Eschneider (Talk | contribs) |
Eschneider (Talk | contribs) |
||
Line 64: | Line 64: | ||
<li class="side_list"> | <li class="side_list"> | ||
− | <a href="#ovv"> | + | <a href="#ovv">Short Summary</a> |
+ | </li> | ||
+ | <li class="side_list"> | ||
+ | <a href="#ovv">Introduction</a> | ||
</li> | </li> | ||
<li class="side_list"> | <li class="side_list"> | ||
− | <a href="#imp"> | + | <a href="#imp">Preventing Copper Export</a> |
</li> | </li> | ||
<li class="side_list"> | <li class="side_list"> | ||
− | <a href="#exp"> | + | <a href="#exp">Copper Import</a> |
+ | </li> | ||
+ | <li class="side_list"> | ||
+ | <a href="#exp">References</a> | ||
</li> | </li> | ||
</div> | </div> | ||
<div class="container"> | <div class="container"> | ||
+ | |||
<div class="main_content"> | <div class="main_content"> | ||
− | + | ||
+ | <div class="title">Copper Accumulation System</div> | ||
<h2><span id="exp"></span>Short Summary</h2> | <h2><span id="exp"></span>Short Summary</h2> | ||
Line 88: | Line 96: | ||
<article>One important goal of nanoFactory is to create an efficient accumulation system for heavy metal ions in order to subsequently form nanoparticles in <i>E. coli</i>. 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. </article> | <article>One important goal of nanoFactory is to create an efficient accumulation system for heavy metal ions in order to subsequently form nanoparticles in <i>E. coli</i>. 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. </article> | ||
<br><br> | <br><br> | ||
− | 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. | + | 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.</article> |
+ | <figure role="group"> | ||
+ | <img class="figure eighty" src="File:T--Bielefeld-CeBiTec--ES--Accumulation-4-Importer.png"> | ||
+ | <figcaption> | ||
+ | <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 | ||
+ | </figcaption> | ||
+ | </figure> | ||
− | |||
− | + | <h2><span id="exp"></span>Preventing Copper Export</h2> | |
− | <h2><span id="exp"></span>Preventing | + | |
<br><br> | <br><br> | ||
<br><br> | <br><br> | ||
− | In order to prevent the loss of the effortfully imported Cu(II) ions by active or passive export, the <i>cus</i> operon from <i>E. coli</i> was knocked out using CRISPR/Cas9. It consists of <i>cusCFBA</i> which belongs to the resistance-nodulation-cell division superfamily (Tseng <i>et al.</i>, 1999) and the two-component regulatory system <i>cusRS</i>. The response regulator <i>cusR</i> and its associated membrane-bound kinase <i>cusS</i> regulate the expression of the opposed directed <i>cusCFBA</i> genes directly upstream of <i>cusRS</i> (Xiao <i>et al.</i>, 2017, Munson <i>et al.</i>, 2000). The export of Cu(I) and also Ag(I) is carried out by the tripartite protein complex CusCBA (Gudipaty <i>et al.</i>, 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 <i>et al.</i>, 2003, Delmar, Su & Yu, 2013). Cu(I) and Ag(I) ions get delivered to CusB by CusF metallochaperones in the periplasmic space (Bagai <i>et al.</i>, 2008). We created a knock out mutant from <i>E. coli DH5α</i> using CRISPR/Cas9 to remove the whole <i>cus</i> 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 <i>E. coli</i> also in anaerobic environments (Outten <i>et al.</i>, 2001). | + | <article>In order to prevent the loss of the effortfully imported Cu(II) ions by active or passive export, the <i>cus</i> operon from <i>E. coli</i> was knocked out using CRISPR/Cas9. It consists of <i>cusCFBA</i> which belongs to the resistance-nodulation-cell division superfamily (Tseng <i>et al.</i>, 1999) and the two-component regulatory system <i>cusRS</i>. The response regulator <i>cusR</i> and its associated membrane-bound kinase <i>cusS</i> regulate the expression of the opposed directed <i>cusCFBA</i> genes directly upstream of <i>cusRS</i> (Xiao <i>et al.</i>, 2017, Munson <i>et al.</i>, 2000). The export of Cu(I) and also Ag(I) is carried out by the tripartite protein complex CusCBA (Gudipaty <i>et al.</i>, 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 <i>et al.</i>, 2003, Delmar, Su & Yu, 2013). Cu(I) and Ag(I) ions get delivered to CusB by CusF metallochaperones in the periplasmic space (Bagai <i>et al.</i>, 2008). We created a knock out mutant from <i>E. coli DH5α</i> using CRISPR/Cas9 to remove the whole <i>cus</i> 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 <i>E. coli</i> also in anaerobic environments (Outten <i>et al.</i>, 2001).</article> |
<br><br> | <br><br> | ||
<br><br> | <br><br> | ||
− | <h2><span id="exp"></span>Copper | + | <h2><span id="exp"></span>Copper Import</h2> |
<br><br> | <br><br> | ||
<br><br> | <br><br> | ||
Line 106: | Line 118: | ||
<br><br> | <br><br> | ||
In order to increase the concentration of copper ions further, the P-type ATPase HmtA from <i>P. aeruginosa</i> can be used. It actively transports copper ions across the cytoplasmic membrane into the cytoplasm (Lewinsohn, Lee & Rees, 2009). As with <i>oprC</i>, the homolog from the non-pathogenic <i>P. brassicacearum 3Re2-7</i> with a protein identity of 80% was used. <i>HmtA</i> 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 <i>hmtA</i> 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. | In order to increase the concentration of copper ions further, the P-type ATPase HmtA from <i>P. aeruginosa</i> can be used. It actively transports copper ions across the cytoplasmic membrane into the cytoplasm (Lewinsohn, Lee & Rees, 2009). As with <i>oprC</i>, the homolog from the non-pathogenic <i>P. brassicacearum 3Re2-7</i> with a protein identity of 80% was used. <i>HmtA</i> 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 <i>hmtA</i> 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. | ||
− | |||
<br><br> | <br><br> | ||
− | |||
Another transportation system is <i>copCD</i> from the <i>copABCD</i> operon for copper homeostasis in <i>Pseudomonas syringae pv. tomato</i>. This operon encodes for a precise regulation system of intracellular Cu(II) concentration and is a homolog to <i>pcoABCD</i> (Lawton <i>et al.</i>, 2016). The two genes exist in various forms like in a <i>copABCD</i> operon, standing alone as a <i>copCD</i> operon or even build up CopCD fusion proteins (Lawton <i> et al.</i>, 2016, Arnesano <i>et al.</i>, 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 <i>et al.</i>, 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 <i>Pseudomonas brassicacearum 3Re2-7</i> and transfered them into <i>E. coli</i> to increase the uptake of Cu(II) and accumulate intracellular copper. | Another transportation system is <i>copCD</i> from the <i>copABCD</i> operon for copper homeostasis in <i>Pseudomonas syringae pv. tomato</i>. This operon encodes for a precise regulation system of intracellular Cu(II) concentration and is a homolog to <i>pcoABCD</i> (Lawton <i>et al.</i>, 2016). The two genes exist in various forms like in a <i>copABCD</i> operon, standing alone as a <i>copCD</i> operon or even build up CopCD fusion proteins (Lawton <i> et al.</i>, 2016, Arnesano <i>et al.</i>, 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 <i>et al.</i>, 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 <i>Pseudomonas brassicacearum 3Re2-7</i> and transfered them into <i>E. coli</i> to increase the uptake of Cu(II) and accumulate intracellular copper. | ||
</article> | </article> |
Revision as of 15:44, 14 October 2018
Copper Accumulation System
Short Summary
Introduction
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.
Preventing Copper Export
Copper Import
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.
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.
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.