Open AccessIsolation and characterization of novel cold‐sensitive dnaA mutants of Escherichia coli
Author(s) -
Guo Lei,
Katayama Tsutomu,
Seyama Yousuke,
Sekimizu Kazuhisa,
Miki Takeyoshi
Publication year - 1999
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1999.tb13684.x
Subject(s) - dnaa , mutant , plasmid , mutagenesis , biology , gene , escherichia coli , genetics , mutation , microbiology and biotechnology , origin of replication
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.