A Phage Variable Region Encodes Anti-CRISPR Proteins Inhibiting All Streptococcus thermophilus 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 Streptococcus thermophilus ( Sth ) phages, a more comprehensive understanding of Acr diversity in Sth 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 Sth CRISPR-Cas systems. These acr 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 Sth Acrs and highlight the phage variable region as a key reservoir of immune-modulating factors.