mCAFE: Microbial Community Analysis and Functional Evaluation in Soils

The mCAFEs project is a proposed 10-year Department of Energy (DOE) multi-institutional Science Focus Area (SFA). It is aimed at understanding the interactions, localization, and dynamics of grass rhizosphere communities at the molecular level (genes, proteins, and metabolites) to predict responses to perturbations and understand the persistence and fate of engineered genes and microbes for secure biosystems design. To do this, advanced fabricated ecosystems (called EcoFABs) are used in combination with gene editing technologies such as CRISPR-Cas and bacterial virus (phage)-based approaches for interrogating gene and microbial functions in situ.

As part of our tool development, we are attempting to pioneer microbial community editing tools for dissecting the function of genes, pathways, and microbes within complex communities.
a) ET-Seq: Environmental Transformation Sequencing (ET-Seq) is a technology developed to assess the ability of individual species within a microbial community to acquire and integrate exogenous DNA without the need to isolate any individual members. In ET-seq, a microbial community is exposed to a randomly integrating mobile genetic element, and in the absence of any selection, total community DNA is extracted and sequenced to determine insertion location and quantity in each host and to quantify the abundance of each community member in a sample

b) DART: The DNA-editing all-in-one RNA-guided CRISPR–Cas transposase (DART) system was developed for locus-specific insertion of DNA into organisms within a microbial community. The DART system uses an RNA-guided CRISPR–Cas Tn7 transposase to deliver cargo DNA into a specific location of a target genome defined by the guide RNA (gRNA) also encoded on the same plasmid. These systems also include barcodes that make them compatible with ET-Seq to allow for the detection and tracking of uniquely edited cells within communities.

c) Phage-mediated organism- and gene-knockout: We isolate phage from various environments with potent activity against organisms of interest to then experimentally probe the role of these organisms in communities. Work on the SFA has developed a means of delivering a Cas9 base editor for gene knockouts in E coli from a three-member consortium.

d) EcoFAB and EcoPODs: We have developed a fabricated ecosystem (EcoFAB) that can serve as a standardized and reproducible analysis platform for both synthetic consortia and natural microbiomes in simulated and natural environments under controlled conditions. The system is particularly suited for the study of rhizosphere microbiology, as it accommodates plant growth and allows live cell imaging. The SFA is currently being piloted in meter-scale contained and controlled ecosystems (called EcoPODs) that enable the extension of m-CAFEs science into more complex environments.

e) Microbial interaction prediction: We aim to demonstrate our community-editing tools by probing microbial interactions within microbial communities. To identify interactions in communities, we have developed a metagenomics-informed abundance correlation network analysis method for the identification of metabolite exchange, particularly vitamins, in microbial communities and have verified these predicted interactions in co-cultures and microcosms. The SFA has similarly developed metabolic models which are able to predict interactions and co-culture growth dynamics between two organisms based on genetic information. These computational models are then iteratively refined through simulations and experimentation.

Together, these efforts lay a critical foundation for understanding the role and function of genes and organisms within mixed microbial communities. These technologies will allow us to manipulate and harness beneficial microbiomes to support sustainable bioenergy, improving our understanding of nutrient cycling in the rhizosphere.

Learn more about the project at https://mcafes.lbl.gov/

Relevant publications

Hessler, T., Huddy, R.J., Sachdeva, R., Lei, S., Harrison, S.T., Diamond, S. and Banfield, J.F., 2023. Vitamin interdependencies predicted by metagenomics-informed network analyses validated in microbial community microcosms. bioRxiv, pp.2023-01

Adler, B.A., Hessler, T., Cress, B.F., Lahiri, A., Mutalik, V.K., Barrangou, R., Banfield, J. and Doudna, J.A., 2022. Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing. Nature Microbiology, pp.1-13