Our idea and design - Tellurium

Idea

Tellurium is a scarce but strategically important element, notably for thin-film photovoltaics. Its recovery from waste streams is challenging because physicochemical remediation is often inefficient and costly. Therefore, we propose bacterial bioremediation of tellurite as an alternative strategy.

We chose Pseudomonas putida S12 as a source for the genetic parts of our system. P. putida S12 is a soil bacterium widely used as a model in microbiology, known for its metabolic versatility and solvent tolerance (Han et al, 2024). This strain harbors a large megaplasmid pTTS12 (~583 kb), which encodes multiple resistance functions, including the terABCD tellurite resistance operon (Kusumawardhani et al, 2022; Kottara et al, 2020). By building on this natural capacity, we aim to improve tellurite recycling by cloning this operon into a plasmid and overexpressing it in E. coli (BL21).

Figure 1: Representation of the megaplasmid containing the terABCD operon of interest. The megaplasmid pTTS12, found in the Pseudomonas putida S12 strain, carries genes involved in solvent resistance and detoxification, as well as the terABCD tellurium resistance operon. This operon provided the genetic parts used in our experiments.

Our E. coli engineered strains are designed with genetic modules to:

• Capture tellurite from solution.
• Reduce tellurite to insoluble elemental tellurium (Te⁰).
• Recover elemental tellurium captured by the bacteria.

Figure 2: Schematic representation of tellurite reduction by bacteria. Bacteria first capture and take up soluble, toxic tellurite ions. Inside the cell, reductive agents convert tellurite (TeO₃²⁻) into elemental tellurium (Te⁰). The resulting tellurium is insoluble and accumulates as dark deposits. The reduction of soluble tellurite into elemental tellurium is a pivotal step, since it transforms a toxic and mobile compound into a recoverable solid.

Design

Gene Collection

Table 1: Schematic representation of the tellurite resistance genes. The table lists the collection of the tellurite resistance genes, with the respective original terABCD DNA sequence source and a link to iGEM terABCD composite part. Displayed in the last row, a schematic representation of the terABCD gene cluster we cloned in the E.coli DH5α and BL21 strains. All genes are preceded by a ribosome binding site (not shown).

Gene Name	Graphical DNA representation	Link iGEM parts	Original Publications
terA		BBa_252F55NC	Kusumawardhani et al.
terB		BBa_258KVW5Q	Kusumawardhani et al.
terC		BBa_258V49XQ	Kusumawardhani et al.
terD		BBa_25EIPUVH	Kusumawardhani et al.
terABCD		BBa_252NUJ3X	Kusumawardhani et al.

AlphaFold Predictions of Ter Proteins

Figure 3: Structural predictions of the TerABCD proteins. The structures were predicted using the AlphaFold server based on the translated amino acid sequence of terA, terB, terC and terD. A. TerA possesses two TerD domains (shown in pink). B. TerB structural prediction, it is predicted to contain six α-helices. C. TerC contains cytoplasmic domains (shown in pink), transmembrane domains (shown in green) and extracellular domains (shown in blue). D. TerD structural prediction. Domain characterization was performed with Pfam, and domain coloring was applied using ChimeraX.

The precise functions and mechanisms of these genes remained unclear. TerB was proposed to act as the central component of tellurite reduction in association with TerC. TerC likely functioned as a membrane protein facilitating tellurite transport into and within the cell (Fig. 3C). TerD appeared to bind tellurite and operate independently of TerC. TerA, a member of the TerD protein family, was believed to bind metal ions and potentially alleviate the metabolic burden associated with tellurite reduction. The uncloned genes terZ and terE were homologous to terA and terD, respectively, and likely shared similar functions (Peng et al., 2022).

Choice of Chassis and cloning strain

Plasmid overview

Both strains carried the same vector: pET28a–terABCD with kanamycin resistance. The construct features a T7 promoter with lacO, lacI repressor, an optimized RBS, and a T7 terminator; we kept the backbone elements used elsewhere in the project for consistency (Figure 4). This design allowed rapid cloning in DH5α and high, tunable expression in BL21(DE3).

Figure 4: Schematic overview of the plasmids designed for the tellurite resistance project. KanR (kanamycin resistance), Ori (origin of replication), LacI promoter + lac operator (IPTG-inducible control), T7 promoter (drives terABCD expression), RBS (ribosome binding site), terA, terB, terC, terD forming the terABCD operon, T7 terminator (transcription termination), rop (plasmid copy number regulator), bom (mobilization sequence). The figure shows the pET28a-terABCD plasmid construct used in E. coli BL21 (DE3). Created in https://BioRender.com

Controls

To benchmark activity, we included one negative control and one positive control:

• E. coli BL21 (DE3) pET28a-mCitrine: Control strain lacking the terABCD plasmid, used to measure baseline tellurite reduction and survival.
• P. putida S12 pTTS12: Control strain that naturally contains the megaplasmid including the TerZABCDE tellurite resistance operon to measure baseline tellurite reduction and survival.

Ex-situ bioremediation approach

We propose on-site, ex-situ treatment of tellurite-impacted soils or sludges using a sealed, gently rotating, aerated rotary drum bioreactor (RDB). The RDB keeps soil, microbes, oxygen, and moisture in continuous contact, allowing our engineered bacteria to reduce soluble Te(IV) to insoluble Te⁰ uniformly across the matrix. The resulting Te⁰ can be flocculated, separated, and sent for refining, creating a circular Te stream. A closely related RDB setup has already achieved ~82% metal removal in polluted soils (Bravo et al, 2020), validating the unit operations and the ex-situ bioaugmentation approach.

Figure 5: Ex-situ rotary drum bioreactor (RDB) for bioremediation of tellurite-contaminated soil and recovery of elemental tellurium. Tellurite-polluted agricultural soil (left) is fed into a sealed, slowly rotating, aerated drum (air inlet shown). Inside the reactor, engineered E. coli BL21 carrying the terABCD operon catalyzes the reduction of soluble tellurite (TeO₃²⁻) to insoluble elemental tellurium (Te⁰) under aerobic conditions. After treatment, the soil exits the reactor (right) as bioremediated soil; a downstream solid–liquid separation step (not depicted) flocculates/filters Te⁰ for recovery. Abbreviations: TeO₃²⁻, tellurite; Te⁰, elemental tellurium; terABCD, tellurite-resistance operon. Schematic, not to scale. (Concept adapted from an RDB soil-bioremediation approach; see Bravo et al., 2020.)

References

• Bravo, G., Vega-Celedón, P., Gentina, J. C., & Seeger, M. (2020). Bioremediation by Cupriavidus metallidurans Strain MSR33 of Mercury-Polluted Agricultural Soil in a Rotary Drum Bioreactor and Its Effects on Nitrogen Cycle Microorganisms. Microorganisms, 8(12), 1952.
• Chasteen, T. G., Fuentes, D. E., Tantaleán, J. C., & Vásquez, C. C. (2009). Tellurite: history, oxidative stress, and molecular mechanisms of resistance. FEMS Microbiology Reviews, 33(4), 820–832.
• Docherty, T., & Docherty, T. (2023, December 6). First Tellurium reports on two major publications highlighting supply issues for tellurium and other critical metals - FIRST TELLURIUM. FIRST TELLURIUM - Essential metals for a sustainable future.
• Kusumawardhani, H., Hosseini, R., Verschoor, J., & De Winde, J. H. (2022). Comparative analysis reveals the modular functional structure of conjugative megaplasmid pTTS12 of Pseudomonas putida S12: A paradigm for transferable traits, plasmid stability, and inheritance? Frontiers in Microbiology, 13.
• Valková, D., Valkovičová, L., Vávrová, S., Kováčová, E., Mravec, J., & Turňa, J. (2007). The contribution of tellurite resistance genes to the fitness of Escherichia coli uropathogenic strains. Open Life Sciences, 2(2), 182–191.
• Whelan, K. F., Colleran, E., & Taylor, D. E. (1995). Phage inhibition, colicin resistance, and tellurite resistance are encoded by a single cluster of genes on the IncHI2 plasmid R478. Journal of Bacteriology, 177(17), 5016–5027.