What do we want to achieve?

Our goal is to produce an E. coli strain that can specifically hunt for the heavy metal cadmium by using a manipulated chemotaxis system in order to both detect and accumulate it. In this way, we can not only detect environmentally harmful cadmium contamination quickly and on a mass scale, but also purify polluted waters. Furthermore, the recovered cadmium can be recycled and purified for industrial usage.

How we CADch!

Chemotaxis



Chemotaxis.

E. coli is adapting the direction of movement to the current environmental conditions. The swimming behavior is regulated by the direction of rotation of the flagellum. If the flagellum moves counterclockwise, the cells move away in one direction. If the flagellum rotates clockwise, the cells start to tumble and rotate. By alternating tumbling and swimming, E. coli can change its swimming direction. In the presence of a chemoattractant gradient, which represents for example nutrients or amino acids, E. coli swarms towards the molecule by increasing the swimming phases. If there is a chemorepellent gradient, like toxic substances, E. coli changes its direction, by alternating tumbling and swimming-phases.

On a molecular basis, E. coli detects molecules with five different transmembrane receptors: tar, tsr, trg, tap and aer[2]. The so-called Methyl-accepting chemotaxis proteins (MCPs) are located in the inner membrane. On the cytoplasmatic side of the MCPs, the histidine kinase CheA is bound to the MCP by CheW[6]. The autophosphorylation of CheA, leads to a signal-cascade. The phosphoryl groups are donated to the response regulator CheY. The phosphorylated CheY then binds to the flagellar switch protein FliM to cause clockwise rotation and cell rotation[8],[7]. The phosphatase CheZ dephosphorylates CheY stopping the tumbling of the cell[5].

Signaling molecules pass through the outer membrane and bind to receptors in the periplasm, resulting in regulation of CheA[6]. Binding of chemorepellent molecules increases CheA activity and promotes tumbling; chemoattractant molecules decrease CheA activity, resulting in movement toward the gradient.

To guarantee a movement to the highest concentration of chemoattractants, the affinity of the MCPs is regulated by CheB and CheR[1]. The methyltransferase CheR mediates the methylation of MCPs, resulting in a higher affinity for signaling molecules[3]. In the phosphorylated state, the esterase CheB demethylates the MCPs and decreases the affinity. The phosphorylation is again mediated by phosphorylated CheA[3]. According to the regulation of the sensitivity of MCPs, E. coli is able to sense different concentrations of signaling molecules.

In our project we redesigned a knockout E. coli strain of all methyl-accepting chemotaxis proteins (Tsr,Trg,Aer,Tar and Tap) originally manufactured and kindly provided by John S. Parkinson from the University of Utah. To achieve chemoattractant behaviour towards cadmium, we implemented mutants of the Tsr (inactive, just there for dimerization and function of Trg) and Trg (mutant for not binding the galactose binding protein) receptors together with the ribose binding protein (RBP) that was redesigned earlier this year[10] to specifically bind cadmium ions (CdRBP1m). This combination of preventing native chemotaxis systems of E. coli and adding a manipulated Cd-ion specific version of it back into the strain, should yield a monospecific chemotaxis behavior. The additional chromoprotein Meff Blue[11] was added to make the cells visible in chemotaxis assays in the most simple way possible. We tested the capacity of the strain to detect cadmium residues which is more thoroughly described in the results section.

REFERENCES
  1. Endres RG, Wingreen NS (2006) Precise adaptation in bacterial chemotaxis through “assistance neighborhoods”. Proc Nat Acad Sci USA 103:13040–13044
  2. Grebe TW, Stock J. Bacterial chemotaxis: the five sensors of a bacterium. Curr Biol. 1998 Feb 26;8(5):R154-7.
  3. Hess, J. F., Oosawa, K., Kaplan, N. & Simon, M. I. Phosphorylation of three proteins in the signalling pathway of bacterial chemotaxis. Cell 53, 79–87 (1988).
  4. Li H, Zhang C, Chen X, You H, Lai L. Tailoring Escherichia coli Chemotactic Sensing towards Cadmium by Computational Redesign of Ribose-Binding Protein. mSystems. 2022 Feb 22;7(1):e0108421.
  5. McEvoy, M. M., Bren, A., Eisenbach, M. & Dahlquist, F. W. Identification of the binding interfaces on CheY for two of its targets, the phosphatase CheZ and the flagellar switch protein FliM. J. Mol. Biol. 289, 1423–1433 (1999).
  6. Salah Ud-Din, A.I.M., Roujeinikova, A. Methyl-accepting chemotaxis proteins: a core sensing element in prokaryotes and archaea. Cell. Mol. Life Sci. 74, 3293–3303 (2017).
  7. Toker, A. S. & Macnab, R. M. Distinct regions of bacterial flagellar switch protein FliM interact with FliG, FliN and CheY. J. Mol. Biol. 273, 623–634 (1997).
  8. Welch, M., Oosawa, K., Aizawa, S.-I. & Eisenbach, M. Phosphorylation-dependent binding of a signal molecule to the flagellar switch of bacteria. Proc. Natl Acad. Sci. USA 90, 8787–8791 (1993).
  9. Zhou Q, Ames P, Parkinson JS. Biphasic control logic of HAMP domain signalling in the Escherichia coli serine chemoreceptor. Mol Microbiol. 2011 May;80(3):596-611.
  10. Li Hengyi; Zhang Changsheng; Chen Xi; You Hantian; Lai Luhua (2022): Tailoring Escherichia coli Chemotactic Sensing towards Cadmium by Computational Redesign of Ribose-Binding Protein. In: mSystems 7 (1), e01084-21. DOI: 10.1128/msystems.01084-21.
  11. Liljeruhm, Josefine; Funk, Saskia K.; Tietscher, Sandra; Edlund, Anders D.; Jamal, Sabri; Wistrand-Yuen, Pikkei et al. (2018): Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. In: Journal of biological engineering 12, S. 8. DOI: 10.1186/s13036-018-0100-0.

Bioremediation



Bioremediation.

In the second part of our project, we aim at establishing a genetically engineered E. coli strain that will be able not only to tolerate, but also sequester large amounts of Cadmium ions form polluted water sources. To this end, we want to overexpress two exogenous “storage” proteins that will be able to bind the heavy-metal ions taken up from the surrounding medium. First however, it is necessary to ensure an increased uptake of Cadmium into the bacterium’s cytoplasma. For the transport across E. coli`s outer membrane we are aided by the unspecific nature of endogenous porins that enable accumulation of many different compounds in the periplasmatic space. For a subsequent uptake into the cytoplasm across the inner membrane, we plan to overexpress the metal ion transporter MntH from B. subtilis, which next to its preferred substrate, manganese, also shows robust affinity for cadmium[1].

To ensure survival of our modified E. coli strains under increased cytoplasmic Cadmium levels, we plan to express one of two proteins, the metallothein hMT2 usually found in the liver of H. sapiens or as an alternative a phythochelatin-synthetase, AtPCS1 from a plant called A. thaliana. Both systems are based on the affinity of proteomic sulphide groups which should coordinate positively charged Cadmium ions, thereby mitigating their cytotoxic effect. While the expression of hMT2, a small cysteine-rich protein will directly ensure this sequestration[2], AtPCS1 will catalyse the condensation of several glutathione into a so called-called phytochelatines[3]. These phytochelatines, like hMT2 are composed to an increased degree of cysteine, following the same mechanism in Cadmium-binding described just now.

Both storage systems will require a heightened level of intracellular L-cysteine to function properly. To achieve an increased synthesis of this specific amino acid, we need to ensure an adequate supply of sulphur. To achieve this, we will overexpress the sulphate importer CysP from B. subtilis in order ensure transport across the inner membrane[4]. As described for Cadmium, uptake of sulphate into the periplasmic space is already taking place through endogenous porins in the outer membrane. Once within the cell, sulphate will be metabolized by an overexpressed variant of CysE*, the wild type of which is endogenous to E. coli. This enzyme is involved in the synthesis of L-cysteine from L-alanine and sulphate ions, with the chosen mutant rendered insensitive to feedback-inhibition by its own product[5]. The increased levels of L-cysteine are theorized to help directly facilitate the overexpression of hMT2 as well as indirectly ensure the functions of AtPCS1 by providing a basis for elevated levels of GSH.

As an addition to our Cadmium storage system, we also hypothesized that the expression of EcCARs could boost the reactivity of proteomic cysteine side chains through the conversion of its sulphide into a polysulphide group. EcCARs takes two L-cysteines as a substrate as well as pyridoxal phosphate to from these special Cys-SSH compounds[6]. By incorporation of these increasingly reactive groups into hMT2 as well as AtPCS1-synthesized phytochelatines, we hope to further increase the level of Cadmium sequestration in our engineered E. coli strains. More importantly, we hope that this will lead to the formation of capped CdS quantum dots, which find their application in the industrial production of solar panels due to their fluorescent/optical properties. The recycling process from cadmium waste into new sustainable resources, which aid the green industrial transformation is a milestone long-term goal of this project.

REFERENCES
  1. Que, Q. & Helmann, J. D. Manganese homeostasis in Bacillus subtilis is regulated by MntR, a bifunctional regulator related to the diphtheria toxin repressor family of proteins. Molecular microbiology 35, 1454–1468; 10.1046/j.1365-2958.2000.01811.x (2000).
  2. Ma, Y., Lin, J., Zhang, C., Ren, Y. & Lin, J. Cd(II) and As(III) bioaccumulation by recombinant Escherichia coli expressing oligomeric human metallothioneins. Journal of hazardous materials 185, 1605–1608; 10.1016/j.jhazmat.2010.10.051 (2011).
  3. Clemens, S. Evolution and function of phytochelatin synthases. Journal of Plant Physiology 163, 319–332; 10.1016/j.jplph.2005.11.010 (2006).
  4. Hryniewicz, M., Sirko, A., Pałucha, A., Böck, A. & Hulanicka, D. Sulfate and thiosulfate transport in Escherichia coli K-12: identification of a gene encoding a novel protein involved in thiosulfate binding. Journal of bacteriology 172, 3358–3366; 10.1128/jb.172.6.3358-3366.1990 (1990).
  5. Wang, C. L., Maratukulam, P. D., Lum, A. M., Clark, D. S. & Keasling, J. D. Metabolic engineering of an aerobic sulfate reduction pathway and its application to precipitation of cadmium on the cell surface. Appl Environ Microbiol 66, 4497–4502; 10.1128/AEM.66.10.4497-4502.2000 (2000).
  6. Sawa, T., Motohashi, H., Ihara, H. & Akaike, T. Enzymatic Regulation and Biological Functions of Reactive Cysteine Persulfides and Polysulfides. Biomolecules 10; 10.3390/biom10091245 (2020).