1887

Abstract

Many products of secondary metabolism are activated by quorum sensing (QS), yet even at cell densities sufficient for QS, their production may be repressed under suboptimal growth conditions via mechanisms that still require elucidation. For many beneficial plant-associated bacteria, secondary metabolites such as phenazines are important for their competitive survival and plant-protective activities. Previous work established that phenazine biosynthesis in 30-84 is regulated by the PhzR/PhzI QS system, which in turn is regulated by transcriptional regulator Pip, two-component system RpeA/RpeB and stationary phase/stress sigma factor RpoS. Disruption of MiaA, a tRNA modification enzyme, altered primary metabolism and growth leading to widespread effects on secondary metabolism, including reduced phenazine production and oxidative stress tolerance. Thus, the mutant provided the opportunity to examine the regulation of phenazine production in response to altered metabolism and growth or stress tolerance. Despite the importance of MiaA for , the most significant effect of disruption on phenazine production was the reduction in the of , and , whereas neither the transcription nor translation of RpeB, a transcriptional regulator of , was affected. Constitutive expression of or in the mutant completely restored phenazine production, but it resulted in further growth impairment. Constitutive expression of RpoS alleviated sensitivity to oxidative stress resulting from RpoS translation inefficiency in the mutant, but it did not restore phenazine production. Our results support the model that cells curtail phenazine biosynthesis under suboptimal growth conditions via RpeB/Pip-mediated regulation of QS.

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2017-01-01
2020-01-17
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References

  1. Chin-A-Woeng TFC, Bloemberg GV, Lugtenberg BJJ. Phenazines and their role in biocontrol by Pseudomonas bacteria. New Phytol 2003;157:503–523 [CrossRef]
    [Google Scholar]
  2. Mavrodi DV, Blankenfeldt W, Thomashow LS. Phenazine compounds in fluorescent Pseudomonas spp. biosynthesis and regulation. Annu Rev Phytopathol 2006;44:417–445 [CrossRef][PubMed]
    [Google Scholar]
  3. Mavrodi DV, Parejko JA, Mavrodi OV, Kwak Y-S, Weller DM et al. Recent insights into the diversity, frequency and ecological roles of phenazines in fluorescent Pseudomonas spp. Environ Microbiol 2013;15:675–686 [CrossRef][PubMed]
    [Google Scholar]
  4. Pierson LS, Pierson EA. Metabolism and function of phenazines in bacteria: impacts on the behavior of bacteria in the environment and biotechnological processes. Appl Microbiol Biotechnol 2010;86:1659–1670 [CrossRef][PubMed]
    [Google Scholar]
  5. Price-Whelan A, Dietrich LE, Newman DK. Rethinking ‘secondary' metabolism: physiological roles for phenazine antibiotics. Nat Chem Biol 2006;2:71–78 [CrossRef][PubMed]
    [Google Scholar]
  6. Pierson LS III, Thomashow LS. Cloning and heterologous expression of the phenazine biosynthetic locus from Pseudomonas aureofaciens 30–84. Mol Plant Microbe Interact 1992;5:330–339 [CrossRef][PubMed]
    [Google Scholar]
  7. Mazzola M, Cook RJ, Thomashow LS, Weller DM, Pierson LS III. Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats. Appl Environ Microbiol 1992;58:2616–2624[PubMed]
    [Google Scholar]
  8. Maddula VS, Zhang Z, Pierson EA, Pierson LS III. Quorum sensing and phenazines are involved in biofilm formation by Pseudomonas chlororaphis (aureofaciens) strain 30-84. Microb Ecol 2006;52:289–301 [CrossRef][PubMed]
    [Google Scholar]
  9. Wang D, Yu JM, Dorosky RJ, Pierson LS III, Pierson EA. The phenazine 2-hydroxy-phenazine-1-carboxylic acid promotes extracellular DNA release and has broad transcriptomic consequences in Pseudomonas chlororaphis 30–84. PLoS One 2016;11:e0148003 [CrossRef][PubMed]
    [Google Scholar]
  10. Dietrich LE, Price-Whelan A, Petersen A, Whiteley M, Newman DK. The phenazine pyocyanin is a terminal signalling factor in the quorum sensing network of Pseudomonas aeruginosa. Mol Microbiol 2006;61:1308–1321 [CrossRef][PubMed]
    [Google Scholar]
  11. Gross H, Loper JE. Genomics of secondary metabolite production by Pseudomonas spp. Nat Prod Rep 2009;26:1408–1446 [CrossRef][PubMed]
    [Google Scholar]
  12. Mentel M, Ahuja EG, Mavrodi DV, Breinbauer R, Thomashow LS et al. Of two make one: the biosynthesis of phenazines. Chembiochem 2009;10:2295–2304 [CrossRef][PubMed]
    [Google Scholar]
  13. Pierson LS III, Gaffney T, Lam S, Gong F. Molecular analysis of genes encoding phenazine biosynthesis in the biological control bacterium Pseudomonas aureofaciens 30–84. FEMS Microbiol Lett 1995;134:299–307[PubMed]
    [Google Scholar]
  14. Wood DW, Gong F, Daykin MM, Williams P, Pierson LS III. N-Acyl-homoserine lactone-mediated regulation of phenazine gene expression by Pseudomonas aureofaciens 30–84 in the wheat rhizosphere. J Bacteriol 1997;179:7663–7670[PubMed][CrossRef]
    [Google Scholar]
  15. Pierson LS III, Keppenne VD, Wood DW. Phenazine antibiotic biosynthesis in Pseudomonas aureofaciens 30-84 is regulated by PhzR in response to cell density. J Bacteriol 1994;176:3966–3974 [CrossRef][PubMed]
    [Google Scholar]
  16. Wang D, Yu JM, Pierson LS III, Pierson EA. Differential regulation of phenazine biosynthesis by RpeA and RpeB in Pseudomonas chlororaphis 30–84. Microbiology 2012;158:1745–1757 [CrossRef][PubMed]
    [Google Scholar]
  17. Wang D, Lee SH, Seeve C, Yu JM, Pierson LS III et al. Roles of the Gac-Rsm pathway in the regulation of phenazine biosynthesis in Pseudomonas chlororaphis 30-84. Microbiologyopen 2013;2:505–524 [CrossRef][PubMed]
    [Google Scholar]
  18. Girard G, Barends S, Rigali S, van Rij ET, Lugtenberg BJ et al. Pip, a novel activator of phenazine biosynthesis in Pseudomonas chlororaphis PCL1391. J Bacteriol 2006;188:8283–8293 [CrossRef][PubMed]
    [Google Scholar]
  19. Schuster M, Hawkins AC, Harwood CS, Greenberg E. The Pseudomonas aeruginosa RpoS regulon and its relationship to quorum sensing. Mol Microbiol 2004;51:973–985[PubMed][CrossRef]
    [Google Scholar]
  20. Selin C, Fernando WGD, De Kievit T. The PhzI/PhzR quorum-sensing system is required for pyrrolnitrin and phenazine production, and exhibits cross-regulation with RpoS in Pseudomonas chlororaphis PA23. Microbiology 2012;158:896–907 [CrossRef][PubMed]
    [Google Scholar]
  21. Whistler CA, Pierson LS III. Repression of phenazine antibiotic production in Pseudomonas aureofaciens strain 30–84 by RpeA. J Bacteriol 2003;185:3718–3725[PubMed][CrossRef]
    [Google Scholar]
  22. Girard G. Regulatory roles of psrA and rpoS in phenazine-1-carboxamide synthesis by Pseudomonas chlororaphis PCL1391. Microbiology 2006;152:43–58 [CrossRef][PubMed]
    [Google Scholar]
  23. Girard G, Rigali S. Role of the phenazine-inducing protein Pip in stress resistance of Pseudomonas chlororaphis. Microbiology 2011;157:398–407 [CrossRef][PubMed]
    [Google Scholar]
  24. Bjork GR. Modified Nucleosides in RNA – Their Formation and Function Boca Raton, Fla: Processing of RNA CRC Press, Inc; 1984; pp.291–330
    [Google Scholar]
  25. Björk GR, Ericson JU, Gustafsson CE, Hagervall TG, Jönsson YH et al. Transfer RNA modification. Annu Rev Biochem 1987;56:263–285 [CrossRef][PubMed]
    [Google Scholar]
  26. Esberg B, Björk G. The methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A) present next to the anticodon contributes to the decoding efficiency of the tRNA. J Bacteriol 1995;177:1967–1975[PubMed][CrossRef]
    [Google Scholar]
  27. Esberg B, Leung HC, Tsui HC, Björk GR, Winkler ME. Identification of the miaB gene, involved in methylthiolation of isopentenylated A37 derivatives in the tRNA of Salmonella typhimurium and Escherichia coli. J Bacteriol 1999;181:7256–7265[PubMed]
    [Google Scholar]
  28. Leung HC, Chen Y, Winkler ME. Regulation of substrate recognition by the MiaA tRNA prenyltransferase modification enzyme of Escherichia coli K-12. J Biol Chem 1997;272:13073–13083 [CrossRef][PubMed]
    [Google Scholar]
  29. Thompson KM, Gottesman S. The MiaA tRNA modification enzyme is necessary for robust RpoS expression in Escherichia coli. J Bacteriol 2014;196:754–761 [CrossRef][PubMed]
    [Google Scholar]
  30. Tsui HC, Feng G, Winkler ME. Transcription of the mutL repair, miaA tRNA modification, hfq pleiotropic regulator, and hflA region protease genes of Escherichia coli K-12 from clustered Esigma32-specific promoters during heat shock. J Bacteriol 1996;178:5719–5731 [CrossRef][PubMed]
    [Google Scholar]
  31. Jackman JE, Alfonzo JD. Transfer RNA modifications: nature's combinatorial chemistry playground. Wiley Interdiscip Rev RNA 2013;4:35–48 [CrossRef][PubMed]
    [Google Scholar]
  32. Li J-N, Esberg B, Curran JF, Björk GR. Three modified nucleosides present in the anticodon stem and loop influence the in vivo aa-tRNA selection in a tRNA-dependent manner. J Mole Biol 1997;271:209–221[PubMed][CrossRef]
    [Google Scholar]
  33. Ericson JU, Björk G. Pleiotropic effects induced by modification deficiency next to the anticodon of tRNA from Salmonella typhimurium LT2. J Bacteriol 1986;166:1013–1021[PubMed][CrossRef]
    [Google Scholar]
  34. Buck M, Griffiths E. Regulation of aromatic amino acid transport by tRNA: role of 2-methylthio-N6-(delta2-isopentenyl)-adenosine. Nucleic Acids Res 1981;9:401–414[PubMed][CrossRef]
    [Google Scholar]
  35. Tsui HC, Leung HC, Winkler ME. Characterization of broadly pleiotropic phenotypes caused by an hfq insertion mutation in Escherichia coli K-12. Mol Microbiol 1994;13:35–49 [CrossRef][PubMed]
    [Google Scholar]
  36. Blum PH. Reduced leu operon expression in a miaA mutant of Salmonella typhimurium. J Bacteriol 1988;170:5125–5133[PubMed][CrossRef]
    [Google Scholar]
  37. Aubee JI, Olu M, Thompson KM. The i6A37 tRNA modification is essential for proper decoding of UUX-leucine codons during rpoS and iraP translation. RNA 2016;22:729–742 [CrossRef][PubMed]
    [Google Scholar]
  38. Sambrook J, Russell DW. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2001
    [Google Scholar]
  39. Larsen RA, Wilson MM, Guss AM, Metcalf WW. Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria. Arch Microbiol 2002;178:193–201 [CrossRef][PubMed]
    [Google Scholar]
  40. Chiang P, Burrows LL. Biofilm formation by hyperpiliated mutants of Pseudomonas aeruginosa. J Bacteriol 2003;185:2374–2378[PubMed][CrossRef]
    [Google Scholar]
  41. Miller JH. Experiments in molecular genetics NY: Cold Spring Harbor Laboratory; 1972
    [Google Scholar]
  42. Heeb S, Blumer C, Haas D. Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0. J Bacteriol 2002;184:1046–1056[PubMed][CrossRef]
    [Google Scholar]
  43. Kulkarni PR, Jia T, Kuehne SA, Kerkering TM, Morris ER et al. A sequence-based approach for prediction of CsrA/RsmA targets in bacteria with experimental validation in Pseudomonas aeruginosa. Nucleic Acids Res 2014;42:6811–6825 [CrossRef][PubMed]
    [Google Scholar]
  44. Maddula VS, Pierson EA, Pierson LS III. Altering the ratio of phenazines in Pseudomonas chlororaphis (aureofaciens) strain 30-84: effects on biofilm formation and pathogen inhibition. J Bacteriol 2008;190:2759–2766 [CrossRef][PubMed]
    [Google Scholar]
  45. Loper JE, Hassan KA, Mavrodi D, Davis EW, Lim CK et al. Comparative genomics of plant-associated Pseudomonas spp.: insights into diversity and inheritance of traits involved in multitrophic interactions. PLoS Genet 2012;8:e1002784 [CrossRef][PubMed]
    [Google Scholar]
  46. Brennan RG, Link TM. Hfq structure, function and ligand binding. Curr Opin Microbiol 2007;10:125–133 [CrossRef][PubMed]
    [Google Scholar]
  47. Sonnleitner E, Hagens S, Rosenau F, Wilhelm S, Habel A et al. Reduced virulence of a hfq mutant of Pseudomonas aeruginosa O1. Microb Pathog 2003;35:217–228[PubMed][CrossRef]
    [Google Scholar]
  48. Sonnleitner E, Schuster M, Sorger-Domenigg T, Greenberg EP, Bläsi U. Hfq-dependent alterations of the transcriptome profile and effects on quorum sensing in Pseudomonas aeruginosa. Mol Microbiol 2006;59:1542–1558 [CrossRef][PubMed]
    [Google Scholar]
  49. Wang G, Huang X, Li S, Huang J, Wei X et al. The RNA chaperone Hfq regulates antibiotic biosynthesis in the rhizobacterium Pseudomonas aeruginosa M18. J Bacteriol 2012;194:2443–2457 [CrossRef][PubMed]
    [Google Scholar]
  50. Wu X-G, Duan H-M, Tian T, Yao N, Zhou H-Y et al. Effect of the hfq gene on 2,4-diacetylphloroglucinol production and the PcoI/PcoR quorum-sensing system in Pseudomonas fluorescens 2P24. FEMS Microbiol Lett 2010;309:16–24 [CrossRef][PubMed]
    [Google Scholar]
  51. Haas D, Keel C, Reimmann C. Signal transduction in plant-beneficial rhizobacteria with biocontrol properties. Antonie van Leeuwenhoek 2002;81:385–395[PubMed][CrossRef]
    [Google Scholar]
  52. Haas D, Défago G. Biological control of soil-borne pathogens by fluorescent pseudomonads. Nat Rev Microbiol 2005;3:307–319 [CrossRef][PubMed]
    [Google Scholar]
  53. Siddiqui IA, Haas D, Heeb S. Extracellular protease of Pseudomonas fluorescens CHA0, a biocontrol factor with activity against the root-knot nematode Meloidogyne incognita. Appl Environ Microbiol 2005;71:5646–5649 [CrossRef][PubMed]
    [Google Scholar]
  54. Zhao J, Leung HE, Winkler ME. The miaA mutator phenotype of Escherichia coli K-12 requires recombination functions. J Bacteriol 2001;183:1796–1800 [CrossRef][PubMed]
    [Google Scholar]
  55. Olekhnovich I, Gussin GN. Effects of mutations in the Pseudomonas putida miaA gene: regulation of the trpE and trpGDC operons in P. putida by attenuation. J Bacteriol 2001;183:3256–3260 [CrossRef][PubMed]
    [Google Scholar]
  56. Ditta G, Stanfield S, Corbin D, Helinski DR. Broad host range DNA cloning system for Gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 1980;77:7347–7351[PubMed][CrossRef]
    [Google Scholar]
  57. Blumer C, Heeb S, Pessi G, Haas D. Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites. Proc Natl Acad Sci USA 1999;96:14073–14078[PubMed][CrossRef]
    [Google Scholar]
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