A novel method has been created to help scientists identify the characteristics or functions linked to genes with unknown roles in microbes. This is a crucial advancement in understanding the contributions and effects of different species in our planet’s varied microbiomes.
A groundbreaking method developed at Lawrence Berkeley National Laboratory (Berkeley Lab) simplifies the process for researchers to uncover the characteristics or functionalities associated with genes of unknown purpose in microbes, which is essential for gaining insights into the contributions of various species.
This new method, known as barcoded overexpression bacterial shotgun library sequencing, or Boba-seq, is detailed in a research paper published on August 5 in Nature Communications.
“There is a significant amount of genetic dark matter — DNA that we can sequence using current techniques but whose functions remain unknown — within the microbial realm. The key question is, how can we effectively study this genetic material to comprehend the microbiomes around us? The primary answer is — through this approach,” explained Adam Arkin, the study’s lead author and a senior faculty scientist in the Biosciences Area at Berkeley Lab.
Boba-seq consists of randomly selecting DNA fragments from specific bacteria and expressing them in host bacterial cells. “The concept is that we can observe how introducing new genes alters the growth characteristics of that bacterium,” commented Yolanda Huang, first author and an assistant professor of Microbiology and Immunology at the University of Buffalo, who conducted her postdoctoral research in Arkin’s lab. “This method of functional genomics enables us to quickly establish a link between a gene or a segment of DNA and its potential function.”
The name “barcode” refers to a short DNA sequence that acts as a unique identifier for a larger DNA fragment, similar to a grocery store barcode that identifies a specific item with a small code. The organism’s entire genome is randomly divided into fragments that contain individual genes or clusters of genes. These fragments are then placed into plasmids — circular DNA structures — each tagged with unique barcodes. The collection of these barcoded plasmids from an organism is referred to as the Boba-seq “library.” This library can be introduced into various bacterial hosts, creating a vast array of genetic variants, which can then be screened for novel behaviors or characteristics.
Arkin and his team in the Biosciences Area are recognized experts in high-throughput methods for gene function analysis and have played a role in developing numerous other techniques that manipulate or silence genes for functional investigation, including RB-TnSeq, CRISPRi, and Dub-Seq.
With Boba-seq, researchers can introduce hundreds of thousands of barcoded fragments into host cells and cultivate them under different conditions to ascertain their functions within a single experiment. For example, if a particular barcode’s cells thrive in antibiotic exposure while others die, it indicates that the gene or genes associated with that fragment provide antibiotic resistance. Thanks to the barcode system, pinpointing the specific fragment responsible for this trait is efficient and cost-effective.
“Yolanda’s advancements with Boba-seq enable us to pinpoint which of the hundreds of thousands of fragments are responsible for the traits or characteristics we wish to study,” Arkin stated. “Our new strategy allows us to create libraries and utilize them with a higher throughput compared to previous overexpression methods.”
Another major advancement with Boba-seq is the ability to test fragments in the same organism they were derived from (or closely related ones), which is crucial for accurately understanding gene functions. Earlier methods were constrained to testing genes only within model organisms like E. coli and yeast, making it challenging or sometimes impossible to assess gene functions of genes from vastly different organisms that may not work effectively in E. coli.
The computational tool developed for analyzing the results from Boba-seq is shared with other researchers through an open-source platform. “I am eager to see how researchers around the globe might adopt Boba-seq, especially for metagenomic research involving gut microbiomes or environmental studies,” remarked co-author Allison Hung, a UC Berkeley graduate student in Arkin’s lab. “Extracting functional information from microbial communities without needing isolation saves considerable time and resources and is vital for examining microbes that are difficult to culture in labs, such as those found in complex ecosystems like ENIGMA.” ENIGMA, which stands for Ecosystems and Networks Integrated with Genes and Molecular Assemblies, is a Department of Energy (DOE) Scientific Focus Area co-led by Arkin that focuses on understanding how microbial communities cycle nutrients and detoxify harmful metal pollutants.
After developing and refining Boba-seq, Arkin’s team applied the method to investigate the genes in Bacteroidales, a group of microbes prevalent in the human gut known for their various roles in our internal microbiome. Bacteroidales also play significant roles in soil ecosystems, breaking down organic matter to recycle nutrients for plants. The researchers generated 305,000 barcoded fragments from six species of Bacteroidales and assessed over 21,000 protein-coding genes simultaneously.
The initial results demonstrated that certain genes encoding enzymes responsible for creating specific lipid molecules grant resistance to the antibiotic ceftriaxone, which belongs to the cephalosporin family. These genes had not been previously associated with antibiotic resistance, indicating a need for further exploration.
The team also identified new functions within carbohydrate metabolism, including an enzyme necessary for the breakdown of glucosamine, a modified sugar found in bones, connective tissue, and the exoskeletons of insects and crustaceans. In the gut, microbes utilize glucosamine as an energy source and to build their cell walls, while human intestinal cells use it to generate the mucus membrane, which is crucial for healthy nutrient absorption and protecting against pathogens.
These findings on Bacteroidales will aid health researchers in gaining deeper insights into gut functionality since this microbial group typically acts as “commensals” that support gut health, but under certain conditions, the nutrients released can also assist pathogenic growth,” Huang noted.
Arkin and his ENIGMA colleagues are currently employing Boba-seq to explore how soil microbes extract energy from complex carbon-based compounds that most life forms are unable to metabolize. In parallel, Huang intends to implement Boba-seq in her new laboratory at the University of Buffalo’s Jacobs School of Medicine & Biomedical Sciences to investigate the genes that bacteria utilize to fend off bacteriophages (viruses that infect bacteria), enhance their ability to colonize the gut, and metabolize complex carbohydrates.
This research received partial funding from the DOE Office of Science. Yolanda Huang is an Astellas Pharmaceuticals Awardee connected to the Life Sciences Research Foundation.
