The resistance plasmid R1 is a conjugative plasmid, which belongs to the incompatibility group IncFII. Thus, it is related to the F plasmid and, among others, plasmid P307 which is responsible for enterotoxin production. Conjugation is mediated by the products of the tra genes, most of which are arranged in a large operon. One of the transfer genes, traM, which encodes a product essential for conjugation, lies outside of this operon. We have shown previously that TraM is a DNA-binding protein (Schwab et al., 1991). Recently, we demonstrated the autoregulatory effect of protein TraM. Autoregulation is allele specific, i.e. R1- or P307-encoded TraM proteins recognize only the cognate DNA. The N-terminal part of TraM forms an amphiphilic -helix, which is the primary DNA-binding domain. Autoregulation-deficient forms of TraM were created by site-specific mutagenesis in the N-terminus of the protein (Schwab et al., 1993). By expressing hybrid P307/R1 TraM proteins we could show that the specificity of DNA-binding resides exclusively in the N-terminal amphiphilic helix. An N-terminal deletion-mutant, a C-terminal deletion mutant, and three point mutations which might affect structural features of the molecule, like two basic amino acids, were tested for DNA-binding and autoregulation functions. After an allelic exchange of the mutated traM sequences from the high copy plasmid to the conjugative plasmid R1-16, two mutants were tested for DNA transfer activity and susceptibility to R17 phage infection.
In order to further investigate the functions of the domains of protein TraM we designed additional TraM deletions and site specific mutations.