The evolution of host manipulation by bacteriophage.

Project Information

PHAGECONTROL

Grant agreement ID: 101041421

DOI

10.3030/101041421

EC signature date 28 January 2022

Start date 1 November 2022

End date 31 October 2027

Funded under

• European Research Council (ERC)

Total cost

€ 2 255 511,00

EU contribution

€ 2 255 511,00

Coordinated by

UNIVERZA V LJUBLJANI
Slovenia

Periodic Reporting for period 2 - PHAGECONTROL (The evolution of host manipulation by bacteriophage.)

Reporting period: 2024-05-01 to 2025-10-31

Summary of the context and overall objectives of the project

Parasites that manipulate their host are among the most fascinating adaptations in biology, that till date have not been discovered in microbial world. This manipulation manifests in behavioral patterns, which benefit the parasite by faciliating its transmission, but which harm the host. Bacteriophages are viruses that target bacteria and the most abundant parasites on the planet. So called Phage RS (Regulatory Switches) can control survival, social interactions and evolution of their bacteria. PHAGECONTROL aims to understand why phages modify bacterial phenotypes, hypothesizing that phage RS act as parasite manipulators. The three main research objetives and their associated research tasks are: 1) Find individual and social host behaviors which are controlled by RS phage; 2) Elucidate molecular mechanisms behind host control by RS phage; 3) Determine evolutionary forces behind host control by RS phage.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Over the past period, we expanded our collection of both bacteria and phages (viruses that infect bacteria), thanks to field sampling and international collaborations. We found that the majority of phages predicted by bioinformatics tools remain inactive within their bacterial hosts. This sheds new light on the smaller group of phages that are active and able to influence their hosts. To identify these active ones, we also developed a protocol for extracting high-quality phage DNA from our model system. We created several new molecular tools to study bacteria–virus interactions. These include a collection of identical bacterial strains infected with different versions of a phage, as well as the same phage infecting different bacterial strains. Some of these bacteria and phages were also fluorescently labelled—either to make them easier to track or to monitor the activation of specific genes and proteins.
We optimized methods to simultaneously cultivate and track growing bacteria and phages using an advanced imaging platform. Currently, we can track two samples at once, and work is ongoing to scale this up and automate image analysis. These methods allow us to visualize how phages impact their bacterial hosts—for example, by changing how bacteria grow, what shape they take, how they respond to antibiotics, or how they interact with other bacteria. We can also observe what happens to the phage DNA inside the bacterial cell.
To better understand phage-bacteria dynamics at molecular level, we developed a software tool called SynPhage. It helps quickly identify unique genes in phage DNA, making it easier to link specific phage genes to changes in bacterial behavior. We also developed methods for extracting and analyzing RNA in order to access gene activity/expression (RNA sequencing) in our model system. This will allow us to pinpoint which bacterial and phage genes play key roles in their interaction. In parallel, we are engineering bacteria by inserting selected phage DNA segments to recreate the effects of entire phages. This alternative strategy helps us identify which genes are essential for the interaction. Genes tested so far appeared neutral but the work is ongoing.
Finally, we began studying how phage–bacteria relationships evolve over time, with a focus on phages that carry bacteriocins—small proteins that can kill other bacteria. We started examining how these bacteriocins affect the fitness of both the bacteria and the phage. To support this, we developed a high-throughput screening method to test how sensitive different bacteria are to these bacteriocins. We discovered that the range of bacteria a phage can infect does not match the range affected by its bacteriocin, an important finding for understanding how phages avoid harming their own potential hosts.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

One of our key contributions is a large, carefully analyzed dataset of prophage elements found in Bacillus subtilis. These viral elements were grouped and studied for how they might help or hinder each other, offering new predictions now ready for experimental testing. This resource is publicly available through Zenodo, making it useful for many researchers studying bacterial evolution and genetics.
To support this work, we developed SynPhage, an open-source software tool that helps scientists explore viral genes and their conservation across genomes. Although created for phage studies, it is also useful for broader gene comparison work. The tool is freely available on GitHub, has already been the subject of a peer-reviewed publication, and was featured in a hands-on workshop with researchers.
We also clarified past findings by showing that a previously reported phage effect on bacterial biofilms was caused by unrelated mutations. We're working with the original authors to publish an updated interpretation, which improves the accuracy of the scientific record.
Another exciting direction is our discovery that viruses within the same bacterial host can exchange genes—like shuffling a deck of cards, leading to chimeric phages. This process, confirmed during a collaborative research visit, could accelerate bacterial evolution and might even be used to design helpful bacterial strains in the future.
On the technology front, we began using a state-of-the-art microscope that lets us watch bacteria and viruses interact in real time. This platform will soon support high-throughput experiments and strengthen collaborations both within and beyond our institution.
Beyond the lab, the project is boosting international collaboration and training. We’ve brought together postdocs and students from around the world, hosted researchers from Japan and across Europe, and supported young scientists through prestigious fellowships. Our team has formed strong partnerships with leading labs in Germany, Denmark, the UK, France, the Czech Republic, and Italy.

Summary of research activities in PHAGECONTROL project