Although retrons have been known, for decades, to be bacterial genetic retroelements that encode a reverse transcriptase (RT) that uses small RNA as a template to produce multicopy single-stranded DNA (msDNA), their role has remained elusive. Now, scientists have identified that some retrons encode toxin proteins, which they keep inactive with the help of a small DNA fragment. When a bacteriophage infects bacteria, the small DNA senses the attack and the toxin is activated.

More specifically, the authors of a new paper show that a retron of Salmonella Typhimurium—Retron-Sen2—encodes an accessory toxin protein (RcaT) which is neutralized by the RT-msDNA antitoxin complex. They go on to show that activation occurs when msDNA biosynthesis is perturbed. This RcaT-containing retron family constitutes a new type of tripartite DNA-containing toxin/antitoxin systems (TAs).

These findings are published in Nature in the paper, “Bacterial retrons encode phage-defending tripartite toxin–antitoxin systems.

“For more than 30 years, we had no clue why bacteria have retrons because no phenotypes had been associated with cells lacking retrons or msDNA,” said Jacob Bobonis, PhD, a former graduate student in the lab of Nassos Typas, PhD, group leader in the Genome Biology Unit and a co-chair of EMBL’s Microbial Ecosystems and Infection Biology transversal themes.

But new information came to light when a previous member of the Typas group found that Salmonella cannot grow in colder temperatures without making msDNA. This pointed to  the finding that Salmonella cells that are unable to make msDNA were also sensitive to a lack of oxygen, preventing them from colonizing a cow’s gut.

“We quickly realized that retrons, while more complicated, looked very similar to other systems in bacteria called toxin/antitoxin systems,” Bobonis explained.

Many bacteria contain hundreds of toxin/antitoxin systems in their genomes. One gene encodes a toxin that stops the growth of the bacterium, with the antitoxin located right next to the toxin. While the two co-exist, bacteria grow. But if the antitoxin is removed, the poison becomes active and inhibits their growth.

“Analogously, in our case, we have the retron reverse transcriptase that makes msDNA, and if we delete it, the ‘toxin’ is activated,” Bobonis said. “We realized that the msDNA together with the reverse transcriptase form a new class of antitoxins. But we still wondered what could be that ‘switch’ to trigger this growth inhibition complex naturally.”

To identify the elusive molecular triggers and blockers of TAs, the team developed TAC/TIC (Toxin Activation/Inhibition Conjugation), a high-throughput reverse genetics approach. By applying TAC/TIC to Retron-Sen2, the authors noted, they identified multiple such proteins of phage origin.

Ultimately, using genetics, proteomics, bioinformatics, they parsed out the mechanism and discovered how viral proteins can activate, as well as block, these systems. They found that retrons can thwart viral invasion on a single-cell level.

“Imagine you have 10 bacteria, and a virus goes in and infects just one of them. The virus replicates itself hundreds of times, eventually breaking the cell so that virus spills over from the infected cell, and goes on to infect the other nine cells (or more if bacteria have duplicated in the meantime). In that case, the bacterial population is killed,” Bobonis explained. “In a cell where the retron is switched on by the virus, the initial infected cell withers, but so does the virus, as it needs the bacterium’s machinery to replicate. Without the initially infected bacterium, the virus falters, and retrons have protected the rest of the population.”

“Bacterial chromosomes contain hundreds of different toxin/antitoxin systems of unknown function that might be leveraged to inhibit phages, and our findings provide an approach to understand how they could do that,” said Typas. “Since these are bacteria’s internal suicide systems, knowing the trigger switches for them means that we have an angle to design artificial toxin triggers to externally activate the toxin and kill the cell,” he continued. “Such new strategies are urgently needed as effective antibiotics become scarce to treat antimicrobial-resistant pathogens. EMBL’s Infection Biology transversal theme aims to better understand antimicrobial resistance and find new ways to curtail, prevent, reverse, or bypass it.”

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