Open access: https://www.pnas.org/doi/full/10.1073/pnas.2513255122
Emergence of antiphage functions from random sequence libraries reveals mechanisms of gene birth
Idan Frumkin, Christopher N. Vassallo, Yi Hua Chen, and Michael T. Laub
122 (42) e2513255122 https://doi.org/10.1073/pnas.2513255122
Abstract
De novo gene birth—the emergence of genes from nongenic sequences—drives biological innovation, yet its adaptive potential remains poorly understood. To investigate this issue, we screened libraries of ~100 million short (semi-)random sequences, mimicking early stages of gene birth, for genes that promote Escherichia coli survival during phage infection. This selection uncovered thousands of functional genes that confer viral resistance through at least two distinct mechanisms: 1) activation of a bacterial regulatory system that remodels the outer membrane, which provides broad-spectrum defense, and 2) transcriptional repression of bacterial outer membrane receptors required for phage adsorption, which provides phage-specific protection. Remarkably, unrelated random genes with no sequence similarity produced similar protective phenotypes, revealing that diverse sequences can converge on equivalent functions. We further showed that T4 phage rapidly evolves to counter these novel defenses, acquiring baseplate mutations that enhance adsorption to resistant hosts. Together, these findings demonstrate that random sequences can rapidly evolve into functional genes with direct fitness benefit, highlighting the evolutionary potential of de novo gene birth in the microbial world.
They transform bacteria with plasmids encoding up to 50 codon long random ORFs (using two different libraries, one of which allow only 9 possible amino acids), then grow these bacteria on agar plates containing T4 phages.
Results
Selection for Functional, (Semi-)Random Genes that Provide Antiphage Defense.
We sought to identify genes derived from (semi-)random nucleotide sequences that enable bacterial cells to survive phage infection. To this end, we implemented a selection strategy to isolate random genes that confer resistance to the T4 phage, a well-characterized model virus that infects E. coli (46). We screened a library of ~100 million plasmids (42), each carrying an inducible promoter driving the expression of two open reading frames.
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Our selection approach builds on a previously established screening protocol for identifying natural antiphage defense systems using plasmid libraries(48) (Fig. 1B). In this method, E. coli MG1655 cells carrying the (semi-)random gene libraries are mixed with T4 phages in a structured medium (soft agar) at a phage concentration that allows individual clones to proliferate into microcolonies before encountering a phage particle. Clones expressing random genes that confer resistance survive the infection and form visible colonies. Unlike liquid-based selection methods, where the strongest resistance phenotypes rapidly dominate, this solid-medium approach enables the detection of a broader spectrum of defense mechanisms, including those with intermediate levels of antiviral protection.
They find ~4500 functional genes in their library of 100 million random genes. That’s a frequency of functional (that give resistance to T4 phage) genes of about 1 in 22 000.
Remarkably, the library encoding only 9 possible amino acids (which has a strong bias towards hydrophobicity) also has a considerably higher fraction of functional genes (~4500) that give resistance to T4 phage, than the library using codons allowing all 20 amino acids (only about 360).