# H pilin cyclisation and pilus biogenesis are promiscuous but electrostatic perturbations impair conjugation efficiency

## TL;DR

The study shows that specific amino acid changes in a bacterial pilus disrupt DNA transfer due to electrostatic interactions with recipient cell membranes.

## Contribution

The work reveals that electrostatic interactions between pilus and recipient membrane phospholipids are critical for efficient conjugation.

## Key findings

- Substituting Asp69 with amino acids of varying side chain sizes affects conjugation efficiency in a size-dependent manner.
- Positive charges at Gly1 or Asp69 abolish conjugation due to unfavorable electrostatic interactions with recipient membranes.
- Conjugation is restored in recipients lacking phosphatidylethanolamine, confirming the role of membrane phospholipids.

## Abstract

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.

Cyclisation of R27 plasmid TrhA pilin occurs via a Gly1–Asp69 bond. Here, authors show Gly1/Lys and Asp69/Lys substitutions impose positive pilus charge, abolishing conjugation into wild-type recipients, rescued in phosphatidylethanolamine-deficient recipients, highlighting phospholipid importance in both donors and recipients.