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Applied and Environmental Microbiology, November 2007, p. 7075-7082, Vol. 73, No. 21
0099-2240/07/$08.00+0     doi:10.1128/AEM.01756-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Genetic Method To Analyze Essential Genes of Escherichia coli{triangledown}

Katarzyna Hupert-Kocurek,{dagger} Jay M. Sage,{ddagger} Magdalena Makowska-Grzyska, and Jon M. Kaguni*

Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824-1319

Received 28 July 2007/ Accepted 31 August 2007

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-ß-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.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824-1319. Phone: (517) 353-6721. Fax: (517) 353-9334. E-mail: kaguni{at}msu.edu

{triangledown} Published ahead of print on 14 September 2007.

{dagger} Present address: Department of Biochemistry, University of Silesia, Jagiellonska 28, 40032 Katowice, Poland.

{ddagger} Present address: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01655-0116.

Applied and Environmental Microbiology, November 2007, p. 7075-7082, Vol. 73, No. 21
0099-2240/07/$08.00+0     doi:10.1128/AEM.01756-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.





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