A new technique developed by Biosciences Area researchers will make it much easier to discover the traits or activities encoded by genes of unknown function in microbes—a key step toward understanding the roles and impact of individual species. The approach, called barcoded overexpression bacterial shotgun library sequencing, or Boba-seq, is described in a paper published in Nature Communications. Initial experiments with the approach have already revealed bacterial functions relevant to human health; future work will investigate functions in ecosystems as well.
“There is so much genetic dark matter—DNA that we can sequence quickly with today’s methods but don’t know the function of—out there in the microbial universe. And the question is, how are we ever going to study all that matter to understand the microbiomes surrounding us?” said lead author Adam Arkin, a senior faculty scientist in the Environmental Genomics and Systems Biology (EGSB) Division. “The fundamental answer is: like this.”
The new technique involves separating the entire genome of an organism of interest into fragments and inserting the fragments into circular packages of DNA (called plasmids) tagged with unique barcodes. The resulting library can then be introduced into different hosts to generate a huge number of genetic variants, which are then screened for new behaviors or properties.
The ability to extract functional information from a microbial community without isolation saves a huge amount of time and resources and will be key for studying microbes that are difficult to culture in a lab, like those living in complex ecosystems currently studied as part of ENIGMA. Short for Ecosystems and Networks Integrated with Genes and Molecular Assemblies, ENIGMA is a Department of Energy (DOE) Science Focus Area co-led by Arkin that aims to understand how microbial communities cycle nutrients through ecosystems and detoxify heavy-metal contaminants.
After building and refining Boba-seq, the team tested it by studying the genes in Bacteroidales, a taxonomic order of microbes that are abundant in the human gut and play a major role in terrestrial soil processes. Results from these proof-of-principle experiments revealed that genes encoding enzymes that build certain lipid molecules endow resistance to ceftriaxone, an antibiotic in the cephalosporin class. These genes have not been previously linked to antibiotic resistance, and warrant further investigation.
Arkin and his ENIGMA colleagues are now using Boba-seq to investigate how soil microbes derive energy from complex carbon-based molecules in the environment that most life forms cannot metabolize.
Additional EGSB researchers who contributed this work include: former Arkin lab postdoc Yolanda Huang (now an assistant professor at the University of Buffalo); research scientist Morgan Price; software developer Omree Gal-Oz; UC Berkeley graduate student Allison Hung; and senior scientist Adam Deutschbauer.
