Interaction between the F plasmid TraA (F-pilin) and TraQ proteins

Elaboration of conjugative (F) pili by F+ strains of Escherichia coli requi res the activities of over a dozen F-encoded DNA transfer (Tra) proteins. T he organization and functions of these proteins are largely unknown. Using the yeast two-hybrid assay, we have begun to analyse binary interactions am ong the Tra proteins required for F-pilus formation. We focus here on inter actions involving F-pilin, the only known F-pilus subunit. Using a library of F tra DNA fragments that contained all the F genes required for F pilus formation in a yeast GAL4 activation domain vector (pACTII), we transformed yeast containing a plasmid (pAS1CYH2traA) encoding a GAL4 DNA-binding doma in-F-pilin fusion. Doubly transformed cells were screened for GAL4-dependen t gene expression. This screen repeatedly identified only a single Tra prot ein, TraQ, previously identified as a likely F-pilin chaperone. The F-pilin -TraQ interaction appeared to be specific, as no transcriptional activation was detected in yeast transformants containing pACTIItraQ plasmids and the Salmonella typhi pED208 traA gene cloned in pAS1CYH2. Two traQ segments is olated in the screen against F-pilin were tested for complementation of a t raQ null allele in E. coli. One, lacking the first 11 (of 94) TraQ amino ac ids, restored DNA donor activity, donor-specific bacteriophage sensitivity and membrane F-pilin accumulation to wild-type levels. The second, lacking the first 21 amino acids, was much less effective in these assays. Both Tra Q polypeptides accumulated in E. coli as transmembrane proteins. The longer , biologically active segment was fused to the GAL4 DNA-binding domain gene of pAS1CYH2 and used to screen the tra fragment library. The only positive s from this screen identified traA segments. The fusion sites between the t raA and GAL4 segments identified the hydrophobic, C-terminal domain IV of F -pilin as sufficient for the interaction. As TraQ is the only Tra protein r equired for the accumulation of inner membrane F-pilin, the interaction pro bably reflects a specific, chaperone-like function for TraQ in E. coli. Att empts to isolate an F-pilin-TraQ complex from E. coli were unsuccessful, su ggesting that the interaction between the two is normally transient, as exp ected from previous studies of the kinetics of TraA membrane insertion and processing to F-pilin.