Expanding the landscape of BREX diversity: uncovering multi-layered functional frameworks and identification of novel BREX-related defense systems.

Despite extensive scrutiny of BREX systems, several overarching questions persist regarding the functional modalities of individual components and the collective mechanistic framework underlying their defense responses. Using comparative genomics and sequence-structure analyses, we comprehensively map the phyletic distribution and domain-level functional annotations of BREX proteins across all subtypes. Our analysis uncovers numerous previously uncharacterized domains with key functional implications and demonstrates that BrxA- and BrxB-like homologs are universally present across all subtypes, thereby redefining the core machinery of BREX. Our survey strikingly expands the BREX landscape by characterizing three newly defined RM-like systems, which we term BREX-Related (BR) systems, and establishes them as new subtypes that share multiple core components with BREX. Furthermore, we identified a novel composite anti-invader system that intriguingly integrates the BREX machinery with components derived from HerA/FtsK-based capture and Dpd defense systems, forming a unique multifaceted defense machinery. Notably, we identify an extensive repertoire of auxiliary and backup effectors recruited alongside the primary effectors across all BREX and BR systems, functioning to reinforce initial restriction responses and counter phage anti-defense mechanisms. Based on these findings, we propose a unified model organized around a multi-modal "sensing-modifying-restricting" functional architecture, defining the fundamental basis of the multi-layered BREX defense systems.