Rapid detection of colicin E9-induced DNA damage using Escherichia coli cells carrying SOS promoter-lux fusions

Mireille Vankemmelbeke; Bryan Healy; Geoffrey R Moore; Colin Kleanthous; Christopher N Penfold; Richard James

doi:10.1128/JB.187.14.4900-4907.2005

Rapid detection of colicin E9-induced DNA damage using Escherichia coli cells carrying SOS promoter-lux fusions

Mireille Vankemmelbeke, Bryan Healy, Geoffrey R Moore, Colin Kleanthous, Christopher N Penfold, Richard James

Biology

Research output: Contribution to journal › Article › peer-review

Abstract

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 language	English
Pages (from-to)	4900-4907
Number of pages	8
Journal	Journal of Bacteriology
Volume	187
Issue number	14
DOIs	https://doi.org/10.1128/JB.187.14.4900-4907.2005
Publication status	Published - Jul 2005

Keywords

PROTEIN-PROTEIN INTERACTION
NUCLEASE DOMAIN
OUTER-MEMBRANE
E9
TRANSLOCATION
ENDONUCLEASE
BINDING
CHANNEL
IDENTIFICATION
MECHANISMS

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10.1128/JB.187.14.4900-4907.2005

Cite this

@article{86397562b16f4e50ac8862bc15946001,

title = "Rapid detection of colicin E9-induced DNA damage using Escherichia coli cells carrying SOS promoter-lux fusions",

abstract = "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.",

keywords = "PROTEIN-PROTEIN INTERACTION, NUCLEASE DOMAIN, OUTER-MEMBRANE, E9, TRANSLOCATION, ENDONUCLEASE, BINDING, CHANNEL, IDENTIFICATION, MECHANISMS",

author = "Mireille Vankemmelbeke and Bryan Healy and Moore, {Geoffrey R} and Colin Kleanthous and Penfold, {Christopher N} and Richard James",

year = "2005",

month = jul,

doi = "10.1128/JB.187.14.4900-4907.2005",

language = "English",

volume = "187",

pages = "4900--4907",

journal = "Journal of Bacteriology",

issn = "0021-9193",

publisher = "American Society for Microbiology",

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TY - JOUR

T1 - Rapid detection of colicin E9-induced DNA damage using Escherichia coli cells carrying SOS promoter-lux fusions

AU - Vankemmelbeke, Mireille

AU - Healy, Bryan

AU - Moore, Geoffrey R

AU - Kleanthous, Colin

AU - Penfold, Christopher N

AU - James, Richard

PY - 2005/7

Y1 - 2005/7

N2 - 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.

AB - 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.

KW - PROTEIN-PROTEIN INTERACTION

KW - NUCLEASE DOMAIN

KW - OUTER-MEMBRANE

KW - E9

KW - TRANSLOCATION

KW - ENDONUCLEASE

KW - BINDING

KW - CHANNEL

KW - IDENTIFICATION

KW - MECHANISMS

U2 - 10.1128/JB.187.14.4900-4907.2005

DO - 10.1128/JB.187.14.4900-4907.2005

M3 - Article

C2 - 15995205

SN - 0021-9193

VL - 187

SP - 4900

EP - 4907

JO - Journal of Bacteriology

JF - Journal of Bacteriology

IS - 14

ER -