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Isolation and characterization of Co1E2 plasmid mutants unable to kill colicin-sensitive cells
Abstract:
After transfer from a mutagenized host, twenty one Co1E2 plasmid mutants were isolated after screening 10,000 clones for abnormal colicin production. Analysis by SDS polyacrylamide slab gel electrophoresis of proteins synthesized after mitomycin C-induction of mutant cultures, indicates that all but two of the mutations are in the structural gene from colicin E2. Of these, nine produce fragments of colicin in both whole cells and minicells and some are suppressed by nonsense suppressors. Studies with a nonsense mutant producing only a small colicin E2 fragment (Co1E2-421) suggest that colicin E2 in not involved in plasmid DNA replication, in the control of its own synthesis, or required for cell death when cells become committed to colicin production. The two plasmid mutants outside the colicin gene segregate plasmid-free cells at 33 degrees, 37 degrees and 43 degrees. One segregates fairly rapidly (about 4% per generation) though the colicin-producing cells make normal amounts of colicin, whilst the other segregates more slowly and the colicin-producing cells make much reduced amounts of colicin.
Insights
Researchers identified 21 colicin E2 plasmid mutants, with most affecting the colicin gene. Studies revealed colicin E2 is not essential for plasmid replication or cell death, while other mutants impact plasmid stability.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Colicin E2 is a bacteriocin produced by certain strains of Escherichia coli.
- Understanding the genetic basis of colicin production and its regulation is crucial for microbial genetics.
- Plasmid mutants offer insights into gene function and cellular processes.
Purpose of the Study:
- To isolate and characterize mutants of the Co1E2 plasmid with altered colicin production.
- To determine the role of the colicin E2 structural gene in plasmid maintenance and cell death.
- To investigate plasmid stability and colicin production in mutants affecting non-colicin genes.
Main Methods:
- Screening of 10,000 clones for abnormal colicin production after transfer from a mutagenized host.
- SDS-PAGE analysis of proteins synthesized after mitomycin C induction in mutant cultures.
- Characterization of plasmid segregation and colicin production in selected mutants.
Main Results:
- Twenty-one Co1E2 plasmid mutants were isolated; 19 had mutations in the colicin E2 structural gene.
- Nine mutants produced colicin E2 fragments, with some mutations suppressed by nonsense suppressors.
- A nonsense mutant (Co1E2-421) indicated colicin E2 is not involved in plasmid replication, autoregulation, or cell death.
- Two mutants outside the colicin gene exhibited plasmid-free cell segregation at various temperatures, affecting colicin production levels.
Conclusions:
- The colicin E2 structural gene is primarily responsible for colicin E2 production.
- Colicin E2 is not essential for plasmid DNA replication, autoregulation, or commitment to cell death.
- Mutations outside the colicin gene can affect plasmid stability and influence colicin production.