Modulation of Hachiman defence by a type II toxin-antitoxin system via balancing trade-off between the fitness cost and antiphage activity

Hachiman systems provide innate antiphage immunity across prokaryotic domains. The system encodes a HamA nuclease and a HamB helicase both of which exhibit great diversity in sequence. Nevertheless, phylogenetic analyses of HamA and HamB proteins revealed similar phylogenetic trees for both proteins, falling into three major types. Close examination of one of the subclades identified a distinct subfamily in which most of these Hachiman systems stands alone, however, Hachiman in the Streptococcus genus is combined with PezAT, a distinct pneumococcal epsilon zeta toxin-antitoxin system, yielding the Pez-Ham system. Investigation of a S. thermophilus Pez-Ham system revealed that only the Hachiman system is required for mediating antiphage defence. Biochemical characterization of encoded proteins, i.e., HamA or HamB individually or in protein complex revealed that the HamA nuclease is inactive alone, but upon the formation of heterologous dimer with HamB, the resulting protein complex effectively cleaves DNAs of various forms with a broad specificity (5’-CNNNG-3’), and the nuclease activity is greatly facilitated by ATP-binding in HamB and to a less degree by ATP hydrolysis. Genetic investigations further showed, while the Pez system did not function in antiphage immunity in E. coli , the system repressed the expression of Hachiman, and thereby balancing the trade-off between the fitness cost and the effectiveness of antiphage defence.