In this work subunits of extracellular filaments, conjugative pili, of the Ti plasmids virulence region and the conjugative resistance plasmids RP4 and R751 were identified. In the latter two cases TrbC propilin is the precursor of the pilus subunit in E. coli. Likewise, its homologue VirB2 propilin is processed to the pilin of the Ti plasmids' T pilus in Agrobacterium tumefaciens. TrbC and VirB2 propilin are truncated post-translationally at the N terminus by the removal of a 36/47-residue leader peptide, respectively. TrbC propilin undergoes a second processing step by the removal of 27 residues at the C terminus by host-encoded functions followed by the cleavage of four additional C‑terminal residues by a plasmid-borne serine protease. The final IncP pilin product of 78 residues is cyclized in a concerted, highly efficient way, comprising an intramolecular covalent head-to-tail peptide bond. The T pilin does not undergo additional truncation but is likewise cyclized in Agrobacterium, where a TraF-like function must be host-encoded, but not in E. coli. Circular structures of these pilins, as verified by mass spectrometry and site directed mutagenesis, represent a novel primary configuration that conform and assemble into the conjugative apparatus.
The RP4 plasmid encoded protease/cyclase TraF was subject to extended mutational analysis in this study. The combined results of TrbC and TraF investigation are used to propose a mechanism for the cyclization reaction of the pilin.