You say yes, I say no / You say stop, and I say go, go, go

When the Beatles sang “Hello Goodbye,” opposites and reversals were played to whimsical effect. With microbes, they hint at something darker, or at least something more complex. Microbes, it appears, don’t necessarily have to follow each other’s rules for what bits of genetic code mean. In the case of phages, one kind of reversal—codon reassignment—could be exploited to weaken bacterial defenses.

Call it codon warfare or translational override. Either way, it enriches our view of metagenomics and even suggests that greater care may need to be taken in synthetic biology, where bits of code have been inserted to prevent the swapping of genetic information between lab-engineered microbes and their cousins in the wild. Such bits of code may be weaker safeguards than once imagined.

While exploring the biological frontier known as “microbial dark matter,” researchers at the U.S. Department of Energy’s Joint Genome Institute (JGI) discovered that deviations in the way microbes read genetic code are fairly common, challenging the view that genetic code is deeply conserved across all domains of life with few exceptions. In particular, these researchers found that stop codons were reassigned up to 10% of the time in some environments.

The researchers, led by Natalia Ivanova, Ph.D., published their results May 23 in Science, in an article entitled “Stop codon reassignments in the wild.”

“By scanning 5.6 trillion base pairs of metagenomic data for stop codon reassignment events, we detected recoding in a substantial fraction of the >1,700 environmental samples examined,” the researchers wrote. “We observed extensive opal and amber stop codon reassignments in bacteriophages and of opal in bacteria.”

Opal and Amber codons are ordinarily read as “stop” signs during gene expression. In some organisms, however, the stop sign is not interpreted as a stop. Instead, it signals the cell’s translational machinery to keep adding amino acids and expand the protein.

The particular observation that caught the team's interest in looking for breakdowns in the canonical genetic code was when Dr. Ivanova came across an anomaly: bacteria with extraordinarily short genes of only 200 base pairs in length. Typically, genes from microbes are about 800–900 base pairs long.

Opal resulted in the bacteria having unbelievably short genes. “When Dr. Ivanova applied a different vocabulary where Opal, instead of being interpreted as a stop, was assumed to encode the amino acid glycine, the genes in the bacteria suddenly appeared to be of normal length,” said senior author Eddy Rubin, Ph.D. Their interpretation of the finding was that “Opal-recoded” organisms, instead of stopping, incorporated an amino acid into the polypeptide, which kept growing and eventually produced normal-sized proteins.

Following this finding they wanted to see how frequently this occurs in nature and looked for similar occurrences in enormous amounts of sequence data from uncultured microbes. The samples included those from far-flung and esoteric locations—marine, fresh water, and terrestrial environments—as well as those from the human mouth and gut.

“We were surprised to find that an unprecedented number of bacteria in the wild possess these codon reassignments, from ‘stop’ to amino-acid encoding ‘sense,’ up to 10% of the time in some environments,” said Dr. Rubin.

Another observation the researchers made was that beyond bacteria, these reassignments were also happening in phage, viruses that attack bacterial cells. Phage infect bacteria, injecting their DNA into the cell and exploiting the translational machinery of the cell to create more of themselves to the point when the bacterial cell explodes, releasing more progeny phage particles to spread to neighboring bacteria and run amok.

“To make this all happen, the established dogma was that phage needed to employ the exact genetic code that the host cell uses, otherwise whatever DNA they inject wouldn't be properly translated,” Dr. Rubin said. “But we observed phage with codon vocabularies that did not match any we found in their bacterial hosts. We scratched our heads at this result, because we wondered about what was up with the host. The dogma tells us that the phage to need to share the same code as the host, but we saw no Amber in bacteria. So what were these phage doing?”

The punch line, Dr. Rubin said, is that the dogma is wrong.

“Phage apparently don't really 'care' about the codon usage of the host. They have ways to get around that, and in fact they use differences to attack the host.” The phage use certain molecular tricks, just those slight changes in the codon table, to suppress the host cell's protective mechanisms to conduct a 'hostile takeover' of the cell. “We call this strategy 'codon warfare',” Dr. Rubin said. “We need to keep this in mind when characterizing environments and how their resident microbes contribute to biochemical and biogeochemical processes. Now that our assumptions about the canonical nature of the codon table are shaken up, we will be able to devise new analysis methods that take this phenomenon of unexpected complexity into consideration so we can obtain a better understanding of how these environments function.”

Additional food for thought, Dr. Rubin noted, is whether adequate controls can effectively be established for those emergent organisms developed through synthetic biology. Some of these organisms have been engineered with an intentionally altered genetic code, designed as a “firewall” to prevent the exchange of genetic information between laboratory-engineered microbes and their cousins in the wild.

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