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Journal of Bacteriology, July 2005, p. 4900-4907, Vol. 187, No. 14
0021-9193/05/$08.00+0     doi:10.1128/JB.187.14.4900-4907.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Rapid Detection of Colicin E9-Induced DNA Damage Using Escherichia coli Cells Carrying SOS Promoter-lux Fusions

Mireille Vankemmelbeke,1 Bryan Healy,1 Geoffrey R. Moore,2 Colin Kleanthous,3 Christopher N. Penfold,1 and Richard James1*

School of Molecular Medical Sciences and Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, United Kingdom,1 School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, United Kingdom,2 Department of Biology, University of York, York YO10 5YW, United Kingdom3

Received 15 February 2004/ Accepted 12 April 2005

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.

* Corresponding author. Mailing address: Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, United Kingdom. Phone: 44-115-8467952. Fax: 44-115-8467951. E-mail: richard.james{at}nottingham.ac.uk.

Journal of Bacteriology, July 2005, p. 4900-4907, Vol. 187, No. 14
0021-9193/05/$08.00+0     doi:10.1128/JB.187.14.4900-4907.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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