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Researchers at Lawrence Berkeley National Laboratory (Berkeley Lab) have developed a novel technique that significantly simplifies the process of identifying the functions of genes within microbial genomes. This advancement, detailed in a recent Nature Communications article published on August 5, presents a promising new method known as barcoded overexpression bacterial shotgun library sequencing, or Boba-seq.
Genetic Dark Matter in Microbes
Microbial genomes contain a vast amount of uncharacterized genetic material, often referred to as "genetic dark matter." The new Boba-seq technique offers a functional genomics approach to rapidly associate specific gene sequences with their potential roles within a microbial community. "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," said Adam Arkin, the lead author and senior faculty scientist at Berkeley Lab's Biosciences Area. "And the question is, how are we ever going to study all that matter to understand the microbiomes surrounding us? The fundamental answer is—like this."
Boba-seq operates by introducing random DNA fragments from a target microbe into host bacterial cells. "The idea is that we can see how the presence of new genes confers a difference in phenotype of growth of that bacterium," explained Yolanda Huang, the first author and now an assistant professor at the University of Buffalo. This method allows scientists to observe how specific genetic fragments influence microbial behavior under various conditions.
The Role of Barcodes in Gene Identification
A key feature of Boba-seq is the use of DNA barcodes—short sequences that uniquely identify each DNA fragment within the experiment. The researchers generate a "library" of these barcoded DNA fragments by dividing the entire genome of a microbe into smaller segments, which are then inserted into plasmids tagged with the unique barcodes. These plasmids are introduced into bacterial hosts, creating a diverse array of genetic variants. The barcode simplifies the identification of the fragments responsible for any observed phenotypic changes, enabling high-throughput screening in a single experiment.
“Yolanda’s innovations with Boba-seq allow us to identify which of hundreds of thousands of fragments are conferring the phenotype or the property that we want,” said Arkin. “Our new strategy allows us to make libraries and use them at a higher throughput than previous overexpression approaches.”
Applications and Implications
One of the significant advantages of Boba-seq is its ability to test genetic fragments in their native or closely related microbial hosts, which is crucial for accurate functional analysis. Previous techniques often relied on model organisms like Escherichia coli or yeast, which are not always compatible with genes from more distantly related microbes.
The computational tools developed alongside Boba-seq are made available as open-source resources, allowing other researchers to apply this technique to their studies. Co-author Allison Hung, a graduate student at UC Berkeley, expressed excitement about the potential applications, particularly in metagenomic studies of complex ecosystems like the human gut or environmental samples. “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,” said Hung. ENIGMA, which stands for Ecosystems and Networks
Integrated with Genes and Molecular Assemblies, is a Department of Energy (DOE) Scientific Focus Area co-directed by Arkin. The program focuses on studying how microbial communities contribute to nutrient cycling within ecosystems and the detoxification of harmful heavy metal contaminants.
Testing Boba-seq in Bacteroidales
Arkin’s team tested Boba-seq by analyzing the genes of Bacteroidales, a taxonomic order of microbes prevalent in the human gut and soil ecosystems. They generated over 305,000 barcoded fragments from six Bacteroidales species, allowing them to evaluate more than 21,000 protein-coding genes simultaneously. These proof-of-principle experiments uncovered previously unknown
gene functions, such as the involvement of lipid-modifying enzymes in antibiotic resistance—a discovery that warrants further investigation.
The team also identified new functions related to carbohydrate metabolism, including an enzyme necessary for the breakdown of glucosamine, a molecule with critical roles in both microbial and human physiology. In the gut, microbes utilize glucosamine as an energy source and for cell wall construction, while human cells use it to maintain the mucus membrane that supports nutrient absorption and pathogen defense.
These findings on Bacteroidales provide health researchers with a deeper understanding of gut function, as this bacterial order typically acts as commensals and plays a crucial role in maintaining gut health, noted Huang. However, under specific conditions, the nutrients produced by Bacteroidales may be exploited by pathogens to fuel their own growth.
Future Directions
Building on these findings, Arkin’s team plans to apply Boba-seq to study how soil microbes process complex carbon molecules that are inaccessible to most life forms. Meanwhile, Huang intends to use Boba-seq in her new lab to explore how bacteria evade bacteriophages, enhance colonization in the gut, and degrade complex carbohydrates.
Original Publication
Huang, Y.Y., Price, M.N., Hung, A. et al. Barcoded overexpression screens in gut Bacteroidales identify genes with roles in carbon utilization and stress resistance. Nat Commun 15, 6618 (2024). https://doi.org/10.1038/s41467-024-50124-3