Summary
A technique is presented by which mutations can be introduced into the Escherichia coli chromosome by gene replacement between the chromosome and a plasmid carrying the mutant gene. The segregational instability of plasmids in E. coli is used with high efficiency to isolate E. coli mutants. The method should be applicable to construction of mutants for any E. coli chromosomal gene provided it is dispensable, and for any E. coli strain provided it is capable of homologous recombination. The use of the method was demonstrated by constructing E. coli mutants for the glycogen branching enzyme gene (glgB) and the β-galactosidase gene (lacZ). The results show that recombination occurs via a reciprocal mechanism indicating that the method should, in a slightly modified form, also be useful in transferring chromosomal mutations onto multicopy plasmids in vivo.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bochner BR, Huang H-C, Schieven GL, Ames BN (1980) Positive selection for loss of tetracycline resistance. J Bacteriol 143:926–933
Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179–207
Chattoraj DK, Cordes K, Berman ML, Das A (1984) Mutagenesis and mutation transfer induced by ultraviolet light in plasmid-cloned DNA. Gene 27:213–222
Damotte M, Cattanéo J, Sigal N, Puig J (1968) Mutants of Escherichia coli K 12 altered in their ability to store glycogen. Biochem Biophys Res Commun 32:916–920
Della Latta P, Bouanchaud D, Novick RP (1978) Partition kinetics and thermosensitive replication of pT169, a naturally occurring multicopy tetracycline resistance plasmid of Staphylococcus aureus. Plasmid 1:366–375
Gay NJ (1984) Construction and characterization of an Escherichia coli strain with a uncI mutation. J Bacteriol 158:820–825
Govons S, Vinopal R, Ingraham J, Preiss J (1969) Isolation of mutants of Escherichia coli B altered in their ability to synthesize glycogen. J Bacteriol 97:970–972
Gutterson NI, Koshland DE Jr (1983) Replacement and amplification of bacterial genes with sequences altered in vitro. Proc Natl Acad Sci USA 80:4894–4898
Hashimoto-Gotoh T, Ishii K (1982) Temperature-sensitive replication plasmids are passively distributed during cell division at non-permissive temperature: a new model for replicon duplication and partitioning. Mol Gen Genet 187:523–525
Haziza C, Stragier P, Patte J-C (1982) Nucleotide sequence of the asd gene of Escherichia coli: absence of a typical attenuation signal. EMBO J 1:379–384
Iglesias A, Trautner TA (1983) Plasmid transformation in Bacillus subtilis: Symmetry of gene conversion in transformation with a hybrid plasmid containing chromosomal DNA. Mol Gen Genet 189:73–76
Ish-Horowicz D, Burke JF (1981) Rapid and efficient cosmid cloning. Nucleic Acids Res 9:2989–2998
Jason M, Schimmel P (1984) Deletion of an essential genes in Escherichia coli (capital) by site-specific recombination with linear DNA fragments. J Bacteriol 159:783–786
Jones IM, Primrose SB, Robinson A, Ellwood DC (1980) Maintenance of some ColEl-type plasmids in chemostat culture. Mol Gen Genet 180:579–584
Joyce CM, Grindley NDF (1984) Method for determining whether a gene of Escherichia coli is essential: application to the polA gene. J Bacteriol 158:636–643
Liljeström P, Pirhonen M, Palva ET (1985) In vivo transfer of chromosomal mutations onto multicopy plasmids by transduction with bacteriophage P1. Gene 40:241–246
Maloy SR, Nunn WD (1981) Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol 145:1110–1112
Maniatis T, Fritsch EF, Sambrook F (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Marinus MG (1973) Location of DNA methylation genes on the Escherichia coli K-12 genetic map. Mol Gen Genet 127:47–55
Matsuyama S-I, Mizushima S (1985) Construction and characterization of a deletion mutant lacking micF, a proposed regulatory gene for ompF synthesis in Escherichia coli. J Bacteriol 162:1196–1202
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring harbor, NY
Okita TW, Rodriguez RL, Preiss J (1981) Biosynthesis of bacterial glycogen: Cloning of the glycogen biosynthetic enzyme structural genes of Escherichia coli. J Biol Chem 256:6944–6952
Preiss J (1984) Bacterial glycogen synthesis and its regulation. Annu rev Microbiol 38:419–458
Saarilahti HT, Palva ET (1985) In vivo transfer of chromosomal mutations onto multicopy plasmids utilizing polA strains: cloning of an ompR2 mutation in Escherichia coli K-12. FEMS Microbiol Lett 26:27–33
Silhavy TJ, Berman ML, Enquist LW (1984) Experiments with gene fusions. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Skogman G, Nilsson J, Gustafsson P (1983) The use of a partition locus to increase stability of tryptophan-operon-bearing plasmids in Escherichia coli. Gene 23:105–115
Summers DK, Sherratt DJ (1984) Multimerization of high copy number plasmids causes instability: ColEl encodes a determinant essential for plasmid monomerization and stabilty. Cell 36:1097–1103
Tabak HF, Flavell RA (1978) A method for the recovery of DNA from agarose gels. Nucleic Acids Res 5:2321–2332
Trieu-Cuot P, Courvalin P (1983) Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3′5″-aminoglycoside phosphotransferase type III. Gene 23:331–341
Twigg AJ, Sherratt D (1980) Trans-complementable copy number mutants of plasmid ColEl. Nature 283:216–218
Vieira J, Messing J (1982) the pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
Weinstock GM, Berman ML, Silhavy TJ (1983) Chimeric genes with β-galactosidase. In: Papas TS, Rosenberg M, Chirikjian JG (eds) Gene amplification and analysis III. Elsevier North-Holland, New York, pp 27–64
Author information
Authors and Affiliations
Additional information
Communicated by D. Goldfarb
Rights and permissions
About this article
Cite this article
Kiel, J.A.K.W., Vossen, J.P.M.J. & Venema, G. A general method for the construction of Escherichia coli mutants by homologous recombination and plasmid segregation. Mol Gen Genet 207, 294–301 (1987). https://doi.org/10.1007/BF00331592
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00331592