Isolation and characterization of novel cold-sensitive dnaA mutants of Escherichia coli

Lei Guo, Tsutomu Katayama, Yousuke Seyama, Kazuhisa Sekimizu, Takeyoshi Miki

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)


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. Copyright (C) 1999 Federation of European Microbiological Societies.

Original languageEnglish
Pages (from-to)357-366
Number of pages10
JournalFEMS microbiology letters
Issue number2
Publication statusPublished - Jul 15 1999

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology
  • Genetics


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