Overproduction, purification and characterization of the F traT protein

A lambda transducing phage (ED lambda 110) which carries the sex factor F surface exclusion genes, traS and traT, was characterized by both genetic and physiochemical techniques. The transducing segment consists of 5.2 kilobases of F tra DNA, and carries the carboxy-terminal one-half of the upstream traG gene, as well as traS, traT, and the adjacent downstream gene traD. These tra proteins could be identified in infected UV-irradiated cells, and the major part of their synthesis was found to occur from the phage's late promoter pR' under Q control. Lysogens for ED lambda 110 were induced and found to greatly overproduce the traT gene product (TraTp), an outer membrane protein normally found in about 20,000 copies per cell, to levels which exceeded the major outer membrane proteins. This led to the development of a simple purification procedure for TraTp, the most important step of which was the construction of an appropriate ompB derivative to eliminate the major outer membrane porin proteins, which have several physical properties in common with TraTp. Purified TraTp was added to mixtures of donor and recipient cells and found to inhibit mating. The specificity of this assay was demonstrated by using an R100-1 donor, which responds to a heterologous surface exclusion system, and by using an altered TraTp containing a missense amino acid substitution. A mechanism by which TraTp mediates surface exclusion is proposed.