Mechanism of T4 phage restriction by plasmid Rts 1. Cleavage of T4 phage DNA by Rts 1-specific enzyme.

Rts 1 is a plasmid which confers upon host bacteria the c a p a c i t y to restrict T4 phage growth at 32°C but not at 42°C.This restriction was not dependent on cellular adenyl cyclase, while other phenotypes of Rts 1, such as the temperature-sensitive effect on host growth, were dependent on this enzyme.At 32"C, in Escherichia coli 2OSO/Rts 1 cells, 55 to 60% of the parental T4 phage D N A became acid-soluble within 5 to 10 min after infection, while in E. coli JC7623/Rts 1 cells, which lack exonuclease I and V, very little T4 D N A became acidsoluble.The capacity of E. coli 2OSO/Rts 1 to degrade infecting T, DNA decreased 70% within 15 min after shifting the temperature to 42°C.Temperature shift up and down experiments in the presence of chloramphenicol showed that the protein responsible f o r T, D N A cleavage is thermosensitive, and once it is denatured it cannot regain its activity by lowering the temperature to 32°C in vivo.Alkaline sucrose g r a d i e n t centrifugat i o n revealed that the infecting Tq D N A was nicked in the presence o f Rts 1 at 32"C, but not at 42°C in E. coli JC7623.The cell-free extract o f E. coli JC7623/Rts 1 grown at 32°C contained an enzyme which converted Tq D N A to smaller but acid-insoluble fragments.This enzyme could not be detected in E. coli JC7623 or E. coli JC7623/Rts 1 grown at 42°C.The enzyme was rapidly inactivated when incubated at 42°C in vitro.A n a l y s i s of the reaction product by gel electrophoresis and alkaline and sodium dodecyl sulfate-neutral sucrose density gradient centrifugation, showed marked heterogeneity o f the in vitro reaction product.This enzyme d i d not cleave either T7 D N A or nonglucosylated T4 D N A under the same experimental conditions.Only M$+ was required f o r i t s activity.These experiments suggest that the plasmid Rts 1 codes f o r a thermosensitive enzyme which specifically degrades glucosylated T, DNA, resulting in the restriction of T, phage growth at 32°C but not at 42°C.This enzyme, named Rts 1 endonuclease, was purified approximately 500-fold.Rts 1 is a drug resistance factor which has various thermosensitive phenotypes.For example, it influences the host growth pattern, depending on the temperature (1, 2), while the conjugal transfer of this plasmid as well as synthesis of covalently closed circular form of Rts 1 is also thermosensitive (3,4).In addition, a linear form of this plasmid, instead of the conventional covalently closed circular form, may exist and replicate as such at 42°C ( 5 ) .This plasmid confers upon its host resistance to kanamycin as well as to cupric ions (4, 6).