Project Details
Projekt
Dissecting the vast diversity of anti-retron mechanisms in phage genomes
Applicant
Athanasios Typas, Ph.D.
Subject Area
Metabolism, Biochemistry and Genetics of Microorganisms
Term
since 2024
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 548567266
Bacteria deploy an impressive variety of immunity systems to counter phage infection. Even within the same type, bacterial defenses are highly diverse and neutralise phages through a plethora of molecular mechanisms, which we only now begin to understand. Retrons, which shortcut phage infection via abortive infection, exemplify this diversity. They are widespread tripartite systems, containing a reverse transcriptase and a fast evolving non-coding DNA (sometimes DNA-RNA hybrid), which together keep in check a multitude of different effector proteins. It is the effector proteins that divide retrons into many different families. The first glimpses of phage-encoded strategies against this avalanche of bacterial defense systems are also starting to emerge. However, current tools and techniques to study anti-defense systems in a systematic way are limited, due to the mosaic nature and poor understanding of phage genomes (full of uncharacterized genes). The former hampers genome-context approaches that have revolutionized the bacterial immunity field. We have circumvented current limitations and used unbiased genetics approaches to expose a vast diversity of phage-encoded anti-retron strategies. The likelihood that the same is true for any immunity system is high. Using functional metagenomics and a forward-genetics screen for the Retron-Sen2 we have identified > 50 distinct phage proteins that inhibit its RcaT toxin, also during phage infection. Half of these proteins have no similarity to known proteins. As part of this proposal, we want to stratify the different mechanisms of action of these proteins, with a focus on identifying direct RcaT binders, which would indicate convergent evolution as a main player in phage anti-retron defense. We will also assess the evolutionary spread of these blocker genes in phage genomes, and use knock-out, -down and -in approaches to assess whether these proteins are necessary and sufficient for anti-defense in native contexts. To define the spectrum of action of such blockers, we will test them against a set of retrons from the same and different families, as well as phage defense systems with related mode-of-actions to RcaT retrons. Finally, we will use experimental evolution to optimize blockers towards a specific retron, and assess how this impacts their spectrum against other retrons or defense systems. Overall, our work will shed light into the diversity of phage-encoded anti-defense strategies, using RcaT retrons as model systems, and will implement versatile methodologies that can be applied to any other defense system and its anti-defense repertoire.
DFG Programme
Priority Programmes
