Evidence that F-plasmid proteins TraV, TraK and TraB assemble. into an envelope-spanning structure in Escherichia coli

We have examined the role of the F-plasmid TraV outer membrane lipoprotein in the assembly of F-pili. Yeast two-hybrid analysis with a traV bait repea tedly identified traK, which is predicted to encode a periplasmic protein, among positive prey plasmids. A traK bait in turn identified traV and traB, which is predicted to encode an inner membrane protein. A traB bait exclus ively identified traK preys. Several additional observations support the hy pothesis that TraV, TraK and TraB form a complex in Escherichia coli that s pans the cell envelope from the outer membrane (TraV) through the periplasm (TraK) to the inner membrane (TraB). First, two-hybrid analyses indicated that TraV and TraB bind to different TraK segments, as required if TraK bri dges a ternary complex. Secondly, all three proteins fractionated with the E. coli outer membrane in tra(+) cells. In contrast, TraB fractionated with the inner membrane in traV or traK mutant cells, and TraK appeared in the osmotic shock fluid from the traV mutant. These results are consistent with a TraV-TraK-TraB complex anchored to the outer membrane via the TraV lipop rotein. Further, in traK mutant cells, TraV failed to accumulate to a detec table level, and the TraB level was significantly reduced, suggesting that TraV and TraB must interact with TraK for either protein to accumulate to i ts normal level. Both TraK and TraV accumulated in traB2[Am] cells; however , the TraB2 amber fragment could be detected by Western blot, and sequence analysis indicated that the fragment retained the TraK-binding domain sugge sted by yeast two-hybrid analysis. We propose that TraV is the outer membra ne anchor for a trans-envelope, Tra protein structure required for the asse mbly of F-pili and possibly for other events of conjugal DNA transfer.