Isolation and characterization of lambda pleu bacteriophages

Abstract

In the Escherichia coli lysogen HfrH73, none of the enzymes coded for by the leucine operon is synthesized due to an insertion of phage .lambda. into cistron leuA. The orientation of .lambda. in the chromosome is ara leuDCB .lambda.JAN leuA. After heat induction of the lysogen, plaque-forming transducing phages of 2 types are formed at low frequency. One type (e.g., .lambda.pleu9) transduces leuD, leuC and leuB strains to prototrophy. The other type (e.g., .lambda.pleu13) transduces leuA strains to prototrophy. [Phage] .lambda.pleu13 forms lysogens at low frequency (.apprx. 0.2%) by integration into the leucine operon. These lysogens are unstable, segregating phage-sensitive clones at high frequency (.apprx. 1%). Phages carrying different portions of the leucine operon were formed by aberrant excision after heat induction of strain CV437 (leuA371 .lambda.pleu13). A phage carrying the entire leucine operon (.lambda.K2) was constructed by a cross between .lambda.pleu9 and .lambda.pleu13. An analysis of leucine-forming enzyme levels in strains lysogenized with .lambda.K2 indicated that leuO and leuP are present and functional in .lambda.K2. The leu-specific mRNA from E. coli hybridizes to the heavy (r) strand of .lambda.K2. The leucine operon of .lambda.G4 pleuABCD (an S7 derivative of .lambda.K2) exists intact on a 7.3 .times. 106-dalton fragment (.lambda.G4EcoRI-B) generated by cleavage with [restriction] endonuclease EcoRI. Heteroduplexes formed between .lambda.G4 and .lambda. show a 5.4 .times. 106-dalton piece of bacterial DNA replacing a 4.5 .times. 106-dalton piece of .lambda. DNA starting at 0.46 fractional unit on the map of .lambda.. Fragment .lambda.G4EcoRI-B has .apprx. 0.6 .times. 106 daltons of .lambda. DNA from the b2 region at one end and .apprx. 1.4 .times. 106 daltons of .lambda. DNA from the int region at the other end.