Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1985 Aug;163(2):688–695. doi: 10.1128/jb.163.2.688-695.1985

Isolation of protease-proficient, recombinase-deficient recA mutants of Escherichia coli K-12.

E S Tessman, P K Peterson
PMCID: PMC219176  PMID: 3160687

Abstract

We isolated recA mutants with altered protease activity and then examined recombinase activity to determine whether the protease and recombinase functions of the RecA protein of Escherichia coli are separable. We found five mutants that had moderately strong constitutive RecA protease activity but no recombinase activity above the delta recA strain background, the first clear-cut examples of mutants of this class, designated Prtc Rec-. We also isolated 65 mutants that were protease-defective toward the LexA repressor and found that all of them were also recombinase deficient. Four of these mutants retained both partial recombinase activity and partial inducible protease activity. The recombinase-defective mutants were much more sensitive than the recA+ strain to crystal violet, kanamycin, and chloramphenicol, indicating altered membrane permeability. The recA (Prtc Rec-) mutants had a subtle alteration in protease specificity, all being defective in spontaneous induction of phages lambda imm434 and 21. They differed from Prtc Rec+ mutants of comparable or even weaker constitutive protease strength, all of which showed dramatic spontaneous induction of these prophages. However, treating a Prtc Rec- mutant with mitomycin C resulted in significant prophage induction. Thus, the RecA proteins of the Prtc Rec- mutants have constitutive protease activity toward the LexA repressor, but have only DNA damage-activable protease activity toward phage repressors. UV-induced mutagenesis from his to his+ was studied for one Prtc Rec- mutant, and induced mutation frequencies as high as those for the recA+ strain were found despite the absence of recombinase activity.

Full text

PDF
695

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blanco M., Herrera G., Collado P., Rebollo J. E., Botella L. M. Influence of RecA protein on induced mutagenesis. Biochimie. 1982 Aug-Sep;64(8-9):633–636. doi: 10.1016/s0300-9084(82)80102-8. [DOI] [PubMed] [Google Scholar]
  2. CLARK A. J., MARGULIES A. D. ISOLATION AND CHARACTERIZATION OF RECOMBINATION-DEFICIENT MUTANTS OF ESCHERICHIA COLI K12. Proc Natl Acad Sci U S A. 1965 Feb;53:451–459. doi: 10.1073/pnas.53.2.451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Clark A. J. Recombination deficient mutants of E. coli and other bacteria. Annu Rev Genet. 1973;7:67–86. doi: 10.1146/annurev.ge.07.120173.000435. [DOI] [PubMed] [Google Scholar]
  4. Cox M. M., Lehman I. R. recA protein of Escherichia coli promotes branch migration, a kinetically distinct phase of DNA strand exchange. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3433–3437. doi: 10.1073/pnas.78.6.3433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Craig N. L., Roberts J. W. Function of nucleoside triphosphate and polynucleotide in Escherichia coli recA protein-directed cleavage of phage lambda repressor. J Biol Chem. 1981 Aug 10;256(15):8039–8044. [PubMed] [Google Scholar]
  6. D'Ari R., Huisman O. Novel mechanism of cell division inhibition associated with the SOS response in Escherichia coli. J Bacteriol. 1983 Oct;156(1):243–250. doi: 10.1128/jb.156.1.243-250.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Devoret R., Pierre M., Moreau P. L. Prophage phi 80 is induced in Escherichia coli K12 recA430. Mol Gen Genet. 1983;189(2):199–206. doi: 10.1007/BF00337804. [DOI] [PubMed] [Google Scholar]
  8. Dutreix M., Bailone A., Devoret R. Efficiency of induction of prophage lambda mutants as a function of recA alleles. J Bacteriol. 1985 Mar;161(3):1080–1085. doi: 10.1128/jb.161.3.1080-1085.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Froehlich B., Epstein W. Escherichia coli mutants in which transcription is dependent on recA function. J Bacteriol. 1981 Sep;147(3):1117–1120. doi: 10.1128/jb.147.3.1117-1120.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Huisman O., D'Ari R. An inducible DNA replication-cell division coupling mechanism in E. coli. Nature. 1981 Apr 30;290(5809):797–799. doi: 10.1038/290797a0. [DOI] [PubMed] [Google Scholar]
  11. Kenyon C. J., Walker G. C. DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli. Proc Natl Acad Sci U S A. 1980 May;77(5):2819–2823. doi: 10.1073/pnas.77.5.2819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Little J. W. Autodigestion of lexA and phage lambda repressors. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1375–1379. doi: 10.1073/pnas.81.5.1375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Little J. W., Mount D. W. The SOS regulatory system of Escherichia coli. Cell. 1982 May;29(1):11–22. doi: 10.1016/0092-8674(82)90085-x. [DOI] [PubMed] [Google Scholar]
  14. Morand P., Blanco M., Devoret R. Characterization of lexB mutations in Escherichia coli K-12. J Bacteriol. 1977 Aug;131(2):572–582. doi: 10.1128/jb.131.2.572-582.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Quillardet P., Moreau P. L., Ginsburg H., Mount D. W., Devoret R. Cell survival, UV-reactivation and induction of prophage lambda in Escherichia coli K12 overproducing RecA protein. Mol Gen Genet. 1982;188(1):37–43. doi: 10.1007/BF00332993. [DOI] [PubMed] [Google Scholar]
  16. Radman M. SOS repair hypothesis: phenomenology of an inducible DNA repair which is accompanied by mutagenesis. Basic Life Sci. 1975;5A:355–367. doi: 10.1007/978-1-4684-2895-7_48. [DOI] [PubMed] [Google Scholar]
  17. Rebollo J. E., Moreau P. L., Blanco M., Devoret R. Restoration of RecA protein activity by genetic complementation. Mol Gen Genet. 1984;195(1-2):83–89. doi: 10.1007/BF00332728. [DOI] [PubMed] [Google Scholar]
  18. Roberts J. W., Roberts C. W. Two mutations that alter the regulatory activity of E. coli recA protein. Nature. 1981 Apr 2;290(5805):422–424. doi: 10.1038/290422a0. [DOI] [PubMed] [Google Scholar]
  19. Rupp W. D., Howard-Flanders P. Discontinuities in the DNA synthesized in an excision-defective strain of Escherichia coli following ultraviolet irradiation. J Mol Biol. 1968 Jan 28;31(2):291–304. doi: 10.1016/0022-2836(68)90445-2. [DOI] [PubMed] [Google Scholar]
  20. Rupp W. D., Wilde C. E., 3rd, Reno D. L., Howard-Flanders P. Exchanges between DNA strands in ultraviolet-irradiated Escherichia coli. J Mol Biol. 1971 Oct 14;61(1):25–44. doi: 10.1016/0022-2836(71)90204-x. [DOI] [PubMed] [Google Scholar]
  21. Shibata T., DasGupta C., Cunningham R. P., Williams J. G., Osber L., Radding C. M. Homologous pairing in genetic recombination. The pairing reaction catalyzed by Escherichia coli recA protein. J Biol Chem. 1981 Jul 25;256(14):7565–7572. [PubMed] [Google Scholar]
  22. Smith K. C., Meun D. H. Repair of radiation-induced damage in Escherichia coli. I. Effect of rec mutations on post-replication repair of damage due to ultraviolet radiation. J Mol Biol. 1970 Aug;51(3):459–472. doi: 10.1016/0022-2836(70)90001-x. [DOI] [PubMed] [Google Scholar]
  23. Tessman E. S., Peterson P. Plaque color method for rapid isolation of novel recA mutants of Escherichia coli K-12: new classes of protease-constitutive recA mutants. J Bacteriol. 1985 Aug;163(2):677–687. doi: 10.1128/jb.163.2.677-687.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Walker G. C. Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev. 1984 Mar;48(1):60–93. doi: 10.1128/mr.48.1.60-93.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Witkin E. M. Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol Rev. 1976 Dec;40(4):869–907. doi: 10.1128/br.40.4.869-907.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yarranton G. T., Sedgwick S. G. Cloned truncated recA genes in E. coli II. Effects of truncated gene products on in vivo recA+ protein activity. Mol Gen Genet. 1982;185(1):99–104. doi: 10.1007/BF00333797. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES