Method for measurement of bacillithiol redox potential changes using the Brx-roGFP2 redox biosensor in Staphylococcus aureus.
Identifieur interne : 000046 ( Main/Exploration ); précédent : 000045; suivant : 000047Method for measurement of bacillithiol redox potential changes using the Brx-roGFP2 redox biosensor in Staphylococcus aureus.
Auteurs : Vu Van Loi [Allemagne] ; Haike Antelmann [Allemagne]Source :
- MethodsX [ 2215-0161 ] ; 2020.
Abstract
Recent advances in the design of genetically encoded redox biosensors, such as redox-sensitive GFP (roGFP) have facilitated the real-time imaging of the intracellular redox potential in eukaryotic cells at high sensitivity and at spatiotemporal resolution. To increase the specificity of roGFP2 for the interaction with the glutathione (GSH)/ glutathione disulfide (GSSG) redox couple, roGFP2 has been fused to glutaredoxin (Grx) to construct the Grx-roGFP2 biosensor. We have previously designed the related Brx-roGFP2 redox biosensor for dynamic measurement of the bacillithiol redox potential (EBSH) in the human pathogen Staphylococcus aureus. Here, we describe the detailed method for measurements of the oxidation degree (OxD) of the Brx-roGFP2 biosensor in S. aureus using the microplate reader. In particularly, we provide details for determination of the EBSH changes during the growth and after oxidative stress. For future biosensor applications at the single cell level, we recommend the design of genome-encoded roGFP2 biosensors enabling stable expression and fluorescence in bacteria.•Brx-roGFP2 is specific for measurements of the bacillithiol redox potential in Staphylococcus aureus cells•Control samples for fully reduced and oxidized states of Brx-roGFP2 are required for calibration during OxD measurements•Easy to measure fluorescence excitation intensities at the 405 and 488 nm excitation maxima using microplate readers.
DOI: 10.1016/j.mex.2020.100900
PubMed: 32420048
PubMed Central: PMC7214941
Affiliations:
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To increase the specificity of roGFP2 for the interaction with the glutathione (GSH)/ glutathione disulfide (GSSG) redox couple, roGFP2 has been fused to glutaredoxin (Grx) to construct the Grx-roGFP2 biosensor. We have previously designed the related Brx-roGFP2 redox biosensor for dynamic measurement of the bacillithiol redox potential (<i>E</i><sub>BSH</sub>) in the human pathogen <i>Staphylococcus aureus</i>. Here, we describe the detailed method for measurements of the oxidation degree (OxD) of the Brx-roGFP2 biosensor in <i>S. aureus</i> using the microplate reader. In particularly, we provide details for determination of the <i>E</i><sub>BSH</sub> changes during the growth and after oxidative stress. For future biosensor applications at the single cell level, we recommend the design of genome-encoded roGFP2 biosensors enabling stable expression and fluorescence in bacteria.•Brx-roGFP2 is specific for measurements of the bacillithiol redox potential in <i>Staphylococcus aureus</i> cells•Control samples for fully reduced and oxidized states of Brx-roGFP2 are required for calibration during OxD measurements•Easy to measure fluorescence excitation intensities at the 405 and 488 nm excitation maxima using microplate readers.</div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">32420048</PMID><DateRevised><Year>2020</Year><Month>09</Month><Day>28</Day></DateRevised><Article PubModel="Electronic-eCollection"><Journal><ISSN IssnType="Print">2215-0161</ISSN><JournalIssue CitedMedium="Print"><Volume>7</Volume><PubDate><Year>2020</Year></PubDate></JournalIssue><Title>MethodsX</Title><ISOAbbreviation>MethodsX</ISOAbbreviation></Journal><ArticleTitle>Method for measurement of bacillithiol redox potential changes using the Brx-roGFP2 redox biosensor in <i>Staphylococcus aureus</i>.</ArticleTitle><Pagination><MedlinePgn>100900</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1016/j.mex.2020.100900</ELocationID><Abstract><AbstractText>Recent advances in the design of genetically encoded redox biosensors, such as redox-sensitive GFP (roGFP) have facilitated the real-time imaging of the intracellular redox potential in eukaryotic cells at high sensitivity and at spatiotemporal resolution. To increase the specificity of roGFP2 for the interaction with the glutathione (GSH)/ glutathione disulfide (GSSG) redox couple, roGFP2 has been fused to glutaredoxin (Grx) to construct the Grx-roGFP2 biosensor. We have previously designed the related Brx-roGFP2 redox biosensor for dynamic measurement of the bacillithiol redox potential (<i>E</i><sub>BSH</sub>) in the human pathogen <i>Staphylococcus aureus</i>. Here, we describe the detailed method for measurements of the oxidation degree (OxD) of the Brx-roGFP2 biosensor in <i>S. aureus</i> using the microplate reader. In particularly, we provide details for determination of the <i>E</i><sub>BSH</sub> changes during the growth and after oxidative stress. For future biosensor applications at the single cell level, we recommend the design of genome-encoded roGFP2 biosensors enabling stable expression and fluorescence in bacteria.•Brx-roGFP2 is specific for measurements of the bacillithiol redox potential in <i>Staphylococcus aureus</i> cells•Control samples for fully reduced and oxidized states of Brx-roGFP2 are required for calibration during OxD measurements•Easy to measure fluorescence excitation intensities at the 405 and 488 nm excitation maxima using microplate readers.</AbstractText><CopyrightInformation>© 2020 The Author(s). Published by Elsevier B.V.</CopyrightInformation></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Van Loi</LastName><ForeName>Vu</ForeName><Initials>V</Initials><AffiliationInfo><Affiliation>Freie Universität Berlin, Institute of Biology-Microbiology, D-14195 Berlin, Germany.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Antelmann</LastName><ForeName>Haike</ForeName><Initials>H</Initials><AffiliationInfo><Affiliation>Freie Universität Berlin, Institute of Biology-Microbiology, D-14195 Berlin, Germany.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2020</Year><Month>04</Month><Day>24</Day></ArticleDate></Article><MedlineJournalInfo><Country>Netherlands</Country><MedlineTA>MethodsX</MedlineTA><NlmUniqueID>101639829</NlmUniqueID><ISSNLinking>2215-0161</ISSNLinking></MedlineJournalInfo><KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">Bacillithiol</Keyword><Keyword MajorTopicYN="N">Genetically encoded roGFP2 biosensors</Keyword><Keyword MajorTopicYN="N">Microplate reader measurements</Keyword><Keyword MajorTopicYN="N">Staphylococcus aureus</Keyword></KeywordList><CoiStatement>The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.</CoiStatement></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2020</Year><Month>01</Month><Day>07</Day></PubMedPubDate><PubMedPubDate 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