1887

Abstract

The promoter of the plasmid-borne genes encoding enzymes for phenol degradation resembles the promoter and is activated by CatR, the regulator of the chromosomally encoded catechol-degradative genes in . In this study, site-directed mutagenesis of the promoter region was performed. The interrupted inverted repeat sequence of the CatR recognition binding site (RBS) of the promoter is highly homologous to that of the promoter. However, the RBS was shown not to be the sole important feature for high-affinity binding of CatR to this site. Mutagenesis of the activation binding site (ABS) of CatR, which overlaps the −35 hexamer sequence TTGGAT of the promoter, revealed that the two G nucleotides in this sequence are important for promoter activity but not for CatR binding. All other substitutions made in the ABS negatively affected both the promoter activity and CatR binding. The spacer sequence of the and promoters between the −10 and −35 hexamers is 19 bp, which is longer than optimal. However, reducing the spacer region of the promoter was not sufficient for CatR-independent promoter activation. An internal binding site (IBS) for CatR is located downstream of the transcriptional start site of the genes and it negatively regulates the operon. A similar IBS was identified in the case of the operon and tested for its functionality. The results indicate a conservation of CatR-mediated regulation mechanisms between the promoter and the promoter. This universal mechanism of CatR-mediated transcriptional activation could be of great importance in enabling catechol-degrading bacteria to expand their substrate range via horizontal transfer of the phenol degradative genes.

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2000-01-01
2019-10-21
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References

  1. Adams, M. H. (1959). Bacteriophages, pp. 445–447. New York: Interscience Publishers.
  2. Aldrich, T. L. & Chakrabarty, A. M. ( 1988; ). Transcriptional regulation, nucleotide sequence, and localization of the promoter of the catBC operon in Pseudomonas putida. J Bacteriol 170, 1297-1304.
    [Google Scholar]
  3. Ansari, A. Z., Chael, M. L. & O’Halloran, T. V. ( 1992; ). Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR. Nature 355, 87-89.[CrossRef]
    [Google Scholar]
  4. Basak, S. & Nagaraja, V. ( 1998; ). Transcriptional activator C protein-mediated unwinding of DNA as a possible mechanism for mom gene activation. J Mol Biol 284, 893-902.[CrossRef]
    [Google Scholar]
  5. Bayley, S. A., Duggleby, C. J., Worsey, M. J., Williams, P. A., Hardy, K. G. & Broda, P. ( 1977; ). Two modes of loss of the TOL function from Pseudomonas putida mt-2. Mol Gen Genet 154, 203-204.[CrossRef]
    [Google Scholar]
  6. Bolker, M., Wulczyn, F. G. & Kahmann, R. ( 1989; ). Role of bacteriophage Mu C protein in activation of the mom gene promoter. J Bacteriol 171, 2019-2027.
    [Google Scholar]
  7. 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.[CrossRef]
    [Google Scholar]
  8. Carter, P., Bedoulle, H. & Winter, G. ( 1985; ). Improved oligonucleotide site-directed mutagenesis using M13 vectors. Nucleic Acids Res 13, 4431-4443.[CrossRef]
    [Google Scholar]
  9. Chugani, S. A., Parsek, M. R., Hershberger, C. D., Murakami, K., Ishihama, A. & Chakrabarty, A. M. ( 1997; ). Activation of the catBCA promoter: probing the interaction of CatR and RNA polymerase through in vitro transcription. J Bacteriol 179, 2221-2227.
    [Google Scholar]
  10. Chugani, S. A., Parsek, M. R. & Chakrabarty, A. M. ( 1998; ). Transcriptional repression mediated by LysR-type regulator CatR bound at multiple binding sites. J Bacteriol 180, 2367-2372.
    [Google Scholar]
  11. Coco, W. M., Rothmel, R. K., Henikoff, S. & Chakrabarty, A. M. ( 1993; ). Nucleotide sequence and initial functional characterization of the clcR gene encoding a LysR family activator of the clcABD operon in Pseudomonas putida. J Bacteriol 175, 417-427.
    [Google Scholar]
  12. Franz, B. & Chakrabarty, A. M. ( 1987; ). Organization and nucleotide sequence determination of a gene cluster involved in 3-chlorocatechol degradation. Proc Natl Acad Sci USA 84, 4460-4464.[CrossRef]
    [Google Scholar]
  13. Goethals, K., Van Montagu, M. & Holsters, M. ( 1992; ). Conserved motifs in a divergent nod box of Azorhizobium caulinodans ORS571 reveal a common structure in promoters regulated by LysR-type proteins. Proc Natl Acad Sci USA 89, 1646-1650.[CrossRef]
    [Google Scholar]
  14. Hanahan, D. ( 1983; ). Studies on the transformation of E. coli with plasmids. J Mol Biol 166, 577-580.
    [Google Scholar]
  15. Harayama, S. & Timmis, K. N. ( 1989; ). Catabolism of aromatic hydrocarbons by Pseudomonas. In Genetics of Bacterial Diversity, pp. 151-174. Edited by D. A. Hopwood & K. E. Chater. London: Academic Press.
  16. Herrmann, H., Janke, D., Kejsna, S. & Roy, M. ( 1988; ). In vivo generation of R68.45-pPGH1 hybrid plasmids conferring a Phl+ (meta-pathway) phenotype. Mol Gen Genet 214, 173-176.[CrossRef]
    [Google Scholar]
  17. Hõrak, R. & Kivisaar, M. ( 1998; ). Expression of the transposase gene tnpA of Tn4652 is positively affected by integration host factor. J Bacteriol 180, 2822-2829.
    [Google Scholar]
  18. Kallastu, A., Hõrak, R. & Kivisaar, M. ( 1998; ). Identification and characterization of IS1411, a new insertion sequence which causes transcriptional activation of the phenol degradation genes in Pseudomonas putida. J Bacteriol 180, 5306-5312.
    [Google Scholar]
  19. Kasak, L., Hõrak, R., Nurk, A., Talvik, K. & Kivisaar, M. ( 1993; ). Regulation of the catechol 1,2-dioxygenase- and phenol monooxygenase-encoding pheBA operon in Pseudomonas putida PaW85. J Bacteriol 175, 8038-8042.
    [Google Scholar]
  20. Kivisaar, M., Hõrak, R., Kasak, L., Heinaru, A. & Habicht, J. ( 1990; ). Selection of independent plasmids determining phenol degradation in Pseudomonas putida and the cloning and expression of genes encoding phenol monooxygenase and catechol 1,2-dioxygenase. Plasmid 24, 25-36.[CrossRef]
    [Google Scholar]
  21. Kukor, J. J. & Olsen, R. H. ( 1990; ). Molecular cloning, characterization, and regulation of a Pseudomonas pickettii PKO1 gene encoding phenol hydroxylase and expression of the gene in Pseudomonas aeruginosa PAO1. J Bacteriol 172, 4624-4630.
    [Google Scholar]
  22. McFall, S. M., Klem, T. J., Fujita, N., Ishihama, A. & Chakrabarty, A. M. ( 1997; ). DNase I footprinting, DNA bending and in vitro transcription analyses of ClcR and CatR on the clcABD promoter: evidence of a conserved transcriptional activation mechanism. Mol Microbiol 24, 965-976.[CrossRef]
    [Google Scholar]
  23. McFall, S. M., Chugani, S. A. & Chakrabarty, A. M. ( 1998; ). Transcriptional activation of the catechol and chlorocatechol operons: variations on a theme. Gene 223, 257-267.[CrossRef]
    [Google Scholar]
  24. van der Meer, J. R., Frijters, A. C. J., Leveau, J. H. J., Eggen, R. I. L., . Zehnder, A. J. B. & de Vos, W. M. ( 1991; ). Characterization of the Pseudomonas sp. strain P51 gene tcbR, a LysR-type transcriptional activator of the tcbCDEF chlorocatechol oxidative operon, and analysis of the regulatory region. J Bacteriol 173, 3700-3708.
    [Google Scholar]
  25. Miller, J. H. (1992). A Short Course in Bacterial Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  26. Murakami, K., Fujita, N. & Ishihama, A. ( 1996; ). Transcription factor recognition surface on the RNA polymerase α subunit is involved in contact with the DNA enhancer element. EMBO J 15, 4358-4367.
    [Google Scholar]
  27. O’Halloran, T. V., Frantz, B., Shin, M. K., Ralston, D. M. & Wright, J. G. ( 1989; ). The MerR heavy metal receptor mediates positive activation in a topologically novel transcription complex. Cell 56, 119-129.[CrossRef]
    [Google Scholar]
  28. Ornston, L. N. ( 1966; ). The conversion of catechol and protocatechuate to β-ketoadipate by Pseudomonas putida. IV. Regulation. J Biol Chem 241, 3800-3810.
    [Google Scholar]
  29. Parkhill, J. & Brown, N. L. ( 1990; ). Site-specific insertion and deletion mutants in the mer promoter–operator region of Tn501; the nineteen base-pair spacer is essential for normal induction of the promoter by MerR. Nucleic Acids Res 18, 5157-5162.[CrossRef]
    [Google Scholar]
  30. Parsek, M. R., Shinabarger, D. L., Rothmel, R. K. & Chakrabarty, A. M. ( 1992; ). Role of CatR and cis,cis-muconate in the activation of the catBC operon involved on benzoate degradation in Pseudomonas putida. J Bacteriol 174, 7798-7806.
    [Google Scholar]
  31. Parsek, M. R., Ye, R. W., Pun, P. & Chakrabarty, A. M. ( 1994; ). Critical nucleotides in the interaction of a LysR-type regulator with its target promoter region. J Biol Chem 269, 11279-11284.
    [Google Scholar]
  32. Parsek, M. R., Kivisaar, M. & Chakrabarty, A. M. ( 1995; ). Differental DNA bending introduced by the Pseudomonas putida LysR-type regulator, CatR, at the plasmid-borne pheBA and chromosomal catBC promoters. Mol Microbiol 15, 819-828.[CrossRef]
    [Google Scholar]
  33. Parsek, M. R., McFall, S. M. & Chakrabarty, A. M. ( 1996; ). Evolution of regulatory systems of biodegradative pathways. In Molecular Biology of Pseudomonads, pp. 135-152. Edited by T. Nakazawa, K. Furukawa, D. Haas & S. Silver. Washington, DC: American Society for Microbiology.
  34. Peters, M., Heinaru, E., Talpsep, E., Wand, H., Stottmeister, U., Heinaru, A. & Nurk, A. ( 1997; ). Acquisition of a deliberately introduced phenol degradation operon, pheBA, by different indigenous Pseudomonas species. Appl Environ Microbiol 63, 4899-4906.
    [Google Scholar]
  35. Rothmel, R. K., Aldrich, T. L., Houghton, J. E., Coco, W. M., Ornston, L. N. & Chakrabarty, A. M. ( 1990; ). Nucleotide sequencing of Pseudomonas putida catR: positive regulator of the catBC operon is a member of LysR family. J Bacteriol 172, 922-931.
    [Google Scholar]
  36. Rothmel, R. K., Shinabarger, D. L., Parsek, M. R., Aldrich, T. L. & Chakrabarty, A. M. ( 1991; ). Functional analysis of the Pseudomonas putida regulatory protein CatR: transcriptional studies and determination of the CatR DNA-binding site by hydroxyl-radical footprinting. J Bacteriol 173, 4717-4724.
    [Google Scholar]
  37. Sharma, R. C. & Schimke, R. T. ( 1996; ). Preparation of electro-competent E. coli using salt-free growth medium. BioTechniques 20, 42-44.
    [Google Scholar]
  38. Shingler, V., Powlowski, J. & Marklund, U. ( 1992; ). Nucleotide sequence and functional analysis of the complete phenol/3,4-dimethylphenol catabolic pathway of Pseudomonas sp. Strain CF600. J Bacteriol 174, 711-724.
    [Google Scholar]
  39. Stefano, J. E. & Gralla, J. D. ( 1982; ). Spacer mutations in the lac ps promoter. Proc Natl Acad Sci USA 79, 1069-1072.[CrossRef]
    [Google Scholar]
  40. Studier, F. W. & Moffatt, B. A. ( 1986; ). Use of bacteriophage T7 polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189, 113-130.[CrossRef]
    [Google Scholar]
  41. Tsuda, M., Minegishi, K.-I. & Iino, T. ( 1989; ). Toluene transposons Tn4651 and Tn4653 are class II transposons. J Bacteriol 171, 1386-1363.
    [Google Scholar]
  42. Wigmore, G. J., DiBerardino, D. & Bayly, R. C. ( 1977; ). Regulation of the enzymes of the meta-cleavage pathway of Pseudomonas putida: a regulatory model. J Gen Microbiol 100, 81-87.[CrossRef]
    [Google Scholar]
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