In the microscopic world of bacteria, an ancient war is being waged. Bacteria build ever more complex defense systems, viruses find new ways to evade them, bacteria create new defenses — and this molecular arms race has been going on for several billion years.
Among the main participants in this story are bacteriophages — viruses that infect and destroy bacteria. Their ability to destroy bacterial cells has long attracted the attention of medicine, especially today, when the resistance of microorganisms to antibiotics is becoming one of the most serious global public health problems.
However, researchers face a remarkable obstacle: we have learned to read the genomes of many phages, but we often still poorly understand what exactly the genes recorded in them do. Most phage genes remain functionally unknown.
Professor Peter Fineran of the University of Otago in New Zealand compares the current understanding of phages to how humanity understood antibiotics in the mid-twentieth century. These unknown regions of the phage genome — a kind of "microbial dark matter" — long remained inaccessible to direct study.
Now a team of researchers at the University of Otago has created a tool that makes it possible to literally illuminate this territory across an entire genome at once.
The method was named phage Tn-seq. The study, conducted by Natalie Kite, Manuela Fuchs, Leah Smith and Peter Fineran, was published on 25 September 2026 in the journal Nature Microbiology — one of the leading publications in the field of microbiology.
The technology is based on a small mobile DNA fragment — the Tn5 transposon, about 5,8 kilobases in length and carrying antibiotic resistance genes.
This transposon is capable of inserting itself into the most diverse regions of the phage genome, like a molecular syringe injecting genetic material into a foreign genome. When such an insertion lands inside a gene, that gene's function is disrupted. The researchers generate thousands of different variants of the virus, each affecting its own region of the genome — a kind of mutant library.
Then the most interesting part begins.
If a phage continues to exist and reproduce after a particular gene has been disrupted, this indicates that this region tolerates change under the given conditions and is not critical for survival. If, however, mutants with damage to a specific gene are practically impossible to detect, this indicates that the gene plays an important or vitally necessary role. The researchers obtain a clear map: which genes the phage can lose, and which are indispensable to it.
Thus, instead of the lengthy study of one gene after another, there emerges a complete map of the vitally important regions of the entire phage genome — a whole functional portrait of the virus, obtained in a single experiment.
To select the desired mutant viruses from a thousand-strong multitude, the researchers linked transposon mutagenesis to the CRISPR–anti-CRISPRsystem. The transposon carries an anti-CRISPR gene that enables the phage to survive the selective pressure of the CRISPR system inside the bacterial cell. After that, deep sequencing shows into which places of the viral genome the transposons have inserted themselves and which genes have remained untouched. This combination of three techniques — transposon mutagenesis, CRISPR selection and sequencing — turns the technology from a set of separate, labor-intensive experiments into a systematic, automatable method of research.
The team tested phage Tn-seq on various species of bacteriophages, including quite exotic ones. The method worked even with the giant phage PCH45, which infects the bacterium Serratia. During infection, this phage creates inside the bacterial cell an unusual structure resembling a cell nucleus, which separates and protects the phage's DNA from the bacterial immune system. The researchers also managed to perform transposon insertions in phages with heavily chemically modified DNA — in genomes that had traditionally been especially difficult for genetic engineering.
But the technology can do more than simply disrupt genes.
The transposon can be turned into a miniature genetic truck.
The researchers loaded additional sequences into it and managed to deliver new genetic material directly into the phage genome. In one of the experiments, the phages received genes for fluorescent proteins and glowed under the microscope. The authors also showed that other anti-CRISPR genes can be introduced, and they even created double mutants — phages with two targeted changes at once, using artificially engineered systems.
This changes the very paradigm of research.
The phage becomes an organism that can not only be observed under the microscope but also, far more quickly, studied functionally and rebuilt at will. Researchers can now pose direct questions: which gene is necessary for the virus to assemble its coat? Which one helps it penetrate into the bacterium? Which regions allow it to overcome bacterial defense? Where can a new genetic sequence be placed so that the phage remains alive and functional?
Each such question could previously require months of a separate, complex series of experiments. Now many of them can be studied simultaneously, which accelerates research dozens of times over.
This work is of particular importance for the future of phage therapy — the use of bacteriophages as medicines.
Bacteriophages are already being considered as potential antimicrobial agents against resistant bacteria, but to create reliable and safe therapeutic variants it is necessary to understand precisely the biology of phages and to be able to modify their genomes in a controlled manner. The authors of the work directly name insufficient knowledge of the functions of phage genes and the difficulties of rapid genetic engineering as some of the main obstacles on the path of development of this promising field.
At the same time, the study itself so far represents primarily a new technological platform, rather than a ready-made treatment. Any specially engineered phage intended for clinical use will require a separate, often multi-year assessment of efficacy, storage stability, safety and tolerability.
And yet an important conceptual shift is taking place here.
For several decades, genomics taught us one thing: how to read a DNA sequence. Now more and more technologies make it possible to ask the next, significantly more complex question: what will happen if each of these sequences is changed?
For bacteriophages this is especially important and timely. Their compact genomes are filled with regions whose functions have yet to be discovered, and whose potential has scarcely been studied.
Phage Tn-seq turns this unknown into an experimental map, into direct answers to direct questions.
The dark matter of the phage world is gradually beginning to reveal its outlines — gene by gene, mutation by mutation, function by function.
And along with this, bacteriophages are increasingly turning from mysterious natural viruses into precise tools that biology is learning to understand, to program and — in the coming years — to use.
