Chemiluminescent Biosensors for Detection of Second Messenger Cyclic di-GMP
Abstract
Bacteria colonize highly diverse and complex environments, from gastrointestinal tracts to soil and plant surfaces. This colonization process is controlled in part by the intracellular signal cyclic di-GMP, which regulates bacterial motility and biofilm formation. To interrogate cyclic di-GMP signaling networks, a variety of fluorescent biosensors for live cell imaging of cyclic di-GMP have been developed. However, the need for external illumination precludes the use of these tools for imaging bacteria in their natural environments, including in deep tissues of whole organisms and in samples that are highly autofluorescent or photosensitive. The need for genetic encoding also complicates the analysis of clinical isolates and environmental samples. Toward expanding the study of bacterial signaling to these systems, we have developed the first chemiluminescent biosensors for cyclic di-GMP. The biosensor design combines the complementation of split luciferase (CSL) and bioluminescence resonance energy transfer (BRET) approaches. Furthermore, we developed a lysate-based assay for biosensor activity that enabled reliable high-throughput screening of a phylogenetic library of 92 biosensor variants. The screen identified biosensors with very large signal changes (~40- and 90-fold) as well as biosensors with high affinities for cyclic di-GMP ($$K_D$$ < 50 nM). These chemiluminescent biosensors then were applied to measure cyclic di-GMP levels in $E. coli$. The cellular experiments revealed an unexpected challenge for chemiluminescent imaging in Gram negative bacteria but showed promising application in lysates. Taken together, this work establishes the first chemiluminescent biosensors for studying cyclic di-GMP signaling and provides a foundation for using these biosensors in more complex systems.
- Authors:
-
[3]
- Univ. of California, Berkeley, CA (United States). Dept. of Chemistry
- USDOE Joint Genome Institute (JGI), Walnut Creek, CA (United States)
- Univ. of California, Berkeley, CA (United States). Dept. of Chemistry, and Dept. of Molecular & Cell Biology
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States). National Energy Research Scientific Computing Center (NERSC); Univ. of California, Oakland, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC)
- OSTI Identifier:
- 1563956
- Alternate Identifier(s):
- OSTI ID: 1543712
- Grant/Contract Number:
- AC02-05CH11231
- Resource Type:
- Accepted Manuscript
- Journal Name:
- ACS Chemical Biology
- Additional Journal Information:
- Journal Volume: 13; Journal Issue: 7; Journal ID: ISSN 1554-8929
- Publisher:
- American Chemical Society (ACS)
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; Biochemistry & Molecular Biology
Citation Formats
Dippel, Andrew B., Anderson, Wyatt A., Evans, Robert S., Deutsch, Samuel, and Hammond, Ming C. Chemiluminescent Biosensors for Detection of Second Messenger Cyclic di-GMP. United States: N. p., 2018.
Web. doi:10.1021/acschembio.7b01019.
Dippel, Andrew B., Anderson, Wyatt A., Evans, Robert S., Deutsch, Samuel, & Hammond, Ming C. Chemiluminescent Biosensors for Detection of Second Messenger Cyclic di-GMP. United States. https://doi.org/10.1021/acschembio.7b01019
Dippel, Andrew B., Anderson, Wyatt A., Evans, Robert S., Deutsch, Samuel, and Hammond, Ming C. Wed .
"Chemiluminescent Biosensors for Detection of Second Messenger Cyclic di-GMP". United States. https://doi.org/10.1021/acschembio.7b01019. https://www.osti.gov/servlets/purl/1563956.
@article{osti_1563956,
title = {Chemiluminescent Biosensors for Detection of Second Messenger Cyclic di-GMP},
author = {Dippel, Andrew B. and Anderson, Wyatt A. and Evans, Robert S. and Deutsch, Samuel and Hammond, Ming C.},
abstractNote = {Bacteria colonize highly diverse and complex environments, from gastrointestinal tracts to soil and plant surfaces. This colonization process is controlled in part by the intracellular signal cyclic di-GMP, which regulates bacterial motility and biofilm formation. To interrogate cyclic di-GMP signaling networks, a variety of fluorescent biosensors for live cell imaging of cyclic di-GMP have been developed. However, the need for external illumination precludes the use of these tools for imaging bacteria in their natural environments, including in deep tissues of whole organisms and in samples that are highly autofluorescent or photosensitive. The need for genetic encoding also complicates the analysis of clinical isolates and environmental samples. Toward expanding the study of bacterial signaling to these systems, we have developed the first chemiluminescent biosensors for cyclic di-GMP. The biosensor design combines the complementation of split luciferase (CSL) and bioluminescence resonance energy transfer (BRET) approaches. Furthermore, we developed a lysate-based assay for biosensor activity that enabled reliable high-throughput screening of a phylogenetic library of 92 biosensor variants. The screen identified biosensors with very large signal changes (~40- and 90-fold) as well as biosensors with high affinities for cyclic di-GMP ($K_D$ < 50 nM). These chemiluminescent biosensors then were applied to measure cyclic di-GMP levels in $E. coli$. The cellular experiments revealed an unexpected challenge for chemiluminescent imaging in Gram negative bacteria but showed promising application in lysates. Taken together, this work establishes the first chemiluminescent biosensors for studying cyclic di-GMP signaling and provides a foundation for using these biosensors in more complex systems.},
doi = {10.1021/acschembio.7b01019},
journal = {ACS Chemical Biology},
number = 7,
volume = 13,
place = {United States},
year = {Wed Feb 21 00:00:00 EST 2018},
month = {Wed Feb 21 00:00:00 EST 2018}
}
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