Use of M13mp phages to study gene regulation, structure and function: cloning and recombinational analysis of genes of the Salmonella typhimurium histidine operon

Abstract

A restriction map was determined for a φ80λd his transducing phage DNA carrying the Salmonella typhimurium histidine operon. DNA fragments containing the promoter/regulatory region and the first two structural genes of the histidine operon ( hisOGD) were identified by their ability to direct regulated synthesis of histidinol dehydrogenase (product of hisD) in a coupled in vitro protein synthesizing system. A 3.1-kb SalI- EcoRI restriction fragment containing the hisOGD region, was subcloned into phage M13mp8 and M13mp9 RF DNAs. Methods are described for shuttling mutant and wild-type bacterial DNA sequences between the M13mp:: his phage and host bacterial genomes. Of novel importance is the use of the phage M13 gene II amber mutation to obtain integration of the M13mp:: his phage genome into the homologous his region of the bacterial chromosome following transduction of recipients lacking an amber suppressor. This method can be used to facilitate allele replacement with genes carried on M13 transducing phages.