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Sublethal effects of photoactive engineered nanomaterials on filamentous bacteriophage infection and E. coli gene expression in freshwater

Wu, Shushan1; Gray, Kimberly1

Published Dec 22, 2025 on Dryad. https://doi.org/10.5061/dryad.1zcrjdg3r

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Abstract

Wide application and release of engineered nanomaterials (ENMs) into the environment require an understanding of their potential ecological impacts, particularly under real environmental conditions. Previously, we reported that low doses of photoactive ENMs exert significant sublethal stress on bacterial outer membranes under light in a freshwater medium, potentially increasing bacterial susceptibility to environmental stress and viral infection, and promoting microbial evolution and diversity. However, little is known about how ENMs may affect bacteriophage infection under environmental conditions. In this study, we investigated how two common photoactive ENMs, n-TiO2 and n-Ag, and their mixtures, on the infection of a filamentous coliphage, bacteriophage f1, at environmentally relevant concentrations under freshwater conditions, particularly by affecting cell surface structures (outer membrane and F-pili) and properties (zeta potential), and by altering the gene expression of the host bacteria. The phage infection was evaluated by plate counting using the double-layer agar method. Using RT-qPCR (reverse transcription quantitative polymerase chain reaction), we probed the mechanisms of how ENMs affect filamentous phage infection by measuring the gene expression of F-pili and membrane proteins (TolA and OmpF) on E. coli, relevant to the phage infection process, after being treated with the ENMs or their mixtures under light and dark conditions. We also investigated how ENMs and their mixtures affect the cell surface and the phage-host contact by visual observation from S/TEM micrographs and zeta potential measurements. Overall, our results provide mechanistic insight into the complex interactions among bacteria, bacteriophage, and ENMs under environmentally relevant conditions and further detail their potential ecological risks.