Mikhail Bogdanov

Bacterial conjugation, the process of horizontal gene transfer between bacteria, is initiated by mating pair formation (MPF) via a conjugative pilus. Conjugation of the IncP RP4 plasmid is mediated by short mating pili. Here, we report the cryo-EM structure of the RP4 pilus at 2.74 Å resolution. Uniquely, both the structural and quantitative mass spectral analyses 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 F- (pKpQIL), H- (R27) or W- (R388) pili, whose biogenesis and DNA transfer is PG-dependent. RP4 is the first example of a lipid-independent functional mating pilus. This discovery suggests that an amphipathic lipid moiety is not universally essential for the biogenesis of conjugative pili and MPF, providing an alternative model for their assembly and function. These data expand our understanding of the diverse bacterial mechanisms employ to transfer genetic material.

Transmembrane (TM) asymmetry of glycerophospholipids in the plasma membrane is well established, yet still a complex and evolving area of study regarding the exact mechanisms, magnitude, and composition. The emerging controversy challenges “Fluid Mosaic Sandwich” model simplicity by combining compositional (different types of lipids on each side) and numerical (different total amounts of phospholipids) asymmetries. While Gram-negative Escherichia coli phosphatidylethanolamine (PE) distribution is asymmetric (mostly cytoplasmic), the TM distribution of phosphatidylglycerol (PG) and cardiolipin (CL) across the inner (cytoplasmic) membrane (IM) is unknown. By integrating novel in vivo mapping techniques for PE, PG, and CL, we made the unexpected observation that their TM distribution does not correspond to the distribution of their total mass between the two IM leaflets. Instead, the underpopulated periplasmic leaflet can be occupied by lipid A, diacylglycerol (DAG), lipoproteins, and glycolipid MPase, whereas CL is found predominantly in the cytoplasmic leaflet of the IM. While PG is significantly enriched in the cytoplasmic leaflet of the IM in logarithmically growing E. coli, its asymmetrical distribution is inverted in stationary-grown cells, cells lacking CL, or cells subjected to growth in a hypotonic medium. These data suggest that cells can minimize the numerical differences in TM distribution by triggering a biosynthetic TM “cleanup” response, preventing membrane collapse. The numerical asymmetric interleaflet distribution of phospholipids may serve specific functional roles, potentially governed by critical threshold levels of lipid A and DAG that determine their compatibility for colocalization within the same leaflet. The overabundance of glycerophospholipids in the cytoplasmic leaflet of the IM may be driven by packing and localization constraints imposed during cell division and impacted by the rate of phospholipid biosynthesis and intermembrane flows. This imbalance may generate or result from the lipid concentration gradients and downhill flows across IM and between IM and outer membrane (OM), and the necessity to maintain appropriate electrostatic balance across the IM.

During conjugation, plasmid DNA is transferred from donor to recipient bacteria via the plasmid-encoded mating pilus, formed as thin helical assemblies of polymerised pilin subunits. In the IncHI1 R27 plasmid-encoded pilus, the TrhA pilin undergoes cyclisation (via a peptide bond between Gly1 and Asp69), essential for conjugation. Gly1 and Asp69 are exposed on the pilus surface and conserved in all TrhA pilins in the Plascad database. Substituting Asp69 with Asn, Ala, Gly, or Arg does not prevent cyclisation or pilus formation, which remains structurally indistinguishable from the wild type. Conjugation efficiency of the Asp69 substitutions across multiple recipient species correlates with side chain size, in the order Asp69Asn > Asp69Ala > Asp69Gly. However, Asp69Arg, as well as Asp69Lys and Gly1Lys substitutions abolish conjugation, likely due to the positively charged pilus surface (opposite to the wild-type negative charge) forming unfavourable electrostatic interactions with the recipient outer membrane’s inner leaflet, composed solely of zwitterionic phosphatidylethanolamine (PE). Consistently, conjugation is rescued in recipients lacking PE. These findings indicate strong selective pressure to maintain Gly1 and Asp69, as efficient DNA transfer depends on precise electrostatic and steric constraints of the pilus surface.