Summary
We describe the molecular cloning of BglII fragments of the hybrid plasmid pRS5 (pSC101 and EcoRI fragments of F; f7, f5, f3 and f6). The clones isolated were examined for the expression of F-specified replication, incompatibility, mobilization and inhibition of T7 bacteriophage multiplication. Proteins directed by the BglII clones were labelled in Escherichia coli K12 maxicells and analyzed by SDS-polyacrylamide gel electrophoresis. The sizes of previously reported proteins, encoded by the replication, incompatibility and leading regions encompassed by these plasmids have been confirmed in this study. In addition, the results demonstrate that a pif gene, which encodes an 80,000 dalton polypeptide essential for the inhibition T7 phage multiplication, is located on the BglII fragment that spans the junction of EcoRI fragments f7 and f5.
Similar content being viewed by others
References
Achtman M, Willetts N, Clark AJ (1971) Beginning a genetic analysis of conjugational transfer determined by the F factor in Escherichia coli by isolation and characterization of transfer-deficient mutants. J Bacteriol 106: 529–538
Austin S, Wierzbicki A (1983) Two mini-F-encoded proteins are essential for equipartition. Plasmid 10: 73–81
Berg DE, Weiss A, Crossland L (1980) Polarity of Tn5 insertion mutations in Escherichia coli J Bacteriol 142: 439–446
Bex F, Karoui H, Rokeach L, Drèze P, Garcia L, Couturier M (1983) Mini-F encoded proteins: identification of a new 10.5 kilodalton species. EMBO J 2: 1853–1861
Bolivar F, Rodriguez RL, Greene PJ, Betlach MC, Heyneker HL, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2: 95–113
Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134: 1141–1156
Ciampi MS, Schmid MB, Roth JR (1982) Transposon Tn10 provides a promoter for transcription of adjacent sequences. Proc Natl Acad Sci USA 79: 5016–5020
Cram DS, DiBerardino D, Skurray RA (1982) Beginning a transcriptional analysis of the leading region in F plasmid DNA transfer. Mol Gen Genet 185: 186–188
Cram D, Ray A, Skurray R (1984a) Transcriptional analysis of the leading region in F plasmid DNA transfer. Plasmid 11: 221–233
Cram D, Ray A, Skurray R (1984b) Molecular analysis of F plasmid pif region specifying abortive infection of T7 phage. Mol Gen Genet 197: 137–142
Crisona NJ, Clark AJ (1977) Increase in conjugational transmission frequency of nonconjugative plasmids. Science 196: 186–187
Ebbers J, Eichenlaub R (1981) Complementation of replication-deficient deletion derivatives of plasmid mini-F. J Bacteriol 147: 736–743
Everett R, Willetts N (1982) Cloning, mutation, and location of the F origin of conjugal transfer. EMBO J 1: 747–753
Fredericq P (1969) The recombination of colicinogenic factors with other episomes and plasmids. In: Wolstenholme GEW, O'Connor M (eds) Bacterial episomes and plasmids. Ciba Foundation Symposium. Churchhill, London, pp 163–178
Gardner RC, Malcolm L, Bergquist PL, Lane HED (1982) IncD, a genetic locus in F responsible for incompatibility with several plasmids of the IncF1 group. Mol Gen Genet 188: 345–352
Gasson M, Willetts NS (1977) Further characterization of the F fertility inhibition systems of “unusual” Fin+ plasmids. J Bacteriol 131: 413–420
Howard-Flanders P, Theriot L (1966) Mutants of Escherichia coli K-12 defective in DNA repair and in genetic recombination. Genetics 53: 1137–1150
Johnson D, Everett R, Willetts NS (1981) Cloning of F DNA fragments carrying the origin of transfer oriT and the fertility inhibition gene finP. J Mol Biol 153: 187–202
Johnson DA, Willetts NS (1983) λ-transducing phages carrying transfer genes isolated from an abnormal prophage insertion into the traY gene of F. Plasmid 9: 71–85
Johnson RC, Yin JCP, Reznikoff WS (1982) Control of Tn5 transposition in Escherichia coli is mediated by protein from the right repeat. Cell 30: 873–882
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. In: Wu R (ed) Methods in enzymology, vol 68. Academic Press, New York, pp 326–328
Kennedy N, Beutin L, Achtman M, Skurray R, Rahmsdorf U, Herrlich P (1977) Conjugation proteins encoded by the F sex factor. Nature 270:580–585
Kilbane JJ, Malamy MH (1980) F factor mobilization of nonconjugative chimeric plasmids in Escherichia coli: General mechanisms and a role for site-specific recA-independent recombination at oriV1. J Mol Biol 143:73–93
Kolodkin AL, Capage MA, Golub EI, Low KB (1983) The F sex factor of Escherichia coli K-12 codes for a single-strand DNA binding protein. Proc Natl Acad Sci USA 80:4422–4426
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 186:193–203
Lane HED (1981) Replication and incompatibility of F and plasmids in the IncFl group. Plasmid 5:100–126
Le Grice SFJ, Matzura H, Marcoli R, Iida S, Bickle TA (1982) The catabolite-sensitive promoter for the chloramphenicol acetyl transferase gene is preceded by two binding sites for the catabolite gene activator protein. J Bacteriol 150:312–318
Monk M, Kinross J (1972) Conditioned lethality of recA and recB derivatives of a strain of Escherichia coli K12 with a temperature-sensitive deoxyribonucleic acid polymerase 1. J Bacteriol 109:981–978
Morrison TG, Malamy MH (1971) T7 translational control mechanisms and their inhibition by F factors. Nature New Biol 231:37–41
Murotsu T, Matsubara K, Sugisaki H, Takanami M (1981) Nine unique repeating sequences in a region essential for replication and incompatibility of the mini-F plasmid. Gene 15:257–271
Puhler A, Klipp W (1981) Fine structure analysis of the gene region for N2-fixation (nif) of Klebsiella pneumoniae. In: Bothe H and Trebst A (eds) Biology of inorganic nitrogen and sulphur. Springer-Verlag, Berlin Heidelberg New York, pp 276–286
Ray A, Skurray R (1983) Cloning and polypeptide analysis of the leading region in F plasmid DNA transfer. Plasmid 9:262–272
Ray A, Skurray R (1984) Stabilization of the cloning vector pA-CYC184 by insertion of F plasmid leading region sequences. Plasmid 11:272–275
Rotman GS, Cooney R, Malamy MH (1983) Cloning of the pif region of the F sex factor and identification of a pif protein product. J Bacteriol 155:254–264
Sancar A, Hack AM, Rupp WD (1979) Simple method for identification of plasmid-coded proteins. J Bacteriol 137:692–693
Sharp PA, Cohen SN, Davidson N (1973) Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. II. Structure of drug resistance (R) factors and F factors. J Mol Biol 75:235–255
Skurray RA, Nagaishi H, Clark AJ (1976) Molecular cloning of DNA from F sex factor of Escherichia coli K12. Proc Natl Acad Sci USA 73:64–68
Skurray RA, Clark AJ, Uhlin BE, Nagaishi H, Hua S (1978a) F sex factor: Physical and functional analysis of cloned EcoRI DNA fragments. In: Schlessinger D (ed) Microbiology-1978. Am Soc Microbiology, Washington, DC, pp 192–196
Skurray RA, Nagaishi H, Clark AJ (1978b) Construction and BamHI analysis of chimeric plasmids containing EcoRI DNA fragments of the F sex factor. Plasmid 1:174–186
Stüber D, Bujard H (1981) Organization of transcriptional signals in plasmids pBR322 and pACYC184. Proc Natl Acad Sci USA 78:167–171
Tolun A, Helinski DR (1981) Direct repeats of the F plasmid incC region express F incompatibility. Cell 24:687–694
Watson LA, Phua SH, Bergquist PL, Lane HED (1982) An M r 29,000 protein is essential for mini-F maintenance in E. coli. Gene 19:173–178
Wehlmann H, Eichenlaub R (1980) Plasmid mini-F encoded proteins. Mol Gen Genet 180:205–211
Willetts N, Skurray R (1980) The conjugation system of F-like plasmids. Annu Rev Genet 14:41–76
Author information
Authors and Affiliations
Additional information
Communicated by W. Arber
Rights and permissions
About this article
Cite this article
Jackson, R., Cram, D., Ray, A. et al. Cloning and analysis of pif, replication and leading regions of the F plasmid. Mol Gen Genet 197, 129–136 (1984). https://doi.org/10.1007/BF00327933
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00327933