Transcription-driven DNA replication of plasmid pAMbeta1 in Bacillus subtilis

pAMbeta1 is a plasmid isolated from Enterococcus faecalis which replicates in Bacillus subtilis by a unidirectional theta mechanism. It has been shown previously that initiation of pAMbeta1 replication requires a plasmid-encoded protein (RepE) and a short origin and is carried out by the host DNA polymerase I. It is not known which primer is used by this polymerase for initiating replication. Here, we report that a transcription fork passing through the origin is a limiting factor for plasmid replication. Transcription that activates the origin is initiated at the repE promoter and is thus regulated by the plasmid copy-number control system. Two lines of evidence suggest that the transcription generates the primer for the DNA polymerase I. First, the transcription must start upstream from the origin and progress in the direction of replication to be effective. Second, 3' ends of RNA transcripts initiated upstream of the origin map within the origin, provided that the Rep protein and an intact origin are present. This is the first report for simultaneous requirement of a transcription fork, a replication protein and the DNA polymerase I in initiation of DNA replication.