Mutational analysis of F-pilin reveals domains for pilus assembly, phage infection and DNA transfer
J Manchak1, K G Anthony, Laura S Frost
1Department of Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E9, Canada.
Abstract:
The F-pilus has been implicated in recipient cell recognition during the establishment of a stable mating pair before conjugation as well as forming part of the conjugative pore for DNA transfer. The F-pilus is the site of attachment of the filamentous phages (M13, f1 and fd), which attach to the F-pilus tip, and the RNA phages, R17 and Qbeta, which attach to different sites exposed on the sides of the pilus. R17 has been shown to undergo eclipse, or capsid release, outside the cell on pili attached to cells. New and existing mutants of traA combined with natural variants of F-pilin were assayed for pilin stability and processing, pilus elongation, transfer, phage sensitivity and R17 eclipse. Phenotypes of these mutants indicated that the F-pilin subunit contains specific regions that can be associated with pilus assembly, phage sensitivity and DNA transport. Mutations involving lysines and phenylalanines within residues 45-60 suggest that these residues might participate in transmitting a signal down the length of the pilus that initiates DNA transfer or R17 eclipse.
Insights
Researchers studied the F-pilus, crucial for bacterial conjugation and phage attachment. Mutations revealed specific F-pilin regions involved in pilus assembly, DNA transfer, and phage interactions, potentially signaling DNA transfer initiation.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The F-pilus is essential for bacterial conjugation, mediating recipient cell recognition and DNA transfer.
- It also serves as an attachment site for various bacteriophages, including filamentous and RNA phages.
- The F-pilus structure and function are critical for these interactions.
Purpose of the Study:
- To investigate the role of specific F-pilin regions in pilus assembly, stability, and function.
- To understand how F-pilus mutations affect phage sensitivity and DNA transfer during conjugation.
- To identify residues involved in signaling DNA transfer or phage R17 eclipse.
Main Methods:
- Generation and analysis of new and existing traA mutants combined with natural F-pilin variants.
- Assays for pilin stability, processing, pilus elongation, and transfer efficiency.
- Evaluation of phage sensitivity and R17 eclipse phenotypes.
Main Results:
- Mutant phenotypes indicated specific F-pilin regions are associated with pilus assembly, phage sensitivity, and DNA transport.
- Mutations affecting lysines and phenylalanines (residues 45-60) were identified.
- These residues may play a role in signal transmission for DNA transfer or R17 eclipse.
Conclusions:
- The F-pilin subunit possesses distinct regions critical for pilus assembly and function.
- Specific amino acid residues within the F-pilin are involved in mediating interactions with phages and facilitating DNA transfer.
- These findings provide insights into the molecular mechanisms governing bacterial conjugation and phage-host interactions.
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