Molecular and General Genetics MGG

, Volume 190, Issue 2, pp 245–254 | Cite as

Deletion mapping of kil and kor functions in the trfA and trfB regions of broad host range plasmid RK2

  • Christopher A. Smith
  • Christopher M. Thomas


Figurski et al. (1982) have reported that certain loci on the broad host range plasmid RK2 (kil functions) can be cloned only in the presence of other trans-acting segments of the plasmid genome (kor functions). They have suggested that the presence of these functions may in part account for the structure of mini RK2 replicons which were constructed in order to define the regions of the plasmid which encode replication/maintenance functions (Thomas et al. 1980). We have therefore investigated the relationship between these two sets of kil and kor loci and the loci implicated in the replication/maintenance of RK2. We find that, whilst the three kil loci reported by Figurski et al. (1982) are absent from these derivatives, a fourth such locus (kilD) is closely linked to trfA, a gene essential for RK2 replication. The kilD locus was probably responsible for the inclusion in mini replicons of a segment of PK2 DNA which carries both korD and korA in addition to trfB, a gene defined by a temperature-sensitive maintenance defect, but which can be deleted leaving a functional RK2 replicon (Thomas 1981 b). The kilB locus is situated on the opposite side of kilD from trfA, all three loci lying within a 3.6 kb segment of RK2 DNA. The korA, korD and trfB functions all map within a 900 bp segment of DNA, while korB requires sequence information at least 1.5 kb from this segment.


Host Range Sequence Information Broad Host Range Part Account Deletion Mapping 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Barth PT (1979) RP4 and R300B as wide host-range cloning vehicles. In: Timmis KN, Pühler A (eds) Plamids of medical, environmental and commercial importance. Elsevier/North-Holland, Amsterdam, p 399–410Google Scholar
  2. Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198Google Scholar
  3. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7:1513–1523Google Scholar
  4. Bolivar R, Rodriguez RL, Green PJ, Betlach MC, Heynecker HC, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113Google Scholar
  5. Burkhardt HJ, Riess G, Pühler A (1979) Relationship of group P1 plasmids revealed by heteroduplex experiments: RP1, RP4, R68 and RK2 are identical. J Gen Microbiol 114:341–348Google Scholar
  6. Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic mini plasmid. J Bacteriol 134:1141–1156Google Scholar
  7. Cho JJ, Panopoulos NJ, Schroth MN (1975) Genetic transfer of Pseudomonas aeruginosa R factors to plant pathogenic Erwinia species. J Bacteriol 122:192–198Google Scholar
  8. Covey C, Richardson D, Carbon J (1976) A method for the deletion of restriction sites in bacterial plasmid deoxyribonucleic acid. Mol Gen Genet 145:155–158Google Scholar
  9. Datta N, Hedges RW (1972) Host range of R factors. J Gen Microbiol 70:453–460Google Scholar
  10. 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 76:1648–1652Google Scholar
  11. Figurski DH, Pohlman RF, Bechhofer DH, Prince AS, Kelton CA (1982) The broad host range plasmid RK2 encodes multiple kil genes potentially lethal to Escherichia coli host cells. Proc Natl Acad Sci USA 79:1935–1939Google Scholar
  12. 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 3:279–288Google Scholar
  13. Kahn M, Kolter R, Thomas C, Figurski D, Meyer R, Remaut E, Helinski DR (1979) Plasmid cloning vehicles derived from plasmids ColE1, F, R6K and RK2. Methods Enzymol 68:268–280Google Scholar
  14. Kolter R, Helinski DR (1978) construction of plasmid R6K derivatives in vitro: characterization of the R6K replication region. Plasmid 1:571–580Google Scholar
  15. Komai N, Nishizawa T, Hayakawa Y, Murotsu T, Matsubara K (1982) Detection and mapping of six miniF-encoded proteins by cloning analysis of dissected miniF segments. Mol Gen Genet 186:193–203Google Scholar
  16. McKenny K, Shimatke H, Court D, Schmeissner U, Brady C, Rosenberg M (1981) A system to study promoter and terminator signals recognised by Escherichia coli RNA polymerase. In: Chirikjian JC, Papas TS (eds) Gene amplification and analysis, vol II. Analysis of nucleic acids by enzymatic methods. Elsevier/North Holland, Amsterdam, p 383–415Google Scholar
  17. Meyer R, Figurski D, Helinski D (1977) Physical and genetic studies with restriction endonucleases on the broad host-range plasmid RK2. Mol Gen Genet 152:129–135Google Scholar
  18. Meyer RJ (1979) Expression of incompatibility by derivatives of the broad host range IncP-1 plasmid RK2. Mol Gen Genet 177:155–161Google Scholar
  19. Olsen RH, Shipley P (1973) Host range and properties of the Pseudomons aeruginosa R factor R1822. J Bacteriol 113:772–780Google Scholar
  20. Sakanyan VA, Yakubor LZ, Alikhanian SI, Stepanov AI (1978) Mapping of RP4 plasmid using deletion mutants of pAS8 hybrid (RP4-ColE1). Mol Gen Genet 165:331–341Google Scholar
  21. Schmidhauser TJ, Filutowicz M, Helinski DR (1983) Replication of derivatives of the broad host-range plasmid RK2 in two distantly related bacteria. Plasmid 9:No 3Google Scholar
  22. Selveraj G, Iyer VN (1981) Genetic transformation of Rhizobium meliloti by plasmid DNA. Gene 15:279–283Google Scholar
  23. Sutcliffe JG (1978) pBR322 restriction map derived from the DNA sequence: accurate DNA size markers up to 4361 nucleotide pairs long. Nucl Acids Res 5:2721–2728Google Scholar
  24. Thomas CM, Meyer R, Helinski DR (1980) Regions of broad host-range plasmid RK2 which are essential for replication and maintenance. J Bacteriol 141:213–222Google Scholar
  25. Thomas CM (1981a) Molecular genetics of broad host range plasmid RK2. Plasmid 5:10–19Google Scholar
  26. Thomas CM (1981b) Complementation analysis of replication and maintenance functions of broad host range plasmids RK2 and RP1. Plasmid 5:277–291Google Scholar
  27. Thomas CM, Hussain AAK, Smith CA (1982) Maintenance of broad host range plasmid RK2 replicons in Pseudomonas aeruginosa. Nature 298:674–676Google Scholar
  28. Tolun A, Helinski DR (1982) Separation of the minimal replication region of the F plasmid into a replication origin segment and a trans-acting segment. Mol Gen Genet 186:372–377Google Scholar

Copyright information

© Springer-Verlag 1983

Authors and Affiliations

  • Christopher A. Smith
    • 1
  • Christopher M. Thomas
    • 1
  1. 1.Department of GeneticsUniversity of BirminghamBirminghamEngland

Personalised recommendations