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

Abstract

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.

Aeromonas trota Is Highly Refractory to Acquire Exogenous Genetic Material

“…Phosphorotioate modification Dnd system is similar to RM systems, where the DndABCDE protein complex acts as a modification module in a sequence-specific manner, which substitutes the non-bridging oxygen with sulfur in the DNA phosphate backbone, and DndFGH, which acts as a cognate restriction module [ 76 ]. A recent report suggests that DndFGH could be a defense system independent of DndABCDE activity [ 105 ]. Both DndABCDE and DndFGH were found in two refractory A. trota strains (9.3 and 9.1).…”