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Published on: November 12, 2012
Isolation and characterization of novel cold-sensitive dnaA mutants of Escherichia coli
1Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Abstract:
We developed an efficient method for isolation of novel dnaA mutations based on PCR mutagenesis in the presence of manganese ion and shuffling of dnaA-carrying plasmids in a dnaA deletion host bacterium. Using this system, we obtained 30 cold-sensitive mutants from 4000 clones carrying plasmids with a mutagenized dnaA gene. All 27 cold-sensitive mutants analyzed were defective in DNA replication; none had a DnaAcos (over-initiation) phenotype. Nucleotide sequencing revealed that novel 15 alleles (mutations in 14 amino acid residues) are responsible for the cold-sensitive phenotype and are all located in the carboxy-terminal half of the DnaA protein.
Insights
Researchers developed a new method to find dnaA gene mutations. This efficient technique identified 15 novel cold-sensitive dnaA alleles, all impacting DNA replication and located in the protein
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- The dnaA gene is crucial for initiating DNA replication in bacteria.
- Understanding dnaA mutations is key to deciphering DNA replication control.
- Previous methods for isolating dnaA mutations were less efficient.
Purpose of the Study:
- To develop an efficient method for isolating novel dnaA mutations.
- To characterize the phenotype and location of newly identified dnaA alleles.
Main Methods:
- Utilized PCR mutagenesis in the presence of manganese ions.
- Employed plasmid shuffling in a dnaA deletion host bacterium.
- Screened for cold-sensitive phenotypes and analyzed DNA replication.
- Determined mutation locations via nucleotide sequencing.
Main Results:
- Isolated 30 cold-sensitive dnaA mutants from 4000 screened clones.
- 27 cold-sensitive mutants analyzed were defective in DNA replication.
- Identified 15 novel dnaA alleles, all located in the carboxy-terminal half of the DnaA protein.
- No over-initiation (DnaAcos) phenotype was observed in the cold-sensitive mutants.
Conclusions:
- The developed method is efficient for isolating dnaA mutations.
- Novel cold-sensitive dnaA alleles primarily affect DNA replication initiation.
- The carboxy-terminal region of DnaA is critical for regulating DNA replication initiation, especially at low temperatures.
