# Experimental Evolution of Magnetic Nanoparticle Resistance in _Escherichia coli_
## 4 Discussion
coli_ is a rod-shaped bacterium, a rough calculation of the increase in the cell area is given by multiplying its length and width. On average FeNP area = 1.25 \(\times\) 10\({}^{6}\) nm\({}^{2}\), control = 6.17 \(\times\) 10\({}^{5}\) nm\({}^{2}\), and ancestor = 5.76 \(\times\) 10\({}^{5}\) nm\({}^{2}\). This represents a 2.18-fold increase in cell area in the FeNP populations compared to their ancestor. The magnitude of the cell size response suggests that this may be playing an important role in FeNP adaptation to metal and antibiotic toxicity. One of the primary mechanisms by which magnetite is known to impact bacteria is via ROS [45]. There is evidence that larger cell size is correlated to ROS resistance. A study of _Lactobacillus_ species found that greater cell size was positively associated with resistance to ROS [46]. Another study of _Mycobacterium_ isolates found that greater size variation was associated with antibiotic resistance [47]. In _E. coli_ MG1655, stress is known to cause an increased cell length; this has also been observed in _M. tuberculosis_ and _M. snegmatis_[48; 49]. Finally, it has been shown that increase in cell length is an adaptation associated with antibiotic resistance due to the synthesis of a modified nucleotide in response to stress (p) ppGpp [50; 51; 52; 53]. Genetics analysis identified selective sweeps in RNA polymerase subunit \(\beta^{\prime}\) (_rpoC_) in some replicates. In addition, others displayed significant polymorphisms (f > 0.500) in RNA polymerase subunit \(\alpha\) (_rpoA_). As RNA polymerase is involved in putting together all the RNA in the cell, mutations in this protein are known to be widely pleiotropic and play major roles in relieving stress and increasing metabolic efficiency in _E. coli_[54; 55]. This may explain why the FeNP populations displayed superior growth compared to the control and ancestral populations in all the stresses applied to them in this study (magnetic, ionic metals, and antibiotics). It can be argued that selective sweeps displayed in specific genes associated with a given environment at best illustrate an indirect demonstration of the causal role of those genes in producing resistant phenotypes. This is a general limitation of experimental evolution as a means of studying microbial adaptation [41; 14]. However, in prior studies we have deployed more direct methods to test whether the genomic variants we discovered should and actually do confer resistance. For example, we have utilized computational modeling of 3-dimensional structures of altered proteins to test their affinity for ionic metals [12; 15; 42]. These studies showed the alteration in the protein caused by the variant resulting from the selective sweep did reduce or increase the affinity to the target metal compared to the controls/ancestor. We also have deployed recombination to move genetic variants discovered by experimental evolution into the genomic background of the ancestor to demonstrate that the variant did produce the resistance observed in the selected populations [42]. We did not perform these experiments in this study, simply due to the abundance of evidence that experimental evolution does reliably uncover adaptive variants. Despite the fact that the FeNP populations displayed an increase in growth in magnetite, iron (II), iron (III), and gallium (III, an iron III analog), we did not find selective sweeps of variants associated with iron resistance in our prior studies [14; 15; 16]. These included mutations in genes such _as fccA_, _fur_, _dnaK_, _murC_, _ptsP_, and _ilvG_. Magnetic contains both iron (II) and iron (III), and these are released in solution [1]. In this study, the \(\gamma\)-magnetic particles were stabilized by a coating of tetramethylammonium hydroxide (CH\({}_{3}\))\({}_{4}\)NOH. In our study, the magnetite nanoparticles were stabilized with PVP (polyvinylpyrrolidone). Coating agents used to improve the stability of magnetite nanoparticles decrease the release of ionic Fe\({}^{2+}\) and Fe\({}^{3+}\) in the medium [56; 57].