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                           <b>Figure 1:</b> 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
 
                           <b>Figure 1:</b> 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

Revision as of 15:48, 14 October 2018

Copper Accumulation System
Accumulation of copper ions in living cells requires increased uptake and inhibition of export systems. We knocked out the dominant export system cusCFBA from Escherichia coli which promotes Cu(I) export from the cytoplasm to the outside. The accumulation system is supplemented by an uptake system consisting of the outer membrane importers copC and oprC as well as the inner membrane transporters copD and hmtA.

Introduction

One important goal of nanoFactory is to create 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 presence 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, non-bonded, dissolved copper in particular is very toxic, so there are also efficient export systems in E. coli to remove excess copper (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 1: 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 active or passive export, the cus operon from 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 (Xiao et al., 2017, Munson et al., 2000). 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 driven by proton-motive force located in the inner cell membrane . CusA is located in the inner cell memrane and pumps driven by proton-motive force Cu(I)/Ag(I) ions through the adapter-like CusB and CusC which sits in the outer membrane (Franke et al., 2003, Delmar, Su & Yu, 2013). Cu(I) and Ag(I) ions get delivered to CusB by CusF metallochaperones in the periplasmic space (Bagai et al., 2008). We created a knock out mutant from E. coli DH5α using CRISPR/Cas9 to remove the whole cus operon to prohibit Cu(I) export. Consequently Cu(I) ions can not leave the periplasmic space. Subsequently the oxidization of Cu(I) to Cu(II) occurs in the oxidative environment of aerobically grown cultures and is supported by multicopper oxidase CueO native to E. coli also in anaerobic environments (Outten et al., 2001).




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 then to the cytoplasm over the inner cell membrane if further needed. 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) a good candidate for this purpose is the outer membrane porin OprC from Pseudomonas aeruginosa (Yoneyama & Nakae, 2001). OprC belongs to the superfamily of TonB-dependent receptors to which TonB binds as a signal protein (Postle & Good, 1983). It could be 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 held back (Quintana, Novoa-Aponte & Argüello, 2017, Yoneyama & Nakae, 1996). We used a homolog from Pseudomonas 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 increase the concentration of copper ions further, 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 result in acute hypersensitivity for Cu(II) and Zn(II), which is a result of Cu(II)/Zn(II) uptake as the expression resulted in increased intracellular concentration of these metal ions (Lewinsohn, Lee & Rees, 2009). Uptake of other cations into the cytoplasm like toxic Ag(I) and Cd(II) ions did not occur (Lewinsohn, 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 and is a homolog to pcoABCD (Lawton et al., 2016). The two genes exist in various forms like in a copABCD operon, standing alone as a copCD operon or even build up CopCD fusion proteins (Lawton et al., 2016, Arnesano et al., 2002) 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 probable 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 Pseudomonas brassicacearum 3Re2-7 and transfered 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 whole cus operon. Then we identified genes involved in copper uptake, hmtA, oprC, copC and copD and cloned them into E. coli. Together this systems provide a highly efficient and also specific accumulation system for copper ions.
Ladomersky, E., & Petris, M. J. (2015). Copper tolerance and virulence in bacteria. Metallomics, 7(6), 957-964.
Lewinson, O., Lee, A. T., & Rees, D. C. (2009). A P-type ATPase importer that discriminates between essential and toxic transition metals. Proceedings of the National Academy of Sciences, 106(12), 4677-4682.
Pederick, V. G., Eijkelkamp, B. A., Begg, S. L., Ween, M. P., McAllister, L. J., Paton, J. C., & McDevitt, C. A. (2015). ZnuA and zinc homeostasis in Pseudomonas aeruginosa. Scientific reports, 5, 13139.
Postle, K., & Good, R. F. (1983). DNA sequence of the Escherichia coli tonB gene. Proceedings of the National Academy of Sciences, 80(17), 5235-5239.
Rensing, C., & Grass, G. (2003). Escherichia coli mechanisms of copper homeostasis in a changing environment. FEMS microbiology reviews, 27(2-3), 197-213.
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