Texas A&M researchers report de novo evolution of an overlapping gene in bacteriophage

A team of researchers from Texas A&M University has discovered a hidden gene, embedded within another gene, in the group of bacteriophages called leviviruses. They report that the hidden gene is rapidly evolving and thus holds the potential for understanding and preventing antibiotic resistance. 

The research was published Nov. 26 in Nature Communications.

Developing new kinds of antibiotics from bacteriophages, types of viruses that can eat bacteria, is an important area of study, especially as resistance to existing antibiotics increases.

The use of phages to treat bacterial infections was pioneered in Soviet Georgia in the 1920s and 1930s, years before modern antibiotics, and their use continued in the Soviet Union and Eastern Europe. 

Today, with the rise in antibiotic resistance and advancement in modern techniques for analyzing phage genomes and structure, scientists are once again trying to engineer bacteriophage therapeutics.

Leviviruses are small, single-stranded RNA (ssRNA) phages whose natural hosts are enterobacteria such as E. coli. Many leviviruses are now known but prior to this study, most of them were found to be missing an annotated lysis gene, known as Sgl. This is the specific gene that kills the bacteria by rupturing its cell wall. 

The A&M research team came up with criteria to identify these hidden genes. From their pipeline, using 244 leviviruses they found 293 genomic possibilities which they then tested in E. coli bacteria. They found 33 Sgls that at least reduced against E. coli growth, plus two evolved Sgls. 

When they tried to identify Sgls using sequence similarity in tens of thousands of recently discovered leviviruses using sequence technology, they found only a "handful of hits." "Almost all of the Sgls discovered in this study share no significant [>50%] sequence similarity with each other or to any of the previously known eight Sgls from classic ssRNA phages," the researchers wrote.

Thus, the authors argue: "Sgls are extremely diverse and remain vastly untapped as a source ... for protein antibiotics."

The A&M team noticed that even for leviviruses that share extensive sequence similarity, the Sgl sequence might be significantly different and embedded in a different location.

"We noticed that lysis genes in different ssRNA phages arose in new locations within the genome and that some of these evolution events had happened in very closely related genomes, which suggests that these evolution events had happened recently and rapidly before coat or rep genes have diverged significantly," lead author Karthik R. Chamakura explained to Current Science Daily. "This is possible because ssRNA phages are 100- to 1,000-fold more error prone in their replication when compared to their hosts, and they can accumulate at ~10,000 virions per cell. When you take these mind-boggling numbers into account it is reasonable to think that new Sgl genes arise rapidly." 

The authors propose mining the ssRNA phage space and using in vitro reconstruction experiments to determine how the Sgl genes evolve, and how fast.

In looking at the de novo evolution, the research team found that the genomes involved were from regions geographically close to each other. Current Science Daily asked Chamakura why that might be the case.

"When we discovered that very closely related ssRNA phage genomes had evolved their respective Sgls in different locations within the genome, we reasoned that these RNA phages had diverged recently and were probably still in close geographic proximity," he said. "Tracking down the source of the metatrancriptomes confirmed that all four of these genomes were sourced from within a 200-km radius. 

"We discovered that there were several mutations ... which resulted in evolution of a new gene in a new location with concomitant loss in the other location, or vice versa in the other phage."

Chamakura said that without the advances in sequencing technologies, this important research would not have been possible.

"Global metatranscriptomic and RNA-inclusive metagenomic studies were instrumental in collecting vast amounts of RNA sequence data and depositing them in public databases," he told Current Science Daily. "Then scientists like S.R. Krishnamurthy reasoned that there might be undiscovered ssRNA phage genomes in the publicly available sequence data. They looked for genetic signatures that resembled known ssRNA phages in the deposited data and extracted those sequences out as new RNA phage genomes. 

"This work led to a major expansion of available ssRNA genomes and laid the foundation for our work." 

Chamakura is a postdoctoral research associate at Texas A&M's Center for Phage Technology.