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ABSTRACT Microbial species are integral in environmental homeostasis and have thus developed diverse strategies to adapt, survive, and proliferate under exogenous stress. The growing demand for nanoscale battery materials has led to increasing environmental concentrations of nanomaterials and their constituent metal ions. Previous work has shown that the Gram-negative bacteriumShewanella oneidensisis able to rapidly evolve resistance to one such nanomaterial, lithiated nickel manganese cobalt oxide (NMC). Yet, the specific stimuli that trigger resistance evolution and the ensuing genomic changes were previously unknown. Here, we demonstrate that the combination of cobalt and nickel ions released from NMC trigger a gene amplification event that enables stable nanomaterial and metal resistance, likely caused by amplification of a cation efflux pump. Amplification copy number is highly dynamic over the period of study, yet amplification events persist over prolonged recovery in the absence of metal stress. Growth rate comparisons reveal no physiological cost associated with a high copy number. The stability of this genomic mutation combined with the lack of observable fitness cost distinguishes this genomic perturbation from previously reported gene amplification events. Finally, the observed amplification event was unique to the combination of cobalt and nickel, which implies that the intracellular targets of these metal ions have a specific interaction that yield resistance. Ultimately, we report a dynamic and specific gene amplification event that allowsS. oneidensisto survive metal stress. This work not only illuminates the broader ecological consequences associated with introducing nanomaterials and metals into the environment but also provides insight into the larger scope of bacterial resistance mechanisms. IMPORTANCEStudying mechanisms of microbial resistance evolution is critical for understanding the role of bacteria in the environment and developing new strategies to combat antimicrobial resistance to modern therapeutics. Technological innovation has often led to the introduction of novel environmental stressors that impart pressure on microbes to evolve resistance. This may ultimately lead to novel resistance genes or gene cassettes in the environmental gene pool, which could further promote antimicrobial resistance in pathogenic organisms. In this study, we highlight the importance of considering gene amplification as a mechanism of bacterial resistance to environmental toxins. We demonstrate that amplification events can exist within a population in the absence of selection pressure and without a clear fitness cost. Identifying specific stimuli that trigger these events will help us understand factors that accelerate bacterial resistance evolution and have the potential to disrupt ecosystem balance. 

Use of complex metal oxide nanoparticles has drastically risen in recent years, especially due to their utility in electric vehicle batteries. However, use of these materials has outpaced our understanding of how they might affect environmental organisms, which they could encounter through release during manufacture, use, and disposal. In particular, little is known about the effects of chronic exposure to complex metal oxide nanoparticles. Here, we have focused on an environmentally-relevant bacterial species, Shewanella oneidensis, which is ubiquitous in nature and responsible for bioremediation of heavy metals, and assessed the toxic effects of nanoscale lithiated nickel manganese cobalt oxide (NMC), which is an emerging battery cathode material for electronic devices. We previously reported that chronic exposure of S. oneidensis to NMC results in the emergence of an adaptive phenotype where the bacteria are able to tolerate otherwise lethal concentrations of NMC. In the present study, we aim to investigate the role of reactive oxygen species (ROS) and changes in phenotype of the NMC-adapted bacterial population. We found that NMC-exposed bacteria possess ROS-containing membrane vesicles, as well as an increased propensity to generate random DNA mutations and harbor other DNA damage. Thus, our data indicate substantial genetic-level variation in bacteria that results from chronic exposure to toxic complex metal oxide nanomaterials.