Bacteriophages must rapidly deploy anti-CRISPR proteins (Acrs) to inactivate the RNA-guided nucleases that enforce CRISPR-Cas adaptive immunity in their bacterial hosts. Listeria monocytogenes temperate phages encode up to three anti-Cas9 proteins, with acrIIA1 always present. AcrIIA1 binds and inhibits Cas9 with its C-terminal domain; however, the function of its highly conserved N-terminal domain (NTD) is unknown. Here, we report that the AcrIIA1^NTD is a critical transcriptional repressor of the strong anti-CRISPR promoter. A rapid burst of anti-CRISPR transcription occurs during phage infection and the subsequent negative feedback by AcrIIA1^NTD is required for optimal phage replication, even in the absence of CRISPR-Cas immunity. In the presence of CRISPR-Cas immunity, full-length AcrIIA1 uses its two-domain architecture to act as a “Cas9 sensor,” tuning acr expression according to Cas9 levels. Finally, we identify AcrIIA1^NTD homologs in other Firmicutes and demonstrate that they have been co-opted by hosts as “anti-anti-CRISPRs,” repressing phage anti-CRISPR deployment.

噬菌体必须迅速部署抗CRISPR蛋白（Acrs），以灭活RNA引导的核酸酶，从而在细菌宿主中增强CRISPR-Cas适应性免疫力。单核细胞增生李斯特氏菌温带噬菌体最多编码三种抗Cas9蛋白，而acrIIA1始终存在。AcrIIA1以其C末端结构域结合并抑制Cas9；但是，其高度保守的N末端域（NTD）的功能尚不清楚。在这里，我们报道AcrIIA1 ^NTD是强抗CRISPR启动子的关键转录阻遏物。在噬菌体感染期间，抗CRISPR转录迅速爆发，随后AcrIIA1 ^NTD产生了负反馈最佳噬菌体复制是必需的，即使在没有CRISPR-Cas免疫的情况下也是如此。在存在CRISPR-Cas免疫的情况下，全长AcrIIA1使用其两个结构域结构充当“ Cas9传感器”，根据Cas9的水平调节acr的表达。最后，我们在其他Firmicutes中鉴定了AcrIIA1 ^NTD同源物，并证明它们已被宿主选作“ anti-anti-CRISPRs”，从而抑制了噬菌体的抗CRISPR部署。