Replication of viral DNA in SPO1-infected Bacillus subtilis: I. Replicative intermediates

Abstract

During virulent infection of Bacillus subtilis 168 M by bacteriophage SPO1, the synthesis of mature progeny viral DNA occurs by the sequential conversion of two rapidly sedimenting intermediate DNA species, both of which are resistant to lysozyme and detergent. The first intermediate (“VF”) is a complex of viral DNA with protein and RNA, which, at neutral pH, sediments 4–6 times as fast as the mature SPO1 DNA molecule. The sedimentation coefficient of “ VF” is rendered indistinguishable from that of the second and slower intermediate (“F”) by the action of pronase. “F” appears to be a high molecular weight concatemer of viral DNA, sedimenting up to 3 times as fast as mature DNA at neutral pH. Sedimentation at alkaline pH incates that both “VF” and “F” contain strands several times the size of mature strands. Both intermediates appear to contain numerous single-strand discontinuities. Examination of the kinetics of appearance of “VF”,“F”, and mature DNA by continuous, pulse, and pulse-chase labeling shows that about 10 min elapse between the onset of viral DNA replication and the first appearance of mature progeny DNA. During this time “VF” accumulates. The mechanism for conversion of “VF” to “F” then becomes active, and acts very rapidly. “F” is then converted to mature DNA by a somewhat slower process.