Long-term metagenomic insights into the roles of antiviral defense systems in stabilizing activated sludge bacterial communities

Bacteria have evolved various antiviral defense systems to protect themselves, but how defense systems respond to the variation of bacteriophages in complex bacterial communities and whether defense systems function effectively in maintaining the stability of bacterial community structure and function remain unknown. Here, we conducted a long-term metagenomic investigation on the composition of bacterial and phage communities of monthly collected activated sludge samples from two full-scale wastewater treatment plants over six years and found that defense systems were widespread in activated sludge, with 91.1% of metagenome-assembled genomes having more than one complete defense system. The stability of the bacterial community was maintained under the fluctuations of the phage community, and defense system abundance and phage abundance were strongly positively correlated; there was a 0–3-month time lag in the responses of defense systems to phage fluctuations. The rapid turnover of CRISPR spacer repertoires further highlighted the dynamic nature of bacterial defense mechanisms. A pan-immunity phenomenon was also observed, with nearly identical metagenome-assembled genomes showing significant differences in defense system composition, which contributed to community stability at the species level. This study provides novel insights into the complexity of phage–bacteria interactions in complex bacterial communities, and reveals the key roles of defense systems in stabilizing bacterial community structure and function.