REPRESSION OF THE TRAM GENE OF PLASMID R100 BY ITS OWN PRODUCT AND INTEGRATION HOST FACTOR AT ONE OF THE 2 PROMOTERS

Plasmid R100 codes for the traM gene, which is required for DNA transfer and whose product has been shown to bind to the four sites, called sbmA to sbmD, upstream of traM. To determine whether the TraM protein regulates the expression of traM, we constructed the plasmids carrying various portions of the region upstream of the initiation codon ATG for traM, which was fused with lacZ in frame, and introduced them into the cells, which did or did not harbor another compatible plasmid carrying traM. We then assayed the beta-galactosidase (LacZ) activity to monitor the expression of the fusion genes and analyzed the traM-specific transcripts made in the cells. Two promoters for traM were identified and designated p(M1) and p(M2). Promoter p(M2) lies upstream of p(M1) and overlaps the sbmC-sbmD region. Promoter p(M1) is constitutively expressed, while p(M2) is much stronger but is repressed almost completely by the TraM protein and partially by integration host factor, whose binding site is near p(M2). The traM gene is likely to be expressed from p(M2) when the TraM protein is at low levels after dilution in the donor cell during cell growth or before its expression in the recipient cell which has just received R100 by conjugation. The expression from p(M2) Could maintain the amount of the TraM protein at a constant level needed to initiate DNA transfer at any time. Integration host factor, which can partially repress the traM gene, may play a role in forming an active complex with the TraM protein at the sbm region to facilitate DNA transfer.