A technique for targeted mutagenesis of the EF-Tu chromosomal gene by M13-mediated gene replacement

A generally applicable system for targeted mutagenesis of a chromosomal sequence is described. The Escherichia coli tufA gene was mutated using a recombinant M13mp9 phage vector carrying a tuf gene. Integration via crossing over with the chromosomal tufA target gene produced an M13 lysogen. These lysogens were screened for resistance to kirromycin. The M13 phage carrying tufA mutations were efficiently retrieved by a genetic procedure. Genetic mapping was performed with the M13 vectors. The same recombinant M13 phage was used for mutagenesis, lysogen formation, gene replacement, retrieval, mapping and sequencing of kirromycin mutants. Three different mutations yielding resistance to kirromycin were found: two of these have previously been found and characterised, while the third mutation, Gly316 Asp, is a new mutant. We also report the identification of a fourth kirromycin-resistant mutant, Gln124 Lys.