SP62, a Viable Mutant of Bacteriophage T4D Defective in Regulation of Phage Enzyme Synthesis

Abstract

SP62 is a mutant of bacteriophage T4D that was discovered because it produces fewer phage than the wild type in the presence of 5-fluorodeoxyuridine. In the absence of phage DNA synthesis, SP62 solubilizes host DNA slower than normal; this may explain the sensitivity to 5-fluorodeoxyuridine. In Escherichia coli B at 37 C in the absence of drugs, SP62 makes DNA at a normal rate and the kinetics of appearance of phage are nearly normal. Under the same conditions, SP62 produces T4 lysozyme (gene e) at a normal rate until 20 min, but then produces it at twice the normal rate until at least 60 min. It has long been known that, when T4 DNA synthesis is blocked (DNA − state) in an otherwise normal infection, the synthesis of a number of early enzymes continues beyond the shutoff time of about 12 min seen in the DNA + state, but still stops at about 20 min. We have termed the 12-min shutoff event S1 and the 20-min shutoff event S2. We show here that, in the DNA + state, SP62 makes four early enzymes normally, i.e., S1 occurs. However, in the DNA − state (where S1 is missing), SP62 continues to make dCTPase (gene 56), dCMP hydroxymethylase (gene 42), and deoxynucleotide kinase (gene 1) for at least an hour; this results in production of up to 13 times the normal level of dCTPase at 60 min after infection, or 6 times the DNA − level. We conclude that SP62 is defective in the second shutoff mechanism, S2, for these three enzymes. In contrast, SP62 causes premature cessation of dTMP synthetase production in the DNA − state; the result is a twofold underproduction of dTMP synthetase. Autoradiograms of pulse-labeled proteins separated by slab-gel electrophoresis in the presence of sodium dodecyl sulfate show that a number of other T4 early proteins, including the products of genes 45, 46, and rIIA, are synthesized longer than normal by SP62 in the DNA − state. Few late proteins are made in the DNA − state, but in autoradiograms examining the DNA + state there is little or no effect of the SP62 mutation on the synthesis of T4 late or early proteins. Circumstantial evidence is presented favoring a role for the gene of SP62 in translation of certain mRNAs. At very high temperatures (above 43 C) in the absence of drugs, phage production, but not DNA synthesis, is much reduced in SP62 infections relative to wild-type T4 infections; this temperature sensitivity is greater on E. coli CR63 than on E. coli B. This property has facilitated recognition of the SP62 genotype and aided in complementation testing and genetic mapping. A later publication will provide evidence that SP62 defines a new T4 gene named regA , which maps between genes 43 and 62.