Rate of translocation of bacteriophage T7 DNA across the membranes of Escherichia coli

Abstract

Translocation of bacteriophage T7 DNA from the capsid into the cell has been assayed by measuring the time after infection that each GATC site on the phage genome is methylated by cells containing high levels of DNA adenine methylase. Methylation at GATC sites on T7 DNA renders both the infecting genome and any newly synthesized molecules sensitive to the restriction enzyme DpnI. In a normal infection at 30 degrees C, translocation of the T7 genome into the cell takes between 9 and 12 min. In contrast, translocation of the entire phage lambda genome or of a T7 genome ejected from a lambda capsid can be detected within the first minute of infection. Entry of the leading end of the T7 genome occurs by a transcription-independent mechanism that brings both Escherichia coli and T7 promoters into the cell. Further translocation of the genome normally involves transcription by the RNA polymerases of both E. coli and T7; the rates of DNA translocation into the cell when catalyzed by each enzyme are comparable to the estimated rates of transcription of the respective enzymes. A GATC site located between the early E. coli promoters and the coding sequences of the first T7 protein made after infection is not methylated before the protein is synthesized, a result supporting the idea (B. A. Moffatt and F. W. Studier, J. Bacteriol. 170:2095-2105, 1988) that only certain proteins are permitted access to the entering T7 DNA. In the absence of transcription, the genomes of most T7 strains do not completely enter the cell. However, the entire genome of a mutant that lacks bp 3936 to 808 of T7 DNA enters the cell in a transcription-independent process at an average overall rate of 50 bp per s.