Cryo-EM structure of the Agrobacterium tumefaciens T-pilus reveals the importance of positive charges in the lumen

• Published in: Structure (London) 2023-04, Vol.31 (4), p.375-384.e4
• Main Authors: Amro, Jaafar, Black, Corbin, Jemouai, Zakaria, Rooney, Nathan, Daneault, Caroline, Zeytuni, Natalie, Ruiz, Matthieu, Bui, Khanh Huy, Baron, Christian
• Format: Article
• Language: English
• Subjects: Agrobacterium tumefaciens; Agrobacterium tumefaciens - chemistry; Agrobacterium tumefaciens - genetics; Agrobacterium tumefaciens - metabolism; Bacterial Proteins - chemistry; Cell Membrane - metabolism; conjugation; Cryoelectron Microscopy; Fimbriae, Bacterial - metabolism; pili; pKM101; plasmid; type IV secretion system; VirB2
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2022.11.007

Agrobacterium tumefaciens is a natural genetic engineer that transfers DNA into plants, which is the most applied process for generation of genetically modified plants. DNA transfer is mediated by a type IV secretion system in the cell envelope and extracellular T-pili. We here report the cryo-electron microscopic structures of the T-pilus at 3.2-Å resolution and of the plasmid pKM101-determined N-pilus at 3-Å resolution. Both pili contain a main pilus protein (VirB2 in A. tumefaciens, TraM in pKM101) and phospholipids arranged in a five-start helical assembly. They contain positively charged amino acids in the lumen, and the lipids are positively charged in the T-pilus (phosphatidylcholine) conferring overall positive charge. Mutagenesis of the lumen-exposed Arg91 in VirB2 results in protein destabilization and loss of pilus formation. Our results reveal that different phospholipids can be incorporated into type IV secretion pili and that the charge of the lumen may be of functional importance. [Display omitted] •We report the cryo-EM structures of two type IV secretion system-determined pili•Both pili comprise major pilin proteins and phospholipids in a five-start helical assembly•The T-pilus contains phosphatidylcholine conferring positive charge to the lumen•The positive charge of the lumen may be of functional importance Amro et al. describe the high-resolution cryo-EM structures of pili from the natural genetic engineer Agrobacterium tumefaciens and from the antimicrobial resistance gene-carrying plasmid pKM101. The Agrobacterium pilus contains a positively charged phospholipid, and the charge of the lumen may be of functional importance for the transfer of DNA to plants.