The role of F plasmid TraF and TrbB in stabilizing the conjugative apparatus

Abstract

The role of two proteins, TrbB and TraF, encoded by the F plasmid, were investigated for their ability to affect F transfer protein localization and stability using cell fractionation by sucrose gradient sedimentation and immunoblot analyses. Bacterial conjugation mediates the horizontal transfer of genetic material throughout the microbial world. The F plasmid contains a transfer region that encodes a type IV secretion system (T4SS) necessary for conjugative pilus assembly and DNA transfer. The F-type T4SS are considerably more complex than the P-type systems because of the properties of pilus retraction and stable mating pair formation. The proteins that are specific to F-T4SS are TraF, -G (C-terminal domain), TraH, -N, -U, -W, TrbB, -C and-I. TraG, -N and -U are important for mating pair stabilization and the remaining proteins are required for pilus assembly and retraction. Several F-T4SS proteins are remarkable for their high cysteine content (TraN, TraU and TraH have 22, 10 and 6 cysteines, respectively). TraF and TrbB, have thioredoxin-folds with TrbB, but not TraF, having a characteristic CxxC motif. Previously, we have shown TrbB has DsbC-like activity and acts as a disulfide bond isomerase since, like DsbC, it can partially complement a dsbA mutation. Whereas TrbB is not essential for conjugation, TraF is required for pilus assembly and transfer, suggesting that it has a role in construction of the T4SS conjugative pore. Using antisera to the F-T4SS proteins we demonstrate that traF and trbB mutations affect the stability of the T4SS complex, particularly TraH and TraV.