1887

Abstract

The ability to monitor the spatial and temporal distribution of signals in complex environments is necessary for an understanding of the function of bacteria in the wild. To this end, an existing recombinase-based transcriptional reporter strategy (recombinase-based expression technology, RIVET) has been extended and applied to the plant-colonizing bacterium SBW25. Central to the project was a rhizosphere-inducible locus, , which functional analyses show is , a histidine-inducible gene that is required for histidine utilization. A transcriptional fusion between and a promoterless site-specific recombinase ( ) results in excision of a chromosomally integrated tetracycline-resistance cassette in a histidine-dependent manner. The dose- and time-responsiveness of the promoterless recombinase to histidine closely mirrored the histidine responsiveness of an identical fusion to promoterless . To demonstrate the effectiveness of the strategy, the activity of was monitored on sugar beet seedlings. Low levels of transcriptional activity were detected in the phyllosphere, rhizosphere and in plant extract, but not in vermiculite devoid of seedlings. The histidine concentration in the rhizosphere was estimated to be 0.6 μg ml. The ecological significance of the locus was examined by competing a deletion mutant against the wild-type during colonization of sugar beet seedlings. No impact on competitive fitness was detected, suggesting that the ability to utilize plant-derived histidine is not essential for bacterial colonization.

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2006-06-01
2024-03-28
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References

  1. Brandl M. T, Quinones B, Lindow S. E. 2001; Heterogeneous transcription of an indoleacetic acid biosynthetic gene in Erwinia herbicola on plant surfaces. Proc Natl Acad Sci U S A 98:3435–3459
    [Google Scholar]
  2. Camilli A, Mekalanos J. J. 1995; Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection. Mol Microbiol 18:671–683 [CrossRef]
    [Google Scholar]
  3. Camilli A, Beattie D. T, Mekalanos J. J. 1994; Use of genetic recombination as a reporter of gene expression. Proc Natl Acad Sci U S A 91:2634–2638 [CrossRef]
    [Google Scholar]
  4. Casavant N. C, Beattie G. A, Phillips G. J, Halverson L. J. 2002; Site-specific recombination-based genetic system for reporting transient or low-level gene expression. Appl Environ Microbiol 68:3588–3596 [CrossRef]
    [Google Scholar]
  5. Casavant N. C, Thompson D, Beattie G. A, Phillips G. J, Halverson L. J. 2003; Use of a site-specific recombination-based biosensor for detecting bioavailable toluene and related compounds on roots. Environ Microbiol 5:238–249 [CrossRef]
    [Google Scholar]
  6. Ditta G, Stanfield S, Corbin D, Helinski D. R. 1980; Broad host range DNA cloning system for Gram negative bacteria: construction of a gene bank of Rhizobium meliloti . Proc Natl Acad Sci U S A 77:7347–7351 [CrossRef]
    [Google Scholar]
  7. Elowitz M. B, Levine A. J, Siggia E. D, Swain P. S. 2002; Stochastic gene expression in a single cell. Science 297:1183–1186 [CrossRef]
    [Google Scholar]
  8. Fiering S, Whitelaw E, Martin D. I. K. 2000; To be or not to be active: the stochastic nature of enhancer action. BioEssays 22:381–387 [CrossRef]
    [Google Scholar]
  9. Gal M, Preston G. M, Massey R. C, Spiers A. J, Rainey P. B. 2003; Genes encoding a cellulose polymer contribute toward the ecological success of Pseudomonas fluorescens SBW25 on plant surfaces. Mol Ecol 12:3109–3121 [CrossRef]
    [Google Scholar]
  10. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580 [CrossRef]
    [Google Scholar]
  11. Horton R. M, Hunt H. D, Ho S. N, Pullen J. K, Pease L. R. 1989; Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77:61–68 [CrossRef]
    [Google Scholar]
  12. Hu L, Allison S. L, Phillips A. T. 1989; Identification of multiple repressor recognition sites in the hut system of Pseudomonas putida . J Bacteriol 171:4189–4195
    [Google Scholar]
  13. Joyner D. C, Lindow S. E. 2000; Heterogeneity of iron bioavailability on plants assessed with a whole-cell GFP-based bacterial biosensor. Microbiology 146:2435–2445
    [Google Scholar]
  14. Lee S. H, Angelichio M. J, Mekalanos J. J, Camilli A. 1998; Nucleotide sequence and spatiotemporal expression of the Vibrio cholerae vieSAB genes during infection. J Bacteriol 180:2298–2305
    [Google Scholar]
  15. Lee S. H, Hava D. L, Waldor M. K, Camilli A. 1999; Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection. Cell 99:625–634 [CrossRef]
    [Google Scholar]
  16. Lenski R. E. 1991; Quantifying fitness and gene stability in microorganisms. Biotechnology 15:173–192
    [Google Scholar]
  17. Leveau J. H. J, Lindow S. E. 2001; Appetite of an epiphyte: quantitative monitoring of bacterial sugar consumption in the phyllosphere. Proc Natl Acad Sci U S A 98:3446–3453 [CrossRef]
    [Google Scholar]
  18. Lindow S. E, Brandl M. T. 2003; Microbiology of the phyllosphere. Appl Environ Microbiol 69:1875–1883 [CrossRef]
    [Google Scholar]
  19. McAdams H. H, Arkin A. 1997; Stochastic mechanisms in gene expression. Proc Natl Acad Sci U S A 94:814–819 [CrossRef]
    [Google Scholar]
  20. Merrell D. S, Camilli A. 2000; Detection and analysis of gene expression during infection by in vivo expression technology. Philos Trans R Soc Lond B 355:587–599 [CrossRef]
    [Google Scholar]
  21. Miller J. 1972 Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  22. Poindexter J. S. 1981; Oligotrophy. Adv Microb Ecol 5:63–89
    [Google Scholar]
  23. Preston G. M, Bertrand N, Rainey P. B. 2001; Type III secretion in plant growth-promoting Pseudomonas fluorescens SBW25. Mol Microbiol 41:999–1014
    [Google Scholar]
  24. Rainey P. B. 1999; Adaptation of Pseudomonas fluorescens to the plant rhizosphere. Environ Microbiol 1:243–257 [CrossRef]
    [Google Scholar]
  25. Rainey P. B, Bailey M. J. 1996; Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome. Mol Microbiol 19:521–533 [CrossRef]
    [Google Scholar]
  26. Rainey P. B, Preston G. M. 2000; In vivo expression technology strategies: valuable tools for biotechnology. Curr Opin Biotechnol 11:440–444 [CrossRef]
    [Google Scholar]
  27. Rietsch A, Wolfgang M. C, Mekalanos J. J. 2004; Effect of metabolic imbalance on expression of type III secretion genes in Pseudomonas aeruginosa . Infect Immun 72:1383–1390 [CrossRef]
    [Google Scholar]
  28. Sambrook J, Fritsch E. F, Maniatis T. 1989 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  29. Simons M, Permentier H. P, Wijffelman C. A, Lugtenberg B. J. J, de Weger L. A. 1997; Amino acid synthesis is necessary for tomato root colonization by Pseudomonas fluorescens strain WCS365. Mol Plant Microbe Interact 10:102–106 [CrossRef]
    [Google Scholar]
  30. Spiers A. J, Kahn S. G, Bohannon J, Travisano M, Rainey P. B. 2002; Adaptive divergence in experimental populations of Pseudomonas fluorescens . I. Genetic and phenotypic bases of wrinkly spreader fitness. Genetics 161:33–46
    [Google Scholar]
  31. Spiers A. J, Bohannon J, Gerhrig S. M, Rainey P. B. 2003; Biofilm formation at the air-liquid interface by the Pseudomonas fluorescens SBW25 wrinkly spreader requires an acetylated form of cellulose. Mol Microbiol 50:15–27 [CrossRef]
    [Google Scholar]
  32. Stanier R. Y, Palleroni N. J, Doudoroff M. 1966; The aerobic pseudomonads: a taxonomic study. J Gen Microbiol 43:159–271 [CrossRef]
    [Google Scholar]
  33. Stark W. M, Grindley N. D. F, Hatfull G. F, Boocock M. R. 1991; Resolvase-catalysed reactions between res sites differing in the central dinucleotide of subsite. EMBO J 10:3541–3548
    [Google Scholar]
  34. Zhang X.-X, Lilley A. K, Bailey M. J, Rainey P. B. 2004a; The indigenous Pseudomonas plasmid pQBR103 encodes plant-inducible genes including three putative helicases. FEMS Microbiol Ecol 51:9–17 [CrossRef]
    [Google Scholar]
  35. Zhang X.-X, Lilley A. K, Bailey M. J, Rainey P. B. 2004b; Functional and phylogenetic analysis of a plant-inducible oligoribonuclease (orn) gene from an indigenous Pseudomonas plasmid. Microbiology 150:2889–2898 [CrossRef]
    [Google Scholar]
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