Rapid mutational analysis of regulatory loci in Escherichia coli K-12 using bacteriophage M13

Abstract

A derivative of bacteriophage M13mp8 , designated M13mp8 /P, was prepared in which the promoter and NH2-terminal codons of bacterial genes may be fused to a portion of beta-galactosidase, resulting in an easily scorable phenotype. Because transcription from the inserted promoter remains responsive to the host regulatory system, it is simple to screen mutagenized phage for isolates with aberrant regulatory phenotypes and to determine the mutational changes by dideoxy sequence analysis. The feasibility of the method was demonstrated by isolation of a large number of mutations in the regulatory regions of two genes, lexA and recA. Base substitutions that altered the phenotype of recombinant phage were identified both in the single LexA repressor binding site of recA and in the two binding sites of lexA, as well as in other sites that likely affect translational efficiency. Our results suggest that this approach will be generally useful for mutational analysis of transcriptional and translational regulatory elements.