Revealing the mysteries within microbial genomes

 

Image designed by Tim Sandle

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 impact of individual species.

 

The approach is called barcoded overexpression bacterial shotgun library sequencing, or Boba-seq. This involves taking random fragments of DNA from bacteria of interest and expressing them in host bacterial cells.

 

The term "barcode" in the name refers to a small sequence of DNA that the scientists use as an identifying tag for a much larger fragment of DNA, much like how a barcode at 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 clusters of several genes, then inserted into plasmids -- circular packages of DNA -- that have been tagged with unique barcodes.

 

The Boba-seq "library" refers to all the barcoded plasmids containing fragments from an organism. This library can be introduced into different bacterial hosts to generate a huge number of genetic variants, which are then screened for new behaviors or properties.

 

With Boba-seq, hundreds of thousands of barcoded fragments can be put into host cells and cultured under varying conditions to determine function in a single experiment. For example, if cells with a certain barcode grow happily when the whole culture is exposed to an antibiotic, but the others perish, you know that the gene or genes in that fragment encoded antibiotic resistance traits. And identifying the fragment responsible for this new ability is cheap and fast, thanks to the barcode.

 

The other significant breakthrough is that Boba-seq fragments can be tested in the same organism that they were pulled from (or a close relative), which is essential for getting an accurate picture of what a gene does. Previous techniques are limited because they only test 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 the genes do.

 

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

 

The technique connects with the ENIGMA project, short for Ecosystems and Networks Integrated with Genes and Molecular Assemblies. This is a U.S. Department of Energy (DOE) Scientific Focus Area co-led by Arkin that is aimed at understanding how microbial communities cycle nutrients through ecosystems and detoxify toxic heavy metal contaminants.

 

After building and refining Boba-seq, the researchers tested the new technique by studying the genes in Bacteroidales, a taxonomic order of microbes that are abundant in the human gut and known to play many roles in our internal microbiome.

Bacteroidales are also major players in terrestrial soil processes, where they degrade organic matter and return the 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.

 

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.

 

The researchers also discovered several new functions in carbohydrate metabolism, including an enzyme needed to metabolize glucosamine, a modified sugar molecule found in bones, connective tissue, and the exoskeletons of insects and crustaceans. In the gut, microbes use glucosamine as an energy molecule and to construct their cell walls, whereas human cells that form the lining of the intestine use it to produce the mucus membrane that helps maintain healthy nutrient uptake and prevent invasion of pathogens.

 

These insights into Bacteroidales will help health researchers better understand gut function, as this order acts as commensals most of the time and really maintain gut health. But in certain states, the nutrient released by Bacteroidales can be used by pathogens to support their own growth.

 

See:

 

Yolanda Y. Huang, Morgan N. Price, Allison Hung, Omree Gal-Oz, Surya Tripathi, Christopher W. Smith, Davian Ho, Héloïse Carion, Adam M. Deutschbauer, Adam P. Arkin. Barcoded overexpression screens in gut Bacteroidales identify genes with roles in carbon utilization and stress resistance. Nature Communications, 2024; 15 (1) DOI: 10.1038/s41467-024-50124-3 

 

Posted by Dr. Tim Sandle, Pharmaceutical Microbiology Resources (http://www.pharmamicroresources.com/)