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Insertion and replication of the Pseudomonas aeruginosa mutator phage D3112
Abstract:
D3112 is a temperate bacteriophage of P. aeruginosa with heterogeneous sequences at one extremity of the virion DNA molecule. Infection of strain PAOl with phage D3112 results in a 40- to 65-fold increase in the frequency of ami mutants resistant to fluoroacetamide. Nine ami::D3112 prophages have been mapped to distinct sites within the ami locus by Southern blotting experiments with a cloned ami+ probe. All prophages have the same restriction map as the D3112 genome extracted from phage particles. The position of D3112 insertions correlates with the phenotype and reversion behavior of the ami mutants. Induction of D3112cts prophages results in amplification of internal prophage segments as discrete restriction fragments before the terminal viral fragments are visible as sharp hybridizing species. This indicates that D3112 replication is accompanied by recombination of prophage termini to numerous sites in the bacterial genome. Chromosomal junction fragments of an ami::D3112cts prophage are maintained through most of the replication cycle but are cleaved shortly before cell lysis, apparently by the viral encapsidation system.
Insights
The temperate bacteriophage D3112 insertion into Pseudomonas aeruginosa increases fluoroacetamide-resistant mutants. D3112 prophage replication involves recombination and specific cleavage before bacterial cell lysis.
Area of Science:
- Microbiology
- Molecular Biology
- Bacteriophage Research
Background:
- Pseudomonas aeruginosa is a common opportunistic pathogen.
- Temperate bacteriophages can integrate into bacterial genomes.
- D3112 is a temperate bacteriophage with unique DNA characteristics.
Purpose of the Study:
- To investigate the mechanism of D3112 bacteriophage integration and replication in P. aeruginosa.
- To understand the genetic basis of increased fluoroacetamide resistance in P. aeruginosa mutants.
- To characterize the role of D3112 prophage in bacterial mutagenesis.
Main Methods:
- Bacteriophage infection and mutant selection.
- Southern blotting for prophage mapping.
- Restriction enzyme analysis of viral DNA.
- Induction of prophages and analysis of replication intermediates.
Main Results:
- D3112 infection increased fluoroacetamide resistance 40-65 fold.
- Nine D3112 prophages were mapped to the ami locus.
- Prophage insertion sites correlated with mutant phenotypes.
- D3112 replication involved recombination and specific cleavage of junction fragments.
Conclusions:
- D3112 integration and replication are complex processes involving recombination.
- The viral encapsidation system plays a role in cleaving prophage DNA before lysis.
- D3112 can serve as a tool to study mutagenesis and genome dynamics in P. aeruginosa.