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Following Cell-fate in E. coli After Infection by Phage Lambda
Published on: October 14, 2011
Mutual adaptation of bacteriophage fd, pfd plasmids and their host strains
K Geider1, R Baldes, P Bellemann
1Max-Planck-Institut für medizinische Forschung, Heidelberg, Germany.
Microbiological Research
|November 1, 1995
Summary
Synthetic plasmids derived from filamentous phage fd exhibit unique adaptations in E. coli, including reduced viral gene expression and metabolic adjustments under stress. These findings offer insights into phage-plasmid interactions and potential cloning vector applications.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Filamentous bacteriophages, like phage fd, can be engineered into synthetic plasmids for molecular biology applications.
- Phage fd gene 2 protein is essential for replication but can be toxic to host cells.
- Bacteriophage genomes, including those of filamentous phages, show a tendency towards genome reduction.
Purpose of the Study:
- To investigate the autonomous replication and genetic stability of a synthetic plasmid (pfdC1) containing the phage fd replication origin and gene 2 in E. coli.
- To analyze host cell adaptations and plasmid behavior under stress conditions, particularly in E. coli strains carrying phage fd gene 2 on an F-episome.
- To explore the potential of engineered bacteriophage vectors for genetic manipulation and cloning.
Main Methods:
- Construction and autonomous replication of synthetic plasmid pfdC1 in E. coli.
- DNA sequencing to identify mutations in pfdC1 compared to the wild-type fd genome.
- Culturing E. coli strains under stress conditions (42°C) with specific plasmid and episomal constructs.
- Analysis of host cell metabolism and plasmid gene expression.
- Investigating gene restoration mechanisms in bacteriophages with transposon insertions.
Main Results:
- The synthetic plasmid pfdC1 replicated autonomously in E. coli, with observed mutations leading to reduced expression of the potentially toxic viral gene 2 protein.
- E. coli strains carrying fd gene 2 on an F-episome and a pfdA-plasmid adapted their metabolism at 42°C without altering plasmid replication or episomal gene expression.
- Filamentous bacteriophages demonstrated genome size reduction (miniphages) and gene restoration capabilities through homologous recombination in recA-host cells.
- Mobilization of pfd-plasmids was hindered by replication-transfer interference in rolling circle replication.
Conclusions:
- Engineered phage fd plasmids exhibit adaptive mutations and host responses that mitigate toxicity and ensure stable replication in E. coli.
- The study highlights the plasticity of bacteriophage genomes and their interaction with host systems.
- The developed vectors demonstrate compatibility and potential utility as cloning vectors in diverse molecular biology applications.
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