Mixed heavy metal stress induces global iron starvation response

Multiple heavy metal contamination is an increasingly common global problem. Heavy metals have the potential to disrupt microbially mediated biogeochemical cycling. However, systems-level studies on the effects of combinations of heavy metals on bacteria are lacking. For this study, we focused on the Oak Ridge Reservation (ORR; Oak Ridge, TN, USA) subsurface which is contaminated with several heavy metals and high concentrations of nitrate. Using a native Bacillus cereus isolate that represents a dominant species at this site, we assessed the combined impact of eight metal contaminants, all at site-relevant concentrations, on cell processes through an integrated multi-omics approach that included discovery proteomics, targeted metabolomics, and targeted gene-expression profiling. The combination of eight metals impacted cell physiology in a manner that could not have been predicted from summing phenotypic responses to the individual metals. Exposure to the metal mixture elicited a global iron starvation response not observed during individual metal exposures. This disruption of iron homeostasis resulted in decreased activity of the iron-cofactor-containing nitrate and nitrite reductases, both of which are important in biological nitrate removal at the site. We propose that the combinatorial effects of simultaneous exposure to multiple heavy metals is an underappreciated yet significant form of cell stress in the environment with the potential to disrupt global nutrient cycles and to impede bioremediation efforts at mixed waste sites. Our work underscores the need to shift from single- to multi-metal studies for assessing and predicting the impacts of complex contaminants on microbial systems.