oriT-processing and regulatory roles of TrwA protein in plasmid R388 conjugation

TrwA protein was purified from an overproducing Escherichia coli strain and characterized as a 53 kDa tetrameric DNA-binding protein. Gel shift assays showed that TrwA bound specifically to the oriT sequence of plasmid R388. DNAse I footprinting analysis defined two DNA regions within oriT (sites A and B) that were protected by TrwA. At low TrwA concentrations only region A was protected (K(D) = 4 x 10(-8) M) while region B required higher TrwA concentrations (K(D) = 4 x 10(-7) M). As a result of its binding to oriT, TrwA was found to perform two biochemical activities related to its role in R388 conjugation. First, TrwA binding to oriT resulted in transcriptional repression of the trwABC operon as indicated by its effect on the beta-galactosidase activity of transcriptional fusions in trwB and trwC, and by direct measurement of the trwA mRNA levels by hybridization. This result was further confirmed by the fact that TrwA overexpression resulted in lowered conjugation frequencies. Second, TrwA enhanced the relaxation activity of TrwC in vitro. This effect was correlated to a 10(5)-fold increase in the frequency of conjugation in vivo and was shown to be independent of the regulation of transcription. Thus, TrwA shows functional similarities to protein TraY of F-like plasmids, that could be correlated to a structural similarity in their DNA-binding motifs.