A new family of small ArdA proteins reveals an antirestriction activity

Antirestriction proteins are known to protect mobile genetic elements from the host’s restriction-modification (RM) systems. In our study, we identified a new family of small proteins, which we named sArdA The sArdA proteins are homologous to DNA-mimicking ArdA proteins but differ in size, being approximately one-third the length of full ArdAs. Moreover, sArdA family contains two subgroups, one of which is structurally similar to the N-terminal end of ArdA, while the other one – to the C-terminal end. Phylogenetic analysis demonstrated that genes encoding these proteins evolved into evolutionarily stable subfamilies, named sArdN and sArdC, respectively. Alphafold structure prediction of sArdA interaction with RM systems revealed four states of EcoKI, which differ the angle between its two M-subunits while interacting with different agents. Interestingly, both sArdN and sArdC triggered the same intermediate closed state of EcoKI indicating the possible new interaction pathways of Ards with RM systems.

For phenotypic studies in Escherichia coli cells, we cloned the sardN gene from the chromosome of Corynebacterium pilbarense and the sardC gene from Lactococcus cremoris . Both genes were shown to protect λ phage DNA from restriction by the type I RM system. However, they revealed specificity to different restriction-modification systems. Specifically, sArdC was more effective against EcoR124II, whereas sArdN was more potent against EcoKI. Furthermore, both genes demonstrated high antimethylation activity against EcoKI. Our current findings suggest the idea that binding specificity of DNA-mimicking proteins to their targets could also be achieved by very short proteins.