Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

A rationally designed c-di-AMP FRET biosensor to monitor nucleotide dynamics

Alex J. Pollock, Philip H. Choi, Shivam A. Zaver, Liang Tong, Joshua J. Woodward
doi: https://doi.org/10.1101/2021.02.10.430713
Alex J. Pollock
aDepartment of Microbiology, University of Washington, Seattle, WA, 98195, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Philip H. Choi
bDepartment of Biological Sciences, Columbia University, New York, NY 10027, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Shivam A. Zaver
aDepartment of Microbiology, University of Washington, Seattle, WA, 98195, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Liang Tong
bDepartment of Biological Sciences, Columbia University, New York, NY 10027, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Joshua J. Woodward
aDepartment of Microbiology, University of Washington, Seattle, WA, 98195, USA
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: jjwoodwa@uw.edu
  • Abstract
  • Full Text
  • Info/History
  • Metrics
  • Preview PDF
Loading

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 single cells in a nondestructive manner. Our initial studies identify a unimodal distribution of c-di-AMP in Bacillus subtilis which decreases rapidly when cells are grown in diluted Luria Broth. Furthermore, we find that B. subtilis mutants lacking either a c-di-AMP phosphodiesterase or cyclase have respectively higher and lower FRET responses, again in a unimodal manner. These findings provide novel insight into 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.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
Back to top
PreviousNext
Posted February 11, 2021.
Download PDF
Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
A rationally designed c-di-AMP FRET biosensor to monitor nucleotide dynamics
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
A rationally designed c-di-AMP FRET biosensor to monitor nucleotide dynamics
Alex J. Pollock, Philip H. Choi, Shivam A. Zaver, Liang Tong, Joshua J. Woodward
bioRxiv 2021.02.10.430713; doi: https://doi.org/10.1101/2021.02.10.430713
Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
A rationally designed c-di-AMP FRET biosensor to monitor nucleotide dynamics
Alex J. Pollock, Philip H. Choi, Shivam A. Zaver, Liang Tong, Joshua J. Woodward
bioRxiv 2021.02.10.430713; doi: https://doi.org/10.1101/2021.02.10.430713

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Bioengineering
Subject Areas
All Articles
  • Animal Behavior and Cognition (2646)
  • Biochemistry (5269)
  • Bioengineering (3678)
  • Bioinformatics (15796)
  • Biophysics (7257)
  • Cancer Biology (5629)
  • Cell Biology (8099)
  • Clinical Trials (138)
  • Developmental Biology (4768)
  • Ecology (7518)
  • Epidemiology (2059)
  • Evolutionary Biology (10578)
  • Genetics (7733)
  • Genomics (10137)
  • Immunology (5194)
  • Microbiology (13915)
  • Molecular Biology (5387)
  • Neuroscience (30785)
  • Paleontology (215)
  • Pathology (879)
  • Pharmacology and Toxicology (1525)
  • Physiology (2254)
  • Plant Biology (5024)
  • Scientific Communication and Education (1041)
  • Synthetic Biology (1388)
  • Systems Biology (4148)
  • Zoology (812)