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Involvement of kil and kor genes in the phenotype of a host-range mutant of RP4

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Summary

Plasmid pRP761 is a derivative of the promiscuous plasmid RP4, which has a Tn76 insert 1.8 kb from its EcoRI site within the trfB region (Barth 1979). This mutation was pleiotropic, having three effects: the plasmid is unstably maintained in E. coli, it reduces the growth rate of its host and it has suffered a reduction in host-range. We show that pRP761 has reduced expression from both its korA and korB genes and that Tn76 has inserted between them. Fragment exchange experiments showed that this is the only mutant region in pRP761 and is therefore solely responsible for the pleiotropic effects. A spontaneous deletion derivative pRP761-6 has lost Tn76 and its adjacent kilA and korA genes: it has reacquired stability, does not inhibit host growth but is still reduced in its host-range. The provision of cloned korA + in trans complements the first two phenotypic effects in pRP761 to a large extent, but neither korA + alone nor korA + with korB + complements the host-range reduction in pRP761 or pRP761-6. A possible explanation for these results is that there is a site between korA and korB, affected by the Tn76 insert, that is essential to stable replication of these plasmids in some of their bacterial hosts.

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References

  • Bachmann BJ (1972) Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev 36:525–557

    Google Scholar 

  • Barth PT (1979) RP4 and R300B as wide host-range plasmid cloning vehicles. In: Timmis KN, Pühler A (eds) Plasmids of medical, environmental and commercial importance. Elsevier/North Holland Biomedical Press, Amsterdam, p 399–410

    Google Scholar 

  • Barth PT, Grinter NJ (1974) Comparison of the deoxyribonucleic acid molecular weights and homologies of plasmids conferring linked resistance to streptomycin and sulphonamides. J Bacteriol 120:618–630

    Google Scholar 

  • Barth PT, Grinter NJ (1977) Map of plasmid RP4 derived by insertion of transposon C. J Mol Biol 113:455–474

    Google Scholar 

  • Barth PT, Datta N, Hedges RW, Grinter NJ (1976) Transposition of a DNA sequence encoding trimethoprim and streptomycin resistances from R483 to other replicons. J Bacteriol 125:800–810

    Google Scholar 

  • Barth PT, Grinter NJ, Bradley DE (1978) Conjugal transfer system of plasmid RP4: analysis by transposon 7 insertion. J Bacteriol 133:43–52

    Google Scholar 

  • Barth PT, Tobin L, Sharpe GS (1981) Development of broad hostrange plasmid vectors. In: Levy SB, Clowes RC, Koenig EL (eds) Molecular biology, pathogenicity and ecology of bacterial plasmids. Plenum Publishing Corp, New York, pp 439–448

    Google Scholar 

  • Bechhofer DH, Figurski DH (1983) Map location and nucleotide sequence of korA, a key regulatory gene of promiscuous plasmid RK2. Nucl Acids Res 11:7453–7469

    Google Scholar 

  • Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198

    Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7:1513–1523

    Google Scholar 

  • Bryan LE, van den Elzen HM, Tseng JT (1972) Transferable drug resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 1:22–29

    Google Scholar 

  • Burkardt HJ, Riess G, Pühler A (1979) Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68, RK2 are identical. J Gen Microbiol 114:341–348

    Google Scholar 

  • Cowan P, Krishnapillai V (1982) Tn7 insertion mutations affecting the host range of the promiscuous IncP-1 plasmid R18. Plasmid 8:164–174

    Google Scholar 

  • Datta N, Hedges RW (1972) Host ranges of R factors. J Gen Microbiol 70:453–460

    Google Scholar 

  • Datta N, Hedges RW, Shaw EJ, Sykes RB, Richmond MH (1971) Properties of an R factor from Pseudomonas aeruginosa. J Bacteriol 108:1244–1249

    Google Scholar 

  • Datta N, Hughes VM, Nugent ME, Richards H (1979) Plasmids and transposons and their stability and mutability in bacteria isolated during an outbreak of hospital infection. Plasmid 2:182–196

    Google Scholar 

  • Figurski D, Helinski DR (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA 76:1648–1652

    Google Scholar 

  • Figurski DH, Pohlman RF, Bechhofer DH, Prince AS, Kelton CA (1982) The broad host-range plasmid RK2 encodes multiple kil genes potentially lethal to E. coli host cells. Proc Natl Acad Sci USA 79:1935–1939

    Google Scholar 

  • Ingram LC, Richmond MH, Sykes RB (1973) Molecular characterization of the R factors implicated in the carbenicillin resistance of a sequence of Pseudomonas aeruginosa strains isolated from burns. Antimicrob Agents Chemother 3:279–288

    Google Scholar 

  • Kleckner N (1977) Translocatable elements in procaryotes. Cell 11:11–23

    Google Scholar 

  • Lanka E, Barth PT (1981) Plasmid RP4 specifies a DNA primase involved in its conjugal transfer and maintenance. J Bacteriol 148:769–781

    Google Scholar 

  • Lanka E, Scherzinger E, Günther E, Schuster H (1979) A DNA primase specified by I-like plasmids. Proc Natl Acad Sci USA 76:3632–3636

    Google Scholar 

  • Lanka E, Lurz R, Fürste JP (1983) Molecular cloning and mapping of SphI restriction fragments of plasmid RP4. Plasmid 10:303–307

    Google Scholar 

  • Mercer AA, Loutit JS (1979) Transformation and transfection of Pseudomonas aeruginosa: effects of metal ions. J Bacteriol 140:37–42

    Google Scholar 

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

    Google Scholar 

  • Olsen RH, Shipley P (1973) Host ranges and properties of the Pseudomonas aeruginosa R factor R1822. J Bacteriol 113:772–780

    Google Scholar 

  • Pohlman RF, Figurski DH (1983a) Conditional lethal mutants of the kilB determinant of broad host-range plasmid RK2. Plasmid 10:82–95

    Google Scholar 

  • Pohlman RF, Figurski DH (1983b) Essential genes of plasmid RK2 in E. coli: trfB region controls a kil gene near trfA. J Bacteriol 156:584–591

    Google Scholar 

  • Schmidhauser TJ, Filutowicz M, Helinski DR (1983) Replication of derivatives of the broad host-range plasmid RK2 in two distantly related bacteria. Plasmid 9:325–330

    Google Scholar 

  • Smith CA, Thomas CM (1983) Deletion mapping of kil and kor functions in the trfA and trfB regions of broad host-range plasmid RK2. Mol Gen Genet 190:245–254

    Google Scholar 

  • Thomas CM (1981) Complementation analysis of replication and maintenance functions of broad host-range plasmids RK2 and RP1. Plasmid 5:277–291

    Google Scholar 

  • Thomas CM, Stalker D, Guiney D, Helinski DR (1979) Essential regions for the replication and conjugal transfer of the broad host-range plasmid RK2. In: Timmis KN, Pühler A (eds) Plasmids of medical, environmental and commercial importance. Elsevier/North Holland Biomedical Press, Amsterdam, p 375–385

    Google Scholar 

  • Thomas CM, Meyer R, Helinski DR (1980) Regions of broad host-range plasmid RK2 which are essential for replication and maintenance. J Bacteriol 141:213–222

    Google Scholar 

  • Thomas CM, Hussain AAK, Smith CA (1982) Maintenance of broad host-range plasmid RK2 replicons in Pseudomonas aeruginosa. Nature (London) 298:674–676

    Google Scholar 

  • Young C, Bechhofer DH, Figurski DH (1984) Gene regulation in plasmid RK2: positive control by korA in the expression of korC. J Bacteriol 157:247–252

    Google Scholar 

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Author notes

  1. Kim Ellis

    Present address: Biochemistry Department, St. Mary's Hospital Medical School, W2, London, UK

  2. David H. Bechhofer

    Present address: Department of Microbiology, The Public Health Research Institute of the City of New York, 10016, New York, NY, USA

Authors and Affiliations

  1. ICI Corporate Bioscience Group, The Heath, WA7 4QE, Runcorn, Cheshire, UK

    Peter T. Barth

  2. Bacteriology Department, Royal Postgraduate Medical School, DuCane Road, W12 OHS, London, UK

    Kim Ellis

  3. Department of Microbiology and Cancer Centre, College of Physicians and Surgeons, 701 West 168th Street, 10032, New York, NY, USA

    David H. Bechhofer & David H. Figurski

Authors
  1. Peter T. Barth
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  2. Kim Ellis
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  3. David H. Bechhofer
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  4. David H. Figurski
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Additional information

Communicated by J. Schell

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Barth, P.T., Ellis, K., Bechhofer, D.H. et al. Involvement of kil and kor genes in the phenotype of a host-range mutant of RP4. Mol Gen Genet 197, 236–243 (1984). https://doi.org/10.1007/BF00330969

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  • DOI: https://doi.org/10.1007/BF00330969

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