Innovative Technology Improves Our Understanding of Bacterial Cell Signaling

Newly developed chemiluminescent biosensors shed light on how bacteria function and colonize diverse environments.

The Science

The molecule cyclic di-GMP plays a key role in controlling cellulose production and biofilm formation. To better understand cyclic di-GMP signaling pathways, the team developed the first chemiluminescent biosensor system for cyclic di-GMP and showed that it could be used to assay cyclic di-GMP in bacterial lysates. (Image courtesy of Hammond Lab, UC Berkeley)

Cyclic di-GMP (Guanine Monophosphate) is found in nearly all types of bacteria and interacts with cell signaling networks that control many basic cellular functions. It plays an important role in regulating microbial cellulose production and biofilm formation, which affects a number of environments, including plants, soil, and the gut. To better understand the dynamics of this molecule, researchers developed the first chemiluminescent biosensors for measuring cyclic di-GMP in bacteria through work enabled by the JGI’s Community Science Program (CSP).

The Impact

Cyclic di-GMP controls microbial colonization, which can affect pathogenic and symbiotic bacterial interactions with animals and plants, or cause metal corrosion in oil pipes. Scientists have tried to study cyclic di-GMP and cellular networks with fluorescent biosensors before, but such sensors require external illumination, making them difficult to use inside deep tissues. Additionally, all plants, many bacterial cultures, and environmental samples auto-fluoresce, making fluorescent biosensors ineffective. Chemiluminescent biosensors do not need external illumination to produce a signal, making them useful in a wider range of environments. This new technology enables researchers to directly measure cyclic di-GMP levels in environmental samples or clinical isolates. As cyclic di-GMP also regulates enzymes involved in cellulose production in microbes, these biosensors may eventually lead to ways to improve cellulose-based biofuel synthesis. Additionally, it opens the door to measuring real-time changes in cyclic di-GMP via live cell imaging.

Summary

From digestive tracts to plant surfaces and soil, bacteria live in complex and varied conditions. A molecule called cyclic di-GMP is a key player in the intracellular signaling network that drives bacterial colonization and motility. By building on a previously developed combined approach, researchers at the University of California, Berkeley and the Joint Genome Institute, a DOE Office of Science User Facility, built a chemiluminescent cyclic di-GMP biosensor that can produce both large signal changes and high signal intensity for imaging. The work was enabled in part by a proposal to the DNA Synthesis CSP call, and the results are described in ACS Chemical Biology.

A high-throughput screen in crude lysates identified chemiluminescent protein biosensors for measuring a key bacterial signal involved in cellulose production and biofilm formation. (Image courtesy of Hammond Lab, UC Berkeley)

Using fluorescent biosensors to monitor the intracellular signaling network is inefficient on auto-fluorescing samples, rendering this approach useless when applied to plants, and several bacterial cultures and environmental samples. Chemiluminescent biosensors typically use luciferases, enzymes that drive oxidative reactions to produce light. Engineered to produce light when bound to a molecule of interest, such sensors allow researchers to easily detect and measure their target molecule. There are two main types of luciferase-based biosensors: 1) complementation of split luciferase (CSL) and, 2) bioluminescence resonance energy transfer (BRET)-based biosensors. In general, CSL biosensors produce large signal changes, but their signal intensity is low due to weak reconstitution of luciferase activity. In contrast, BRET biosensors have high signal intensity, but produce small signal changes in response to the molecule of interest. The team adapted a previously developed combination CSL-BRET approach called “Nano-lantern” to develop their new biosensor for cyclic di-GMP.

The team created functional biosensors by finding an insertion site for the E. coli cyclic di-GMP binding protein, EcYcgR, within the Nano-lantern scaffold. They then mined sequence databases for homologous YcgR proteins and chose a set of 92 proteins for functional characterization as candidate biosensors. These 92 homologues were synthesized as codon optimized sequences and cloned into the Nano-lantern scaffold. Because the biosensors are not hindered by autofluorescence, the library was able to be quickly screened for biosensor activity in bacterial lysates without further purification. The best biosensor candidates were then used to develop a high-throughput, lysate-based assay to screen enzymes for cyclic di-GMP synthesis activity. Ultimately, the team aims to examine cyclic di-GMP dynamics in plant-pathogen interactions or gut bacteria—environments where fluorescent biosensors are ineffective. Given that cyclic di-GMP regulates enzymes involved in cellulose production in microbes, these biosensors may eventually lead to ways to improve cellulose-based biofuel synthesis.

BER Contact

Daniel Drell, Ph.D.
Program Manager
Biological Systems Sciences Division
Office of Biological and Environmental Research
Office of Science
US Department of Energy
daniel.drell@science.doe.gov

PI Contact

Ming C. Hammond, Ph.D.
Assistant Professor
University of California, Berkeley
Department of Chemistry
mingch@berkeley.edu

Funding

The work conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. This work was supported in part by NIH grants DP2 OD008677 and R01 GM124589, NIH training grant T32 GM066698, and UC Berkeley College of Chemistry Summer Research Stipend.

Publication

Dippel AB et al. Chemiluminescent biosensors for detection of second messenger cyclic di-GMP. ACS Chem Biol. February 21, 2018. doi: 1021/acschembio.7b01019.