MUTATIONAL ANALYSIS OF ESSENTIAL INCP-ALPHA PLASMID TRANSFER GENES TRAF AND TRAG AND INVOLVEMENT OF TRAF IN PHAGE SENSITIVITY

Although the broad-host-range IncP plasmids can vegetatively replicate in diverse gram-negative bacteria, the development of shuttle vector systems has established that the host range for IncP plasmid conjugative transfer is greater than the range of bacteria that sustain IncP replicons. Towards understanding IncP plasmid conjugation and the connection between IncP conjugation and Agrobacterium tumefaciens T-DNA transfer to plants, two sets of mutants were generated in the larger transfer region (Tra1) of the IncPalpha plasmid RK2. Mutagenesis strategies were chosen to minimize transcriptional polar effects. Mutant Tra1 clones were mapped, sequenced, and processed to reconstruct 49.5-kb Tra2-containing plasmid derivatives in order to assay for transfer activity and IncP plasmid-specific phage sensitivity. Focusing on the activities of the gene products of traF and traG in Escherichia coli, we found that mutations in traF abolished transfer activity and rendered the host cells phage resistant and mutations in traG abolished transfer activity but had no effect on phage sensitivity. Complementation of these mutant derivatives with corresponding trans-acting clones carrying traF or traG restored transfer activity and, in the case of the traF mutant, the phage sensitivity of the host cell. We conclude that in E. coli, both TraF and TraG are essential for IncP plasmid transfer and that TraF is necessary (but not sufficient) for donor-specific phage sensitivity, and sequencing data suggest that both TraF and TraG are membrane spanning.