Reappraisal of the DNA phosphorothioate modification machinery: uncovering neglected functional modalities and identification of new counter-invader defense systems

The DndABCDE systems catalysing the unusual phosphorothioate (PT) DNA backbone modification, and the DndFGH systems, which restrict invasive DNA, have enigmatic and paradoxical features. Using comparative genomics and sequence-structure analyses, we show that the DndABCDE module is commonly functionally decoupled from the DndFGH module. However, the modification gene-neighborhoods encode other nucleases, potentially acting as the actual restriction components or suicide effectors limiting propagation of the selfish elements. The modification module's core consists of a coevolving gene-pair encoding the DNA-scanning apparatus - a DndD/CxC-clade ABC ATPase and DndE with two ribbon-helix-helix (MetJ/Arc) DNA-binding domains. Diversification of DndE's DNA-binding interface suggests a multiplicity of target specificities. Additionally, many systems feature DNA cytosine methylase genes instead of PT modification, indicating the DndDE core can recruit other nucleobase modifications. We show that DndFGH is a distinct counter-invader system with several previously uncharacterized domains, including a nucleotide kinase. These likely trigger its restriction endonuclease domain in response to multiple stimuli, like nucleotides, while blocking protective modifications by invader methylases. Remarkably, different DndH variants contain a HerA/FtsK ATPase domain acquired from multiple sources, including cellular genome-segregation systems and mobile elements. Thus, we uncovered novel HerA/FtsK-dependent defense systems that might intercept invasive DNA during replication, conjugation, or packaging. Bacteria defend against selfish genetic elements by distinguishing their genetic material through special chemical modifications and using specific enzymes to break down viral DNA. This study explores the Dnd defense system, revealing several of its poorly understood facets. The Dnd modification system, utilizing sulfur to distinguish bacterial from viral DNA, cooperates with various anti-viral and cell-suicide nuclease enzymes to limit viral infection. While previously considered its restriction component, DndFGH emerges as an independent defense system, recognizing signals like nucleotides and DNA to thwart protective modifications of invader DNA. DndH, featuring diverse versions of the HerA/FtsK ATPase domain, helped unveil several unrecognized bacterial defense systems. This discovery illuminates sophisticated bacterial defenses against viral threats during crucial cellular processes. Graphical Abstract