Identification of Potential Corynebacterium ammoniagenes Purine Gene Regulators Using the pur-lacZ Reporter in Escherichia coli

  • Published : 2004.12.01

Abstract

This study has developed Corynebacterium ammoniagenes (c. ammoniagenes) purine gene transcriptional reporters (purF-lacZ and purE-lacZ) that function in Escherichia coli (E. coli) DH5a. After transformation of a C. ammoniagenes gDNA library into E. coli cells harboring either purF-lacZ or purE-lacZ, C. ammoniagenes clones were obtained that repress purF-lacZ and purE-lacZ gene expression. The potential purE and purF regulatory genes are homologous to the genes encoding transcription regulators, the regulatory subunit of RNA polymerase, and genes for purine nucleotide biosynthesis of various bacteria. The C. ammoniagenes purE-lacZ and purF-lacZ reporters were repressed by adenine and guanine within E. coli, indicating similarity in the regulatory mechanism of purine biosynthesis in C. ammoniagenes and E. coli. Gene regulation of pur-lacZ by adenine and guanine was partly abolished in cells expressing potential purine regulatory genes, indicating functionality of the purine gene regulators in repression of purE-lacZ and purF-lacZ. The purE-lacZ and purF-lacZ reporters can be used for the screening of genes involved in the regulation of the de novo synthesis of the purine nucleotides.

Keywords

References

  1. Andersen, L., M. Kilstrup, and J. Neuhard. 1989. Pyrimidine, purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K12. Involvement of the glnLG and purR genes in the regulation of codA expression. Arch. Microbiol. 152: 115-118
  2. Blattner, F. R., G. Plunkett III, C. A. Bloch, N. T. Perna, V. Burland, M. Riley, J. Collado-Vides, J. D. Glasner, C. K. Rode, G. F. Mayhew, J. Gregor, N. W. Davis, H. A. Kirkpatrick, M. A. Goeden, D. J. Rose, B. Mau, and Y. Shao. 1997. The complete genome sequence of Escherichia coli K-12. Science 277: 1453-1474
  3. Choi, K. Y. and H. Zalkin. 1990. Regulation of Escherichia coli pyrC by the purine regulon repressor protein. J. Bacteriol. 172: 3201-3207
  4. Choi, K. Y. and H. Zalkin. 1992. Structural characterization and corepressor binding of Escherichia coli purine repressor. J. Bacteriol. 174: 6207-6214
  5. Choi, K. Y. and H. Zalkin. 1994. Role of the purine repressor hinge sequences in repressor function. J. Bacteriol. 176: 1767-1772
  6. Choi, K. Y., F. Lu, and H. Zalkin. 1994. Mutagenesis of amino acid residues required for binding of corepressors to the purine repressor. J. Biol. Chem. 269: 24066- 24072
  7. Chung, S. O., J. H. Lee, S. Y. Lee, and D. S. Lee. 1996. Genomic organization of purK and purE in Brevibacterium ammoniagenes ATCC6872: Pure locus provides a clue for genomic evolution. FEMS Microbiol. Lett. 137: 265-268
  8. Ebbole, D. J. and H. Zalkin. 1987. Cloning and characterization of a 12-gene cluster from Bacillus subtilis encoding nine enzymes for de novo purine nucleotide synthesis. J. Biol. Chem. 262: 8274-8287
  9. Ebbole D. J. and H. Zalkin. 1989. Bacillus subtilis pur operon expression and regulation. J. Bacteriol. 171: 2136-2141
  10. Ebbole, D. J. and H. Zalkin. 1986. Interaction of a putative repressor protein with an extended control region of the Bacillus subtilis pur operon. J. Biol. Chem. 264: 3553-3561
  11. Eikmanns, B. J., E. Klentz, W. Liebl, and H. A. Shm. 1991. A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for cloning, controlled gene expression, and promoter probing. Gene 2: 93-98
  12. Got, J. S. and C. S. Gollub. 1959. Purine analogs as feedback inhibitors. Proc. Soc. Exp. Biol. Med. 101: 641-643
  13. Han, J. K., S. O. Chung, J. H. Lee, and S. M. Byun. 1997. 6- Mercaptoguanosine resistance is related with purF gene encoding 5-phosphoribosyl-1-pyrophosphate amidotransferase in inosine-5-monophosphate overproducing Brevibacterium ammoniagenes. Biotechnol. Lett. 19: 79-83
  14. He, B., A. Shiau, K. Y. Choi, H. Zalkin, and J. M. Smith. 1990. Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction. J. Bacteriol. 172: 4555-4562
  15. Meng, L. M., M. Kilstruop, and P. Nygaard. 1990. Autoregulation of PurR repressor synthesis and involvement of purR in the regulation of purB, purC, purL, purMN, and guaBA expression in Escherichia coli. Eur. J. Biochem. 187: 373-379
  16. Jeon, K. S., G. E. Ji, and I. K. Hwang. 2004. Assay of $\beta$-glucosidase activity of bifidobacteria and the hydrolysis of isoflavone glycosides by Bifidobacterium sp. Int-57 in soymilk fermentation. J. Microbiol. Biotechnol. 12: 8-13
  17. Kim, H.-J., J.-S. Park, H.-S. Lee, and Y. Kim. 2004. Utilization of lacZ to isolate regulatory genes from Corynebacterium glutamicum. J. Microbiol. Biotechnol. 12: 336-339
  18. Micralv, P., J. E. Bernhard, K. Eva, L. Wolfgang, and S. Hermann. 1996. Promoters from Corynebacterium glutamicum: Cloning, molecular analysis and search for a consensus motif. Microbiology 142: 1297-1309
  19. Miller, J. H. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., U.S.A
  20. Mongkolsuk, S., E. J. Duvall, and P. S. Lovett. 1985. Transcription termination signal for the cat-86 indicator gene in a Bacillus subtilis promoter-cloning plasmid. Gene 37: 83-90
  21. Nagy, M. 1970. Regulation of the biosynthesis of purine nucleotides in Schizosaccharomyces pombe: I. Properties of the phosphoribosylpyrophosphate: glutamine amidotransferase of the wild strain and of a mutant desensitized towards feedback modifiers. Biochim. Biophys. Acta 198: 471-481
  22. Paik, J. E. and B. B. R. Lee. 2003. Isolation of transcription initiation signals from Corynebacterium ammoniagenes and comparison of their gene expression levels in C. ammoniagenes and Escherichia coli. Biotechnol. Lett. 25: 1311-1316
  23. Patek, M., B. J. Eikmanns, J. Patek, and H. Sahm. 1996. Promoters from Corynebacterium glutamicum: Cloning, molecular analysis and search for a consensus motif. Microbiology 142: 1297-1309
  24. Patek, M., G. Muth, and W. Wohlleben. 2003. Function of Corynebacterium glutamicum promoters in Escherichia coli, Streptomyces lividans, and Bacillus subtilis. J. Biotechnol. 104: 325-334
  25. Patek, M., J. Nesvera, A. Guyonvarch, O. Reyes, and G. Leblon. 2003. Promoters of Corynebacterium glutamicum. J. Biotechnol. 104: 311-323
  26. Perna, N. T., G. Plunkett III, V. Burland, B. Mau, J. D. Glasner, D. J. Rose, G. F. Mayhew, P. S. Evans, J. Gregor, H. A. Kirkpatrick, G. Posfai, J. Hackett, S. Klink, A. Boutin, Y. Shao, L. Miller, E. J. Grotbeck, N. W. Davis, A. Lim, E. Dimalanta, K. Potamousis, J. Apodaca, T. S. Anantharaman, J. Lin, G. Yen, D. C. Schwartz, R. A. Welch, and F. R. Blattner. 2001. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409: 529-533
  27. Rappu, P., B. S. Shin, H. Zalkin, and P. Mäntsälä. 1999. A role for a highly conserved protein of unknown function in regulation of Bacillus subtilis purA by the purine repressor. J. Bacteriol. 181: 3810-3815
  28. Rasko, D. A., J. Ravel, O. A. Okstad, E. Helgason, R. Z. Cer, L. Jiang, K. A. Shores, D. E. Fouts, N. J. Tourasse, S. V. Angiuoli, J. Kolonay, W. C. Nelson, A. B. Kolsto, C. M. Fraser, and T. D. Read. 2004. The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1. Nucleic Acids Res. 32: 977-988
  29. Rolfes, R. J. and H. Zalkin. 1990. Autoregulation of Escherichia coli purR requires two control sites downstream of the promoter. J. Bacteriol. 172: 5758-5766
  30. Saxild, H. H., K. Brunstedt, K. I. Nielsen, H. Jarmer, and P. Nygaard. 2001. Definition of the Bacillus subtilis PurR operator using genetic and bioinformatic tools and expansion of the PurR regulon with glyA, guaC, pbuG, Xpt-pbuX, yqhZ-folD, and pbuO. J. Bacteriol. 183: 6175-6183
  31. Saxild, H. H. and P. Nygaard. 1998. Gene-enzyme relationships of the purine biosynthetic pathway in Bacillus subtilis. Mol. Gen. Genet. 211: 160-167
  32. Shin, B. S., A. Stein, and H. Zalkin. 1997. Interaction of Bacillus subtilis purine repressor with DNA. J. Bacteriol. 179: 7394-7402
  33. Steiert, J. G., R. J. Rofels, H. Zalkin, and G. V. Stauffer. 1990. Regulation of Escherichia coli glyA gene by the purR gene product. J. Bacteriol. 172: 3799-3830
  34. Weng, M., P. Nagy, and H. Zalkin. 1995. Identification of the Bacillus subtilis pur operon repressor. Proc. Natl. Acad. Sci. USA 92: 7455-7459
  35. Weng, M. and H. Zalkin. 2000. Mutations in the Bacillus subtilis purine repressor that perturb PRPP effector function in vitro and in vivo. Curr. Microbiol. 41: 56-59
  36. Wilson, H. R. and C. L. Turnbough, Jr. 1990. Role of the purine repressor in the regulation of pyrimidine gene expression in Escherichia coli K-12. J. Bacteriol. 172: 3208-3213
  37. Zalkin, H. and J. E. Dixon. 1992. De novo purine nucleotide biosynthesis. Prog. Nucl. Acid Res. 42: 259-287
  38. Zalkin, H. 1999. De novo purine nucleotide synthesis, pp. 335-341. In A. L. Sonenshein, J. A. Hoch, and R. Losick (eds.), Bacillus subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics. American Society for Microbiology, Washington, D.C., U.S.A
  39. Zalkin, H. and P. Nyggard. 1999. Biosynthesis of purine nucleotides, pp. 561-579. In F. C. Neidhardt, R. Curtis III, J. L. Ingraham, E. C. C. Lin, K. B.Low, B. Magasanik, W. S. Rennikoff, M. Riley, M. Schaechter, and H. E. Umbarger (eds.), Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd Ed. American Society for Microbiology, Washington, D.C., U.S.A