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Measurement of in vivo expression of nrdA and nrdB genes of Escherichia coli by using IacZ gene fusions

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By using a promoter probe plasmid we investigated expression of the linked nrdA and nrdB genes coding for the two different subunits of the ribonucleoside diphosphate reductase enzyme of Escherichia coli. For this reason, nrdA-lacZ, nrdAB-lacZ and nrdB-lacZ fusions were constructed. Results obtained indicate that the nrdB gene has a promoter from which it may be transcribed independently of the nrdA gene. Furthermore, the nrdB gene may also be transcribed from the nrdA promoter. The expression of the nrdB gene is about 14-fold higher from the nrdA promoter than from its own promoter. The induction of both nrdA and nrdB genes by DNA-damaging agents in the wild-type strain as well as in several SOS mutants was also studied; nrdA gene expression was increased by these treatments in RecA–, RecA−, and LexAInd− strains, although in both RecA− and LexAInd− mutants the nrdA gene expression was considerably lower than that in RecA– cells. nrdB gene expression was stimulated by DNA damage only when its transcription was from the nrdA promoter, but there was no effect when nrdB was transcribed from its own promoter. In addition, the basal level of nrdA-lacZ and nrdAB-lacZ fusions was reduced in strains containing either RecA− and LexAInd− mutations or a multicopy plasmid carrying the lexA – gene, whereas the presence of a LexA5lDef mutation increased the constitutive expression of both fusions. On the contrary, the basal level of the nrdB-lacZ fusion remained constant in all these strains. Together these results indicate that induction of the SOS response enhances expression of the nrd genes from the nrdA promoter.

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References

  • Barbé J, Vericat JA, Cairó J, Guerrero R (1985) Further characterization of SOS system induction in recBC mutants of Escherichia coli. Mutat Res 146:23–32

    Google Scholar 

  • Barbé J, Villaverde A, Cairó J, Guerrero R (1986) ATP hydrolysis during SOS induction in Escherichia coli. J Bacteriol 167:1055–1057

    Google Scholar 

  • Barbé J, Villaverde A, Guerrero R (1987) Induction of the SOS response by hydroxyurea in Escherichia coli K12. Mutat Res 192:105–108

    Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1525

    Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Google Scholar 

  • Brown NC, Reichard P (1969) Role of effector binding in allosteric control of ribonucleoside diphosphate reductase. J Mol Biol 46:39–55

    Google Scholar 

  • Carlson J, Fuchs JA, Messing J (1984) Primary structure of the Escherichia coli ribonucleoside diphosphate reductase operon. Proc Natl Acad Sci USA 81:4294–4297

    Google Scholar 

  • Casaregola S, D'Ari R, Huisman O (1982) Quantitative evaluation of recA gene expression in Escherichia coli. Mol Gen Genet 185:430–439

    Google Scholar 

  • Clark DJ, Maaløe O (1967) DNA replication and the division cycle of Escherichia coli. J Mol Biol 23:99–112

    Google Scholar 

  • Craig NL, Roberts JW (1980) E. coli recA protein-directed cleavage of phage λ repressor requires polynucleotide. Nature 283:26–30

    Google Scholar 

  • Filpula D, Fuchs JA (1977) Regulation of ribonucleoside diphosphate reductase synthesis in Escherichia coli: increased enzyme synthesis as a result of inhibition of deoxyribonucleic acid synthesis. J Bacteriol 130:107–113

    Google Scholar 

  • Fontecave M, Eliasson R, Reichard P (1989) Oxygen-sensitive ribonucleoside triphosphate reductase is present in anaerobic Escherichia coli. Proc Natl Acad Sci USA 86:2147–2151

    Google Scholar 

  • Fuchs JA (1977) Coordinate control of the synthesis of ribonucleoside diphosphate reductase components in Escherichia coli. J Bacteriol 130:957–959

    Google Scholar 

  • Gibert I, Llagostera M, Barbé J (1988) Regulation of ubiG gene expression in Escherichia coli. J Bacteriol 170:1346–1349

    Google Scholar 

  • Hanke PD, Fuchs JA (1983a) Regulation of ribonucleoside diphosphate reductase mRNA synthesis in Escherichia coli. J Bacteriol 154:1040–1045

    Google Scholar 

  • Hanke PD, Fuchs JA (1983b) Characterization of the mRNA coding for ribonucleoside diphosphate reductase in Escherichia coli. J Bacteriol 156:1192–1197

    Google Scholar 

  • Hanke PD, Fuchs JA (1984) Requirement of protein synthesis for the induction of ribonucleoside diphosphate reductase mRNA in Escherichia coli. Mol Gen Genet 193:327–331

    Google Scholar 

  • Hantke K (1988) Characterization of an iron sensitive Mudl mutant in E. coli lacking the ribonucleoside reductase subunit B2. Arch Microbiol 149:344–349

    Google Scholar 

  • Ihara M, Yamamoto K, Ohnishi T (1987) Induction of phr gene expression by irradiation of ultraviolet light in Escherichia coli. Mol Gen Genet 209:200–202

    Google Scholar 

  • Jamison CS, Adler HI (1987) Mutations in Escherichia coli that effect sensitivity to oxygen. J Bacteriol 169:5087–5094

    Google Scholar 

  • Kren B, Fuchs JA (1987) Characterization of the ftsB gene as an allele of the nrdB gene in Escherichia coli. J Bacteriol 169:14–18

    Google Scholar 

  • Lieberman HB, Witkin EM (1983) DNA degradation, UV sensitivity and SOS-mediated mutagenesis in strains of Escherichia coli deficient in single-strand binding protein: effects of mutation and treatments that alter levels of exonuclease V or RecA protein. Mol Gen Genet 190:92–100

    Google Scholar 

  • Little JW (1984) Autodigestion of lexA and phage λ repressor. Proc Natl Acad Sci USA 58:1903–1910

    Google Scholar 

  • Little JW, Mount DW (1982) The SOS regulatory system of Escherichia coli. Cell 29:11–22

    Google Scholar 

  • Little JW, Edmiston SH, Pazelli LZ, Mount DW (1980) Cleavage of the Escherichia coli lexA protein by the recA protease. Proc Natl Acad Sci USA 77:3225–3229

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Mulligan ME, Hawley DK, Entriken R, McClure RM (1984) Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity. Nucleic Acids Res 12:789–800

    Google Scholar 

  • Oishi M, Smith CL, Friefeld B (1978) Molecular events and molecules that lead to induction of prophage and SOS functions. Cold Spring Harbor Symp Quant Biol 43:897–907

    Google Scholar 

  • Peterson KR, Mount DW (1987) Differential repression of SOS genes by unstable LexA41 (Tsl-1) protein causes a “split-phenotype” in Escherichia coli K-12. J Mol Biol 193:27–40

    Google Scholar 

  • Platz A, Sjöberg BM (1980) Construction and characterization of hybrid plasmids containing the Escherichia coli nrd region. J Bacteriol 143:561–568

    Google Scholar 

  • Quigley NB, Reeves PR (1987) Chloramphenicol resistance cloning vector based on pUC9. Plasmid 17:54–57

    Google Scholar 

  • Salles B, Defais M (1984) Signal of induction of RecA protein in Escherichia coli. Mutat Res 131:53–59

    Google Scholar 

  • Samuni A (1975) A direct spectrophotometric assay and determination of Michaelis constants for the β-lactamase reaction. Anal Biochem 63:17–26

    Google Scholar 

  • Schneider K, Beck CF (1987) New expression vectors for identifying and testing signal structures for initiation and termination of transcription. Methods Enzymol 153:452–461

    Google Scholar 

  • Sinha NK, Snustad DP (1972) Mechanism of inhibition of deoxyribonucleic acid synthesis in Escherichia coli by hydroxyurea. J Bacteriol 112:1321–1334

    Google Scholar 

  • Taschner PEM, Verest JGJ, Woldring CL (1987) Genetic and morphological characterization of ftsB and nrdB mutants of Escherichia coli. J Bacteriol 169:19–25

    Google Scholar 

  • Tessman ES, Peterson P (1985) Plaque color method for rapid isolation of novel recA mutants of Escherichia coli K-12: new classes of protease-constitutive recA mutants. J Bacteriol 163:677–687

    Google Scholar 

  • Thelander L, Reichard P (1979) Reduction of ribonucleotides. Annu Rev Biochem 48:133–158

    Google Scholar 

  • Thomas A, Lloyd RG (1983) Control of recA dependent activation in Escherichia coli: a possible role for the recF product. J Gen Microbiol 129:681–686

    Google Scholar 

  • Tuggle KC, Fuchs JA (1986) Regulation of the operon encoding ribonucleotide reductase in Escherichia coli: evidence for both positive and negative control. EMBO J 5:1077–1085

    Google Scholar 

  • von Döbeln U, Reichard P (1976) Binding of substrates to Escherichia coli ribonucleotide reductase. J Biol Chem 251:3616–3622

    Google Scholar 

  • Walker GW (1984) Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. Microbiol Rev 48:90–93

    Google Scholar 

  • Weinstock GM, McEntee K (1981) RecA protein dependent proteolysis of bacteriophage lambda repressor. Characterization of the reaction and stimulation by DNA-binding proteins. J Biol Chem 256:10883–10888

    Google Scholar 

Download references

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Authors and Affiliations

  1. Department of Genetics and Microbiology, Faculty of Sciences, Autonomous University of Barcelona, Bellaterra, 08193, Barcelona, Spain

    Isidre Gibert, Sebastian Calero & Jordi Barbé

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  1. Isidre Gibert
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  2. Sebastian Calero
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  3. Jordi Barbé
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Communicated by R. Devoret

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Gibert, I., Calero, S. & Barbé, J. Measurement of in vivo expression of nrdA and nrdB genes of Escherichia coli by using IacZ gene fusions. Mol Gen Genet 220, 400–408 (1990). https://doi.org/10.1007/BF00391745

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  • DOI: https://doi.org/10.1007/BF00391745

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