Physiological and proteomic insights into silver nanoparticle-induced stress responses and resistance mechanisms in aerobic denitrifying Enterobacter cloacae HNR

This study combines physiological and proteomic analyses to explore the silver nanoparticles (Ag NPs) induced stress response and resistance mechanisms in the aerobic denitrifying Enterobacter cloacae strain HNR (strain HNR). Results demonstrated concentration-dependent inhibition: at 0.30 mM Ag NPs, bacterial growth (OD 600 ) decreased to 79.7 % of the control, while nitrate removal efficiency dropped to 52.4 %, accompanied by a 6.4-fold increase in nitrite accumulation. Activities of nitrate reductase and nitrite reductase were reduced to 34.3 % and 41.6 %, respectively. Ag NPs also compromised motility, induced oxidative stress (33.11 % rise in intracellular reactive oxygen species), and disrupted membrane integrity (10.4 % lactate dehydrogenase release). Conversely, extracellular polymeric substances (EPS) production surged by 25.6 % under low Ag NPs (0.01 mM), primarily driven by polysaccharide synthesis (55.5 % increase). Proteomics revealed 1987 differentially expressed proteins (978 upregulated, 1009 downregulated). Downregulated pathways (DNA repair, ribosome biosynthesis, glycerophospholipid metabolism, ABC transporters, oxidative phosphorylation, etc.) indicated that Ag NPs disrupted bacterial genome stability, inhibit synthesis of protein, cell membrane, and interfered with material transport and energy production. In response, strain HNR upregulated the sulfur relay system, tyrosine metabolism, and pentose and glucuronate interconversions to combat oxidative stress and energy metabolism inhibition. Additionally, strain HNR increased EPS production and cell surface positive electroactivity by upregulating the starch and sucrose metabolism and the cationic antimicrobial peptide resistance pathways to counteract the adsorption and contact of Ag NPs and silver ion. These findings will guide improvements in aerobic denitrification processes in NP-polluted aquatic environments and facilitate the broader application of aerobic denitrification technologies.