1887

Abstract

The stringent response (SR) is a regulatory mechanism that enables bacteria to adapt to nutrient stress through the production of the alarmone (p)ppGpp. The aim of the current study was to understand how the SR affects the antifungal (AF) activity of PA23. Two SR mutants were generated, PA23 and PA23, that no longer produced (p)ppGpp. Both mutants exhibited increased inhibition of and elevated pyrrolnitrin (PRN), lipase and protease production. Phenazine (PHZ) levels, on the other hand, remained unchanged. Through transcriptional fusion analysis we discovered that (PRN) activity was increased in the SR mutants, whereas (PHZ) activity was equal to that of the wild-type. We also examined how the sigma factor RpoS impacts PA23-mediated antagonism. Similar to the SR mutants, an mutant of PA23, called PA23, exhibited enhanced AF activity and increased expression of PRN, protease and lipase. However, PHZ production and expression of were dramatically reduced. Consistent with what has been reported for other bacteria, the SR exerted positive control over expression. In addition, providing restored the SR phenotype to that of the wild-type. Collectively, our findings indicate that this global stress response impacts production of PA23 AF compounds via regulation of transcription and has an overall negative influence on antagonism.

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2012-01-01
2024-03-29
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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, 828[PubMed]
    [Google Scholar]
  2. Baehler E., Bottiglieri M., Péchy-Tarr M., Maurhofer M., Keel C. ( 2005). Use of green fluorescent protein-based reporters to monitor balanced production of antifungal compounds in the biocontrol agent Pseudomonas fluorescens CHA0. J Appl Microbiol 99:24–38 [View Article][PubMed]
    [Google Scholar]
  3. Bradford M. M. ( 1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254 [View Article][PubMed]
    [Google Scholar]
  4. Cashel M. ( 1994). Detection of ppGpp and pppGpp accumulation patterns in Escherichia coli mutants. Methods in Molecular Genetics vol. 3A341–356 Adolph K. W. New York, NY: Academic press;
    [Google Scholar]
  5. Choi K. H., Gaynor J. B., White K. G., Lopez C., Bosio C. M., Karkhoff-Schweizer R. R., Schweizer H. P. ( 2005). A Tn7-based broad-range bacterial cloning and expression system. Nat Methods 2:443–448 [View Article][PubMed]
    [Google Scholar]
  6. Cupp-Enyard C. ( 2008). Sigma’s non-specific protease activity assay: casein as a substrate. J Vis Exp 19: [View Article][PubMed]
    [Google Scholar]
  7. Erickson D. L., Lines J. L., Pesci E. C., Venturi V., Storey D. G. ( 2004). Pseudomonas aeruginosa relA contributes to virulence in Drosophila melanogaster. . Infect Immun 72:5638–5645 [View Article][PubMed]
    [Google Scholar]
  8. 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[PubMed]
    [Google Scholar]
  9. Gambello M. J., Kaye S., Iglewski B. H. ( 1993). LasR of Pseudomonas aeruginosa is a transcriptional activator of the alkaline protease gene (apr) and an enhancer of exotoxin A expression. Infect Immun 61:1180–1184[PubMed]
    [Google Scholar]
  10. Ge Y.-H., Pei D.-L., Li W.-W., Zhao Y.-H., Xu Y.-Q. ( 2006). Insertional mutation of the rpoS gene contributes to alteration in biosynthesis of antifungal agents in Pseudomonas sp. M18. Biol Control 39:186–192 [View Article]
    [Google Scholar]
  11. Girard G., van Rij E. T., Lugtenberg B. J., Bloemberg G. V. ( 2006a). Regulatory roles of psrA and rpoS in phenazine-1-carboxamide synthesis by Pseudomonas chlororaphis PCL1391. Microbiology 152:43–58 [View Article][PubMed]
    [Google Scholar]
  12. Girard G., Barends S., Rigali S., van Rij E. T., Lugtenberg B. J. J., Bloemberg G. V. ( 2006b). Pip, a novel activator of phenazine biosynthesis in Pseudomonas chlororaphis PCL1391. J Bacteriol 188:8283–8293 [View Article][PubMed]
    [Google Scholar]
  13. Gomez-Escribano J. P., Martin J. F., Hesketh A., Bibb M. J., Liras P. ( 2008). Streptomyces clavuligerus relA-null mutants overproduce clavulanic acid and cephamycin C: negative regulation of secondary metabolism by (p)ppGpp. Microbiol 154:744–755 [View Article]
    [Google Scholar]
  14. Haas D., Défago G. ( 2005). Biological control of soil-borne pathogens by fluorescent pseudomonads. Nat Rev Microbiol 3:307–319 [View Article][PubMed]
    [Google Scholar]
  15. Heeb S., Haas D. ( 2001). Regulatory roles of the GacS/GacA two-component system in plant-associated and other gram-negative bacteria. Mol Plant Microbe Interact 14:1351–1363 [View Article][PubMed]
    [Google Scholar]
  16. Heeb S., Valverde C., Gigot-Bonnefoy C., Haas D. ( 2005). Role of the stress sigma factor RpoS in GacA/RsmA-controlled secondary metabolism and resistance to oxidative stress in Pseudomonas fluorescens CHA0. FEMS Microbiol Lett 243:251–258 [View Article][PubMed]
    [Google Scholar]
  17. Hoang T. T., Karkhoff-Schweizer R. R., Kutchma A. J., Schweizer H. P. ( 1998). A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86 [View Article][PubMed]
    [Google Scholar]
  18. Holloway B. W., Krishnapillai V., Morgan A. F. ( 1979). Chromosomal genetics of Pseudomonas. . Microbiol Rev 43:73–102[PubMed]
    [Google Scholar]
  19. 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]
  20. Jin W., Kim H. K., Kim J. Y., Kang S. G., Lee S. H., Lee K. J. ( 2004). Cephamycin C production is regulated by relA and rsh genes in Streptomyces clavuligerus ATCC27064. J Biotechnol 114:81–87 [View Article][PubMed]
    [Google Scholar]
  21. Lindow S. E., Brandl M. T. ( 2003). Microbiology of the phyllosphere. Appl Environ Microbiol 69:1875–1883 [View Article][PubMed]
    [Google Scholar]
  22. Miller J. H. ( 1972). Experiments in Molecular Genetics351–355 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  23. Park J. Y., Oh S. A., Anderson A. J., Neiswender J., Kim J. C., Kim Y. C. ( 2011). Production of the antifungal compounds phenazine and pyrrolnitrin from Pseudomonas chlororaphis O6 is differentially regulated by glucose. Lett Appl Microbiol 52:532–537 [View Article][PubMed]
    [Google Scholar]
  24. Poritsanos N., Selin C., Fernando W. G. D., 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]
  25. Potrykus K., Cashel M. ( 2008). (p)ppGpp: still magical?. Annu Rev Microbiol 62:35–51 [View Article][PubMed]
    [Google Scholar]
  26. Preston M. J., Seed P. C., Toder D. S., Iglewski B. H., Ohman D. E., Gustin J. K., Goldberg J. B., Pier G. B. ( 1997). Contribution of proteases and LasR to the virulence of Pseudomonas aeruginosa during corneal infections. Infect Immun 65:3086–3090[PubMed]
    [Google Scholar]
  27. 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]
  28. Sarniguet A., Kraus J., Henkels M. D., Muehlchen A. M., Loper J. E. ( 1995). The sigma factor σs affects antibiotic production and biological control activity of Pseudomonas fluorescens Pf-5. Proc Natl Acad Sci U S A 92:12255–12259 [View Article][PubMed]
    [Google Scholar]
  29. Savchuk S. C., 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]
  30. Schnider-Keel U., Seematter A., Maurhofer M., Blumer C., Duffy B., Gigot-Bonnefoy C., Reimmann C., Notz R., Défago G. & other authors ( 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 182:1215–1225 [View Article][PubMed]
    [Google Scholar]
  31. 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]
  32. Sun J., Hesketh A., Bibb M. ( 2001). Functional analysis of relA and rshA, two relA/spoT homologues of Streptomyces coelicolor A3(2). J Bacteriol 183:3488–3498 [View Article][PubMed]
    [Google Scholar]
  33. van Delden C., Comte R., Bally A. M. ( 2001). Stringent response activates quorum sensing and modulates cell density-dependent gene expression in Pseudomonas aeruginosa. . J Bacteriol 183:5376–5384 [View Article][PubMed]
    [Google Scholar]
  34. Washio K., Lim S. P., Roongsawang N., Morikawa M. ( 2010). Identification and characterization of the genes responsible for the production of the cyclic lipopeptide arthrofactin by Pseudomonas sp. MIS38. Biosci Biotechnol Biochem 74:992–999 [View Article][PubMed]
    [Google Scholar]
  35. West S. E., Schweizer H. P., Dall C., Sample A. K., Runyen-Janecky L. J. ( 1994). Construction of improved EscherichiaPseudomonas 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]
  36. Whistler C. A., Pierson L. S. III ( 2003). Repression of phenazine antibiotic production in Pseudomonas aureofaciens strain 30-84 by RpeA. J Bacteriol 185:3718–3725 [View Article][PubMed]
    [Google Scholar]
  37. Whistler C. A., Corbell N. A., Sarniguet A., Ream W., Loper J. E. ( 1998). The two-component regulators GacS and GacA influence accumulation of the stationary-phase sigma factor σs and the stress response in Pseudomonas fluorescens Pf-5. J Bacteriol 180:6635–6641[PubMed]
    [Google Scholar]
  38. Winkler U. K., Stuckmann M. ( 1979). Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. . J Bacteriol 138:663–670[PubMed]
    [Google Scholar]
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