Mutagenesis of the metJBLF gene cluster with transposon Tn5: localization of the metF transcription unit.

Mutants of a specialized lambda dmet transducing phage bearing the metJBLF gene cluster of Escherichia coli K12 were constructed using transposon Tn5. Two of these mutants, lambda dmet128::Tn5, MW77 and lambda dmet128::Tn5, 3-1, were used to locate precisely as well as confirm the existence of the metF transcription unit (approximately 1,000 base pairs in size). The introduction of new restriction sites within the metJBLF gene cluster due to the Tn5 insertion events allowed the metF transcription unit to be cloned into the high copy number plasmid pBR322. Analyses of the structures of two of these recombinant plasmids, pTJ77H and pTJ13-1H, are presented. Expression of the plasmid borne metF allele in cells grown in the absence, or presence, of exogenous L methionine (0.2 mM) demonstrates that the amplification of the metF copy number does not abolish met regulon mediated control of the gene's activity.