Rapid detection of colicin E9-induced DNA damage using Escherichia coli cells carrying SOS promoter-lux fusions.
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.
Centre for Biomolecular Sciences, University of Nottingham, University Park, UK.
Mireille
M
Vankemmelbeke
Bryan
B
Healy
Geoffrey R
GR
Moore
Colin
C
Kleanthous
Christopher N
CN
Penfold
Richard
R
James
D003087:Colicins / Q000235:genetics*; D004249:DNA Damage; D004269:DNA, Bacterial / Q000235:genetics*; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics*; D017930:Genes, Reporter; D007700:Kinetics; D008163:Luminescent Measurements; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013014:SOS Response, Genetics / Q000235:genetics*
D003087:Colicins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C488859:immE9 protein, E coli; D004229:Dithiothreitol