Regulation of mobilization of the broad-host-range plasmid pTF-FC2

The mobilization region of the broad-host-range plasmid pTF-FC2 consists of an oriT and five genes arranged in two operons that are divergently transcribed from the oriT. The transcriptional starts of both operons were identified and the quantity of transcript from the mobC-mobE promoter (P1) was at least 10-fold greater than that from the mobA-mobB promoter (P2). A translational fusion between the first protein of each operon and a lacZ reporter gene was constructed and used to demonstrate that mob gene expression was autoregulated. Analysis of the oriT resulted in the detection of a putative integration host factor (IHF)-binding site and an intrinsically bent region. In the absence of IHF, the mobilization frequency and expression from P1 were reduced. The presence of ssi sites on both strands within the oriT region was demonstrated by using an M13 phage mutant, defective in its mechanism for priming DNA replication. Initiation of DNA synthesis at the oriT did not require a plasmid-encoded primase.