Genetic Method To Analyze Essential Genes of <i>Escherichia coli</i>

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.