Phospholipid-independent biogenesis and function of the RP4 conjugation pilus

Area of Science:

• Microbiology
• Structural Biology
• Molecular Biology

Background:

• Bacterial conjugation facilitates horizontal gene transfer through mating pair formation (MPF) mediated by conjugative pili.
• The IncP RP4 plasmid utilizes short mating pili for its conjugation process.
• Understanding pilus biogenesis is crucial for comprehending bacterial genetic exchange mechanisms.

Purpose of the Study:

• To determine the high-resolution cryo-electron microscopy (cryo-EM) structure of the RP4 pilus.
• To investigate the role of lipid modification in the assembly and function of conjugative pili.
• To compare the RP4 pilus with other characterized pili regarding lipid dependency.

Main Methods:

• Cryo-electron microscopy (cryo-EM) was employed to resolve the RP4 pilus structure at 2.74 Å resolution.
• Quantitative mass spectrometry was utilized to analyze the composition and modifications of pilin subunits.
• Functional assays were performed using an *E. coli pgsA* mutant to assess pilus biogenesis and DNA transfer dependency on phosphatidylglycerol (PG).

Main Results:

• The cryo-EM structure revealed the detailed architecture of the RP4 pilus.
• Structural and mass spectral analyses demonstrated that the cyclic TrbC pilin subunit of RP4 is not lipidated.
• RP4 conjugation occurred in an *E. coli pgsA* mutant lacking phosphatidylglycerol (PG), unlike F-, H-, and W-pili, which require PG for biogenesis and DNA transfer.

Conclusions:

• The RP4 pilus represents the first identified functional mating pilus that is lipid-independent.
• This finding challenges the universal requirement of amphipathic lipid moieties for conjugative pilus assembly and MPF.
• The study provides an alternative model for pilus assembly and function, expanding the understanding of bacterial genetic material transfer mechanisms.