# Experimental Evolution of Magnetic Nanoparticle Resistance in _Escherichia coli_
The fundamental requirement for iron plays a crucial role in microbial pathogenesis, thus many organisms utilize iron sequestration as a defense against infection [9; 10]. Yet, despite iron being an essential metal, it can also be extremely toxic under aerobic conditions [11]. There are multiple mechanisms of excess iron toxicity (see Table 1). For this reason, iron homeostasis is tightly regulated. Such tight regulation associated with the bacterial requirement to acquire iron from its environment can potentially be used as an evolutionary trap. Evolutionary traps are defined as a situation in which an organism prefers resources that reduce its fitness [12]. Thus, in an environment where iron is present in excess, we can ask the question whether bacteria have the capacity to control either the uptake, storage, or efflux of iron at rates sufficient to avoid toxicity? Alternatively, it is important to ask: can and how do bacteria evolve resistance to excess iron? In addition, if they can, what correlated traits will result from the adaptation? We have already answered some of these questions in a series of experiments utilizing ionic iron II, iron III, and the iron analog gallium III [13; 14; 15]. These studies utilized experimental evolution and examined mechanisms of iron and gallium resistance through the evaluation of phenotypic and genomic changes in _E. coli_ K-12 MG1655. We found that this strain of _E. coli_ was capable of evolving resistance to excess iron and gallium primarily through changes in the uptake of iron (or gallium). In addition, we found that iron (II)- and iron (III)--resistant populations showed unanticipated correlated resistances to a range of antibiotics including ampicillin, chloramphenicol, rifampicin, sulfanilamide, and tetracycline. Our results suggested that _E. coli_ K-12 could respond to excess iron by both physiological acclimation and evolutionary adaptation [13]. This was manifested by both patterns of genomic and gene expression changes exhibited in the iron (II)-, iron (III)-, and Gallium (III)-resistant populations compared to their ancestors and controls (grown in the absence of excess iron or gallium). In this study, we examine whether experimental evolution against spherical magnetite nanoparticles (FeNPs) will result in similar outcomes as those observed resulting from selection for ionic iron (II, III) resistance. Nanoparticles (NPs) are particles between 1 and 100 nanometers in size [16]. NPs are broadly classified into various categories depending on their morphology, size, and chemical properties [17; 18]. Metallic ferrous nanoparticles are one of the most studied nanomaterials against multidrug-resistant bacteria [19]. The three most common naturally occurring forms of metallic ferrous NPs are hematite (\(\alpha\)-Fe\({}_{2}\)O\({}_{3}\)), maghemite (\(\gamma\)-Fe\({}_{2}\)O\({}_{3}\)), and magnetite (Fe\({}_{3}\)O\({}_{4}\) or FeO\(\cdot\)Fe\({}_{2}\)O\({}_{3}\)). Magnetite is a black iron oxide, commonly called Hercules stone, and possesses the strongest magnetic behavior [20]. Magnetite is considered as a charge frustrated iron oxide due to the distribution of both iron (II) and (III) in crystallographic sublattice sites [21]. \begin{table}
\begin{tabular}{c c} \hline \hline
**Mechanism** & **Fe** \\ \hline Reactive oxygen species & + \\ Disruption of transcription/translation & + \\ Damage to cell wall/membrane & + \\ Interfering with respiration & + \\ Release of cellular components & + \\ Binding to thiol groups & + \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Mechanisms of excess iron and silver toxicity: Mechanisms of cellular damage are listed resulting from excess iron toxicity. As these systems are common to virtually all bacteria, there is a strong potential that resistance mechanisms might be conserved across wide varieties of taxa. Magnetite NPs exist in nature, and engineered FeNPs are widely used in a range of applications due to their advanced optical properties [18]. FeNPs can be used to determine oxygen concentrations [22; 23; 24].