DNA replication of bacteriophage phi29. Effect of two viral genes on the association of phage chromosomes with the host cell membrane.

The kinetics of DNA arrest and the maintenance of the association of viral chromosomes with the cell membrane were examined by temperature-shift experiments using temperature-sensitive mutants in two early bacteriophage phi29 genes required for phage DNA replication. phi29 ts2(35), a mutant in cistron 2 whose product (protein P2) is continuously required for associating phage DNA with the Bacillus subtilis membrane, does not stop phage DNA synthesis immediately after a shift to the nonpermissive temperature. In contrast, bacteria infected with phi29 ts3(28), a mutant in cistron 3 (which codes for protein P3), stop synthesizing phage DNA immediately after transfer to the nonpermissive temperature. Parental phage DNA in phi29 ts2(35) infections rapidly dissociates from the cell membrane after a shift to 45 degrees, whereas phi29 ts3(28) DNA remains associated with the membrane after the shift to the nonpermissive temperature and then slowly dissociates. Thus the rapid dissociation of parental phage phi29 chromosomes from the membrane is dependent on a functional protein P3. These findings are discussed in terms of possible modes of action of these two proteins and suggest that protein P2 operates as a linker of phage chromosomes to the membrane, whereas protein P3 participates directly in the initiation or in the polymerization of viral DNA molecules.