# Experimental Evolution of Magnetic Nanoparticle Resistance in _Escherichia coli_
## 5 Conclusions  
This is the first study to utilize experimental evolution to investigate magnetite nanoparticle resistance in _E. coli_ or any other bacterium. _E. coli_ K-12 MG1655 evolved resistance to magnetite nanoparticles at 25 days of selection. Resistance to magnetite nanoparticles conferred resistance to both metals and antibiotics. Increased cell length and wide resulted many have played a significant role in adaptation to magnetite. Genomic analysis revealed hard selective sweeps in the _rpoA_ and _rpoC_ made important contributions to magnetite nanoparticle resistance. These results have important consequences for the future use of magnetite nanoparticles as antimicrobials as de novo evolution against these materials resulted quickly and occurred due to relatively simple genomic changes. This is an issue that will continue to be of significance regarding the use of nanomaterials as antimicrobials as we outlined in an earlier review [66].  
Conceptualization of this study was by [PERSON]; methodology, A.J.E. and K.S.; laboratory culture maintenance and phenotypic assays L.R., C.S., D.C.; genomic library preparation and sequencing A.J.E., L.R., C.S., D.C., S.B.; software analysis J.L.G.J.; validation of ICP-OES K.S., formal analysis J.L.G.J., investigation J.L.G.J. and A.J.E.; resources, J.L.G.J.; data curation J.L.G.J.; writing--original draft preparation A.J.E., J.L.G.J., writing--review and editing, J.L.G.J.; visualization, J.L.G.J.; supervision A.J.E.; project administration J.L.G.J.; funding acquisition, J.L.G.J. All authors have read and agreed to the published version of the manuscript.  
This research was funded by BEACON: An NSF Center for the Study of Evolution in Action (National Science Foundation Cooperative Agreement No. DBI-0939454), Characterizing the Evolutionary Behavior of Bacteria in the Presence of Iron Nanoparticles, NSF No. CBET-1602593. And National Institutes of Health, Maximizing Access to Research Careers (MARC USTAR: 1T34 GM113860-01A1).  
The SRA accession number for sequencing data is PRJNA694183 (FeNP-resistant, FeNP\({}_{1-5}\)\({}_{\text{D28}}\) and controls, C\({}_{1-5}\)\({}_{\text{D28}}\)). Finally, the phenotypic data from these studies will be submitted into DRYAD ([[https://datadryad.org/](https://datadryad.org/)]([https://datadryad.org/](https://datadryad.org/)) accessed on 17 March 2021) upon acceptance of this manuscript for publication.  
[PERSON] gave valuable aid in submitting the sequencing data to SRA. The authors would like to thank Analytical Services Laboratory (ASL) at NCAT for elemental analysis using ICP.  
The authors declare no conflict of interest.  
**Figure A1.** The actual amount of ionic iron (II) compared to the theoretical amount of iron (II) at 0 and 24 h in the bacterial medium.  
Figure A2: The amount of ionic iron (III) at 0 and 24 h resulting from iron (III) in DMB medium.  
\begin{table}
\begin{tabular}{c c c} \hline
**Gene** & **Position** & **Description** \\ \hline _catT\(\leftarrow\)_ & 41,809 & putative transporter \\ _yadL\(\leftarrow\)_ & 151,696 & putative fimbrial-like adhesin protein \\ _afuC\(\leftarrow\)_ & 277,859 & CP4-6 prophage; putative ferric transporter subunit \\ _yagG\(\rightarrow\)_ & 286,569 & CP4-6 prophage; putative sugar transporter \\ \hline \end{tabular}
\end{table}
Table 6: Position and description of minor polymorphisms, control populations, day 28.