol 5:1469–1481 [View Article][PubMed]
     [Google Scholar]
  32. Schuster M., Greenberg E. P. 2006; A network of networks: quorum-sensing gene regulation in Pseudomonas aeruginosa . Int J Med Microbiol 296:73–81 [View Article][PubMed]
     [Google Scholar]
  33. Selin C., Habibian R., Poritsanos N., Athukorala S. N., Fernando D., de Kievit T. R. 2010; Phenazines are not essential for Pseudomonas chlororaphis PA23 biocontrol of Sclerotinia sclerotiorum, but do play a role in biofilm formation. FEMS Microbiol Ecol 71:73–83 [View Article][PubMed]
     [Google Scholar]
  34. Selin C., Fernando W. G., de Kievit T. 2012; The PhzI/PhzR quorum-sensing system is required for pyrrolnitrin and phenazine production, and exhibits cross-regulation with RpoS in Pseudomonas chlororaphis PA23. Microbiology 158:896–907 [View Article][PubMed]
     [Google Scholar]
  35. Selin C., Manuel J., Fernando W. G. D., de Kievit T. 2014; Expression of the Pseudomonas chlororaphis strain PA23 Rsm system is under control of GacA, RpoS, PsrA, quorum sensing and the stringent response. Biological Control 69:24–33 [View Article]
     [Google Scholar]
  36. Spiro S. 1994; The FNR family of transcriptional regulators. Antonie van Leeuwenhoek 66:23–36 [View Article][PubMed]
     [Google Scholar]
  37. Ugidos A., Morales G., Rial E., Williams H. D., Rojo F. 2008; The coordinate regulation of multiple terminal oxidases by the Pseudomonas putida ANR global regulator. Environ Microbiol 10:1690–1702 [View Article][PubMed]
     [Google Scholar]
  38. West S. E., Schweizer H. P., Dall C., Sample A. K., Runyen-Janecky L. J. 1994; Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa . Gene 148:81–86 [View Article][PubMed]
     [Google Scholar]
  39. Winteler H. V., Haas D. 1996; The homologous regulators ANR of Pseudomonas aeruginosa and FNR of Escherichia coli have overlapping but distinct specificities for anaerobically inducible promoters. Microbiology 142:685–693 [View Article][PubMed]
     [Google Scholar]
  40. Ye R. W., Haas D., Ka J. O., Krishnapillai V., Zimmermann A., Baird C., Tiedje J. M. 1995; Anaerobic activation of the entire denitrification pathway in Pseudomonas aeruginosa requires Anr, an analog of Fnr. J Bacteriol 177:3606–3609 [View Article][PubMed]
     [Google Scholar]
  41. Yoon S. S., Karabulut A. C., Lipscomb J. D., Hennigan R. F., Lymar S. V., Groce S. L., Herr A. B., Howell M. L., Kiley P. J. et al. 2007; Two-pronged survival strategy for the major cystic fibrosis pathogen, Pseudomonas aeruginosa, lacking the capacity to degrade nitric oxide during anaerobic respiration. EMBO J 26:3662–3672 [View Article][PubMed]
     [Google Scholar]
  42. Zhang Y. (2004); Biocontrol of Sclerotinia stem rot of canola by bacterial antagonists and study of biocontrol mechanisms involved. MSc thesis University of Manitoba; Winnipeg:
  43. Zhang Y., Fernando W. G., de Kievit T. R., Berry C., Daayf F., Paulitz T. C. 2006; Detection of antibiotic-related genes from bacterial biocontrol agents with polymerase chain reaction. Can J Microbiol 52:476–481 [View Article][PubMed]
     [Google Scholar]
  44. Zimmermann A., Reimmann C., Galimand M., Haas D. 1991; Anaerobic growth and cyanide synthesis of Pseudomonas aeruginosa depend on anr, a regulatory gene homologous with fnr of Escherichia coli . Mol Microbiol 5:1483–1490 [View Article][PubMed]
     [Google Scholar]
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The global regulator ANR is essential for Pseudomonas chlororaphis strain PA23 biocontrol
Microbiology 162, 2159 (2016); https://doi.org/10.1099/mic.0.000391
/content/journal/micro/10.1099/mic.0.000391
/content/journal/micro/10.1099/mic.0.000391
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