title: Functional assays of oxidative stress using genetically engineered Escherichia coli strains.
abstract: Oxidative stress may be induced in bacteria by exogenous biocidal agents and is involved in endogenous metabolism. The oxyR operon is a main sensor of oxidative stress and oxyR-deficient bacteria show enhanced sensitivity to oxidative stress and increased accumulation of intracellular reactive oxygen species (ROS). Flow cytometric functional assays in bacteria are limited by the impaired penetration of vital dyes trough the cell wall. Escherichia coli B WP2 strains possess an altered cell-wall lipopolysaccharide that leads to increased membrane permeability. Flow cytometric analysis of WP2 strains is a convenient alternative for cytometric assays of bacterial function. This unit presents protocols for flow cytometric studies of intracellular oxidative stress in two E. coli B WP2 strains, wild-type or deficient in the oxyR function, using ROS-sensitive fluorogenic substrates. Support Protocols describe preparation of phage C21 stock for bacterial verification, verification of the WP2 phenotype, and verification of the deficiency in oxyR function.
authors.affiliation: Universidad de Valencia, Valencia, Spain.
authors.forename: Guadalupe
authors.initials: G
authors.lastname: Herrera
authors.forename: Alicia
authors.initials: A
authors.lastname: Martínez
authors.forename: José-Enrique
authors.initials: JE
authors.lastname: O'Cornor
authors.forename: Manuel
authors.initials: M
authors.lastname: Blanco
mesh_terms: D004268:DNA-Binding Proteins / Q000172:deficiency; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D005818:Genetic Engineering; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000032:analysis*; D012097:Repressor Proteins
chemical_list: D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D012097:Repressor Proteins; C499299:oxyR protein, E coli