Analysis of T4 bacteriophage deletion mutants that lack td and frd genes

The roles of bacteriophage T4-encoded thymidylate synthase and dihydrofolate reductase as virion structural components have been further investigated. Two mutants, del(63-32)7 and del(63-32)9, bearing deletions in the gene 63 to 32 region of the T4 genome, were characterized by Southern blotting analysis, as well as by enzyme and immunological assays. Our results have confirmed the original report of Homyk and Weil (Virology 61:505-523, 1974) that del7 and del9 each carries a deletion of about 4.0 kilobases, which totally eliminates the frd gene, encoding dihydrofolate reductase, and the td gene, encoding thymidylate synthase. With the well-characterized deletion mutants, along with newly prepared antisera against T4-encoded thymidylate synthase and dihydrofolate reductase, we have reevaluated the experimental results supporting the idea that T4-induced dihydrofolate reductase and thymidylate synthase are essential T4 baseplate components and antigenic determinants of phage particles. These deletion mutant phages are not targets for neutralization by antisera against either dihydrofolate reductase or thymidylate synthase purified from cloned genes. Furthermore, these newly prepared antisera also cannot neutralize the infectivity of T4D. Those results suggest that the phage-neutralizing components in the old antisera used in the earlier studies were not antibodies against either dihydrofolate reductase or thymidylate synthase but were antibodies against minor components of the purified enzyme preparations. Study of the biological properties of the deletion mutants indicates that T4-induced thymidylate synthase and dihydrofolate reductase play significant roles in growth of the phage beyond their known roles in nucleotide biosynthesis, even though they are apparently not essential for phage viability. The deletion mutants should be useful in defining these roles.