Utilizing redox-sensitive GFP fusions to detect in vivo redox changes in a genetically engineered prokaryote
Wilhad Hans Reuter,
Thorsten Masuch,
Na Ke,
Marine Lenon,
Meytal Radzinski,
Vu Van Loi,
Guoping Ren,
Paul Riggs,
Haike Antelmann,
Dana Reichmann,
Lars I. Leichert,
Mehmet Berkmen
Affiliations
Wilhad Hans Reuter
New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA
Thorsten Masuch
New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA; Ruhr University Bochum, Institute of Biochemistry and Pathobiochemistry, Microbial Biochemistry, Universitätsstr. 150, 44780, Bochum, Germany
Na Ke
New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA
Marine Lenon
New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA
Meytal Radzinski
The Hebrew University of Jerusalem, The Alexander Silberman Institute of Life Sciences, Safra Campus Givat Ram, Jerusalem, 91904, Israel
Vu Van Loi
Institute for Biology-Microbiology, Freie Universität Berlin, 14195, Berlin, Germany
Guoping Ren
New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA
Paul Riggs
New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA
Haike Antelmann
Institute for Biology-Microbiology, Freie Universität Berlin, 14195, Berlin, Germany
Dana Reichmann
The Hebrew University of Jerusalem, The Alexander Silberman Institute of Life Sciences, Safra Campus Givat Ram, Jerusalem, 91904, Israel
Lars I. Leichert
Ruhr University Bochum, Institute of Biochemistry and Pathobiochemistry, Microbial Biochemistry, Universitätsstr. 150, 44780, Bochum, Germany
Mehmet Berkmen
New England Biolabs, 240 County Road, Ipswich, MA, 01938, USA; Corresponding author.
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. Keywords: roGFP, Redox, SHuffle, Disulfide bond formation
