The plasmid R64 thin pilus identified as a type IV pilus

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• The IncI1 plasmid R64 carries genes essential for thin pilus formation.
• Understanding the genetic basis of pilus biogenesis is crucial for microbial pathogenesis and conjugation studies.

Purpose of the Study:

• To determine the complete nucleotide sequence of the pil region of the IncI1 plasmid R64.
• To identify genes and protein products involved in R64 thin pilus biogenesis.
• To classify the R64 thin pilus within known pilus families and investigate its role in conjugation.

Main Methods:

• DNA sequencing of the pil region of plasmid R64.
• Maxicell protein expression analysis to identify pil gene products.
• Globomycin treatment to determine lipoprotein localization.
• Bioinformatic analysis for sequence homology.
• Construction of frameshift mutations in pilR and pilU genes.

Main Results:

• The pil region of R64 contains 14 genes (pilI-pilV) involved in thin pilus formation.
• Eight pil gene products were identified, including a lipoprotein (PilN).
• Homology searches revealed similarities to type IV pilus biogenesis proteins.
• PilS and PilV were identified as potential prepilins, and PilU as a prepilin peptidase.
• PilR and PilU are essential for R64 liquid mating.

Conclusions:

• The R64 thin pilus is a type IV pilus, specifically group IVB.
• The R64 pilus biogenesis system shares common ancestry with other type IVB systems like V. cholerae's Tcp and E. coli's Bfp.
• The study elucidates the genetic and molecular mechanisms underlying R64 thin pilus assembly and function.