Involvement of extracellular DNA (eDNA) in biofilm architecture and extracellular polymeric matrix stabilization in...

Bacterial biofilms are structured communities encased in extracellular polymeric substances (EPS), with extracellular DNA (eDNA) as a key structural and regulatory component. This study investigates the role of eDNA in biofilm architecture and EPS stabilization in Pseudomonas putida KT2440. Spectrophotometric and gel-based analyses confirmed eDNA release through controlled cell lysis, and supplementation with exogenous eDNA (1 μg/ml) accelerated biofilm maturation. Biofilm biomass increased from 8.89 ± 1.26 μm³/μm² at 36 h in the control to 12.89 ± 9.42 μm³/μm² at 24 h under eDNA supplementation. Cell size analysis confirmed eDNA supplementation produced dense biofilms with smaller cells (0.98-1.95 μm), whereas DNase treatment resulted in elongated (1.49-2.43 μm) chain-like cells. Gene expression revealed downregulation of ftsZ (∼0.15 fold at 24 h; ∼0.31 fold at 36 h) and mreB (∼0.22 fold at 24 h; ∼0.39 fold at 36 h), indicating reduced cell division and elongation. Increased cellular density led to highest EPS yield (505.3 ± 12.9 mg/l) under eDNA supplementation. Protein levels (146.2 ± 8.7 mg/g) were highest under DNase treatment, suggesting compensatory matrix remodeling. Amide I analysis revealed decreased β-sheet (52.8% to 38.4%) and increased β-turn (18.1% to 22.6%) upon eDNA supplementation, partially corroborated with CD spectroscopy. ¹H NMR analysis indicated interactions of eDNA with polysaccharides, with weaker association with lipids. These findings demonstrated crucial involvement of eDNA in regulating biofilm formation, matrix composition, cell size, and cytoskeletal gene expression in P. putida KT2440, providing insight into eDNA-mediated biofilm regulation with implications for targeted modulation of biofilms in medical and biotechnological applications.