Phospholipid-independent biogenesis of a functional RP4 conjugation pilus

Conjugation, the process of DNA transfer between bacteria, is initiated universally by the formation of a mating pair formation (MPF) via a conjugative pilus. Conjugation of the IncP RP4 plasmid is mediated by short, non-retractable, rigid mating pili. Here, we report the cryo-EM structure of the RP4 pilus at 2.75 Å resolution. Uniquely, and consistently with quantitative mass spectral analysis, this revealed that the cyclic TrbC pilin subunit is not lipidated. Consistently, an E. coli pgsA mutant lacking phosphatidylglycerol (PG) can serve as a donor of RP4 but not of R27, encoding the H-pilus consisting of PG-associated cyclic pilin subunits (TrhA). The RP4 is the first example of a lipid-independent functional mating pilus. This discovery not only challenges the long-held assumption that an amphipathic lipid moiety is essential for the construction of conjugative pili and for MPF, but also expanding our understanding of the diverse mechanisms that bacteria employ to transfer genetic material.