PMID:10427718

Citation	Guo, L, Katayama, T, Seyama, Y, Sekimizu, K and Miki, T (1999) Isolation and characterization of novel cold-sensitive dnaA mutants of Escherichia coli. FEMS Microbiol. Lett. 176:357-66
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.
Links	PubMed
Keywords	Adaptation, Physiological; Alleles; Bacterial Proteins/genetics; Cold Temperature; DNA Replication; DNA, Bacterial/biosynthesis; DNA-Binding Proteins/genetics; Escherichia coli/genetics; Escherichia coli/isolation & purification; Escherichia coli/physiology; Mutagenesis; R Factors; Sequence Analysis, DNA
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