Molecular and General Genetics MGG

, Volume 214, Issue 3, pp 379–388

Mapping of the multiple regulatory sites for putP and putA expression in the putC region of Escherichia coli

  • Toshifumi Nakao
  • Ichiro Yamato
  • Yasuhiro Anraku


The effects of regulatory proteins on the expression of putP and putA were studied using put-lacZ fusion genes. The expression of the putP-lacZ gene was activated by the glnG gene product and the catabolite gene activator protein (CAP). The putA gene product inhibited activation of putP-lacZ gene expression by CAP or the glnG gene product and its inhibition was greater in the absence of proline. The expression of the putA-lacZ gene was activated by CAP and repressed by the glnG gene product. The putA gene product acted as a repressor in the absence of proline, but not in its presence. Studies using put-lacZ fusion genes with upstream deletions showed that the region required for the activation of putP by CAP was within 234 bp upstream of the translational initiation site and that that for the activation of putP was within 107 bp upstream of the translational initiation site of the putA gene. This supported the suggested locations of CAP binding sites. The region required for induction of putP and putA expression by proline was located at the Hpal site 182 bp upstream of the translational starting site of putA, suggesting that a sequence of dyad symmetry located 1 bp to the left of the HpaI site is a candidate for the binding site of the putA gene product.

Key words

putP putA putC Escherichia coli Promoter regions of putA and putP 



L-azetidine-2-carboxylic acid




catabolite gene activator protein


nitrogen regulator I


DNA replicative form






2,3,5-triphenyl tetrazolium chloride




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  1. Ames GF-L, Nikaido K (1985) Nitrogen regulation in Salmonella typhimurium. Identification of ntrC protein-binding site and definition of a consensus binding sequence. EMBO J 4:539–547Google Scholar
  2. Bochner BR, Savageau MA (1971) Generalized indicator plate for genetic, metabolic, and taxonomic studies with microorganisms. Appl Environ Microbiol 33:434–444Google Scholar
  3. Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179–207Google Scholar
  4. Casadaban MJ, Chou J, Cohen SN (1980) In vitro gene fusions that join an enzymatically active β-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol 143:971–980Google Scholar
  5. Cohen SN, Chang ACY, Boyer HW, Hellings RB (1973) Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci USA 70:3240–3244Google Scholar
  6. Condamine H (1971) Mutans des voies be biosynthèse et de dégradation de la proline chez E. coli K12. Ann Inst Pasteur (Paris) 120:9–22Google Scholar
  7. Davis BD, Mingioli ES (1959) Mutants of Escherichia coli requiring methionine or vitamin B12. J Bacteriol 60:17–28Google Scholar
  8. De Lucia P, Cairns J (1969) Isolation of an E. coli strain with a mutation affecting DNA polymerase. Nature 224:1164–1133Google Scholar
  9. Dendinger S, Brill WJ (1970) Regulation of proline degradation in S. typhimurium. J Bacteriol 103:144–152Google Scholar
  10. Grothe S, Krogsrad RL, McClellan DJ, Milner JL, Wood JM (1986) Proline transport and osmotic stress response in Escherichia coli K-12. J Bacteriol 166:253–259Google Scholar
  11. Kajie S (1985) On the membrane-associated nitrite reductase system of Escherichia coli. Doctoral thesis, University of TokyoGoogle Scholar
  12. Léon P, Romero D, Garciarrubio A, Bastarrachea F, Covarrubias AA (1985) Glutamine synthetase-constitutive mutation affecting the glnALG upstream promoter of Escherichia coli. J Bacteriol 164:1032–1038Google Scholar
  13. Maloy SR (1987) The proline utilization operon. In: Neidhart FC, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE (eds). Escherichia coli and Salmonella typhimurium cellular and molecular biology. Am Soc Microbiol, Washington, DC, pp 1513–1519Google Scholar
  14. Maloy SR, Nunn WD (1981) Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol 145:1110–1112Google Scholar
  15. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  16. Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–79Google Scholar
  17. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  18. Mogi T, Yamamoto H, Nakao T, Yamato I, Anraku Y (1986) Genetic and physical characterization of putP, the proline carrier gene of Escherichia coli K 12. Mol Gen Genet 202:35–41Google Scholar
  19. Nakao T, Yamato I, Anraku Y (1987a) Nucleotide sequence of putP, the proline carrier gene of Escherichia coli K12. Mol Gen Genet 208:70–75Google Scholar
  20. Nakao T, Yamato I, Anraku Y (1987b) Nucleotide sequence of putC, the regulatory region for the put regulon of Escherichia coli K12. Mol Gen Genet 210:364–368Google Scholar
  21. Ninfa AJ, Reitzer LJ, Magasanik B (1987) Identification of transcription at the bacterial glnAp2 promoter by purified E. coli components is facilitated by enhancers. Cell 50:1039–1046Google Scholar
  22. Ow DW, Ausubel FM (1983) Regulation of nitrogen metabolism genes by nifA gene product in Klebsiella pneumoniae. Nature 301:307–313Google Scholar
  23. Pabo CO, Sauer RT (1984) Protein-DNA recognition. Annu Rev Biochem 53:293–321Google Scholar
  24. Prival MJ, Magasanik B (1971) Resistance to catabolite repression of histidase and proline oxidase during nitrogen-limited growth of Klebsiella aerogenes. J Biol Chem 246:6288–6296Google Scholar
  25. Raibaud O, Schwartz M (1984) Positive control of transcription initiation in bacteria. Annu Rev Genet 18:173–206Google Scholar
  26. Silhavy TJ, Berman ML, Enquist LW (1984) Experiments with gene fusions. Cold Springer Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  27. Wood JM (1981) Genetics of L-proline utilization in Escherichia coli. J Bacteriol 146:895–901Google Scholar
  28. Wood JM, Zadworny D (1979) Characterization of an inducible porter required for L-proline catabolism by Escherichia coli K12. Can J Biochem 57:1191–1199Google Scholar
  29. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: Nucleotide sequence of the M13 mp18 and pUC19 vectors. Gene 33:103–119Google Scholar

Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • Toshifumi Nakao
    • 1
  • Ichiro Yamato
    • 1
  • Yasuhiro Anraku
    • 1
  1. 1.Department of Biology, Faculty of ScienceUniversity of TokyoTokyoJapan

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