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

doi:10.1016/j.redox.2019.101280

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^*

^*Corresponding author for this work

Department of Biological Chemistry

Research output: Contribution to journal › Article › peer-review

13 Scopus citations

Abstract

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.

Original language	American English
Article number	101280
Journal	Redox Biology
Volume	26
DOIs	https://doi.org/10.1016/j.redox.2019.101280
State	Published - Sep 2019

Bibliographical note

Funding 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.

Publisher Copyright:
© 2019 The Authors

Keywords

Disulfide bond formation
Redox
SHuffle
roGFP

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10.1016/j.redox.2019.101280

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title = "Utilizing redox-sensitive GFP fusions to detect in vivo redox changes in a genetically engineered prokaryote",

abstract = "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 = "Disulfide bond formation, Redox, SHuffle, roGFP",

author = "Reuter, {Wilhad Hans} and Thorsten Masuch and Na Ke and Marine Lenon and Meytal Radzinski and {Van Loi}, Vu and Guoping Ren and Paul Riggs and Haike Antelmann and Dana Reichmann and Leichert, {Lars I.} and Mehmet Berkmen",

note = "Funding 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. Publisher Copyright: {\textcopyright} 2019 The Authors",

year = "2019",

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doi = "10.1016/j.redox.2019.101280",

language = "American English",

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journal = "Redox Biology",

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AU - Reuter, Wilhad Hans

AU - Masuch, Thorsten

AU - Ke, Na

AU - Lenon, Marine

AU - Radzinski, Meytal

AU - Van Loi, Vu

AU - Ren, Guoping

AU - Riggs, Paul

AU - Antelmann, Haike

AU - Reichmann, Dana

AU - Leichert, Lars I.

AU - Berkmen, Mehmet

N1 - Funding 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. Publisher Copyright: © 2019 The Authors

PY - 2019/9

Y1 - 2019/9

N2 - 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.

AB - 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.

KW - Disulfide bond formation

KW - Redox

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KW - roGFP

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JO - Redox Biology

JF - Redox Biology

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ER -

Utilizing redox-sensitive GFP fusions to detect in vivo redox changes in a genetically engineered prokaryote

Abstract

Bibliographical note

Keywords

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