Understanding the role of Simple Sequence Repeats in type III Restriction Modification System

Abstract

Microbial evolution has equipped bacteria with various mechanisms to differentiate between self and foreign DNA, one is the Restriction-Modification (R-M) system. Type III RM systems provide innate immunity while functioning as phase-variable regulons, or “Phasevarions,” in host-adapted bacteria, forming heterotrimeric Mod2Res1 complexes. This phase variability is mediated by simple sequence repeats (SSRs) within the mod gene, which undergoes indel mutations due to replication slippage, leading to stochastic switching of global gene expression. This study investigates the role of hypermutable SSRs in a putative type III restriction modification system of Mycoplasma bovis, a minimal genome bacterium and a globally relevant bovine pathogen. Bioinformatic analyses were used to identify candidate SSR motifs for testing mutability in genes where indel instability was assessed as a proxy for SSR polymorphism-driven phase variation. Previous homology-based studies were used to test the plasticity of two different loops by looking into their solubility and protein activity. This hypothesis was further validated in EcoP15I, which has no SSRs in the structure. These findings provide insights into how SSR-driven phase variation influences the function and evolution of the type III restriction modification system.