A Rationally Designed c-di-AMP Förster Resonance Energy Transfer Biosensor To Monitor Nucleotide Dynamics
| العنوان: | A Rationally Designed c-di-AMP Förster Resonance Energy Transfer Biosensor To Monitor Nucleotide Dynamics |
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| المؤلفون: | Shivam A. Zaver, Liang Tong, Joshua J. Woodward, Alex J. Pollock, Philip H. Choi |
| المصدر: | J Bacteriol |
| بيانات النشر: | American Society for Microbiology, 2021. |
| سنة النشر: | 2021 |
| مصطلحات موضوعية: | Models, Molecular, Protein Conformation, Mutant, Biosensing Techniques, Bacillus subtilis, Microbiology, Bacterial Proteins, Fluorescence Resonance Energy Transfer, Nucleotide, Molecular Biology, chemistry.chemical_classification, biology, Nucleotides, Effector, Gene Expression Regulation, Bacterial, Protein engineering, biology.organism_classification, Listeria monocytogenes, Förster resonance energy transfer, Biochemistry, chemistry, Mutation, Second messenger system, Biosensor, Dinucleoside Phosphates, Research Article |
| الوصف: | 3′3′-Cyclic di-AMP (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. c-di-AMP is essential in many bacteria and regulates a diverse array of effector proteins controlling pathogenesis, cell wall homeostasis, osmoregulation, and central metabolism. Despite the ubiquity and importance of c-di-AMP, methods to detect this signaling molecule are limited, particularly at single-cell resolution. In this work, crystallization of the Listeria monocytogenes c-di-AMP effector protein Lmo0553 enabled structure-guided design of a Förster resonance energy transfer (FRET)-based biosensor, which we have named CDA5. CDA5 is a fully genetically encodable, specific, and reversible biosensor which allows the detection of c-di-AMP dynamics both in vitro and within live cells in a nondestructive manner. Our initial studies identified a distribution of c-di-AMP in Bacillus subtilis populations first grown in Luria broth and then resuspended in diluted Luria broth compatible with fluorescence analysis. Furthermore, we found that B. subtilis mutants lacking either a c-di-AMP phosphodiesterase and cyclase have higher and lower FRET responses, respectively. These findings provide novel insight into the c-di-AMP distribution within bacterial populations and establish CDA5 as a powerful platform for characterizing new aspects of c-di-AMP regulation. IMPORTANCE c-di-AMP is an important nucleotide second messenger for which detection methods are severely limited. In this work we engineered and implemented a c-di-AMP-specific FRET biosensor to remedy this dearth. We present this biosensor, CDA5, as a versatile tool to investigate previously intractable facets of c-di-AMP biology. |
| تدمد: | 1098-5530 0021-9193 |
| DOI: | 10.1128/jb.00080-21 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::0cfaac610cbcb686b6a3e1764f93cf26 https://doi.org/10.1128/jb.00080-21 |
| Rights: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....0cfaac610cbcb686b6a3e1764f93cf26 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 10985530 00219193 |
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| DOI: | 10.1128/jb.00080-21 |