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A specific protease encoded by the conjugative DNA transfer systems of IncP and Ti plasmids is essential for pilus
Insights
The TraF protease of broad-host-range plasmid RP4 is crucial for bacterial conjugation. It processes the TrbC prepilin protein, enabling pilus synthesis and phage propagation.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Broad-host-range plasmid RP4 utilizes a conjugative transfer apparatus for DNA dissemination.
- TraF is an essential component of this apparatus, functioning as a protease localized to the cytoplasmic membrane.
- TraF shares similarities with prokaryotic and eukaryotic signal peptidases, suggesting a conserved proteolytic mechanism.
Purpose of the Study:
- To investigate the proteolytic activity and function of the RP4 TraF protease.
- To identify the substrates and processing pathways mediated by TraF.
- To elucidate the role of TraF in conjugative transfer and pilus biogenesis.
Main Methods:
- Mutational analysis of the RP4 TraF gene.
- Biochemical characterization of TraF proteolytic activity.
- Identification and analysis of TraF substrates, including TrbC.
Main Results:
- RP4 TraF exhibits protease activity similar to signal and LexA-like peptidases.
- TrbC, the RP4 prepilin, was identified as a primary target of TraF protease activity.
- TraF-mediated cleavage of TrbC is essential for pilus synthesis, conjugative transfer, and bacteriophage PRD1 propagation.
Conclusions:
- TraF protease plays a critical role in the maturation of the RP4 pilus subunit, TrbC.
- This processing event is a key step in the cascade leading to pilus assembly and conjugative DNA transfer.
- TraF activity is vital for the successful propagation of the donor-specific bacteriophage PRD1.
Abstract:
TraF, an essential component of the conjugative transfer apparatus of the broad-host-range plasmid RP4 (IncP), which is located at the periplasmic side of the cytoplasmic membrane, encodes a specific protease. The traF gene products of IncP and Ti plasmids show extensive similarities to prokaryotic and eukaryotic signal peptidases. Mutational analysis of RP4 TraF revealed that the mechanism of the proteolytic cleavage reaction resembles that of signal and LexA-like peptidases. Among the RP4 transfer functions, the product of the Tra2 gene, trbC, was identified as a target for the TraF protease activity. TrbC is homologous to VirB2 of Ti plasmids and thought to encode the RP4 prepilin. The maturation of TrbC involves three processing reactions: (i) the removal of the N-terminal signal peptide by Escherichia coli signal peptidase I (Lep), (ii) a proteolytic cleavage at the C terminus by an as yet unidentified host cell enzyme, and (iii) C-terminal processing by TraF. The third reaction of the maturation process is critical for conjugative transfer, pilus synthesis, and the propagation of the donor-specific bacteriophage PRD1. Thus, cleavage of TrbC by TraF appears to be one of the initial steps in a cascade of processes involved in export of the RP4 pilus subunit and pilus assembly mediated by the RP4 mating pair formation function.