A new family of small ArdA proteins reveals antirestriction activity.

Antirestriction proteins 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, the sArdA family contains two subgroups, one of which is structurally similar to the N-terminal end of ArdA, whereas the other one matches the C-terminal end. Both the N-terminal and C-terminal domains of ArdA appear capable of independent expression. 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 in the angle between its two M-subunits while interacting with different ArdAs or DNA. Interestingly, both sArdN and sArdC triggered the same intermediate closed state of EcoKI, indicating 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 protected λ phage DNA from restriction by the type I RM system. However, they revealed specificities to different restriction-modification systems. Specifically, sArdC was more effective against EcoR124II, whereas sArdN was more potent against EcoKI. Furthermore, both genes demonstrated antimethylation activity against EcoKI. Our current findings suggest the idea that the binding specificity of DNA-mimicking proteins to their targets could also be achieved by very short proteins.IMPORTANCEOur current findings suggest that the binding specificity of DNA-mimicking proteins to their targets could also be achieved by very short proteins. The ability of these DNA-mimicking proteins to specifically inhibit different DNA-binding proteins makes them a promising tool for regulating a range of intracellular processes, including gene expression.