Molecular and General Genetics MGG

, Volume 215, Issue 3, pp 483–489 | Cite as

Mutation of the promoter and LexA binding sites of cea, the gene encoding colicin E1

  • Bernard Salles
  • George M. Weinstock
Article

Summary

Three mutations were introduced into the cea promoter using oligonucleotide directed mutagenesis. The resulting mutant promoter has the Escherichia coli consensus sequences at its-35 and-10 positions, separated by the optimal spacing. In addition, a plasmid with a mutation in one of the two LexA repressor binding sites in the cea regulatory region was isolated that decreases homology with the consensus LexA binding site. The effects of these mutations on cea expression were studied in cea-lacZ protein fusions. The promoter-up mutant, when present in a multicopy plasmid, showed a shorter induction lag when compared to the wild-type cea gene, and there was less of an effect of the catabolite repression system on cea expression. However, when present in a single copy in the bacterial chromosome, catabolite repression and an induction delay were observed, despite the increased strength of the promoter. The operator mutant showed a slightly higher basal level of expression, but was still repressible. Induction occurred with a shortened lag period, but the effects were not as great as with the promoter mutant. These results support the idea that tight repression by LexA contributes to the delay in cea induction.

Key words

SOS response ColE1 plasmid Catabolite repression Gene fusion RecA 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Casadaban MJ (1976) Transposition and fusion of the lac genes to selected promoters in Escherichia coli using bacteriphage lambda and Mu. J Mol Biol 104:541–555Google Scholar
  2. Chan PT, Ohmori H, Tomizawa J, Lebowitz J (1985) Nucleotide sequence and gene organization of ColE1 DNA. J Biol Chem 260:8925–8935Google Scholar
  3. Ebina Y, Kishi F, Miki T, Kagamiyama H, Nakazawa T, Nakazawa A (1981) The nucleotide sequence surrounding the promoter region of the colicin E1 gene. Gene 15:119–126Google Scholar
  4. Ebina Y, Takahara Y, Kishi F, Nakazawa A, Brent R (1983) LexA protein is a repressor of the colicin E1 gene. J Biol Chem 258:13258–13261Google Scholar
  5. Guidi-Rontani C, Danchin A, Ullmann A (1984) Transcriptional control of polarity in Escherichia coli by cAMP. Mol Gen Genet 195:96–100Google Scholar
  6. Konisky J (1982) Colicins and other bacteriocins with established modes of action. Annu Rev Microbiol 36:125–144Google Scholar
  7. Lotz W (1978) Effect of guanosine tetraphosphate on in vitro protein synthesis directed by E1 and E3 colicinogenic factors. J Bacteriol 135:707–712Google Scholar
  8. Mandecki W, Goldman RA, Powell BS, Caruthers MH (1985) lac up-promoter mutants with increased homology to the consensus promoter sequence. J Bacteriol 164:1353–1355Google Scholar
  9. Markham BE, Harper JE, Mount DW, Sancar GB, Sancar A, Rupp WD, Kenyon CJ, Walker GC (1984) Analysis of mRNA synthesis following induction of the Escherichia coli SOS system. J Mol Biol 178:237–248Google Scholar
  10. McClure WR (1985) Mechanism and control of transcription initiation in prokaryotes. Annu Rev Biochem 54:171–204Google Scholar
  11. Nakazawa A, Tamada T (1972) Stimulation of colicin E1 synthesis by cyclic 3′,5′-adenosine monophosphate in mitomycin C-induced Escherichia coli. Biochem Biophys Res Commun 46:1004–1010Google Scholar
  12. Parker RC (1983) The 5′-terminal ribonucleotide of an in vitro ColE1 transcript is not complementary to the DNA template. Gene 26:127–136Google Scholar
  13. Sabik JF, Suit JL, Luria SE (1983) cea-kil operon of the ColE1 plasmid. J Bacteriol 153:1479–1485Google Scholar
  14. Salles B, Weisemann JM, Weinstock GM (1987) Temporal control of colicin E1 induction. J Bacteriol 169:5028–5034Google Scholar
  15. Silhavy TJ, Berman ML, Enquist LW (1984) Experiments with gene fusions. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  16. Waleh NS, Johnson PH (1985) Structural and functional organization of the colicin E1 operon. Proc Natl Acad Sci USA 82:8389–8393Google Scholar
  17. Walker GC (1987) The SOS response of Escherichia coli. In: Neidhardt FC (ed) Escherichia coli and Salmonella typhimurium. Cellular and Molecular Biology. American Society for Microbiology, Washington DC, pp 1346–1357Google Scholar
  18. Weisemann JM, Weinstock GM (1985a) Use of transcription and translation signals from the colicin E1 gene to express DNA sequences in Escherichia coli. Genet Anal Technol 2:9–16Google Scholar
  19. Weisemann JM, Weinstock GM (1985b) Direct selection of mutations reducing transcription or translation of the recA gene of Escherichia coli with a recA-lacZ protein fusion. J Bacteriol 163:748–755Google Scholar
  20. Weisemann JM, Funk C, Weinstock GM (1984) Measurement of in vivo expression of the recA gene of Escherichia coli by using lacZ gene fusions. J Bacteriol 160:112–121Google Scholar
  21. Wertman KF, Mount DW (1985) Nucleotide sequence binding specificity of the LexA repressor of Escherichia coli K-12. J Bacteriol 163:376–384Google Scholar
  22. Yang HL, Heller K, Gellert M, Zubay G (1979) Differential sensitivity of gene expression in vitro to inhibitors of DNA gyrase. Proc Natl Acad Sci USA 76:3304–3308Google Scholar
  23. Zhang SP, Faro A, Zubay G (1985) Mitomcycin-induced lethality of Escherichia coli cells containing the ColE1 plasmid: involvement of the kil gene. J Bacteriol 163:174–179Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Bernard Salles
    • 1
  • George M. Weinstock
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Texas Medical SchoolHoustonUSA

Personalised recommendations