Experimental evolution of Pseudomonas putida under silver ion versus nanoparticle stress

Whether the antibacterial properties of silver nanoparticles (AgNPs) are simply due to the release of silver ions (Ag⁺) or, additionally, nanoparticle-specific effects, is not clear. We used experimental evolution of the model environmental bacterium Pseudomonas putida to ask whether bacteria respond differently to Ag⁺ or AgNP treatment. We pre-evolved five cultures of strain KT2440 for 70 days without Ag to reduce confounding adaptations before dividing the fittest pre-evolved culture into five cultures each, evolving in the presence of low concentrations of Ag⁺, well-defined AgNPs or Ag-free controls for a further 75 days. The mutations in the Ag⁺ or AgNP evolved populations displayed different patterns that were statistically significant. The non-synonymous mutations in AgNP-treated populations were mostly associated with cell surface proteins, including cytoskeletal membrane protein (FtsZ), membrane sensor and regulator (EnvZ and GacS) and periplasmic protein (PP_2758). In contrast, Ag⁺ treatment was selected for mutations linked to cytoplasmic proteins, including metal ion transporter (TauB) and those with metal-binding domains (ThiL and PP_2397). These results suggest the existence of AgNP-specific effects, either caused by sustained delivery of Ag⁺ from AgNP dissolution, more proximate delivery from cell-surface bound AgNPs, or by direct AgNP action on the cell's outer membrane.