Understanding the in vivo redox biology of cells is a complex albeit important biological problem. Studying redox processes within living cells without physical disruption or chemical modifications is essential in determining the native redox states of cells. In this study, the previously characterized reduction-oxidation sensitive green fluorescent protein (roGFP2) was used to elucidate the redox changes of the genetically engineered Escherichia coli strain, SHuffle. SHuffle cells were demonstrated to be under constitutive oxidative stress and responding transcriptionally in an OxyR-dependent manner. Using roGFP2 fused to either glutathione (GSH)- or hydrogen peroxide (H2O2)- sensitive proteins (glutaredoxin 1 or Orp1), the cytosolic redox state of both wild type and SHuffle cells based on GSH/GSSG and H2O2 pools was measured. These probes open the path to in vivo studies of redox changes and genetic selections in prokaryotic hosts.
Bibliographical noteFunding Information:
The authors would like to thank Tobias Dick for the roGFP plasmids and help with the roGFP mechanistic figure, Jonathan Winkler for the soxS and dps promoter reporter plasmids and Bruno Manta for his helpful comments on the manuscript. We thank ISF ( 1537/18 ) and BSF ( 2015056 ) for the financial support. We further acknowledge the support by an ERC Consolidator grant (GA 615585 ) MYCOTHIOLOME and grants from the Deutsche Forschungsgemeinschaft ( AN746/4–1 and AN746/4–2 ) within the SPP1710, by the SFB973 project C08 N and by the SFB/TR84 project B06 to H.A.
© 2019 The Authors
- Disulfide bond formation