Abstract

Introduction Enterococcus faecalis survives antibiotic exposure through coordinated regulatory programs that extend beyond single resistance determinants, yet the systems-level organization of transcriptional and post-transcriptional control under clinically relevant antibiotic stress remains incompletely defined.Methods Here, we analyzed RNA-seq profiles of E. faecalis V583 exposed to vancomycin or ceftriaxone and reconstructed antibiotic-activated, integrative transcriptional regulatory networks (TRNs) by projecting differentially expressed genes onto a curated TF-target template and explicitly incorporating antisense ncRNA-mRNA predicted links.Results Both antibiotics activated a substantial shared response dominated by envelope remodeling/proteostasis and bioenergetic-nucleotide economy, while vancomycin recruited a broader condition-specific program. Despite differing scale (Van-TRN: 533 nodes/587 edges; Cef-TRN: 375 nodes/431 edges), both networks displayed sparse, hub-centered hierarchies and strong nesting, with similar to 90% of ceftriaxone regulation contained within the vancomycin scaffold. From the shared ncRNA layer, we prioritized two inducible cis-antisense candidates, ncRNA3340 and ncRNA3683, exhibiting modular, multi-branch predicted folds, dual-site cis targeting, and energetics consistent with strong cis pairing and weaker but favorable trans interactions. Both ncRNAs were globally conserved across 687 clinical isolates, with low variability concentrated outside interaction-defined binding regions. Trans-target prediction points to ncRNA3340 as potentially associated with genome maintenance and cell-surface mRNAs, and ncRNA3683 with GA-like domain, glycine-alanine-rich transcripts, and a bacteriocin locus. Finally, qPCR corroborated the antibiotic-induced transcriptional changes observed for both ncRNAs.Conclusion Together, these results provide an integrated TRN framework that connects network rewiring to conserved ncRNA-centered candidate control during vancomycin and ceftriaxone stress responses, prioritizing testable ncRNA regulons for downstream functional validation.