The construction in vitro of derivatives of bacteriophage lambda carrying the amidase genes of Pseudomonas aeruginosa

Abstract

The amidase genes of Pseudomonas aeruginosa were inserted into a λ replacement vector following cleavage with the restriction endonuclease HindIII. The recombinant λami was detected by enhanced growth of Escherichia coli around plaques of the recombinant phage on minimal medium containing acetamide as the nitrogen source. Low levels of amidase activity were detected in E. coli cultures infected with λami and these were sufficient to allow growth with acetamide as nitrogen source. Lysis-defective derivatives of λami were made by introducing Q ^-, S ^- mutations. Cultures of E. coli infected with λamiQ ^- S ^- synthesised amidase as the major protein. The amidase produced by these cultures was identical to that produced by PAC strains of P. aeruginosa in substrate specificity, thermal stability and immunological crossreaction.