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Accumulation

Short Summary

Increasing the accumulation of copper ions in living cells requires enhanced copper uptake as well as inhibition of copper export systems. We knocked out cusCFBA, the dominant copper export system from Escherichia coli which facilitates Cu(I) export from the cytoplasm out of the cell. In addition, we supplemented the native copper accumulation system of E. coli by expression of the outer membrane importers copC and oprC as well as the inner membrane transporters copD and hmtA.

Introduction

One important objective of nanoFactory is to construct an efficient accumulation system for heavy metal ions in order to subsequently form nanoparticles in E. coli. As a proof of principle we concentrated on the accumulation of Cu(II) ions. Besides the knockout of copper exporters, the main component for such a system is a highly efficient uptake system.

Since copper is toxic to all types of cells (Ladomersky & Petris, 2015), the existence of transporters for the active and selective uptake of copper ions may initially come as a surprise, but copper also carries out important functions in many cells. As a cofactor for many different enzymes such as superoxide dismutase it is primarily involved in electron transfer, dioxygen transport and activation (Solomon et al., 2014). Accordingly, there must be natural absorption mechanisms for the trace element both into the periplasm and the cytoplasm. Nevertheless, as non-bonded, dissolved copper in particular is very toxic, there are efficient export systems in E. coli to keep a tightly controlled copper homeostasis and remove excess copper from the cell (Rensing & Grass, 2003). In order to create an efficient accumulation system for copper ions, highly selective uptake systems must be expressed on the one hand and existing export systems must be suppressed on the other.

**Figure 2:** Overview on the different copper accumulation components we worked with. The active copper export system cusCBA is knocked out and prevents Cu(I) loss. Uptake is promoted from the outer membrane importer OprC supported by the inner membrane importers CopD and HmtA. CopC is a metallochaperone delivering Cu(II) to CopD. Toxic Cu(I) ions in the periplasm are oxidated to Cu(II) in the periplasm by the native copper oxidase Cue

Preventing Copper Export

In order to prevent the loss of the effortfully imported Cu(II) ions by transport or diffusion, the cus operon of E. coli was knocked out using CRISPR/Cas9. It consists of cusCFBA which belongs to the resistance-nodulation-cell division superfamily (Tseng et al., 1999) and the two-component regulatory system cusRS. The response regulator cusR and its associated membrane-bound kinase cusS regulate the expression of the opposed directed cusCFBA genes directly upstream of cusRS (Munson et al., 2000, Xiao et al., 2017). The export of Cu(I) and also Ag(I) is carried out by the tripartite protein complex CusCBA (Gudipaty et al., 2012) which is spanning through both the inner and outer membrane of the cell (Delmar, Su & Yu, 2013). CusA is a pump for Cu(I)//Ag(I) ions located in the inner cell membrane. It pumps Cu(I)/Ag(I) ions, driven by proton-motive force, through the adapter-like CusB and CusC which span the outer membrane (Franke et al., 2003, Delmar, Su & Yu, 2013). Cu(I)/Ag(I) ions from the periplasmic space are delivered to CusB by CusF metallochaperones (Bagai et al., 2008).

We constructed a knock out mutant of E. coli DH5α using CRISPR/Cas9 to remove the whole cus operon ultimately prohibiting Cu(I) export. Consequently Cu(I) ions can not leave the periplasmic space. Subsequently the oxidization of Cu(I) to Cu(II) occurs spontaneously in the oxidative environment of aerobically grown cultures and is catalyzed by multicopper oxidase CueO which is native to E. coli (Outten et al., 2001).

**Figure 1:** Structure of the *E. coli cus* operon. The two-component regulatory system *cusRS* is located directly downstream of the *cusCFBA* coding sequences.

Copper Import

The absorption of copper takes place in two steps: In the gram-negative chassis organism E. coli, Cu(II) ions are first transported across the outer cell membrane into the periplasm and subsequently into the cytoplasm over the inner cell membrane if necessary. Due to its toxicity an accumulation of copper ions in the periplasm has many advantages over accumulating it in the cytoplasm, where damage to the DNA and several proteins might occur (Ladomersky & Petris, 2015).

As comparable uptake system are not known in E. coli (Rensing & Grass, 2003) we identified the outer membrane porin OprC from Pseudomonas aeruginosa as a good candidate for this purpose (Yoneyama & Nakae, 2001). OprC belongs to the superfamily of TonB-dependent receptors to which TonB binds as a signal protein (Postle & Good, 1983). It was shown that OprC allows the selective uptake of Cu(I/II) into the periplasmic space whilst other toxic ions like Ag(I) and Hg(II) are not imported (Yoneyama & Nakae, 1996, Quintana, Novoa-Aponte & Argüello, 2017). For our project we used a OprC homolog of P. brassicacearum 3Re2-7 (strain not pulished yet, for more information please contact Prof. Dr. Alfred Pühler) with 71% protein identity to avoid working with an organism from risk group two.

In order to further increase the intracellular concentration of copper ions, the P-type ATPase HmtA from P. aeruginosa can be used. It actively transports copper ions across the cytoplasmic membrane into the cytoplasm (Lewinsohn, Lee & Rees, 2009). As with oprC the homolog from the non-pathogenic P. brassicacearum 3Re2-7 with a protein identity of 80% was used. HmtA expression was found to lead to acute hypersensitivity for Cu(II) and Zn(II), which is a result of Cu(II)/Zn(II) uptake consequently leading to increased intracellular concentrations of these metal ions (Lewinsohn, Lee & Rees, 2009). Uptake of other cations into the cytoplasm like toxic Ag(I) and Cd(II) ions is not facilitated by hmtA expression (Lewinson, Lee & Rees, 2009). As hmtA expression depends on the extracellular Zn(II) concentration and Zn(II) is transported as well as Cu(II) by HmtA (Pederick et al., 2015), it can be assumed that HmtA is a zinc importer which also transports copper ions due to the similarity in size and charge of Zn(II) and Cu(II) ions.

Another transportation system is copCD from the copABCD operon for copper homeostasis in Pseudomonas syringae pv. tomato. This operon encodes for a precise regulation system of intracellular Cu(II) concentration (Lawton et al., 2016). The two genes copC and copD exist in various forms like in a copABCD operon, standing alone as a copCD operon or even build up CopCD fusion proteins (Arnesano et al., 2002, Lawton et al., 2016) and their function is not fully understood yet. One proposed function is the uptake of Cu(II) ions into the cytoplasm (Cha & Cooksey, 1993). CopC is a putative metallochaperone and exists in different forms with one or two copper binding sites (Lawton et al., 2016) and CopD is a inner membrane protein that is presumably able to pump Cu(II) into the cell. We identified both genes in the genome of P. brassicacearum 3Re2-7 and transferred them into E. coli to increase the uptake of Cu(II) and accumulate intracellular copper.

Conclusion

Our copper accumulation system works in two ways. We prevent the export of Cu(I) by knocking out the native E. coli copper export cus operon. In addition we identified genes involved in copper uptake, hmtA, oprC, copC and copD, and cloned them into E. coli. This combined system provides and represents a highly efficient and specific accumulation system for copper ions.

All results of our accumulation system can be found here.

The results of the accumulation experiments can be found here

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Franke, S., Grass, G., Rensing, C., & Nies, D. H. (2003). Molecular analysis of the copper-transporting efflux system CusCFBA of Escherichia coli. Journal of bacteriology, 185(13), 3804-3812.
Gudipaty, S. A., Larsen, A. S., Rensing, C., & McEvoy, M. M. (2012). Regulation of Cu (I)/Ag (I) efflux genes in Escherichia coli by the sensor kinase CusS. FEMS microbiology letters, 330(1), 30-37.
Ladomersky, E., & Petris, M. J. (2015). Copper tolerance and virulence in bacteria. Metallomics, 7(6), 957-964.
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 All results of our accumulation system can be found <a href="https://2018.igem.org/Team:Bielefeld-CeBiTec/Accumulation_Results" target="_blank">here</a>.
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+<h2>The results of the accumulation experiments can be found <a href"https://2018.igem.org/Team:Bielefeld-CeBiTec/Accumulation_Results">here</a>
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