Restoration by Chloramphenicol of Bacteriophage Production in Escherichia coli B Infected with a Ligase-Deficient Amber Mutant

Abstract

The addition of chloramphenicol (CM) 5 min after infection of the nonpermissive host Escherichia coli B with the ligase-negative T4 amber, T4 AmH39X, allowed replication of parental deoxyribonucleic acid (DNA) and the production of high-molecular-weight progeny DNA, composed mostly of subunits with a D 2 /D 1 of 0.6. When CM was removed after the accumulation of a large pool of this DNA, most of the infected bacteria were able to produce viable progeny phage, with an average yield of approximately 15 bacteriophage per bacterium. This phenomenon is called CM rescue of the ligase-negative T4 Am. CsCl and sucrose gradient analyses showed both the resulting phage and DNA extracted from them to be similar to the phage and DNA produced on the permissive host. The total transfer of the parental label to progeny phages was as high as 20%. In contrast, in bacteria not treated with CM or in bacteria to which CM was added after phage-coded nucleases had already been synthesized, both parental and progeny (newly synthesized) DNA was composed of very short fragments. Phage which are produced under conditions other than those of CM rescue are dead, light in CsCl, and contain only very short fragments of DNA. Parent-to-progeny transfer in this case is below 1%. When light radio-active parental DNA was used to infect heavy bacteria, DNA replicating in the CM rescue conditions assumed only a hybrid density. After removal of CM and maturation, the parental DNA was incorporated into progeny molecules in fragments constituting approximately 7 to 10% of its mass. This pattern of distribution is essentially what is observed in similar experiments in the permissive host. The role of ligase as an enzyme which compensates for the lethal action of phage-coded nuclease and which is stringently required for the repair of single-stranded nicks is emphasized. The possibility of specific sites for a unique cutting enzyme is discussed in connection with the hypothesis of a circularly permuted assembly of sets.