Skip to main content
Log in

Isolation and characterization of a conditional replication mutant of the antibiotic resistance factor R1 affected in the gene of the replication protein RepA

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

Summary

In vitro mutagenesis with hydroxylamine of a ParD- miniderivative of R1, pAB174, yielded mutants that were less stable in the cell than pAB174. Some of these mutants had a thermosensitive phenotype. The replication of pAB2623, one of the thermosensitive mutants, was inhibited in the cell at the restrictive temperature of 42° C. The efficiency of the RepA protein of pAB2623 to promote replication of R1 in an in vitro assay was greatly reduced. Sequence analysis indicated that the repA gene of pAB2623 contains, close to its 3′ end, two GC-AT transitions, separated by a single base, that change two consecutive codons of the gene. These results indicate that the phenotype of the mutant is the consequence of a mutated RepA protein and is consistent with the requirement of RepA for the in vivo replication of this plasmid.

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

Access this article

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

References

  • Bachmann BJ (1972) Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev 36:525–555

    Google Scholar 

  • Bravo A, de Torrontegui G, Diaz R (1987) Identification of components of a new stability system of plasmid R1, ParD, that is close to the origin of replication of this plasmid. Mol Gen Genet 210:101–110

    Google Scholar 

  • Brawner ME, Jaskunas SR (1982) Identification of polypeptides encoded by the replication region of R100. J Mol Biol 159:35–55

    Google Scholar 

  • Chou P, Fasman GD (1978) Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol 57:45–148

    Google Scholar 

  • Clark JD, Maaloe O (1967) DNA replication and the cell cycle in Escherichia coli. J Mol Biol 23:99–112

    Google Scholar 

  • Clewell DB, Helinski DR (1969) Supercoiled circular DNA-protein complex in Escherichia coli. Purification and induced conversion to an open circular DNA form. Proc Natl Acad Sci USA 62:1159–1162

    Google Scholar 

  • Cohen SN, Chang ACY, Hsu L (1972) Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci USA 69:2110–2114

    Google Scholar 

  • Diaz R, Ortega S (1984) Initiation of plasmid R1 replication is independent of transcription by host RNA polymerase. Nucleic Acids Res 13:5175–5183

    Google Scholar 

  • Diaz R, Staudenbauer WL (1982) Origin and direction of mini-R1 plasmid replication in cell extracts of Escherichia coli. J Bacteriol 150:1077–1084

    Google Scholar 

  • Diaz R, Nordström K, Staudenbauer WL (1981) Plasmid R1 DNA replication dependent on protein synthesis in cell free extracts of E. coli. Nature 219:1077–1079

    Google Scholar 

  • Donachie WD, Begg KJ, Vicente M (1976) Cell length, cell growth and cell division. Nature 264:328–333

    Google Scholar 

  • Freese E, Bautz E, Bautz-Freese E (1961) The chemical and mutagenic specificity of hydroxylamine. Proc Natl Acad Sci USA 47:845–855

    Google Scholar 

  • Garnier J, Osguthorpe DJ, Robson B (1978) Analysis of the accuracy and implications of simple methods for predicting secondary structure of globular proteins. J Mol Biol 120:97–120

    Google Scholar 

  • Gronenborn B (1976) Overproduction of phage lambda repressor under control of the lac promoter of E. coli. Mol Gen Genet 148:243–250

    Google Scholar 

  • Humphreys GO, Willshaw GA, Smith HR, Anderson ES (1976) Mutagenesis of plasmid DNA with hydroxylamine, isolation of mutants of multi-copy plasmids. Mol Gen Genet 145:101–108

    Google Scholar 

  • Ike Y, Hashimoto H, Motohashi K, Fujisawa N, Mitsuhashi S (1980) Isolation and characterization of a composite plasmid Rs201 mutant temperature sensitive for replication. J Bacteriol 141:577–583

    Google Scholar 

  • Kollek R, Oertel W, Goebel W (1987) Isolation and characterization of the minimal fragment required for autonomous replication of a copy mutant (pKN102) of the antibiotic resistance factor R1. Mol Gen Genet 162:51–58

    Google Scholar 

  • Lennox ES (1955) Transduction of linked genetic characters of the host by bacteriophage P1. Virology 1:190–206

    Google Scholar 

  • Light J, Molin S (1981) Replication control functions of plasmid R1 act as inhibitors of expression of a gene required for replication. Mol Gen Genet 184:56–61

    Google Scholar 

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

    Google Scholar 

  • Masai H, Arai KI (1987) RepA and DnaA proteins are required for initiation of R1 plasmid replication in vitro and interact with the oriR sequence. Proc Natl Acad Sci USA 84:4781–4785

    Google Scholar 

  • Masai H, Kaziro Y, Arai KI (1983) Definition of oriR, the minimurn DNA segment essential for initiation of R1 plasmid replication in vitro. Proc Natl Acad Sci USA 80:6814–6818

    Google Scholar 

  • Messing J, Vieira J (1982) A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene 19:269–276

    Google Scholar 

  • Miki T, Easton MA, Rownd RH (1980) Cloning of replication, incompatibility, and stability functions of R plasmid NR1. J Bacteriol 141:87–99

    Google Scholar 

  • Molin S, Stougaard P, Uhlin BE, Gustafsson P, Nordström K (1979) Clustering of genes involved in replication, incompatibility and stable maintenance of the resistance plasmid R1. J Bacteriol 138:70–79

    Google Scholar 

  • Mott JE, Grant RA, Ho YS, Platt T (1985) Maximizing gene expression from plasmid vectors containing the P1 promoter: strategies for overproducing transcription termination factor. Proc Natl Acad Sci USA 82:88–92

    Google Scholar 

  • Nordström K, Molin S, Light J (1984) Control of replication of bacterial plasmid: Genetics, molecular biology and physiology of the plasmid R1 system. Plasmid 12:71–90

    Google Scholar 

  • Ogura T, Hiraga S (1983) Mini-F plasmid genes that couple host cell division to plasmid proliferation. Proc Natl Acad Sci USA 80:4784–4788

    Google Scholar 

  • Ortega S, Lanka E, Diaz R (1986) The involvement of host replication proteins and of specific origin sequences in the in vitro replication of miniplasmid R1 DNA. Nucleic Acids Res 14:428–435

    Google Scholar 

  • Rosen J, Ryder T, Inokuchi H, Ohtsubo H, Ohtsubo E (1980) Genes and sites involved in replication and incompatibility of an R100 plasmid derivative based on nucleotide sequence analysis. Mol Gen Genet 179:527–537

    Google Scholar 

  • Rosenberg M, Ho YS, Shatzman A (1983) The use of pKC-30 and its derivatives for controlled expression of genes. Methods Enzymol 101:123–138

    Google Scholar 

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

    Google Scholar 

  • Stougaard P, Molin S (1981) Vertical dye-buoyant density gradients for rapid analysis and preparation of plasmid DNA. Anal Biochem 118:191–193

    Google Scholar 

  • Tsuchimoto S, Ohtsubo H, Ohtsubo E (1988) Two genes pemK and pemI, responsible for the stable maintenance of resistance plasmid R100. J Bacteriol 170:1461–1466

    Google Scholar 

  • Uhlin BE, Nordström K (1978) A runaway replication mutant of plasmid R1drd-19. Temperature dependent loss of copy number control. Mol Gen Genet 165:167–179

    Google Scholar 

  • Wechsler JA, Nusslein V, Otto B, Klein A, Bonhoeffer F, Hezzman R, Glover L, Schaller H (1973) Isolation and characterization of thermosensitive Escherichia coli mutants defective in deoxyribonucleic acid replication. Bacteriol 113:1381–1388

    Google Scholar 

  • Womack JE (1977) Inhibition of thymidine phosphorylase in vivo provides a rapid method for switching DNA labeling. Mol Gen Genet 158:11–15

    Google Scholar 

  • Womble D, Rownd RH (1987) Regulation of incFII plasmid DNA replication. A quantitative model for plasmid NR1 replication in the bacterial division cycle. J Mol Biol 192:529–542

    Google Scholar 

  • Yoshikawa M (1974) Identification and mapping of the replication genes of an R-factor, R100-1 integrated into the host chromosome of Escherichia coli K-12. J Bacteriol 118:1123–1131

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Additional information

Communicated by R. Devoret

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ortega, S., de Torrontegui, G. & Díaz, R. Isolation and characterization of a conditional replication mutant of the antibiotic resistance factor R1 affected in the gene of the replication protein RepA. Mol Gen Genet 217, 111–117 (1989). https://doi.org/10.1007/BF00330949

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00330949

Key words

Navigation