Comparative and functional genomic analysis of foreign DNA defense mechanisms in <i>Enterococcus faecium</i>

<i>Enterococcus faecium</i> is notoriously difficult to study genetically due to the poor understanding of barriers preventing foreign DNA uptake, such as the proteins that modify type I restriction modification (RM) system activity. Here, we compared <i>E. faecium</i> repertoires of the HsdS specificity subunit (dictating the DNA motif that is adenine methylated) from type I RM systems among 805 globally reported <i>E. faecium</i> isolates. We showed there were eight distinct HsdS types, with four dominant variants that were also significantly enriched in the hospital-associated clade A1 <i>E. faecium</i> lineage. Adenine methylome analysis of a subset of eight representative <i>E. faecium</i> strains revealed that only two exhibited functional type I RM systems, with the activity corroborated by the construction of type I RM deletion mutants. To investigate this surprising finding, we assessed the contribution of the anti-restriction protein ArdA that specifically inhibits type I RM function. The <i>E. faecium</i> ST796 clinical isolate AUS0233 has one intact type I RM system, no adenine methylation, and two distinct <i>ardA</i> paralogs. When heterologously expressed in <i>Staphylococcus aureus</i> JE2, both <i>E. faecium ardA</i> variants were functional, each inhibiting the function of the two type I RM systems in <i>S. aureus</i>. However, the deletion of one or both versions of <i>ardA</i> in <i>E. faecium</i> AUS0233 did not change the transformation efficiency with exogenous DNA, suggesting ArdA in <i>E. faecium</i> AUS0233 is not controlling type I RM. This study highlights the complexity of DNA defense mechanisms in <i>E. faecium</i> and suggests that unidentified factors control the acquisition of foreign DNA.IMPORTANCE<i>Enterococcus faecium</i> has mechanisms of DNA methylation and targeted DNA degradation (called restriction modification [RM]) that hinder foreign DNA uptake, thus influencing the acquisition of important phenotypes such as antibiotic resistance. Restriction barriers also frustrate efforts for laboratory genetic manipulation used to study this pathogen. From PacBio analysis of <i>E. faecium</i> strains, it was observed that the majority of <i>E. faecium</i> do not adenine methylate DNA despite genome analysis indicating they have intact type I RM methylation systems. One explanation for this observation is that <i>E. faecium</i> produces anti-restriction factors such as ArdA, which can inhibit type I RM systems. However, the deletion of both <i>ardA</i> alleles did not improve the efficiency of DNA uptake. These findings build our foundational knowledge of how <i>E. faecium</i> controls foreign DNA and show there is additional complexity surrounding these systems to be discovered.