title: A bacterial biosensor for oxidative stress using the constitutively expressed redox-sensitive protein roGFP2.
abstract: A highly specific, high throughput-amenable bacterial biosensor for chemically induced cellular oxidation was developed using constitutively expressed redox-sensitive green fluorescent protein roGFP2 in E. coli (E. coli-roGFP2). Disulfide formation between two key cysteine residues of roGFP2 was assessed using a double-wavelength ratiometric approach. This study demonstrates that only a few minutes were required to detect oxidation using E. coli-roGFP2, in contrast to conventional bacterial oxidative stress sensors. Cellular oxidation induced by hydrogen peroxide, menadione, sodium selenite, zinc pyrithione, triphenyltin and naphthalene became detectable after 10 seconds and reached the maxima between 80 to 210 seconds, contrary to Cd(2+), Cu(2+), Pb(2+), Zn(2+) and sodium arsenite, which induced the oxidation maximum immediately. The lowest observable effect concentrations (in ppm) were determined as 1.0 × 10(-7) (arsenite), 1.0 × 10(-4) (naphthalene), 1.0 × 10(-4) (Cu(2+)), 3.8 × 10(-4) (H(2)O(2)), 1.0 × 10(-3) (Cd(2+)), 1.0 × 10(-3) (Zn(2+)), 1.0 × 10(-2) (menadione), 1.0 (triphenyltin), 1.56 (zinc pyrithione), 3.1 (selenite) and 6.3 (Pb(2+)), respectively. Heavy metal-induced oxidation showed unclear response patterns, whereas concentration-dependent sigmoid curves were observed for other compounds. In vivo GSH content and in vitro roGFP2 oxidation assays together with E. coli-roGFP2 results suggest that roGFP2 is sensitive to redox potential change and thiol modification induced by environmental stressors. Based on redox-sensitive technology, E. coli-roGFP2 provides a fast comprehensive detection system for toxicants that induce cellular oxidation.
authors.affiliation: Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan.
authors.forename: Carlos R
authors.initials: CR
authors.lastname: Arias-Barreiro
authors.affiliation: Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Okazaki
authors.affiliation: Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan.|Environmental Risk Management Authority, PO BOX 131, Wellington 6140, New Zealand.
authors.forename: Apostolos
authors.initials: A
authors.lastname: Koutsaftis
authors.affiliation: Faculty of Allied Health Sciences/UKM Medical Molecular Biology Institute, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, Kuala Lumpur 50300, Malaysia.
authors.forename: Salmaan H
authors.initials: SH
authors.lastname: Inayat-Hussain
authors.affiliation: Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Tani
authors.affiliation: Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan.
authors.forename: Maki
authors.initials: M
authors.lastname: Katsuhara
authors.affiliation: Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan.|Department of Materials Science and Chemical Engineering, Shizuoka University, Hamamatsu 432-8561, Japan.
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Kimbara
authors.affiliation: Institute of Plant Science and Resources, Okayama University, Kurashiki 710-0046, Japan.
authors.forename: Izumi C
authors.initials: IC
authors.lastname: Mori
mesh_terms: D018407:Bacterial Physiological Phenomena; D015374:Biosensing Techniques; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress
chemical_list: D049452:Green Fluorescent Proteins