Team:Bielefeld-CeBiTec/Accumulation

Copper Accumulation System

Overview



One important goal of nanoFactory is to create an efficient accumulation system for heavy metal ions, in this case of copper, in order to subsequently form nanoparticles in Escherichia coli. 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 and dioxygen transport and activation (Solomon et al., 2014). Accordingly, there must be natural absorption mechanisms for this trace element into both 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 get rid of excess copper (Rensing & Grass, 2003). In order to create an efficient accumulation system for copper ions, efficient and selective uptake systems must be expressed on the one hand and existing export systems must be suppressed on the other.

Import

The absorption of copper takes place in two steps: In the gram-negative chassis organism E. coli, copper is first transported across the outer cell membrane into the periplasm and transported to the cytoplasm over the inner cell membrane if further needed. Due to its toxicity an accumulation of copper in the periplasm has many advantages over accumulating it in the cytoplasm, where DNA damage might be a problem (Ladomersky & Petris, 2015). For this purpose, the transport of copper into the periplasm, the porin OprC from Pseudomonas brassicacearum is used, as comparable uptake systems in E. coli are not known (Rensing & Grass, 2003). In the closely related pathogenic organism Pseudomonas aeruginosa (71% protein identity) it could be shown that OprC allows for a selective uptake of Cu(II) ions along a concentration gradient, while toxic ions such as Ag(I), Cu(I) or Hg(II) are held back (Yoneyama & Nakae, 1996). OprC belongs to the superfamily of TonB-dependent receptors to which TonB binds as a signal protein (Postle & Good, 1983).


Figure 1: Copper accumulation system of nanoFactory in E. coli. If bound to TonB, OprC enables Cu(I,II) uptake into the periplasm. CueO causes the oxidization of Cu(I) to Cu(II) in the periplasm and HmtA transports Cu(II) to the cytoplasm whilst consuming ATP. At the same time toxic Cu(I) is transported from the cytoplasm to the periplasm and bound by CusC or oxidized by CueO. The CusCBA-system, that is normally transporting Cu(I) from the periplasm out of the cell, is deleted.


In order to increase the concentration of copper ions further, the P-type ATPase HmtA of P. aeruginosa can be used. It actively transports copper ions across the cytoplasmic membrane into the cytoplasm (Lewinsohn, Lee & Rees, 2009). As with the OprC-gene the homologue from the non-pathogenic P. brassicacearum with a protein identity of 80% is 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 the HmtA-transporter (Pederick et al., 2015), it can be assumed that HmtA is a zinc importer, which also transports copper ions due to the similarity of size and charge of Zn(II) and Cu(II) ions.

Export

In order to prevent the loss of imported Cu(II) ions by active or passive export, the genes CusABCFRS from copper homeostasis were knocked out with CRISPR/Cas9. CusR and CusS together build up a two-component system, which regulates the expression of the cusCFBA operon. (Xiao et al., 2017). CusCFBA genes encode for Ag(I)- and Cu(I)-exporting efflux proteins.
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.
Solomon, E. I., Heppner, D. E., Johnston, E. M., Ginsbach, J. W., Cirera, J., Qayyum, M., ... & Tian, L. (2014). Copper active sites in biology. Chemical reviews, 114(7), 3659-3853.
Xiao, M., Zhu, X., Fan, F., Xu, H., Tang, J., Qin, Y., ... & Zhang, X. (2017). Osmotolerance in Escherichia coli is improved by activation of copper efflux genes or supplementation with sulfur containing amino acids. Applied and environmental microbiology, AEM-03050.
Yoneyama, H., & Nakae, T. (1996). Protein C (OprC) of the outer membrane of Pseudomonas aeruginosa is a copper-regulated channel protein. Microbiology, 142(8), 2137-2144.