A Rationally Designed c-di-AMP Förster Resonance Energy Transfer Biosensor To Monitor Nucleotide Dynamics
- 8 September 2021
- journal article
- research article
- Published by American Society for Microbiology in Journal of Bacteriology
- Vol. 203 (19), JB0008021
- https://doi.org/10.1128/jb.00080-21
Abstract
3’3’-cyclic di-adenosine monophosphate (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 for the detection of c-di-AMP dynamics both in vitro and within live cells in a nondestructive manner. Our initial studies identify a distribution of c-di-AMP in Bacillus subtilis populations first grown in Luria Broth and then resuspended in diluted Luria Broth compatible with florescence analysis. Furthermore, we find that B. subtilis mutants lacking either a c-di-AMP phosphodiesterase or cyclase have respectively higher and lower FRET responses. 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 engineer and implement 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.Keywords
Funding Information
- HHS | National Institutes of Health (1R21AI153820-01, 1R21AI137758-01)
- HHS | National Institutes of Health (AI116669)
- HHS | U.S. Public Health Service (T32GM007270)
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