Critical Anti-CRISPR Locus Repression by a Bi-functional Cas9 Inhibitor

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-antiCRISPRs,'' repressing phage anti-CRISPR deployment.