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  • Title: Isolation and characterization of novel cold-sensitive dnaA mutants of Escherichia coli.
    Author: Guo L, Katayama T, Seyama Y, Sekimizu K, Miki T.
    Journal: FEMS Microbiol Lett; 1999 Jul 15; 176(2):357-66. PubMed ID: 10427718.
    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.
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