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ColE9 is a plasmid-encoded protein antibiotic produced by Escherichia coli and closely related species that kills E. coli cells expressing the BtuB receptor. The 15-kDa cytotoxic DNase domain of colicin E9 preferentially nicks double-stranded DNA at thymine bases and shares a common active-site structural motif with a variety of other nucleases, including the H-N-H homing endonucleases and the apoptotic CAD proteins of eukaryotes. Studies of the mechanism by which the DNase domain of ColE9 reaches the cytoplasm of E. coli cells are limited by the lack of a rapid, sensitive assay for the DNA damage that results. Here, we report the development of an SOS promoter-lux fusion reporter system for monitoring DNA damage in colicin-treated cells and illustrate the value of this reporter system in experiments that probe the mechanism and time required for the DNase domain of colicin E9 to reach the cytoplasm.

Original publication

DOI

10.1128/JB.187.14.4900-4907.2005

Type

Journal article

Journal

J Bacteriol

Publication Date

07/2005

Volume

187

Pages

4900 - 4907

Keywords

Colicins, DNA Damage, DNA, Bacterial, Dithiothreitol, Escherichia coli, Escherichia coli Proteins, Genes, Reporter, Kinetics, Luminescent Measurements, Plasmids, Promoter Regions, Genetic, Recombinant Fusion Proteins, SOS Response (Genetics)