Genetic analysis of bacteriophage P22 lysozyme structure

The suppression patterns of 11 phage P22 mutants bearing different amber mutations in the gene encoding lysozyme (19) were determined on six different amber suppressor strains. Of the 60 resulting single amino acid substitutions, 18 resulted in defects in lysozyme activity at 30 degrees; an additional seven were defective at 40 degrees. Revertants were isolated on the "missuppressing" hosts following UV mutagenesis; they were screened to distinguish primary- from second-site revertants. It was found that second-site revertants were recovered with greater efficiency if the UV-irradiated phage stocks were passaged through an intermediate host in liquid culture rather than plated directly on the nonpermissive host. Eleven second-site revertants (isolated as suppressors of five deleterious substitutions) were sequenced: four were intragenic, five extragenic; three of the extragenic revertants were found to have alterations near and upstream from gene 19, in gene 13. Lysozyme genes from the intragenic revertant phages were introduced into unmutagenized P22, and found to confer the revertant plating phenotype.