R27 pilus biogenesis is promiscuous and electrostatic perturbations impair functionality

During conjugation, plasmid DNA is transferred from donor to recipient bacteria via the plasmid-encoded mating pilus, built as thin helical assemblies of polymerised pilin subunits. The structure of the IncHI1 R27 plasmid encoded pilus revealed that the mature ThrA pilin is cyclized by a peptide bond formed between Gly1 (G1) and Asp69 (D69). Loss of cyclization abolished pilus biogenesis and conjugation. Here we report that G1 and D69 are completely conserved in all TrhA pilins in the Plascad database. Substituting D69 with N, A, G or R showed that cyclization is a promiscuous process as all mutants cyclised and polymerised into pili. However, the D69R substitution specifically altered the electrostatic potential of the pilus surface, resulting in very low conjugation efficiency. Moreover, conjugation efficiency of the other substitutions into Escherichia coli MG1655, Enteropathogenic E. coli , Klebsiella pneumoniae , Enterobacter cloacae, Citrobacter rodentium and Citrobacter amalonaticus showed a consistent trend towards correlation with the side chain size: TrhA D69N > D69A > D69G. These results suggest that while cyclisation is promiscuous, there is a strong selective pressure to maintain D as the carboxy terminal residue for TrhA cyclisation.