1887

Abstract

The regulation of the -hydroxybenzoate hydroxylase gene () of WCS358 involved in the catabolism of -hydroxybenzoic acid (PHB) to the central intermediate protocatechuate was studied. Protocatechuic acid (PCA) is then degraded via the β-ketoadipate pathway to form tricarboxylic acid intermediates. In several Gram-negative bacteria has been found genetically linked to a regulator called which activates expression in response to PHB. In this study the identification and characterization of the - locus of WCS358 is presented. The -hydroxybenzoate hydroxylase (PobA) is highly identical to other identified PobA proteins, whereas the regulatory protein PobC did not display very high identity to other PobR proteins studied and belonged to the AraC family of regulatory proteins, hence it has been designated PobC. Using the promoter transcriptionally fused to a promoterless gene it was observed that induction via PobC occurred very efficiently when PHB was present and to a lesser but still significant level also in the presence of PCA. This PobC-PCA response was genetically demonstrated by making use of ::Tn and ::Tn mutants of strain WCS358 constructed in this study. In mutants both the -hydroxybenzoic and PCA response were not observed, whereas in the mutant, which lacks a functional protocatechuate 3,4-dioxygenase, the protocatechuic-acid-dependent activation was still observed. Finally, the activation of by PHB varied according to the concentration and it was observed that in the ::Tn regulatory mutant of strain WCS358 the promoter activity was reduced. PcaR is a regulator involved in the regulation of several loci of the β-ketoadipate pathway, one of which is . It was postulated that the reduction of activation in ::Tn mutants was because there was no expression of the gene encoding the PHB transport protein resulting in lower levels of PHB present inside the cell.

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2001-06-01
2020-10-01
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References

  1. Beringer J. E., Beynon J. L., Buchanan-Wollaston A. V., Johnston A. W. B.. 1978; Transfer of the drug-resistance transposon Tn 5 to Rhizobium . Nature276:633–634[CrossRef]
    [Google Scholar]
  2. Better M., Lewis B., Corbin D., Ditta G., Helinsky D. R.. 1983; Structural relationships among Rhizobium meliloti promoters. Cell35:479–485[CrossRef]
    [Google Scholar]
  3. Birnboim H. C.. 1983; A rapid alkaline method for the isolation of plasmid DNA. Methods Enzymol100:243–255
    [Google Scholar]
  4. Bolton G. W., Nester E. W., Gordon M. P.. 1986; Plant phenolic compounds induce expression of the Agrobacterium tumefaciens loci needed for virulence. Science232:983–985[CrossRef]
    [Google Scholar]
  5. Brunel F., Davison J.. 1988; Cloning and sequencing of Pseudomonas genes encoding vanillate demethylase. J Bacteriol170:4924–4930
    [Google Scholar]
  6. Bullock W. O., Fernandez J. M., Short J. M.. 1987; XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with β-galactosidase selection. BioTechniques5:376–382
    [Google Scholar]
  7. Caetano-Annoles G.. 1993; Amplifying DNA with arbitrary oligonucleotide primers. PCR Methods Appl3:85–92[CrossRef]
    [Google Scholar]
  8. Corbin D., Ditta G., Helinski D. R.. 1982; Clustering of nitrogen fixation ( nif ) genes in Rhizobium meliloti . J Bacteriol149:4759–4764
    [Google Scholar]
  9. DiMarco A. A., Ornston L. N.. 1994; Regulation of p -hydroxybenzoate hydroxylase synthesis by PobR bound to an operator in Acinetobacter calcoaceticus . J Bacteriol176:4277–4284
    [Google Scholar]
  10. DiMarco A. A., Averhoff B., Ornston L. N.. 1993; Identification of the transcriptional activator pobR and characterization of its role in the expression of pobA , the structural gene for p -hydroxybenzoate hydroxylase in Acinetobacter calcoaceticus . J Bacteriol175:4499–4506
    [Google Scholar]
  11. Figursky D. H., Helinsky D. R.. 1979; Replication of an origin containing derivative of plasmid RK2 dependent on a plasmid function provided in trans . Proc Natl Acad Sci USA76:1648–1652[CrossRef]
    [Google Scholar]
  12. Geels F. P., Schippers B.. 1983; Reduction in yield depression in high frequency potato cropping soil after seed tuber treatments with antagonistic fluorescent Pseudomonas spp. Phytopathol Z108:207–221[CrossRef]
    [Google Scholar]
  13. Gerischer U., Segura A., Ornston L. N.. 1998; PcaU, a transcriptional activator of genes for protocatechuate utilization in Acinetobacter . J Bacteriol180:1512–1524
    [Google Scholar]
  14. Hanahan D.. 1983; Studies of transformation of Escherichia coli with plasmids. J Mol Biol166:557–580[CrossRef]
    [Google Scholar]
  15. Kok R. G., D’Argenio D. A., Ornston L. N.. 1997; Combining localized PCR mutagenesis and natural transformation in direct genetic analysis of a transcriptional regulator gene, pobR . J Bacteriol179:4270–4276
    [Google Scholar]
  16. Kok R. G., D’Argenio D. A., Ornston L. N.. 1998; Mutation analysis of pobR and pcaU , closely related transcriptional activators in Acinetobacter . J Bacteriol180:5058–5069
    [Google Scholar]
  17. Magazin M., Moores J. C., Leong J.. 1986; Cloning of the gene coding for the outer membrane receptor protein for ferric-pseudobactin, a siderophore from a plant-growth-promoting Pseudomonas strain .. J Biol Chem261:795–799
    [Google Scholar]
  18. Maniatis T., Fritsch E. F., Sambrook J.. 1982; Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  19. Marugg J. D., Hoekstra W. P. M., Schippers B., Weisbeek P. J., van Spanje M.. 1985; Isolation and analysis of genes involved in siderophore biosynthesis in plant-growth-stimulating Pseudomonas putida WCS358. J Bacteriol164:563–570
    [Google Scholar]
  20. van der Meer J. F., de Vos W. M., Harayama S., Zehender A. J. B.. 1992; Molecular mechanisms of genetic adaptation to xenobiotic compounds. Microbiol Rev56:677–694
    [Google Scholar]
  21. Melchers L. A., Regensburg-Tuink A. J. G., Schilperoot R. A., Hooykaas P. J. J.. 1989; Specificity of signal molecules in the activation of Agrobacterium virulence gene expression. Mol Microbiol3:969–977[CrossRef]
    [Google Scholar]
  22. Messing J.. 1983; New vectors for cloning. Methods Enzymol101:20–78
    [Google Scholar]
  23. Miller J. H.. 1972; Experiments in Molecular Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  24. Moores J. C., Magazin M., Ditta G. S., Leong J.. 1984; Cloning of genes involved in the biosynthesis of pseudobactin, a high-affinity iron transport agent of a plant growth-promoting Pseudomonas strain. J Bacteriol157:53–58
    [Google Scholar]
  25. Nichols N. N., Harwood C. S.. 1995; Repression of 4-hydroxybenzoate transport and degradation by benzoate: a new layer of regulatory control in the Pseudomonas putida β-ketoadipate pathway. J Bacteriol177:7033–7040
    [Google Scholar]
  26. O’Sullivan D. J., O’Gara F.. 1992; Traits of fluorescent Pseudomonas spp. involved in suppression of plant root pathogens. Microbiol Rev56:662–676
    [Google Scholar]
  27. O’Toole G. A., Kolter R.. 1998; Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol28:449–461[CrossRef]
    [Google Scholar]
  28. Overhage J., Kresse A. U., Priefert H., Sommer H., Krammer G., Rabenhorst J., Steinbüchel A.. 1999a; Molecular characterization of the genes pcaG and pcaH encoding protocatechuate-3,4-dioxygenase, which are essential for vanillin catabolism in Pseudomonas sp. strain HR199. Appl Environ Microbiol65:951–960
    [Google Scholar]
  29. Overhage J., Priefert H., Steinbüchel A.. 1999b; Biochemical and genetic analyses of ferulic acid catabolism in Pseudomonas sp. strain HR199 . . Appl Environ Microbiol65:4837–4847
    [Google Scholar]
  30. Parke D.. 1993; Positive regulation of phenolic catabolism in Agrobacterium tumefaciens by the pcaQ gene in response to β-carboxy-cis, cis-muconate. J Bacteriol175:3529–3535
    [Google Scholar]
  31. Parke D.. 1996; Acquisition, reorganization, and merger of genes: novel management of the β-ketoadipate pathway in Agrobacterium tumefaciens . FEMS Microbiol Lett146:3–12
    [Google Scholar]
  32. Priefert H., Rabenhorst J., Steinbüchel A.. 1997; Molecular characterisation of genes of Pseudomonas sp. strain HR199 involved in bioconversion of vanillin to protocatechuate. J Bacteriol179:2595–2607
    [Google Scholar]
  33. Romero-Steiner S., Parales R. E., Harwood C. S., Houghton J. E.. 1994; Characterisation of the pcaR regulatory gene from Pseudomonas putida , which is required for the complete degradation of p -hydroxybenzoate. J Bacteriol176:5771–5779
    [Google Scholar]
  34. Sanger F., Nicklen S., Coulson A. R.. 1977; DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA74:5463–5467[CrossRef]
    [Google Scholar]
  35. Spaink H. P., Okker R. J. H., Wijffelmann C. A., Pees E., Lugtenberg B. J. J.. 1987; Promoter in the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI. Plant Mol Biol9:27–39[CrossRef]
    [Google Scholar]
  36. Stachel S. E., An G., Flores C., Nester E. W.. 1985; A Tn 3 lacZ transposon for the random generation of β-galactosidase gene fusions: application to the analysis of gene expression of Agrobacterium tumefaciens . EMBO J4:891–898
    [Google Scholar]
  37. Staskawicz B., Dahlbeck D., Keen N., Napoli C.. 1987; Molecular characterisation of cloned avirulence genes from race 0 and race 1 of Pseudomonas syringae pv. glycinea . J Bacteriol169:5789–5794
    [Google Scholar]
  38. Toms A., Wood J. M.. 1970; The degradation of trans -ferulic acid by Pseudomonas acidovorans . Biochemistry9:337–343[CrossRef]
    [Google Scholar]
  39. Venturi V., Zennaro F., Degrassi G., Okeke B. C., Bruschi C. V.. 1998; Genetics of ferulic acid bioconversion to protocatechuic acid in plant-growth-promoting Pseudomonas putida WCS358. Microbiology144:965–973[CrossRef]
    [Google Scholar]
  40. Wong C. M., Dilworth M. J., Glenn A. R.. 1994; Cloning and sequencing show that 4-hydroxybenzoate hydroxylase (PobA) is required for uptake of 4-hydroxybenzoate in Rhizobium leguminosarum . Microbiology140:2775–2786[CrossRef]
    [Google Scholar]
  41. Yanisch-Perron C., Vieira J., Messing J.. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene33:103–119[CrossRef]
    [Google Scholar]
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