From phenotype to genotype and back again: large scale functional characterization of microbial dark matter by combining activity-based cell sorting, isotope labeling, and genomic sequencing
EMSL Project ID
49972
Abstract
Two long-standing problems in microbial ecology are: (1) identifying the factors driving metabolic activity of microbes in their natural environment and (2) determining the substrates that are fueling that activity. At the moment, no technology allows researchers to approach these questions in a high-throughput, easily replicable, and inexpensive way. Here, we propose new methodologies for large-scale single-cell manipulation, observation, and experimentation that together offer a direct link between genomic information of individual uncultured cells and their in situ biochemical function and biosynthetic activity. We will develop and employ these technologies, in collaboration with JGI and EMSL researchers, using a model system comprising a group of Yellowstone National Park hot springs. These hot springs harbor several uncharacterized archaeal and bacterial taxa that are of interest for numerous reasons. They are hypothesized to be involved in shaping biogeochemical cycles, they are adapted to extreme conditions (high temperature, high concentration of heavy metals), and they may have biotechnological potential (cellulose degradation). Specifically, we will employ bioorthogonal non-canonical amino acid tagging (BONCAT) in combination with fluorescence-activated cell sorting (FACS; at JGI and EMSL) to separate translationally active microbes from complex microbial communities. Cells active under conditions of high interest to the DOE BER mission (e.g. cellulose degradation; resistance to heavy metals; production or degradation of hydrocarbon gases) will be taxonomically identified using 16S rRNA gene analyses as well as targeted genome amplification and genomic sequencing (at JGI). Complementary to these studies targeting the entire microbial community, we will use stable isotope probing in combination with nano-scale secondary ion mass spectrometry (nanoSIMS; at EMSL) mapping of individual cells to determine specific sources of bio-elements and energy. In addition, uncultured taxa of high interest will be analyzed by stochastic optical reconstruction microscopy (STORM; at EMSL) to determine gene expression patterns under close to in situ conditions. We expect successful completion of this project to lead to a dramatic shift in the design and execution of future microbial ecophysiology studies. Furthermore, this project will be the first to shed light on the in situ biogeochemical roles and drivers of metabolic activity of several yet uncultured microbial lineages predicted to be of high relevance to the DOE-BER and JGI-EMSL missions.
Project Details
Project type
FICUS Research
Start Date
2017-10-01
End Date
2021-12-31
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members