The prokaryotic Gabija complex senses both viral transcription and DNA metabolism for antiviral defense

Abstract The perpetual arms race between bacteria and phages has led to the development of ingenious bacterial defense systems, including an immense array of unknown systems in addition to the well-known restriction-modification and CRISPR/Cas systems, one of the most dominant of which is the Gabija system, which exists in 8.5% of sequenced bacteria and archaea. The Gabija system consists of the GajA and GajB proteins. We revealed that GajA is a DNA nicking endonuclease that senses NTP depletion by phage transcription. The DNA binding of GajA is strictly inhibited by NTP and the antiviral defense of GajA requires the assistance from GajB, which senses DNA termini produced from phage DNA replication and recombination to hydrolyze (d)A/(d)GTP. The GajB gene encodes two colinear forms of proteins. The full-length GajBʹ protein exhibits stronger ATP hydrolysis activity and is sufficient to support bacteriophage resistance. GajA forms tetramers in vitro and tightly binds to GajB and/or GajBʹ to form stable complexes in vivo and in vitro. However, the 1:1 GajA/B or GajA/Bʹ complex has no antiviral activity. To constitute a functional Gabija complex, fine regulation at the protein expression level is required to maintain the molecular ratio between GajB/GajBʹ and GajA below 1:1. Through (i) multiple sensing of phage pivot activities including transcription and DNA replication and recombination and (ii) fine stoichiometry regulation of the DNA processing complex, the Gabija system exhibits a delicate and concise mechanism distinct from any known prokaryotic antiviral system.