A phage-encoded counter-defense inhibits an NAD-degrading anti-phage defense system

Abstract

Bacteria contain a diverse array of genes that provide defense against predation by phages. Anti-phage defense genes are frequently located on mobile genetic elements and spread through horizontal gene transfer. Despite the many anti-phage defense systems that have been identified, less is known about how phages overcome the defenses employed by bacteria. The integrative and conjugative element ICEBs1 in Bacillus subtilis contains a gene, spbK, that confers defense against the temperate phage SP{beta} through an abortive infection mechanism. Using genetic and biochemical analyses, we found that SpbK is an NADase that is activated by binding to the SP{beta} phage portal protein YonE. The presence of YonE stimulates NADase activity of the TIR domain of SpbK and causes cell death. We also found that the SP{beta}-like phage {Phi}3T has a counter-defense gene that prevents SpbK-mediated abortive infection and enables the phage to produce viable progeny, even in cells expressing spbK. We made SP{beta}-{Phi}3T hybrid phages that were resistant to SpbK-mediated defense and identified a single gene in {Phi}3T (phi3T_120, now called nip for NADase inhibitor from phage) that was both necessary and sufficient to block SpbK-mediated anti-phage defense. We found that Nip binds to the TIR (NADase) domain of SpbK and inhibits NADase activity. Our results provide insight into how phages overcome bacterial immunity by inhibiting enzymatic activity of an anti-phage defense protein. Author SummaryBacterial viruses (bacteriophages or phages) are widespread and abundant across the planet. Bacteria have a variety of immune systems, often found on mobile genetic elements, to combat phage predation. Phages can overcome these immune systems by mutating to avoid recognition or by producing molecules that prevent the immune system from working. We determined how an anti-phage defense system encoded by an integrative and conjugative element recognizes phage infection to cause cell death prior to the generation of phage progeny. We also identified a phage gene that prevents this defense system from functioning. The phage-encoded counter-defense protein inhibits the enzymatic activity of the anti-phage defense protein, enabling evasion of immunity and production of infectious phage. There are likely many different phage-encoded counter-defense genes yet to be discovered.