In vitro Real-time Measurement of the Intra-bacterial Redox Potential

Abstract

All bacteria that live in oxygenated environments have to deal with oxidative stress caused by some form of exogenous or endogenous reactive oxygen species (ROS) (Imlay, 2013). Large quantities of ROS damage DNA, lipids and proteins which can eventually lead to bacterial cell death (Imlay, 2013). In contrast, smaller quantities of ROS can play more sophisticated roles in cellular signalling pathways affecting almost every process in the bacterial cell e.g. metabolism, stress responses, transcription, protein synthesis, etc. Previously, inadequate analytical methods prevented appropriate analysis of the intra-bacterial redox potential. Herein, we describe a method for the measurement of real-time changes to the intra-bacterial redox potential using redox-sensitive GFP (roGFP2) (van der Heijden et al., 2015). The roGFP2 protein is engineered to contain specific cysteine residues that form an internal disulfide bridge upon oxidation which results in a slight shift in protein conformation (Hanson et al., 2004). This shift results in two distinct protein isoforms with different fluorescence excitation spectra after excitation at 405 nm and 480 nm respectively. Consequently, the corresponding 405/480 nm ratio can be used as a measure for the intra-bacterial redox potential. The ratio-metric analysis excludes variations due to differences in roGFP2 concentrations and since the conformational shift is reversible the system allows for measurement of oxidizing as well as reducing conditions. In this protocol we describe the system by measuring the intra-bacterial redox potential inside Salmonella typhimurium (S. typhimurium) however this system can be adjusted for use in other Gram-negative bacteria.

Figure 2. Real-time intra-bacterial redox potential of S. typhimurium after two subsequent challenges with 1 mM hydrogen peroxide

Each upward arrow indicates a challenge with hydrogen peroxide.

Figure 1. Example layout of a 96-well imaging plate

In lane 1 all bacteria express roGFP2 while in lane 2 contains well with empty-vector containing bacteria to determine the background fluorescence (BG) for each condition. The two adjacent well e.g. A1 and A2 undergo an identical challenge with oxidizing agents. In row G, 100 mM of hydrogen peroxide is added and these results are used as maximum oxidized values for normalization. In row H, 10 mM DTT is added and these results are used as maximum reduced values for normalization.