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The plasmid R64 thin pilus identified as a type IV pilus
Abstract:
The entire nucleotide sequence of the pil region of the IncI1 plasmid R64 was determined. Analysis of the sequence indicated that 14 genes, designated pilI through pilV, are involved in the formation of the R64 thin pilus. Protein products of eight pil genes were identified by the maxicell procedure. The pilN product was shown to be a lipoprotein by an experiment using globomycin. A computer search revealed that several R64 pil genes have amino acid sequence homology with proteins involved in type IV pilus biogenesis, protein secretion, and transformation competence. The pilS and pilV products were suggested to be prepilins for the R64 thin pilus, and the pilU product appears to be a prepilin peptidase. These results suggest that the R64 thin pilus belongs to the type IV family, specifically group IVB, of pili. The requirement of the pilR and pilU genes for R64 liquid mating was demonstrated by constructing their frameshift mutations. Comparison of three type IVB pilus biogenesis systems, the pil system of R64, the toxin-coregulated pilus (tcp) system of Vibrio cholerae, and the bundle-forming pilus (bfp) system of enteropathogenic Escherichia coli, suggests that they have evolved from a common ancestral gene system.
Insights
The R64 plasmid
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.