Uncovering the mysteries of microbial genomes with a novel high-throughput approach

A new technique developed at Lawrence Berkeley National Laboratory (Berkeley Lab) will make it much easier for researchers to discover the traits or activities encoded by genes of unknown function in microbes, a key step toward understanding the roles and impacts of individual species.

The approach, called barcoded bacterial overexpression library sequencing, or Boba-seq, is described in a paper published Aug. 5 in Nature Communications.

“There is so much genetic dark matter—DNA that we can sequence rapidly with current methods but whose function we don’t know—in the microbial universe. And the question is, how do we study all of this matter to understand the microbiomes around us? The fundamental answer is this,” said lead author Adam Arkin, a senior research scientist in Berkeley Lab’s Department of Biosciences.

Boba-seq involves taking random fragments of DNA from bacteria of interest and expressing them in host bacterial cells.

“The idea is to see how the presence of new genes confers a difference in the growth phenotype of this bacterium,” said first author Yolanda Huang, an assistant professor of microbiology and immunology at the University at Buffalo who was a postdoctoral researcher in Arkin’s lab at the time of the study. “This is a functional genomics approach that we can use to quickly link a gene or a piece of DNA sequence to a potential function.”

The term “barcode” in the name refers to a small sequence of DNA that scientists use as an identification label for a much larger piece of DNA, much like a barcode in a grocery store identifies a specific item with a small code. The entire genome of the organism being studied is randomly separated into fragments containing single genes or groups of multiple genes, then inserted into plasmids (circular packages of DNA) that have been labeled with unique barcodes.

The Boba-seq “library” refers to the set of barcoded plasmids containing fragments of an organism. This library can be introduced into different bacterial hosts to generate a large number of genetic variants, which are then screened for novel behaviors or properties.

Arkin and his colleagues in the biosciences field are leading experts in high-throughput techniques for studying gene function and have helped invent a number of other techniques that insert or silence genes to study their function, including RB-TnSeq, CRISPRi and Dub-Seq.

With Boba-seq, hundreds of thousands of barcoded fragments can be introduced into host cells and cultured under a variety of conditions to determine their function in a single experiment. For example, if cells with a certain barcode grow well when the entire culture is exposed to an antibiotic, but the others die, the gene or genes in that fragment are known to encode antibiotic resistance traits. And identifying the fragment responsible for this new ability is inexpensive and rapid, thanks to the barcode.

“Yolanda’s innovations with Boba-seq allow us to identify among hundreds of thousands of fragments those that confer the phenotype or property we want,” Arkin said. “Our new strategy allows us to create libraries and use them at a higher throughput than previous overexpression approaches.”

The other major advance is that Boba-seq fragments can be tested in the organism from which they were extracted (or a close relative), which is essential for getting an accurate picture of what a gene does. Previous techniques were limited because they only tested genes inside model organisms like E. coli and yeast. Genes from organisms very different from E. coli are often not functional in E. coli, making it difficult or impossible to get a clear picture of what genes do.

The computational tool used to process the results of the laboratory work involved in Boba-seq is available to other researchers on an open source platform.

“I’m excited to see how others around the world might use Boba-seq, particularly for metagenomic studies of the gut or the environment,” said Allison Hung, a co-author of the study and a graduate student at UC Berkeley in Arkin’s lab. “The ability to extract functional information from a microbial community without isolation saves a tremendous amount of time and resources, and will be critical for studying microbes that are difficult to culture in the lab, such as those living in complex ecosystems currently being studied in ENIGMA.”

ENIGMA, short for Ecosystems and Networks Integrated with Genes and Molecular Assemblies, 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 toxic heavy metal contaminants.

After developing and refining the Boba-seq technique, Arkin’s team tested the new technique by studying genes in the Bacteroidales, a taxonomic order of microbes abundant in the human gut and known to play many roles in our internal microbiome. Bacteroidales also play a major role in terrestrial soil processes, where they degrade organic matter and return nutrients to plants. The team generated 305,000 barcoded fragments from libraries of six Bacteroidales species and evaluated more than 21,000 protein-coding genes in parallel.

The results of these proof-of-principle experiments revealed that genes encoding enzymes that build certain lipid molecules confer resistance to ceftriaxone, a cephalosporin antibiotic. These genes have not previously been linked to antibiotic resistance and deserve further study.

The team also discovered several new functions in carbohydrate metabolism, including an enzyme required for the metabolism of glucosamine, a modified sugar molecule found in the bones, connective tissue and exoskeleton of insects and crustaceans. In the gut, microbes use glucosamine as an energy molecule and to build their cell walls, while human cells that form the intestinal wall use it to produce the mucous membrane that helps maintain proper nutrient absorption and prevent pathogen invasion.

This information about Bacteroidales will help health researchers better understand how the gut works, because the order acts as “commensals most of the time and actually maintains gut health,” Huang said. “But in some states, the nutrient released by Bacteroidales can be used by pathogens to support their own growth.”

Arkin and his ENIGMA colleagues are now using Boba-seq to study how soil microbes get energy from complex carbon-based molecules in the environment that most life forms can’t metabolize. In parallel, Huang plans to use Boba-seq in his new lab at the University at Buffalo’s Jacobs School of Medicine & Biomedical Sciences to study the genes that bacteria use to evade attack by bacteriophages (viruses that infect bacteria), increase the efficiency of colonization in the gut, and break down complex carbohydrates.