TY - JOUR
T1 - Microbial genotoxicity bioreporters based on sulA activation
AU - Biran, Alva
AU - Ben Yoav, Hadar
AU - Yagur-Kroll, Sharon
AU - Pedahzur, Rami
AU - Buchinger, Sebastian
AU - Shacham-Diamand, Yosi
AU - Reifferscheid, Georg
AU - Belkin, Shimshon
PY - 2011/7
Y1 - 2011/7
N2 - A bacterial genotoxicity reporter strain was constructed in which the tightly controlled strong promoter of the Escherichia coli SOS response gene sulA was fused to the alkaline phosphatase-coding phoA reporter gene. The bioreporter responded in a dose-dependent manner to three model DNA-damaging agents-hydrogen peroxide, nalidixic acid (NA), and mitomycin C (MMC)-detected 30-60 min after exposure. Detection thresholds were 0.15 μM for MMC, 7.5 μM for nalidixic acid, and approximately 50 μM for hydrogen peroxide. A similar response to NA was observed when the bioreporter was integrated into a specially designed, portable electrochemical detection platform. Reporter sensitivity was further enhanced by single and double knockout mutations that enhanced cell membrane permeability (rfaE) and inhibited DNA damage repair mechanisms (umuD, uvrA). The rfaE mutants displayed a five- and tenfold increase in sensitivity to MMC and NA, respectively, while the uvrA mutation was advantageous in the detection of hydrogen peroxide. A similar sensitivity was displayed by the double rfaE/uvrA mutant when challenged with the pre-genotoxic agents 2-amino-3-methylimidazo[4,5-f]quinoline and 2-aminoanthracene following metabolic activation with an S9 mammalian liver fraction.
AB - A bacterial genotoxicity reporter strain was constructed in which the tightly controlled strong promoter of the Escherichia coli SOS response gene sulA was fused to the alkaline phosphatase-coding phoA reporter gene. The bioreporter responded in a dose-dependent manner to three model DNA-damaging agents-hydrogen peroxide, nalidixic acid (NA), and mitomycin C (MMC)-detected 30-60 min after exposure. Detection thresholds were 0.15 μM for MMC, 7.5 μM for nalidixic acid, and approximately 50 μM for hydrogen peroxide. A similar response to NA was observed when the bioreporter was integrated into a specially designed, portable electrochemical detection platform. Reporter sensitivity was further enhanced by single and double knockout mutations that enhanced cell membrane permeability (rfaE) and inhibited DNA damage repair mechanisms (umuD, uvrA). The rfaE mutants displayed a five- and tenfold increase in sensitivity to MMC and NA, respectively, while the uvrA mutation was advantageous in the detection of hydrogen peroxide. A similar sensitivity was displayed by the double rfaE/uvrA mutant when challenged with the pre-genotoxic agents 2-amino-3-methylimidazo[4,5-f]quinoline and 2-aminoanthracene following metabolic activation with an S9 mammalian liver fraction.
KW - Bioluminescence
KW - Electrochemistry
KW - Environmental monitoring
KW - Escherichia coli
KW - Genotoxicity detection
KW - Whole-cell biosensors
UR - http://www.scopus.com/inward/record.url?scp=79958121788&partnerID=8YFLogxK
U2 - 10.1007/s00216-011-5007-2
DO - 10.1007/s00216-011-5007-2
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C2 - 21533638
AN - SCOPUS:79958121788
SN - 1618-2642
VL - 400
SP - 3013
EP - 3024
JO - Analytical and Bioanalytical Chemistry
JF - Analytical and Bioanalytical Chemistry
IS - 9
ER -