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Effect of Producing Different Phenazines on Bacterial Fitness and Biological Control in Pseudomonas chlororaphis 30-84

  • Yu, Jun Myoung (Department of Horticultural Sciences, Texas A&M University) ;
  • Wang, Dongping (Department of Plant Pathology and Microbiology, Texas A&M University) ;
  • Pierson, Leland S. III (Department of Plant Pathology and Microbiology, Texas A&M University) ;
  • Pierson, Elizabeth A. (Department of Horticultural Sciences, Texas A&M University)
  • Received : 2017.12.25
  • Accepted : 2018.01.07
  • Published : 2018.02.01

Abstract

Pseudomonas chlororaphis 30-84 is a biological control agent selected for its ability to suppress diseases caused by fungal pathogens. P. chlororaphis 30-84 produces three phenazines: phenazine-1-carboxylic acid (PCA), 2-hydroxy-phenazine-1-carboxylic acid (2OHPCA) and a small amount of 2-hydroxy-phenazine (2OHPHZ), and these are required for fungal pathogen inhibition and wheat rhizosphere competence. The two, 2-hydroxy derivatives are produced from PCA via the activity of a phenazine-modifying enzyme encoded by phzO. In addition to the seven biosynthetic genes responsible for the production of PCA, many other Pseudomonas strains possess one or more modifying genes, which encode enzymes that act independently or together to convert PCA into other phenazine derivatives. In order to understand the fitness effects of producing different phenazines, we constructed isogenic derivatives of P. chlororaphis 30-84 that differed only in the type of phenazines produced. Altering the type of phenazines produced by P. chlororaphis 30-84 enhanced the spectrum of fungal pathogens inhibited and altered the degree of take-all disease suppression. These strains also differed in their ability to promote extracellular DNA release, which may contribute to the observed differences in the amount of biofilm produced. All derivatives were equally important for survival over repeated plant/harvest cycles, indicating that the type of phenazines produced is less important for persistence in the wheat rhizosphere than whether or not cells produce phenazines. These findings provide a better understanding of the effects of different phenazines on functions important for biological control activity with implications for applications that rely on introduced or native phenazine producing populations.

Keywords

References

  1. Alhede, M., Kragh, K. N., Qvortrup, K., Allesen-Holm, M., van Gennip, M., Christensen, L. D., Jensen, P. O., Nielsen, A. K., Parsek, M. and Wozniak, D. 2011. Phenotypes of non-attached Pseudomonas aeruginosa aggregates resemble surface attached biofilm. PLoS One 6:e27943. https://doi.org/10.1371/journal.pone.0027943
  2. Baron, S. S., Terranova, G. and Rowe, J. J. 1989. Molecular mechanism of the antimicrobial action of pyocyanin. Curr. Microbiol. 18:223-230. https://doi.org/10.1007/BF01570296
  3. Bellin, D. L., Sakhtah, H., Rosenstein, J. K., Levine, P. M., Thimot, J., Emmett, K., Dietrich, L. E. and Shepard, K. L. 2014. Integrated circuit-based electrochemical sensor for spatially resolved detection of redox-active metabolites in biofilms. Nat. Commun. 5:3256.
  4. Berg, G., Fritze, A., Roskot, N. and Smalla, K. 2001. Evaluation of potential biocontrol rhizobacteria from different host plants of Verticillium dahliae kleb. J. Appl. Microbiol. 91:963-971. https://doi.org/10.1046/j.1365-2672.2001.01462.x
  5. Cezairliyan, B., Vinayavekhin, N., Grenfell-Lee, D., Yuen, G. J., Saghatelian, A. and Ausubel, F. M. 2013. Identification of Pseudomonas aeruginosa phenazines that kill Caenorhabditis elegans. PLoS Pathog. 9:e1003101. https://doi.org/10.1371/journal.ppat.1003101
  6. Chin-A-Woeng, T. F., Bloemberg, G. V. and Lugtenberg, B. J. 2003. Phenazines and their role in biocontrol by Pseudomonas bacteria. New Phytol. 157:503-523. https://doi.org/10.1046/j.1469-8137.2003.00686.x
  7. Chin-A-Woeng, T. F., Bloemberg, G. V., van der Bij, A. J., van der Drift, K. M., Schripsema, J., Kroon, B., Scheffer, R. J., Keel, C., Bakker, P. A. and Tichy, H.-V. 1998. Biocontrol by phenazine-1-carboxamide-producing Pseudomonas chlororaphis pcl1391 of tomato root rot caused by Fusarium oxysporum f. Sp. radicis-lycopersici. Mol. Plant-Microbe Interact. 11:1069-1077. https://doi.org/10.1094/MPMI.1998.11.11.1069
  8. Chin-A-Woeng, T. F., Thomas-Oates, J. E., Lugtenberg, B. J. and Bloemberg, G. V. 2001a. Introduction of the phzh gene of Pseudomonas chlororaphis pcl1391 extends the range of biocontrol ability of phenazine-1-carboxylic acid-producing Pseudomonas spp. strains. Mol. Plant-Microbe Interact. 14:1006-1015. https://doi.org/10.1094/MPMI.2001.14.8.1006
  9. Chin-A-Woeng, T. F., van den Broek, D., de Voer, G., van der Drift, K. M., Tuinman, S., Thomas-Oates, J. E., Lugtenberg, B. J. and Bloemberg, G. V. 2001b. Phenazine-1-carboxamide production in the biocontrol strain Pseudomonas chlororaphis pcl1391 is regulated by multiple factors secreted into the growth medium. Mol. Plant-Microbe Interact. 14:969-979. https://doi.org/10.1094/MPMI.2001.14.8.969
  10. Das, T., Kutty, S. K., Kumar, N. and Manefield, M. 2013a. Pyocyanin facilitates extracellular DNA binding to Pseudomonas aeruginosa influencing cell surface properties and aggregation. PLoS One 8:e58299. https://doi.org/10.1371/journal.pone.0058299
  11. Das, T., Kutty, S. K., Tavallaie, R., Ibugo, A. I., Panchompoo, J., Sehar, S., Aldous, L., Yeung, A. W., Thomas, S. R. and Kumar, N. 2015. Phenazine virulence factor binding to extracellular DNA is important for Pseudomonas aeruginosa biofilm formation. Sci. Rep. 5:8398. https://doi.org/10.1038/srep08398
  12. Das, T. and Manefield, M. 2012. Pyocyanin promotes extracellular DNA release in Pseudomonas aeruginosa. PLoS One 7:e46718. https://doi.org/10.1371/journal.pone.0046718
  13. Das, T., Sehar, S. and Manefield, M. 2013b. The roles of extracellular DNA in the structural integrity of extracellular polymeric substance and bacterial biofilm development. Environ. Microbiol. Rep. 5:778-786. https://doi.org/10.1111/1758-2229.12085
  14. Das, T., Sharma, P. K., Busscher, H. J., van der Mei, H. C. and Krom, B. P. 2010. Role of extracellular DNA in initial bacterial adhesion and surface aggregation. Appl. Environ. Microbiol. 76:3405-3408. https://doi.org/10.1128/AEM.03119-09
  15. Delaney, S. M., Mavrodi, D. V., Bonsall, R. F. and Thomashow, L. S. 2001. Phzo, a gene for biosynthesis of 2-hydroxylated phenazine compounds in Pseudomonas aureofaciens 30-84. J. Bacteriol. 183:318-327. https://doi.org/10.1128/JB.183.1.318-327.2001
  16. Flaishman, M., Eyal, Z., Voisard, C. and Haas, D. 1990. Suppression of Septoria tritici by phenazine-or siderophore-deficient mutants of Pseudomonas. Curr. Microbiol. 20:121-124. https://doi.org/10.1007/BF02092884
  17. Flemming, H.-C. and Wingender, J. 2010. The biofilm matrix. Nat. Rev. Microbiol. 8:623-633. https://doi.org/10.1038/nrmicro2415
  18. Ghosh, P. K. and Maiti, T. K. 2016. Structure of extracellular polysaccharides (eps) produced by rhizobia and their functions in legume-bacteria symbiosis. Achiev. Life Sci. 10:136-143. https://doi.org/10.1016/j.als.2016.11.003
  19. Gibson, J., Sood, A. and Hogan, D. A. 2009. Pseudomonas aeruginosa-candida albicans interactions: localization and fungal toxicity of a phenazine derivative. Appl. Environ. Microbiol. 75:504-513. https://doi.org/10.1128/AEM.01037-08
  20. Gloag, E. S., Turnbull, L., Huang, A., Vallotton, P., Wang, H., Nolan, L. M., Mililli, L., Hunt, C., Lu, J. and Osvath, S. R. 2013. Self-organization of bacterial biofilms is facilitated by extracellular DNA. Proc. Natl. Acad. Sci. U.S.A. 110:11541-11546. https://doi.org/10.1073/pnas.1218898110
  21. Gu, M. and Imlay, J. A. 2011. The soxrs response of Escherichia coli is directly activated by redox-cycling drugs rather than by superoxide. Mol. Microbiol. 79:1136-1150. https://doi.org/10.1111/j.1365-2958.2010.07520.x
  22. Gunn, J. S., Bakaletz, L. O. and Wozniak, D. J. 2016. What's on the outside matters: the role of the extracellular polymeric substance of gram-negative biofilms in evading host immunity and as a target for therapeutic intervention. J. Biol. Chem. 291:12538-12546. https://doi.org/10.1074/jbc.R115.707547
  23. Haas, D. and Defago, G. 2005. Biological control of soil-borne pathogens by fluorescent pseudomonads. Nat. Rev. Microbiol. 3:307-319. https://doi.org/10.1038/nrmicro1129
  24. Hassett, D., Charniga, L., Bean, K., Ohman, D. and Cohen, M. S. 1992. Response of Pseudomonas aeruginosa to pyocyanin: mechanisms of resistance, antioxidant defenses, and demonstration of a manganese-cofactored superoxide dismutase. Infect. Immun. 60:328-336.
  25. Haynes, W. C., Stodola, F. H., Locke, J. M., Pridham, T. G., Conway, H. F., Sohns, V. E. and Jackson, R. W. 1956. Pseudomonas aureofaciens kluyver and phenazine ${\alpha}$-carboxylic acid, its characteristic pigment. J. Bacteriol. 72:412.
  26. Jayathilake, P. G., Jana, S., Rushton, S., Swailes, D., Bridgens, B., Curtis, T. and Chen, J. 2017. Extracellular polymeric substance production and aggregated bacteria colonization influence the competition of microbes in biofilms. Front. Microbiol. 8:1865. https://doi.org/10.3389/fmicb.2017.01865
  27. Liu, G. Y. and Nizet, V. 2009. Color me bad: microbial pigments as virulence factors. Trends Microbiol. 17:406-413. https://doi.org/10.1016/j.tim.2009.06.006
  28. Maddula, V. S., Pierson, E. A. and Pierson, L. S., III. 2008. Altering the ratio of phenazines in Pseudomonas chlororaphis (aureofaciens) strain 30-84: effects on biofilm formation and pathogen inhibition. J. Bacteriol. 190:2759-2766. https://doi.org/10.1128/JB.01587-07
  29. Maddula, V. S., Zhang, Z., Pierson, E. A. and Pierson, L. S., III. 2006. Quorum sensing and phenazines are involved in biofilm formation by Pseudomonas chlororaphis (aureofaciens) strain 30-84. Microb. Ecol. 52:289-301. https://doi.org/10.1007/s00248-006-9064-6
  30. Mann, E. E. and Wozniak, D. J. 2012. Pseudomonas biofilm matrix composition and niche biology. FEMS Microbiol. Rev. 36:893-916. https://doi.org/10.1111/j.1574-6976.2011.00322.x
  31. Mavrodi, D. V., Blankenfeldt, W. and Thomashow, L. S. 2006. Phenazine compounds in fluorescent Pseudomonas spp. biosynthesis and regulation. Annu. Rev. Phytopathol. 44:417-445. https://doi.org/10.1146/annurev.phyto.44.013106.145710
  32. Mavrodi, D. V., Bonsall, R. F., Delaney, S. M., Soule, M. J., Phillips, G. and Thomashow, L. S. 2001. Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa pao1. J. Bacteriol. 183:6454-6465. https://doi.org/10.1128/JB.183.21.6454-6465.2001
  33. Mavrodi, D. V., Mavrodi, O. V., Parejko, J. A., Bonsall, R. F., Kwak, Y.-S., Paulitz, T. C., Thomashow, L. S. and Weller, D. M. 2012a. Accumulation of the antibiotic phenazine-1-carboxylic acid in the rhizosphere of dryland cereals. Appl. Environ. Microbiol. 78:804-812. https://doi.org/10.1128/AEM.06784-11
  34. Mavrodi, D. V., Peever, T. L., Mavrodi, O. V., Parejko, J. A., Raaijmakers, J. M., Lemanceau, P., Mazurier, S., Heide, L., Blankenfeldt, W. and Weller, D. M. 2010. Diversity and evolution of the phenazine biosynthesis pathway. Appl. Environ. Microbiol. 76:866-879. https://doi.org/10.1128/AEM.02009-09
  35. Mavrodi, O. V., Mavrodi, D. V., Parejko, J. A., Thomashow, L. S. and Weller, D. M. 2012b. Irrigation differentially impacts populations of indigenous antibiotic-producing Pseudomonas spp. in the rhizosphere of wheat. Appl. Environ. Microbiol. 78:3214-3220. https://doi.org/10.1128/AEM.07968-11
  36. Mazzola, M., Cook, R. J., Thomashow, L. S., Weller, D. M. and Pierson, L. S., III. 1992. Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats. Appl. Environ. Microbiol. 58:2616-2624.
  37. Miller, W. G., Leveau, J. H. and Lindow, S. E. 2000. Improved gfp and inaz broad-host-range promoter-probe vectors. Mol. Plant-Microbe Interact. 13:1243-1250. https://doi.org/10.1094/MPMI.2000.13.11.1243
  38. Morales, D. K., Jacobs, N. J., Rajamani, S., Krishnamurthy, M., Cubillos-Ruiz, J. R. and Hogan, D. A. 2010. Antifungal mechanisms by which a novel Pseudomonas aeruginosa phenazine toxin kills candida albicans in biofilms. Mol. Microbiol. 78:1379-1392. https://doi.org/10.1111/j.1365-2958.2010.07414.x
  39. Mulcahy, H., Charron-Mazenod, L. and Lewenza, S. 2008. Extracellular DNA chelates cations and induces antibiotic resistance in Pseudomonas aeruginosa biofilms. PLoS Pathog. 4:e1000213. https://doi.org/10.1371/journal.ppat.1000213
  40. O'Toole, G. A. and Kolter, R. 1998. Initiation of biofilm formation in Pseudomonas fluorescens wcs365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol. Microbiol. 28:449-461. https://doi.org/10.1046/j.1365-2958.1998.00797.x
  41. Okshevsky, M. and Meyer, R. L. 2015. The role of extracellular DNA in the establishment, maintenance and perpetuation of bacterial biofilms. Crit. Rev. Microbiol. 41:341-352. https://doi.org/10.3109/1040841X.2013.841639
  42. Ownley, B. H., Weller, D. and Thomashow, L. S. 1992. Influence of in situ and in vitro ph on suppression of Gaeumannomyces graminis var. tritici by Pseudomonas fluorescens 2-79. Phytopathology 82:178-184. https://doi.org/10.1094/Phyto-82-178
  43. Parejko, J. A., Mavrodi, D. V., Mavrodi, O. V., Weller, D. M. and Thomashow, L. S. 2012. Population structure and diversity of phenazine-1-carboxylic acid producing fluorescent Pseudomonas spp. from dryland cereal fields of central washington state (USA). Microb. Ecol. 64:226-241. https://doi.org/10.1007/s00248-012-0015-0
  44. Pierson, E. A., Wood, D. W., Cannon, J. A., Blachere, F. M. and Pierson, L. S., III. 1998. Interpopulation signaling via n-acylhomoserine lactones among bacteria in the wheat rhizosphere. Mol. Plant-Microbe Interact. 11:1078-1084. https://doi.org/10.1094/MPMI.1998.11.11.1078
  45. Pierson, L. S., III, Gaffney, T., Lam, S. and Gong, F. 1995. Molecular analysis of genes encoding phenazine biosynthesis in the biological control bacterium pseudomonas aureofaciens 30-84. FEMS Microbiol. Lett. 134:299-307.
  46. Pierson, L. S., III and Pierson, E. A. 2010. Metabolism and function of phenazines in bacteria: impacts on the behavior of bacteria in the environment and biotechnological processes. Appl. Microbiol. Biotechnol. 86:1659-1670. https://doi.org/10.1007/s00253-010-2509-3
  47. Pierson, L. S., III and Thomashow, L. S. 1992. Cloning and heterologous expression of the phenazine biosynthetic locus from Pseudomonas aureofaciens 30-84. Mol. Plant-Microbe Interact. 5:330-339. https://doi.org/10.1094/MPMI-5-330
  48. Price-Whelan, A., Dietrich, L. E. and Newman, D. K. 2006. Rethinking 'secondary' metabolism: physiological roles for phenazine antibiotics. Nat. Chem. Biol. 2:71-78. https://doi.org/10.1038/nchembio764
  49. Ramos, I., Dietrich, L. E., Price-Whelan, A. and Newman, D. K. 2010. Phenazines affect biofilm formation by Pseudomonas aeruginosa in similar ways at various scales. Res. Microbiol. 161:187-191. https://doi.org/10.1016/j.resmic.2010.01.003
  50. Sambrook, J. and Russell, D. W. 2001. Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.
  51. Selin, C., Habibian, R., Poritsanos, N., Athukorala, S. N., Fernando, D. and De Kievit, T. R. 2009. 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.
  52. Steinberg, N. and Kolodkin-Gal, I. 2015. The matrix reloaded: How sensing the extracellular matrix synchronizes bacterial communities. J. Bacteriol. 197:2092-2103. https://doi.org/10.1128/JB.02516-14
  53. Thomashow, L. S. and Weller, D. M. 1988. Role of a phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici. J. Bacteriol. 170:3499-3508. https://doi.org/10.1128/jb.170.8.3499-3508.1988
  54. Turner, J. M. and Messenger, A. J. 1986. Occurrence, biochemistry and physiology of phenazine pigment production. Adv. Microb. Physiol. 27:211-275.
  55. Wang, D., Yu, J. M., Dorosky, R. J., Pierson, L. S., III and Pierson, E. A. 2016. The phenazine 2-hydroxy-phenazine-1-carboxylic acid promotes extracellular DNA release and has broad transcriptomic consequences in Pseudomonas chlororaphis 30-84. PLoS One 11:e0148003. https://doi.org/10.1371/journal.pone.0148003
  56. Wang, Y. and Newman, D. K. 2008. Redox reactions of phenazine antibiotics with ferric (hydr) oxides and molecular oxygen. Envrion. Sci. Technol. 42:2380-2386. https://doi.org/10.1021/es702290a
  57. Wang, Y., Wilks, J. C., Danhorn, T., Ramos, I., Croal, L. and Newman, D. K. 2011. Phenazine-1-carboxylic acid promotes bacterial biofilm development via ferrous iron acquisition. J. Bacteriol. 193:3606-3617. https://doi.org/10.1128/JB.00396-11
  58. Wei, Q. and Ma, L. Z. 2013. Biofilm matrix and its regulation in Pseudomonas aeruginosa. Int. J. Mol. Sci. 14:20983-21005. https://doi.org/10.3390/ijms141020983
  59. Weller, D. 1983. Colonization of wheat roots by a fluorescent pseudomonad suppressive to take-all. Phytopathology 73:1548-1553. https://doi.org/10.1094/Phyto-73-1548
  60. Whitchurch, C. B., Tolker-Nielsen, T., Ragas, P. C. and Mattick, J. S. 2002. Extracellular DNA required for bacterial biofilm formation. Science 295:1487-1487. https://doi.org/10.1126/science.295.5559.1487
  61. Wilkinson, H., Cook, R. and Alldredge, J. 1985. Relation of inoculum size and concentration to infection of wheat roots by Gaeumannomyces graminis var. tritici. Phytopathology 75: 98-103. https://doi.org/10.1094/Phyto-75-98
  62. Wood, D. W., Gong, F., Daykin, M. M., Williams, P. and Pierson, L. S., III. 1997. N-acyl-homoserine lactone-mediated regulation of phenazine gene expression by Pseudomonas aureofaciens 30-84 in the wheat rhizosphere. J. Bacteriol. 179:7663-7670. https://doi.org/10.1128/jb.179.24.7663-7670.1997
  63. Yu, J. M. 2016. Regulation and ecological roles of phenazine biosynthesis in the biological control strain Pseudomonas chlororaphis 30-84. Ph.D. thesis. Texas A&M University, College Station, TX, USA.
  64. Yu, J. M., Wang, D., Pierson, L. S., III and Pierson, E. A. 2017. Disruption of MiaA provides insights into the regulation of phenazine biosynthesis under suboptimal growth conditions in Pseudomonas chlororaphis 30-84. Microbiology 163:94-108. https://doi.org/10.1099/mic.0.000409
  65. Zhou, L., Jiang, H.-X., Sun, S., Yang, D.-D., Jin, K.-M., Zhang, W. and He, Y.-W. 2016. Biotechnological potential of a rhizosphere Pseudomonas aeruginosa strain producing phenazine-1-carboxylic acid and phenazine-1-carboxamide. World J. Micriobiol. Biotech. 32:50. https://doi.org/10.1007/s11274-015-1987-y