1887

Abstract

The biocontrol agent F113 undergoes phenotypic variation during rhizosphere colonization, and this variation has been related to the activity of a site-specific recombinase encoded by the gene. Here, it is shown that a second recombinase encoded by the gene is also implicated in phenotypic variation. A putative gene from this strain was cloned, and sequence analysis confirmed that it encoded a site-specific recombinase of the integrase family. Mutants affected in the or genes produced a very low quantity of phenotypic variants compared to the wild-type strain, both under prolonged cultivation in the laboratory and after rhizosphere colonization, and they were severely impaired in competitive root colonization. Overexpression of the genes encoding either recombinase resulted in a substantial increment in the production of phenotypic variants under both culture and rhizosphere colonization conditions, implying that both site-specific recombinases are involved in phenotypic variation. Overexpression of the gene suppressed the phenotype of a mutant, but overexpression of the gene had no effect on the phenotype of an mutant. Genetic analysis of the phenotypic variants obtained after overexpression of the genes encoding both the recombinases showed that they carried mutations in the / genes, which are necessary to produce a variety of secondary metabolites. These results indicate that the Gac system is affected by the activity of the site-specific recombinases. Transcriptional fusions of the and genes with a promoterless gene showed that both genes have a similar expression pattern, with maximal expression during stationary phase. Although the expression of both genes was independent of diffusible compounds present in root exudates, it was induced by the plant, since bacteria attached to the root showed enhanced expression.

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2005-03-01
2024-12-06
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References

  1. Achouak W., Conrod S., Cohen V., Heulin T. 2004; Phenotypic variation of Pseudomonas brassicacearum as a plant root-colonization strategy. Mol Plant–Microbe Interact 17:872–879 [CrossRef]
    [Google Scholar]
  2. Bertani G. 1951; Studies on lysogenesis. 1. The mode of phage liberation by lysogenic Escherichia coli . J Bacteriol 62:293–300
    [Google Scholar]
  3. Blakely G., Colloms S., May G., Burke M., Sherratt D. 1991; Escherichia coli XerC recombinase is required for chromosomal segregation at cell division. New Biol 3:789–798
    [Google Scholar]
  4. Blakely G., May G., McCulloch R., Arciszewska L. K., Burke M., Lovett S. T., Sherratt D. J. 1993; Two related recombinases are required for site-specific recombination at dif and cer in Escherichia coli K12. Cell 75:351–361 [CrossRef]
    [Google Scholar]
  5. Blumer C., Heeb S., Pessi G., Haas D. 1999; Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites. Proc Natl Acad Sci U S A 96:14073–14078 [CrossRef]
    [Google Scholar]
  6. Boyer H. W., Roulland-Dussoix D. 1969; A complementation analysis of the restriction and modification of DNA in Escherichia coli . J Mol Biol 41:459–472 [CrossRef]
    [Google Scholar]
  7. Chin-a-Woeng T. F. C., Bloemberg G. V., Mulders I. H. M., Dekkers L. C., Lugtenberg B. J. J. 2000; Root colonization by phenazine-1-carboxamide-producing bacterium Pseudomonas chlororaphis PCL1391 is essential for biocontrol of tomato foot and root rot. Mol Plant–Microbe Interact 13:1340–1345 [CrossRef]
    [Google Scholar]
  8. Colloms S. D., Sykora P., Szatmari G., Sherratt D. J. 1990; Recombination at ColE1 cer requires the Escherichia coli xerC gene product, a member of the λ integrase family of site-specific recombinases. J Bacteriol 172:6973–6980
    [Google Scholar]
  9. Cornet F., Mortier I., Patte J., Louarn J. M. 1994; Plasmid pSC101 harbors a recombination site, psi, which is able to resolve plasmid multimers and to substitute for the analogous chromosomal Escherichia coli site dif . J Bacteriol 176:3188–3195
    [Google Scholar]
  10. Dekkers L. C., Phoelich C. C., Van Der Fits L., Lugtenberg B. J. J. 1998; A site-specific recombinase is required for competitive root colonization by Pseudomonas fluorescens WCS365. Proc Natl Acad Sci U S A 95:7051–7056 [CrossRef]
    [Google Scholar]
  11. Dekkers L. C., Mulders I. H. M., Phoelich C. C., Chin-a-Woeng T. F. C., Wijfjes A. H. M., Lugtenberg B. J. J. 2000; The sss colonization gene of the tomato–Fusarium oxysporum f.sp.radicis-lycopersici biocontrol strain Pseudomonas fluorescens WCS365 can improve root colonization of other wild-type Pseudomonas spp. bacteria. Mol Plant–Microbe Interact 13:1177–1183 [CrossRef]
    [Google Scholar]
  12. Delany I. R., Walsh U. F., Ross I., Fenton A. M., Corkery D. M., O'Gara F. 2001; Enhancing the biocontrol efficacy of Pseudomonas fluorescens F113 by altering the regulation and production of 2,4-diacetylphloroglucinol. Improved Pseudomonas biocontrol inoculants. Plant Soil 232:195–205 [CrossRef]
    [Google Scholar]
  13. Dombrecht B., Vanderleyden J., Michiels J. 2001; Stable RK2-derived cloning vectors for the analysis of gene expression and gene function in Gram-negative bacteria. Mol Plant–Microbe Interact 14:426–430 [CrossRef]
    [Google Scholar]
  14. Dove S. L., Dorman C. J. 1994; The site-specific recombination system regulating expression of the type-1 fimbrial subunit gene of Escherichia coli is sensitive to changes in DNA supercoiling. Mol Microbiol 14:975–988 [CrossRef]
    [Google Scholar]
  15. Duffy B. K., Defago G. 2000; Controlling instability in gacS–gacA regulatory genes during inoculant production of Pseudomonas fluorescens biocontrol strains. Appl Environ Microbiol 66:3142–3150 [CrossRef]
    [Google Scholar]
  16. Fahraeus G. 1957; The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J Gen Microbiol 16:374–381 [CrossRef]
    [Google Scholar]
  17. Ferreira H., Butler-Cole B., Burgin A., Baker R., Sherratt D. J., Arciszewska L. K. 2003; Functional analysis of the C-terminal domains of the site-specific recombinases XerC and XerD. J Mol Biol 330:15–27 [CrossRef]
    [Google Scholar]
  18. Figurski D. H., Helinski D. R. 1979; Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76:1648–1652 [CrossRef]
    [Google Scholar]
  19. Groth A. C., Calos M. P. 2004; Phage integrases, biology and applications. J Mol Biol 335:667–678 [CrossRef]
    [Google Scholar]
  20. Hendricks E. C., Szerlong H., Hill T., Kuempel P. 2000; Cell division, guillotining of dimer chromosomes and SOS induction in resolution mutants (dif,xerC and xerD) of Escherichia coli . Mol Microbiol 36:973–981 [CrossRef]
    [Google Scholar]
  21. Hofte M., Dong Q. G., Kourambas S., Krishnapillai V., Sherratt D., Mergeay M. 1994; The sss gene product, which affects pyoverdin production in Pseudomonas aeruginosa 7NSK2, is a site-specific recombinase. Mol Microbiol 14:1011–1020 [CrossRef]
    [Google Scholar]
  22. Hrabak E. M., Willis D. K. 1992; The lemA gene required for pathogenicity of Pseudomonas syringae pv. syringae on bean is a member of a family of two-component regulators. J Bacteriol 174:3011–3020
    [Google Scholar]
  23. Kalogeraki V. S., Winans S. C. 1997; Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria. Gene 188:69–75 [CrossRef]
    [Google Scholar]
  24. 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 A 89:1562–1566 [CrossRef]
    [Google Scholar]
  25. Marco M. L., Legac J., Lindow S. E. 2003; Conditional survival as a selection strategy to identify plant-inducible genes of Pseudomonas syringae . Appl Environ Microbiol 69:5793–5801 [CrossRef]
    [Google Scholar]
  26. Miller J. H. 1972 Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  27. Naseby D. C., Lynch J. M. 1999; Effects of Pseudomonas fluorescens F113 on ecological functions in the pea rhizosphere are dependent on pH. Microb Ecol 37:248–256 [CrossRef]
    [Google Scholar]
  28. Sacherer P., Defago G., Haas D. 1994; Extracellular protease and phospholipase-C are controlled by the global regulatory gene gacA. in the biocontrol strain Pseudomonas fluorescens CHA0. FEMS Microbiol Lett 116155–160 [CrossRef]
    [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. Sánchez-Contreras M., Martin M., Villacieros M., O'Gara F., Bonilla I., Rivilla R. 2002; Phenotypic selection and phase variation occur during alfalfa root colonization by Pseudomonas fluorescens F113. J Bacteriol 184:1587–1596 [CrossRef]
    [Google Scholar]
  31. Schafer A., Tauch A., Jager W., Kalinowski J., Thierbach G., Puhler A. 1994; Small mobilizable multipurpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome ofCorynebacterium glutamicum . Gene 145:69–73 [CrossRef]
    [Google Scholar]
  32. Scher F. M., Baker R. 1982; Effect of Pseudomonas putida and a synthetic iron chelator on induction of soil suppressiveness to Fusarium wilt pathogens. Phytopathology 72:1567–1573 [CrossRef]
    [Google Scholar]
  33. 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 Microbiol 58:353–358
    [Google Scholar]
  34. Simons M., Vanderbij A. J., Brand I., Deweger L. A., Wijffelman C. A., Lugtenberg B. J. J. 1996; Gnotobiotic system for studying rhizosphere colonization by plant growth-promoting Pseudomonas bacteria. Mol Plant–Microbe Interact 9:600–607 [CrossRef]
    [Google Scholar]
  35. Thompson J. D., Higgins D. G., Gibson T. J. 1994; clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680 [CrossRef]
    [Google Scholar]
  36. Tominaga A., Ikemizu S., Enomoto M. 1991; Site-specific recombinase genes in three Shigella subgroups and nucleotide-sequences of a pinB gene and an invertible B segment fromShigella boydii . J Bacteriol 173:4079–4087
    [Google Scholar]
  37. Van Den Broek D., Chin-a-Woeng T. F. C., Eijkemans K., Mulders I. H. M., Bloemberg G. V., Lugtenberg B. J. J. 2003; Biocontrol traits of Pseudomonas spp. are regulated by phase variation. Mol Plant–Microbe Interact 16:1003–1012 [CrossRef]
    [Google Scholar]
  38. Villacieros M., Power B. 8 other authors Sánchez-Contreras M. 2003; Colonization behaviour of Pseudomonas fluorescens and Sinorhizobium meliloti in the alfalfa (Medicago sativa) rhizosphere. Plant Soil 251:47–54 [CrossRef]
    [Google Scholar]
  39. Wright B. E. 2004; Stress-directed adaptive mutations and evolution. Mol Microbiol 52:643–650 [CrossRef]
    [Google Scholar]
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