title: Isolation and characterization of novel cold-sensitive dnaA mutants of Escherichia coli.
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.
authors.affiliation: Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: L
authors.initials: L
authors.lastname: Guo
authors.forename: T
authors.initials: T
authors.lastname: Katayama
authors.forename: Y
authors.initials: Y
authors.lastname: Seyama
authors.forename: K
authors.initials: K
authors.lastname: Sekimizu
authors.forename: T
authors.initials: T
authors.lastname: Miki
mesh_terms: D000222:Adaptation, Physiological; D000483:Alleles; D001426:Bacterial Proteins / Q000235:genetics*; D003080:Cold Temperature; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D016296:Mutagenesis; D011815:R Factors; D017422:Sequence Analysis, DNA
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria