A Phage Variable Region Encodes Anti-CRISPR Proteins Inhibiting All <i>Streptococcus thermophilus</i> CRISPR Immune Systems

Bacteria and archaea utilize CRISPR-Cas systems to defend against invading mobile genetic elements (MGEs) such as phages and plasmids. In turn, MGEs have evolved anti-CRISPR (Acr) proteins to counteract these defenses. While several type II-A Acrs have been identified in <i>Streptococcus thermophilus</i> (<i>Sth</i>) phages, a more comprehensive understanding of Acr diversity in <i>Sth</i> phages has yet to be explored. Guided by the genomic context of known Acrs, we systematically screened uncharacterized phage proteins and identified several novel Acrs that inhibit type I-E, type II-A or type III-A <i>Sth</i> CRISPR-Cas systems. These <i>acr</i> genes are clustered within a variable phage genomic region, indicating a hotspot for anti-defense activity. We also identified neighboring proteins with predicted enzymatic or structural domains that may modulate phage-host interactions through Acr-independent mechanisms. Together, our findings expand the known repertoire of <i>Sth</i> Acrs and highlight the phage variable region as a key reservoir of immune-modulating factors.