Functional assays of oxidative stress using genetically engineered Escherichia coli strains

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.