The role of gene 49 in DNA replication and head morphogenesis in bacteriophage T4

Abstract

Mutations in T4 phage genes 33 and 55, genes necessary for the synthesis of late phage-induced proteins, cause the replicating phage DNA to appear in lysates in a form that sediments much more rapidly than the normal 200 s form observed during wild-type infection. We now find that an amber mutation in gene 49 produces this same effect. A temperature-sensitive mutation in gene 49 produces an intermediate DNA form, both at permissive and restrictive temperatubes, indicating that the unusual form of the DNA does not preclude its being converted to phage particles. Mutation in no other late gene tested produces this phenotype. However, we find that inhibition of late protein synthesis with chloramphenicol can partially simulate the effect of gene 33, 55 and 49 mutations. Mutations in gene 49 also prevent the formation of mature 63 s DNA. When the rapidly sedimenting form of replicating phage DNA is isolated and tested as substrate, extracts prepared after T4 infection convert it to slower sedimenting structures. The reaction produces 200 s DNA and apparently terminates with the formation of molecules that sediment between 45 and 65 s. Early-mutant extracts and gene 33-defective and 55-defective extracts do not contain this activity. All late-mutant extracts tested do contain the activity, except those prepared after infection with either of the gene 49 mutants. Suppression of the amber mutation in gene 49 results in a tenfold increase in activity, but the suppressed extracts still possess only 0.1 the wild-type activity. Extracts prepared after ts49 infection also show about 0.1 the wild-type activity, regardless of the temperature of infection or assay. Mixing experiments show that gene 49-defective extracts do not contain an inhibitor of the wild-type activity. Mixed infection with various ratios of 49 - to 49 + phage produces extracts containing activity approximately proportional to the number of 49 + gene copies. Lysates from certain of these mixed infections contain both forms of intracellular DNA and therefore complementation is not as complete as expected of a catalytic function. Gene 49 appears to control a product which, when functional, produces 200 s DNA in lysates of infected cells, but when absent results in the appearance of the more rapidly sedimenting DNA form. This gene product may be an enzyme that functions to alter the structure of the intracellular DNA so that it can be cleaved or encapsulated to form phage particles. Since the activity that we detect in extracts is sensitive to anti-T4 antiserum but not to pre-immune serum, it may be a component of the phage particle itself.