Skip to main content
SpringerLink
Log in

A novel type of E. coli mutants with increased chromosomal copy number

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Summary

We have isolated E. coli mutants which can grow at 30°C but not at 42°C and are able to harbor the oriC plasmid (minichromosome) at a higher copy number than the parental wild-type strain at the permissive temperature. The mutants were found to contain higher amounts of chromosomal DNA per mg protein than the wild-type, whether or not they harbor the plasmid. Experimental results suggest that the higher amount of chromosomal DNA is due to a higher copy number of chromosomes and not to a larger amount of DNA per chromosome. These properties in each of the mutants are caused by a single mutation at the rpoB or rpoC gene that code for the β or β′ subunit of RNA polymerase, respectively. The mutations are thought to affect the regulation of replication of oriC-bearing replicons, that is, the E. coli chromosome and oriC plasmids, but not the miniF plasmid.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

SDS:

sodium dodecyl sulfate

kb:

kilobase pairs

TCA:

trichloroacetic acid

References

  • Bachmann BJ, Low KB (1980) Linkage map of Escherichia coli K-12. Microbiol Rev 44:1–56

    Google Scholar 

  • Bagdasarian MM, Izakowska M, Bagdasarian M (1977) Suppression of the DnaA phenotype by mutations in the rpoB cistron of ribonucleic acid polymerase in Salmonella typhimurium and Escherichia coli. J Bacteriol 130:577–582

    Google Scholar 

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

    Google Scholar 

  • Bradford M (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 

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

    Google Scholar 

  • Cohen SN, Chang ACY (1973) Recircularization and autonomous replication of a sheared R-factor DNA segment in Escherichia coli transformants. Proc Natl Acad Sci USA 70:1293–1297

    Google Scholar 

  • Donachie WD (1968) Relationship between cell size and time of initiation of DNA replication. Nature (London) 219:1077–1079

    Google Scholar 

  • Fuller RS, Kaguni JM, Kornberg A (1981) Enzymatic replication of the origin of the Escherichia coli chromosome. Proc Natl Acad Sci USA 78:7370–7374

    Google Scholar 

  • Giles KW, Myers A (1965) An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature (London) 206:93

    Google Scholar 

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

    Google Scholar 

  • Hatfield D, Hofnung M, Schwartz M (1969) Genetic analysis of the maltose A region in Escherichia coli. J Bacteriol 98:559–567

    Google Scholar 

  • Helmstetter CE, Cooper S (1968) DNA synthesis during the division cycle of rapidly growing Escherichia coli B/r. J Mol Biol 31:507–518

    Google Scholar 

  • Hiraga S, Saitoh T (1974) Initiation of DNA replication in Escherichia coli. I. Characteristics of the initiation process in dna mutants. Mol Gen Genet 132:49–62

    Google Scholar 

  • Hirota Y, Ryter A, Jacob F (1968) Thermosensitive mutants of E. coli affected in the processes of DNA synthesis and cellular division. Cold Spring Harbor Symp Quant Biol 33:677–693

    Google Scholar 

  • Hopkins AS, Murray NE, Brammar WJ (1976) Characterization of λtrp-transducing bacteriophages made in vitro. J Mol Biol 107:549–569

    Google Scholar 

  • Horiuchi T, Nagata T (1973) Mutations affecting growth of the Escherichia coli cell under a condition of DNA polymerase I-deficiency. Mol Gen Genet 123:89–110

    Google Scholar 

  • Ikeda H, Tomizawa J (1965) Transducing fragments in generalized transduction by phage P1. I. Molecular origin of the fragmonts. J Mol Biol 14:85–109

    Google Scholar 

  • Ikeuchi T, Yura T, Yamagishi H (1975) Genetic and physical studies of lambda transducing bacteriophage carrying the beta subunit gene of the Escherichia coli ribonucleic acid polymerase. J Bacteriol 122:1247–1256

    Google Scholar 

  • Ishihama A, Fukuda R (1980) Autogenous and post-transcriptional regulation of RNA polymerase synthesis. Mol Cell Biochem 31:177–196

    Google Scholar 

  • Kline BC (1973) Role of DNA transcription in the initiation of Escherichia coli sex factor (F) DNA replication. Biochem Biophys Res Commun 50:280–288

    Google Scholar 

  • Kuempel PL, Duerr SA, Seeley NR (1977) Terminus region of the chromosome in Escherichia coli inhibits replication forks. Proc Natl Acad Sci USA 74:3927–3931

    Google Scholar 

  • Lark KG (1972) Evidence for the direct involvement of RNA in the initiation of DNA replication in Escherichia coli 15T-. J Mol Biol 64:47–60

    Google Scholar 

  • Low B (1968) Formation of merodiploids in matings with a class of Rec- recipient strains of Escherichia coli K12. Proc Natl Acad Sci USA 60:160–167

    Google Scholar 

  • Maaløe O, Kjeldgaard NO (1966) Control of macromolecular synthesis. WA Benjamin Inc, New York

    Google Scholar 

  • Messer W (1972) Initiation of deoxyribonucleic acid replication in Escherichia coli B/r: Chronology of events and transcriptional control of initiation. J Bacteriol 112:7–12

    Google Scholar 

  • von Meyenburg K, Hansen FG, Riise E, Bergmans HEN, Meijer M, Messer W (1979) Origin of replication, oriC, of the Escherichia coli K12 chromosome: Genetic mapping and minichromosome replication. Cold Spring Harbor Symp Quant Biol 43:121–128

    Google Scholar 

  • Meyers JA, Sanchez D, Elwell LP, Falkow S (1976) Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J Bacteriol 127:1529–1573

    Google Scholar 

  • Miki T (1978) Genetic and physical analyses of the replication origin and dnaA gene of the Escherichia coli chromosome by molecular cloning and cleavage mapping with restriction endonucleases. Annu Rep Inst Virus Res Kyoto Univ 21:1–26

    Google Scholar 

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

    Google Scholar 

  • Nakamura Y, Yura T (1973) Localization of the structural gene for the β′ subunit of RNA polymerase in Escherichia coli. Biochem Biophys Res Commun 53:645–652

    Google Scholar 

  • Newman CN, Kubitschek HE (1978) Variation in periodic replication of the chromosome in Escherichia coli B/rTT. J Mol Biol 121:461–471

    Google Scholar 

  • Ogura T, Miki T, Hiraga S (1980) Copy-number mutants of the plasmid carrying the replication origin of the Escherichia coli chromosome: Evidence for a control region of replication. Proc Natl Acad Sci USA 77:3993–3997

    Google Scholar 

  • Ogura T, Hiraga S (1983a) Partition mechanism of F plasmid: Two plasmid gene-encoded products and a cis-acting region are involved in partition. Cell 32:351–360

    Google Scholar 

  • Ogura T, Hiraga S (1983b) MiniF plasmid genes that couple host cell division to plasmid proliferation. Proc Natl Acad Sci USA

  • Osawa T, Nakamura Y, Ikeuchi T, Yura T (1975) Specialized transduction of a structural gene of RNA polymerase in E. coli. III. Identification of the host gene of λdrif + phage. Jpn J Genet 50:489–490

    Google Scholar 

  • Pritchard RH, Barth PT, Collins J (1969) Control of DNA synthesis in bacteria. Symp Soc Gen Microbiol XIX; p 263–297

    Google Scholar 

  • Pritchard RH, Grover NB (1981) Control of plasmid replication and its relationship to incompatibility. In: Levy SB, Clowes RS, Koenig EL (eds) Molecular biology, pathogenecity, and ecology of bacterial plasmid. Plenum Publishing Corporation, New York, p 271–278

    Google Scholar 

  • Rasmussen KV, Atlung T, Kerszman G, Hansen GE, Hansen FG (1983) Conditional change of DNA replication control in an RNA polymerase mutant of Escherichia coli. J Bacteriol 154:443–451

    Google Scholar 

  • Sompayrac L, Maaløe O (1973) Autorepressor model for control of DNA replication. Nature (London) New Biol 241:133–135

    Google Scholar 

  • Timmis K, Cabello F, Cohen SN (1975) Cloning, isolation, and characterization of replication regions of complex plasmid genomes. Proc Natl Acad Sci USA 72:2242–2246

    Google Scholar 

  • Timmis K, Cabello F, Cohen SN (1978) Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6. Mol Gen Genet 162:121–137

    Google Scholar 

  • Uhlin BE, Nordström K (1977) R plasmid gene dosage effects in Escherichia coli K-12: Copy mutants of the R plasmid R1drd19 Plasmid 1:1–7

    Google Scholar 

  • Wada C, Yura T (1974) Phenethyl alcohol resistance in Escherichia coli. III. A temperature-sensitive mutation (dnaP) affecting DNA replication. Genetics 77:199–220

    Google Scholar 

  • Womble DD, Taylor DP, Rownd RH (1977) Method for obtaining more-accurate covalently closed circular plasmid-to-chromosome ratios from bacterial lysates by dye-buoyant density centrifugation. J Bacteriol 130:148–153

    Google Scholar 

  • Yamaguchi K, Tomizawa J (1980) Establishment of Escherichia coli cells with an integrated high copy number plasmid. Mol Gen Genet 178:525–533

    Google Scholar 

  • Yanofsky C, Platt T, Crawford IP, Nichols BP, Christie GE, Horowitz H, Van Cleemput M, Wu AM (1981) The complete nucleotide sequence of the tryptophan operon of Escherichia coli. Nucleic Acids Res 9:6647–6668

    Google Scholar 

  • Zyskind JW, Smith DW (1977) Novel Escherichia coli dnaB mutant: Direct involvement of the dnaB252 gene product in the synthesis of an origin-ribonucleic acid species during initiation of a round of deoxyribonucleic acid replication. J Bacteriol 129:1476–1486

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute for Virus Research, Kyoto University, 606, Kyoto, Japan

    Masafumi Tanaka, Haruo Ohmori & Sota Hiraga

Authors
  1. Masafumi Tanaka
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Haruo Ohmori
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sota Hiraga
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by M. Takanami

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tanaka, M., Ohmori, H. & Hiraga, S. A novel type of E. coli mutants with increased chromosomal copy number. Molec Gen Genet 192, 51–60 (1983). https://doi.org/10.1007/BF00327646

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00327646

Keywords

Search

Navigation