title: Genetic method to analyze essential genes of Escherichia coli.
abstract: The genetic analysis of essential genes has been generally restricted to the use of conditional mutations, or inactivating chromosomal mutations, which require a complementing plasmid that must either be counterselected or lost to measure a phenotype. These approaches are limited because they do not permit the analysis of mutations suspected to affect a specific function of a protein, nor do they take advantage of the increasing abundance of structural and bioinformatics data for proteins. Using the dnaC gene as an example, we developed a genetic method that should permit the mutational analysis of other essential genes of Escherichia coli and related enterobacteria. The method consists of using a strain carrying a large deletion of the dnaC gene, which is complemented by a wild-type copy expressed from a plasmid that requires isopropyl-beta-d-thiogalactopyranoside for maintenance. Under conditions in which this resident plasmid is lost, the method measures the function of a dnaC mutation encoded by a second plasmid. This methodology should be widely applicable to the genetic analysis of other essential genes.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319, USA.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Hupert-Kocurek
authors.forename: Jay M
authors.initials: JM
authors.lastname: Sage
authors.forename: Magdalena
authors.initials: M
authors.lastname: Makowska-Grzyska
authors.forename: Jon M
authors.initials: JM
authors.lastname: Kaguni
mesh_terms: D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D020043:Genes, Essential; D005821:Genetic Techniques; D009154:Mutation; D010957:Plasmids
chemical_list: C071702:DnaC protein, E coli; D029968:Escherichia coli Proteins