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Cloning and nucleotide sequence determination of twelve mutant dnaA genes of Escherichia coli

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Summary

Plasmids carrying different regions of the wild-type dnaA gene were used for marker rescue analysis of the temperature sensitivity of twelve strains carrying dnaA mutations. The different dnaA(Ts) mutations could be unambiguously located within specific regions of the dnaA gene. The mutant dnaA genes were cloned on pBR322-derived plasmids and on nucleotide sequencing by dideoxy chain termination the respective mutations were determined using M13 clones carrying the relevant parts of the mutant dnaA gene. Several of the mutant dnaA genes were found to have two mutations. The dnaA5, dnaA46, dnaA601, dnaA602, dnaA604, and dnaA606 genes all had identical mutations corresponding to an amino acid change from alanine to valine at amino acid 184 in the DnaA protein, close to the proposed ATP binding site, but all carried one further mutation giving rise to an amino acid substitution. The dnaA508 gene also had two mutations, whereas dnaA167, dnaA203, dnaA204, dnaA205, and dnaA211 each had only one. The pairs dnaA601/602, dnaA604/606, and dnaA203/204 were each found to have identical mutations. Plasmids carrying the different dnaA mutant genes intact were introduced into the respective dnaA mutant strains. Surprisingly, these homopolyploid mutant strains were found to be temperature resistant in most cases, indicating that a high intracellular concentration of the mutant DnaA protein can compensate for the decreased activity of the protein.

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References

  • Abe M, Tomizawa J (1971) Chromosome replication in E. coli K12 mutant affected in DNA initiation. Genetics 69:1–15

    Google Scholar 

  • Andrup L, Atlung T, Ogasawara N, Yoshikawa H, Hansen FG (1988) Interaction of the Bacillus subtilis DnaA-like protein with the Escherichia coli DnaA protein. J Bacteriol 170:1333–1338

    Google Scholar 

  • Atlung T (1981) Analysis of seven dnaA suppressor loci in E. coli. ICN-UCLA Symp Mol Cell Biol 22:297–314

    Google Scholar 

  • Atlung T (1984) Allele-specific suppression of dnaA(ts) mutations in Escherichia coli. Mol Gen Genet 197:125–128

    Google Scholar 

  • Atlung T, Clausen E, Hansen FG (1985) Autoregulation of the dnaA gene of Escherichia coli. Mol Gen Genet 200:442–450

    Google Scholar 

  • Atlung T, Løbner-Olesen A, Hansen FG (1987) Overproduction of DnaA protein stimulates initiation of chromosome and minichromosome replication in E. coli. Mol Gen Genet 206:51–59

    Google Scholar 

  • Bachmann BJ (1990) Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev 54:130–197

    Google Scholar 

  • Beyersmann D, Messer W, Schlicht M (1974) Mutants of E. coli B/r defective in DNA initiation: dnaI, a new gene for replication. J Bacteriol 118:783–789

    Google Scholar 

  • Braun RE, O'Day K, Wright A (1985) Autoregulation of the DNA replication gene dnaA in E. coli. Cell 40:159–169

    Google Scholar 

  • Braun RE, O'Day K, Wright A (1987) Cloning and characterization of dnaA(Cs), a mutation which leads to overinitiation of DNA replication in Escherichia coli K-12. J Bacteriol 169:3898–3903

    Google Scholar 

  • Carl P (1970) E. coli mutants with temperature sensitive synthesis of DNA. Mol Gen Genet 109:107–122

    Google Scholar 

  • Eberle H, van de Merwe W, Madden K, Kampo G, Wright L, Donlon K (1989) The nature of an intragenic suppressor of the Escherichia coli dnaA508 temperature-sensitive mutation. Gene 84:237–245

    Google Scholar 

  • Fry DC, Kuby SA, Mildvan AS (1986) ATP-binding site of adenylate kinase: Mechanistic implications of its homology with ras-encoded p21, F1-ATPase, and other nucleotide-binding proteins. Proc Natl Acad Sci USA 83:907–911

    Google Scholar 

  • Fujita MQ, Yoshikawa H, Ogasawara N (1989) Structure of the dnaA region of Pseudomonas putida: Conservation among three bacteria, Bacillus subtilis, Escherichia coli and P. putida. Mol Gen Genet 215:381–387

    Google Scholar 

  • Fujita MQ, Yoshikawa H, Ogasawara N (1990) Structure of the dnaA region of Micrococcus luteus: Conservation and variation among eubacteria. Gene 93:73–78

    Google Scholar 

  • Fuller RS, Funnell BE, Kornberg A (1984) The dnaA protein complex with the E. coli chromosomal origin (oriC) and other sites. Cell 38:889–900

    Google Scholar 

  • Hansen EB, Hansen FG, von Meyenburg K (1982) The nucleotide sequence of the dnaA gene and the first part of the dnaN gene of Escherichia coli K12. Nucleic Acids Res 10:7373–7385

    Google Scholar 

  • Hansen EB, Atlung T, Hansen FG, Skovgaard O, von Meyenburg K (1984) Fine structure genetic map and complementation analysis of mutations in the dnaA gene of Escherichia coli. Mol Gen Genet 196:387–396

    Google Scholar 

  • Hansen FG, Rasmussen KV (1977) Regulation of the dnaA product in E. coli. Mol Gen Genet 155:219–225

    Google Scholar 

  • Hansen FG, von Meyenburg K (1979) Characterization of the dnaA, gyrB and other genes in the dnaA region of the Escherichia coli chromosome on specialized transducing phages λ-tna. Mol Gen Genet 175:135–144

    Google Scholar 

  • Hansen FG, Koefoed S, von Meyenburg K, Atlung T (1981) Transcription and translation events in the oriC region of the E. coli chromosome. ICN-UCLA Symp Mol Cell Biol 22:37–55

    Google Scholar 

  • Hansen FG, Hansen EB, Atlung T (1982) The nucleotide sequence of the dnaA gene promoter and of the adjacent rpmH gene, coding for the ribosomal protein L34, of Escherichia coli. EMBO J 1:1043–1048

    Google Scholar 

  • Hansen FG, Atlung T, Braun RE, Wright A, Hughes P, Kohiyama M (1991a) Initiator (DnaA) protein concentration as a function of growth rate in Escherichia coli and Salmonella typhimurium. J Bacteriol 173:5194–5199

    Google Scholar 

  • Hansen FG, Christensen BB, Atlung T (1991b) The initiator titration model: computer simulation of chromosome and minichromosome control. Res Microbiol 142:161–167

    Google Scholar 

  • Hirota Y, Mordoh J, Jacob F (1970) On the process of cellular division in Escherichia coli. III. Thermosensitive mutants of Escherichia coli altered in the process of DNA initiation. J Mol Biol 53:369–387

    Google Scholar 

  • Hwang DS, Kaguni JM (1988) Interaction of dnaA46 protein with a stimulatory protein in replication from the Escherichia coli chromosomal origin. J Biol Chem 263:10633–10640

    Google Scholar 

  • Ingmer H, Atlung T (1992) Expression and regulation of a dnaA homologous gene isolated from Pseudomonas putida. Mol Gen Genet 232:431–439

    Google Scholar 

  • Kellenberger-Gujer G, Podhajska AJ, Caro L (1978) A cold sensitive dnaA mutant of E. coli which overinitiates chromosome replication at low temperature. Mol Gen Genet 162:9–16

    Google Scholar 

  • Kogoma T, Kline BC (1987) Integrative suppression of dnaA(Ts) mutations mediated by plasmid F in E. coli is a DnaA-dependent process. Mol Gen Genet 210:262–269

    Google Scholar 

  • Kohiyama M, Cousin D, Ryter A, Jacob F (1966) Mutants thermosensibles d'E. coli K-12. I. Isolement et characterisation rapide. Ann Inst Pasteur 110:465–486

    Google Scholar 

  • Kücherer C, Lother H, Kölling R, Schauzu MA, Messer W (1986) Regulation of transcription of the chromosomal dnaA gene of Escherichia coli. Mol Gen Genet 205:115–121

    Google Scholar 

  • Lobner-Olesen A, Skarstad K, Hansen FG, von Meyenburg K, Boye E (1989) The DnaA protein determines the initiation mass of Escherichia coli K-12. Cell 57:881–889

    Google Scholar 

  • Mahaffy JM, Zyskind JW (1989) A model for the initiation of replication in Escherichia coli. J Theor Biol 140:453–477

    Google Scholar 

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

    Google Scholar 

  • Norrander J, Kempe T, Messing J (1983) Construction of improved M13 vectors using oligodeoxy-nucleotide directed mutagenesis. Gene 26:101–106

    Google Scholar 

  • Ogasawara N, Fujita MQ, Moriya S, Fukuoka T, Hirano M, Yoshikawa H (1990) Comparative anatomy of oriC of eubacteria. In: Drlica K, Riley M (eds) The bacterial chromosome. American Society for Microbiology, Washington, DC, pp 287–295

    Google Scholar 

  • Ohmori H, Kimura M, Nagata T, Sakakibara Y (1984) Structural analysis of the dnaA and dnaN genes of E. coli. Gene 28:159–170

    Google Scholar 

  • Sakakibara Y, Mizukami T (1980) A temperature sensitive Escherichia coli mutant defective in DNA replication: dnaN, a new gene adjacent to the dnaA gene. Mol Gen Genet 178:541–553

    Google Scholar 

  • Samitt CE, Hansen FG, Miller JF, Schaechter M (1989) In vivo studies of DnaA binding to the origin of replication of Escherichia coli. EMBO J 8:989–993

    Google Scholar 

  • Sanger F, Nicklen S, Coulsen R (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    Google Scholar 

  • Sekimizu K, Bramhill D, Kornberg A (1987) ATP activates dnaA protein in initiating replication of plasmids bearing the origin of the E. coli chromosome. Cell 50:259–265

    Google Scholar 

  • Sevastopoulos CG, Wehr CT, Glaser DA (1977) Large-scale automated isolation of Escherichia coli mutants with thermosensitive DNA replication. Proc Natl Acad Sci USA 74:3485–3489

    Google Scholar 

  • Skarstad K, von Meyenburg K, Hansen FG, Boye E (1988) Coordination of chromosome replication initiation in Escherichia coli: effects of different dnaA alleles. J Bacteriol 170:852–858

    Google Scholar 

  • Skovgaard O (1990) Nucleotide sequence of a Proteus mirabilis fragment homologous to the 60K-rnpA-rpmH-dnaA-dnaNrecF-gyrB region of Escherichia coli. Gene 93:27–34

    Google Scholar 

  • Skovgaard O, Hansen FG (1987) Comparison of dnaA nucleotide sequences of Escherichia coli, Salmonella typhimurium, and Serratia marcescens. J Bacteriol 169:3976–3981

    Google Scholar 

  • von Meyenburg K, Hansen FG (1987) Regulation of chromosome replication. In: Neidhardt FC, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella typhimurium: Cellular and molecular biology. American Society for Microbiology, Washington, DC, pp 1555–1577

    Google Scholar 

  • von Meyenburg K, Jørgensen BB, Nielsen J, Hansen FG (1982) Promoters of the atp operon coding for the membrane-bound ATP synthase of Escherichia coli mapped by Tn10 insertion mutations. Mol Gen Genet 188:240–248

    Google Scholar 

  • von Meyenburg K, Hansen FG, Atlung T, Boe L, Clausen IG, van Deurs B, Hansen EB, Jorgensen BB, Jorgensen F, Koppes L, Michelsen O, Nielsen J, Pedersen PE, Rasmussen KV, Riise E, Skovgaard O (1985) Facets of the chromosomal origin of replication oriC of E. coli. In: Schaechter M, Neidhardt FC, Ingraham J, Kjeldgaard NO (eds) The molecular biology of bacterial growth. Jones & Bartlett, Boston, pp 260–281

    Google Scholar 

  • Wechsler JA, Gross JD (1971) Escherichia coli mutants temperature sensitive for DNA synthesis. Mol Gen Genet 113:273–284

    Google Scholar 

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Authors and Affiliations

  1. Department of Microbiology, The Technical University of Denmark, DK-2800, Lyngby, Denmark

    Flemming G. Hansen, Sos Koefoed & Tove Atlung

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  1. Flemming G. Hansen
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  2. Sos Koefoed
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  3. Tove Atlung
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Communicated by R. Devoret

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Hansen, F.G., Koefoed, S. & Atlung, T. Cloning and nucleotide sequence determination of twelve mutant dnaA genes of Escherichia coli . Molec. Gen. Genet. 234, 14–21 (1992). https://doi.org/10.1007/BF00272340

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  • DOI: https://doi.org/10.1007/BF00272340

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