1887

Abstract

Results from a random mutagenesis procedure on the PcaU binding site from followed by and screening are presented. PcaU is an IclR-type transcriptional regulator from the soil bacterium and is required for the regulated expression of enzymes for protocatechuate and quinate degradation encoded by the operon. It binds to a 45 bp area located in the intergenic region which consists of three perfect 10 bp sequence repeats forming one palindrome (R1, R2) and an additional direct sequence repeat (R3). selection for gene expression revealed that mutations within the three sequence motifs are tolerated to different extents. The functional requirement for conserved nucleotides was greatest in the external half of the palindrome (R1). Four positions within and directly adjacent to this 10 bp sequence never acquired a mutation, and are therefore considered to be the most important for transcriptional regulation by PcaU. Transcriptional output is affected in different ways; for some of these changes there is a correlation with a reduction in the affinity of PcaU for these sites. Two of these positions were also preserved when screening was performed for PcaU binding alone. Additional conserved residues are detected by the approach, indicating that the regions of the PcaU binding site involved in binding differ, at least in part, from those required for functional gene expression.

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2008-03-01
2024-03-28
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References

  1. Alting-Mees M. A., Short J. M. 1989; pBluescript II: gene mapping vectors. Nucleic Acids Res 17:9494
    [Google Scholar]
  2. Amaya E., Khvorova A., Piggot P. J. 2001; Analysis of promoter recognition in vivo directed by σ F of Bacillus subtilis by using random-sequence oligonucleotides. J Bacteriol 183:3623–3630
    [Google Scholar]
  3. Bairoch A., Apweiler R., Wu C. H., Barker W. C., Boeckmann B., Ferro S., Gasteiger E., Huang H., Lopez R. other authors 2005; The Universal Protein Resource (UniProt. Nucleic Acids Res 33:D154–D159
    [Google Scholar]
  4. Brzostowicz P. C., Reams A. B., Clark T. J., Neidle E. L. 2003; Transcriptional cross-regulation of the catechol and protocatechuate branches of the β -ketoadipate pathway contributes to carbon source-dependent expression of the Acinetobacter sp. strain ADP1 pobA gene. Appl Environ Microbiol 69:1598–1606
    [Google Scholar]
  5. Carey J. 1991; Gel retardation. Methods Enzymol 208:103–117
    [Google Scholar]
  6. Crooks G. E., Hon G., Chandonia J. M., Brenner S. E. 2004; WebLogo: a sequence logo generator. Genome Res 14:1188–1190
    [Google Scholar]
  7. Dal S., Steiner I., Gerischer U. 2002; Multiple operons connected with catabolism of aromatic compounds in Acinetobacter sp. strain ADP1 are under carbon catabolite repression. J Mol Microbiol Biotechnol 4:389–404
    [Google Scholar]
  8. Dal S., Trautwein G., Gerischer U. 2005; Transcriptional organization of genes for protocatechuate and quinate degradation from Acinetobacter sp. strain ADP1. Appl Environ Microbiol 71:1025–1034
    [Google Scholar]
  9. Freede P., Brantl S. 2004; Transcriptional repressor CopR: use of SELEX to study the copR operator indicates that evolution was directed at maximal binding affinity. J Bacteriol 186:6254–6264
    [Google Scholar]
  10. Freemont P. S., Lane A. N., Sanderson M. R. 1991; Structural aspects of protein–DNA recognition. Biochem J 278:1–23
    [Google Scholar]
  11. Gerischer U., Segura A., Ornston L. N. 1998; PcaU, a transcriptional activator of genes for protocatechuate utilization in Acinetobacter . J Bacteriol 180:1512–1524
    [Google Scholar]
  12. Gerischer U., Jerg B., Fischer R. 2008; Spotlight on the Acinetobacter baylyi β -ketoadipate pathway: multiple levels of regulation. In Acinetobacter Molecular Biology pp 203–230 Edited by Gerischer U. Norfolk, UK: Caister Scientific Press;
    [Google Scholar]
  13. Grodberg J., Dunn J. J. 1988; ompT encodes the Escherichia coli outer membrane protease that cleaves T7 RNA polymerase during purification. J Bacteriol 170:1245–1253
    [Google Scholar]
  14. Halford S. E., Marko J. F. 2004; How do site-specific DNA-binding proteins find their targets?. Nucleic Acids Res 32:3040–3052
    [Google Scholar]
  15. Hanahan D. 1983; Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580
    [Google Scholar]
  16. Harwood C. S., Parales R. E. 1996; The β -ketoadipate pathway and the biology of self-identity. Annu Rev Microbiol 50:553–590
    [Google Scholar]
  17. Juni E., Janik A. 1969; Transformation of Acinetobacter calco-aceticus ( Bacterium anitratum . J Bacteriol 98:281–288
    [Google Scholar]
  18. Kanack K. J., Runyen-Janecky L. J., Ferrell E. P., Suh S. J., West S. E. 2006; Characterization of DNA-binding specificity and analysis of binding sites of the Pseudomonas aeruginosa global regulator, Vfr, a homologue of the Escherichia coli cAMP receptor protein. Microbiology 152:3485–3496
    [Google Scholar]
  19. Kolb A., Busby S., Buc H., Garges S., Adhya S. 1993; Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem 62:749–795
    [Google Scholar]
  20. Krell T., Molina-Henares A. J., Ramos J. L. 2006; The IclR family of transcriptional activators and repressors can be defined by a single profile. Protein Sci 15:1207–1213
    [Google Scholar]
  21. Molina-Henares A. J., Krell T., Eugenia Guazzaroni M., Segura A., Ramos J. L. 2006; Members of the IclR family of bacterial transcriptional regulators function as activators and/or repressors. FEMS Microbiol Rev 30:157–186
    [Google Scholar]
  22. Mulder N. J., Apweiler R., Attwood T. K., Bairoch A., Bateman A., Binns D., Bradley P., Bork P., Bucher P. other authors 2005; InterPro, progress and status in 2005. Nucleic Acids Res 33:D201–D205
    [Google Scholar]
  23. Pan B., Unnikrishnan I., LaPorte D. C. 1996; The binding site of the IclR repressor protein overlaps the promoter of aceBAK . J Bacteriol 178:3982–3984
    [Google Scholar]
  24. Popp R., Kohl T., Patz P., Trautwein G., Gerischer U. 2002; Differential DNA binding of transcriptional regulator PcaU from Acinetobacter sp. strain ADP1. J Bacteriol 184:1988–1997
    [Google Scholar]
  25. Prentki P., Krisch H. M. 1984; In vitro insertional mutagenesis with a selectable DNA fragment. Gene 29:303–313
    [Google Scholar]
  26. Sambrook J., Russell D. 2001 Molecular Cloning: a Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  27. Siehler S. Y., Dal S., Fischer R., Patz P., Gerischer U. 2007; Multiple-level regulation of genes for protocatechuate degradation in Acinetobacter baylyi includes cross-regulation. Appl Environ Microbiol 73:232–242
    [Google Scholar]
  28. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. 1990; Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89
    [Google Scholar]
  29. Trautwein G., Gerischer U. 2001; Effects exerted by transcriptional regulator PcaU from Acinetobacter sp. strain ADP1. J Bacteriol 183:873–881
    [Google Scholar]
  30. Tropel D., van der Meer J. R. 2004; Bacterial transcriptional regulators for degradation pathways of aromatic compounds. Microbiol Mol Biol Rev 68:474–500
    [Google Scholar]
  31. Vaneechoutte M., Young D. M., Ornston L. N., De Baere T., Nemec A., Van Der Reijden T., Carr E., Tjernberg I., Dijkshoorn L. 2006; Naturally transformable Acinetobacter sp. strain ADP1 belongs to the newly described species Acinetobacter baylyi . Appl Environ Microbiol 72:932–936
    [Google Scholar]
  32. Walker J. R., Altamentova S., Ezersky A., Lorca G., Skarina T., Kudritska M., Ball L. J., Bochkarev A., Savchenko A. 2006; Structural and biochemical study of effector molecule recognition by the E. coli glyoxylate and allantoin utilization regulatory protein AllR. J Mol Biol 358:810–828
    [Google Scholar]
  33. Zhang R. G., Kim Y., Skarina T., Beasley S., Laskowski R., Arrowsmith C., Edwards A., Joachimiak A., Savchenko A. 2002; Crystal structure of Thermotoga maritima 0065, a member of the IclR transcriptional factor family. J Biol Chem 277:19183–19190
    [Google Scholar]
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