Stable integration of the Mrx1-roGFP2 biosensor to monitor dynamic changes of the mycothiol redox potential in Corynebacterium glutamicum
| Autor: | Jörn Kalinowski, Vu Van Loi, Andreas Nerlich, Andreas C. Hocke, Haike Antelmann, Maren Mieth, Quach Ngoc Tung, Johanna Milse, Tobias Busche |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Models Molecular Mrx1 mycoredoxin-1 GSSG glutathione disulfide Antioxidant Protein Conformation medicine.medical_treatment Clinical Biochemistry Mutant Intracellular Space Gene Expression Biosensing Techniques medicine.disease_cause Grx1-roGFP2 glutaredoxin-fused roGFP2 biosensor Biochemistry HOCl hypochloric acid Corynebacterium glutamicum EMSH mycothiol redox potential chemistry.chemical_compound LMW thiol low molecular weight thiol 0302 clinical medicine PCR polymerase chain reaction Genes Reporter GSH glutathione Trx thioredoxin ECF extracytoplasmic function IPTG isopropyl- β-D-thiogalactopyranoside lcsh:QH301-705.5 roGFP2 redox-sensitive green fluorescent protein Mycothiol redox potential lcsh:R5-920 biology MSH mycothiol CLSM confocal laser scanning microscopy MSSM mycothiol disulfide Mpx mycothiol peroxidase Catalase Brx bacilliredoxin H2O2 hydrogen peroxide Mtr mycothiol disulfide reductase lcsh:Medicine (General) Oxidation-Reduction RCS reactive chlorine species Research Paper TrxR thioredoxin reductase SDS sodium dodecyl sulfate Recombinant Fusion Proteins Green Fluorescent Proteins Oxidative phosphorylation SEM standard error of the mean Mycothiol Models Biological Brx-roGFP2 bacilliredoxin-fused roGFP2 biosensor 03 medical and health sciences Structure-Activity Relationship ROS reactive oxygen species Bacterial Proteins Live cell imaging Mrx1-roGFP2 mycoredoxin-1-fused roGFP2 biosensor medicine CHP cumene hydroperoxide Amino Acid Sequence TL transmitted light PAGE polyacrylamide gel electrophoresis LB Luria Bertani Mrx1-roGFP2 Organic Chemistry KatA catalase Hydrogen Peroxide BSH bacillithiol EGT ergothioneine OxD oxidation degree BSSB bacillithiol disulfide Oxidative Stress 030104 developmental biology CBB Coomassie Brilliant Blue chemistry lcsh:Biology (General) OD500 optical density at 500 nm DTT dithiothreitol Tpx thiol peroxidase Mutation biology.protein NEM N-ethylmaleimide bacteria SigH RNA polymerase sigma-H factor Reactive Oxygen Species 030217 neurology & neurosurgery Oxidative stress NaOCl sodium hypochlorite |
| Zdroj: | Redox Biology Redox Biology, Vol 20, Iss, Pp 514-525 (2019) |
| ISSN: | 2213-2317 |
| Popis: | Mycothiol (MSH) functions as major low molecular weight (LMW) thiol in the industrially important Corynebacterium glutamicum. In this study, we genomically integrated an Mrx1-roGFP2 biosensor in C. glutamicum to measure dynamic changes of the MSH redox potential (EMSH) during the growth and under oxidative stress. C. glutamicum maintains a highly reducing intrabacterial EMSH throughout the growth curve with basal EMSH levels of ~− 296 mV. Consistent with its H2O2 resistant phenotype, C. glutamicum responds only weakly to 40 mM H2O2, but is rapidly oxidized by low doses of NaOCl. We further monitored basal EMSH changes and the H2O2 response in various mutants which are compromised in redox-signaling of ROS (OxyR, SigH) and in the antioxidant defense (MSH, Mtr, KatA, Mpx, Tpx). While the probe was constitutively oxidized in the mshC and mtr mutants, a smaller oxidative shift in basal EMSH was observed in the sigH mutant. The catalase KatA was confirmed as major H2O2 detoxification enzyme required for fast biosensor re-equilibration upon return to non-stress conditions. In contrast, the peroxiredoxins Mpx and Tpx had only little impact on EMSH and H2O2 detoxification. Further live imaging experiments using confocal laser scanning microscopy revealed the stable biosensor expression and fluorescence at the single cell level. In conclusion, the stably expressed Mrx1-roGFP2 biosensor was successfully applied to monitor dynamic EMSH changes in C. glutamicum during the growth, under oxidative stress and in different mutants revealing the impact of Mtr and SigH for the basal level EMSH and the role of OxyR and KatA for efficient H2O2 detoxification under oxidative stress. Highlights • Construction of stably integrated Mrx1-roGFP2 in C. glutamicum. • Highly reducing basal EMSH of ~− 296 mV along the growth curve. • Mtr and SigH contribute to basal EMSH in C. glutamicum. • KatA and OxyR required for EMSH regeneration under oxidative stress. • Live imaging revealed stable biosensor expression and fluorescence. |
| Databáze: | OpenAIRE |
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