Common Divalent Cations Elicit Sublethal Effects of Negatively Charged Nanoplastics on <i>Shewanella oneidensis</i> by Compromising Membrane Protein Functionality

The toxicity of negatively charged nanoplastics (NNP) to bacteria is generally subtler than that of positively charged counterparts, owing to limited NNP-cell interaction. This study hypothesized that common environmental cations (Na⁺, Mg²⁺, Ca²⁺) could enhance interaction between NNP and <i>Shewanella oneidensis</i>, thereby inducing biological effects. Settling experiments and dynamic light scattering analyses showed that NNP-cell interaction increased in the order of Ca²⁺ ≈ Mg²⁺ > Na⁺, which can be attributed to the decreased electrostatic repulsion, as confirmed by extended Derjaguin-Landau-Verwey-Overbeek theory calculations. Although coexposure to NNP and cations did not result in significant lethality, extracellular electron transfer (EET) to insoluble electron acceptors was significantly inhibited by coexposing to NNP and Ca²⁺ (NNP+Ca²⁺, by 37%) or Mg²⁺ (NNP+Mg²⁺, by 20%), but not by NNP alone or NNP and Na⁺ treatments. Two-dimensional correlation spectroscopy indicated that membrane proteins predominantly mediate bacterial interactions with NNP. Physical membrane damage and structural alterations of membrane proteins were observed following coexposure to NNP+Ca²⁺ and NNP+Mg²⁺, impairing the direct EET pathways. Transcriptomic and physiological analyses further revealed that NNP+Ca²⁺ upregulated persister marker genes (<i>spoT</i>, <i>ppx</i>, <i>relA</i>) and induced ATP depletion, triggering cellular dormancy and suppressing membrane protein-mediated processes. By contrast, NNP+Mg²⁺ exposure activated protective responses, including two-component systems and flagellar assembly, consistent with the milder impairment of EET. Notably, these effects were absent in treatments with either cations alone or NNP alone. These findings reveal an overlooked ecological impact of NNP and underscore the potential for distinct bacterial responses to NNP in varying aquatic environments.