Isolation and characterization of novel cold-sensitivednaAmutants ofEscherichia coli
Open Access
- 1 July 1999
- journal article
- Published by Oxford University Press (OUP) in FEMS Microbiology Letters
- Vol. 176 (2) , 357-366
- https://doi.org/10.1111/j.1574-6968.1999.tb13684.x
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.Keywords
This publication has 20 references indexed in Scilit:
- Novel alleles of the Escherichia coli dnaA geneJournal of Molecular Biology, 1997
- Domains of DnaA Protein Involved in Interaction with DnaB Protein, and in Unwinding the Escherichia coli Chromosomal OriginJournal of Biological Chemistry, 1996
- The 4784V missense mutation of the dnaA5 and dnaA46 alleles confers a defect in ATP binding and thermoiability in initiation of Escherichia coli DNA replicationMolecular Microbiology, 1996
- Mapping the cAMP receptor protein contact site on the α subunit of Escherichia coli RNA polymeraseMolecular Microbiology, 1992
- Control of segregation of chromosomal DNA by sex factor F in Escherichia coliJournal of Molecular Biology, 1992
- Structure of the dnaA region of Micrococcus luteus: conservation and variations among eubacteriaGene, 1990
- Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosomeCell, 1988
- pHG165: A pBR322 copy number derivative of pUC8 for cloning and expressionPlasmid, 1986
- The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sitesCell, 1984
- The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primersGene, 1982