Cadmium adsorption by E. coli with surface displayed CadR

Abstract

CadR is a metal-binding protein first isolated from rhizobacterium Pseudomonas putida and specifically recognizes Cd²⁺. Escherichia coli cells surface engineered with CadR shows a high Cd²⁺ adsorption capacity of 19.5 μmol g⁻¹ cells. The surface engineered E. coli cells also show higher tolerance towards cadmium contamination for up to 100 mM and could potentially be utilized as a bio-remediation treatment of cadmium contamination.

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Cadmium is a non-essential toxic heavy metal and ranks as the sixth most toxic substance for significant human health hazard, according to the U.S. Poison and Disease registry.¹ Cadmium intoxication symptoms range from respiratory tract and kidney dysfunction to the notorious “Itai-Itai” disease, which causes osteoporosis.^2–5 Evidence also shows that some Cd²⁺-containing compounds are carcinogenic.^6–10 Cadmium pollution mainly comes from mine drainage, electro-plants and battery industry waste.^11,12 Compared with other metal ions, Cd²⁺ is more easily absorbed by plants and further enriched in animals through the food chain, thus cadmium pollution is a priority in the World Health Organization's study of food contamination.^13–16 Japan and China are the countries that report the most environmental cadmium exposure, partly due to their habit of eating rice.^17–23 The conventional cleanup of Cd²⁺ usually includes chemical precipitation, which is often ineffective for diluted waste water, and the precipitation itself could be a secondary polluting source.^24–26

Bioremediation is a waste managing technology, involving using natural or genetically-modified microorganisms and/or plants for environmental cleanup and has been applied in ameliorating cadmium pollution.^27,28 The efforts vary from the screening of marine microalgae to the cultivation of plant growth-promoting rhizobacteria.^29–33 However, heavy metal ions like Cd²⁺ are not readily absorbed or captured by natural organisms,^34–39 therefore we want to develop a genetically engineered microorganism with a fast growth rate, high Cd²⁺ tolerance and high surface area–volume ratio to obtain high cadmium removal efficiency.

CadR is a MerR family Cd²⁺-binding protein, first isolated from rhizobacterium Pseudomonas putida 06909, which regulates the cellular Cd²⁺ concentration by regulating the expression level of CadA, a Cd²⁺ efflux ATPase.^40–42 CadR contains 147 amino acids and three domains: the DNA binding domain, the metal binding domain and the coupling domain. Three cysteine residues (Cys 77, 112, 119) and its histidine rich C-terminus are predicted as the possible Cd²⁺ binding sites. The sensitive and specific recognition of Cd²⁺ by CadR has been used to develop Cd²⁺ sensors.^43–45

Herein, we want to report the construction of a surface-fused truncated CadR–OmpA on E. coli (Fig. 1).^46–48 These genetically-engineered E. coli cells show high Cd²⁺ tolerance and high adsorption capacity for Cd²⁺. This system may be further developed for the potential bio-remediation of Cd²⁺ contamination.

Fig. 1 Schematic illustration of CadR displayed on an E. coli cell surface via the membrane protein OmpA. The yellow-green columns represent the membrane-spanning domain of OmpA and the blue schematic is CadR displayed on the outer membrane of the cell.

To optimize the best CadR fraction for surface-display, we first tested Cd²⁺ binding affinity with full-length and truncated CadRs. The truncations were positioned to avoid the perturbing of the metal binding domain and are schematically illustrated in Fig. 2a. TC21 truncated the random coil region of 21 amino acids at the C-terminus, whereas TC68 truncated the DNA-binding domain of 68 amino acids at the N-terminus. Electrophoretic mobility shift assays showed that both the full-length CadR and TC21 bind its promoter pcadR (Fig. 2b) and the DNA–protein complex dissociated upon the addition of Cd²⁺ (Fig. 2c). Metal ions, such as Ni²⁺, Cu²⁺ and Cr³⁺, were ineffective at dissociating CadR from the CadR–DNA complex, whereas Zn²⁺ was less effective than Cd²⁺(Fig. 2d). TC68 lost the binding ability of pcadR due to the truncation of the DNA binding domain. Isothermal titration microcalorimetry (ITC) was used to measure the change in the observed enthalpy (ΔH_obs) by titrating Cd²⁺ (0.25 mM) into the TC21 solution (0.05 mM) (Fig. 2e). When the molar ratio of TC21 to Cd²⁺ reached ∼1 : 2, the ΔH_obs values reached a platform, which agreed with the previous report of CadR dimer formation upon Cd²⁺ binding.^40,42 The association constant of TC21 with Cd²⁺ is about 1.47 × 10⁷ M⁻¹. The ITC measurements of CadR gave similar results as TC21 (Fig. S2†), while TC68 showed weaker binding, which may be caused by the misfolding or instability of the protein under the ITC conditions (Fig. S3†). The Tris-buffer and His-tag had negligible influences on the ITC results through the four control titrations of Cd²⁺ to buffer, His-tag labeled proteins and GST-labeled protein (Fig. S4–S6†). When three key cysteine residues (C77, C112, C119) at the C-terminus were mutated to serine, ITC measurements showed dramatically decreased Cd²⁺ binding, which confirmed the importance of these cysteine residues (Fig. S8–S10†). The cysteine residues could tolerate a limited oxidative environment because all our experiments were carried out in air, and no reducing agents were added for dialysis and ITC experiments.

Fig. 2 (a) A schematic illustration of the design of truncated CadRs. (b) Electrophoretic mobility shift assays of the binding of (i) CadR, (ii) TC21 and (iii) TC68 to pcadR. (c) The concentration dependent dissociation of CadR–pcadR complex by Cd²⁺. (d) The electrophoretic mobility shift assays of other metal ions with CadR–pcadR. (e) ITC titration for TC21 with Cd²⁺.

Following the protocols reported by Zhao et al.,⁴⁸ we fused CadR, TC21 and TC68 with a C-terminus FLAG-tag to the C-terminus of OmpA(N1-159) (Fig. 1). After inducing with arabinose, the recombinant fusion proteins' expression in the membrane fraction was confirmed by SDS-PAGE with correct molecular weights (Fig. 3a). Moreover, immunoblotting analysis verified the expression of TC68–OmpA expression with anti-FLAG antibodies (Fig. 3b). Then the immunofluorescence experiments further confirmed the successful expression of fusion proteins on the E. coli extracellular surfaces (Fig. 3c).

Fig. 3 Characterization of the surface-displayed proteins. (a) SDS-PAGE analysis of surface-displayed protein. The red boxes indicate the three proteins, OmpA–CadR (lane 4, 34.8 kDa), OmpA–TC21 (lane 5, 33.6 kDa) and OmpA–TC68 (lane 6, 28.0 kDa). Lanes 1–3 show the membrane fraction from the uninduced bacteria, OmpA–CadR (lane 1), OmpA–TC21 (lane 2) and OmpA–TC68 (lane 3). The supernatant protein fraction from the induced (lane 7) or uninduced (lane 8) OmpA–TC68 bacteria are used as controls. (b) The immunoblotting (anti-FLAG antibody) of OmpA–TC68–FLAG in the membrane fraction of the induced bacteria (lane 3), supernatant (lane 2) and the membrane fraction of the uninduced control (lane 1). (c) The immunofluorescence labelling of E. coli cells using anti-FLAG antibody and FITC conjugated anti-mouse IgG antibody. (i) Arabinose induced OmpA–CadR bacteria, (ii) induced OmpA–TC21 bacteria, (iii) induced OmpA–TC68 bacteria and (iv) uninduced OmpA–TC68 bacteria as control.

The engineered E. coli cells were further tested for their cadmium adsorption ability. After 2 hours' induction with 0.05% arabinose in LB broth, the bacteria were treated with Cd²⁺ (60 μmol L⁻¹) overnight. The harvested cells were washed three times with water and digested by microwave, and then Cd²⁺ concentration was measured by ICP-MS. The Cd²⁺ adsorption efficiencies of the surface engineered bacteria were significantly higher than E. coli cells without CadR fusion (Fig. 4a). The TC68–OmpA fusion construct showed the highest adsorption capacity of about 19.5 μmol g⁻¹ cells. Compared with CadR and TC21, TC68's small size may significantly improve the overall display efficiency. Thanks to CadR's selectivity towards Cd²⁺, the TC68 engineered E. coli cells showed 100-fold higher selectivity towards Cd²⁺ over Cu²⁺, Ni²⁺ and Cr³⁺, and 10-fold higher selectivity over Zn²⁺ (Fig. 4b).

Fig. 4 (a) Cd²⁺ adsorption capacity with the three engineered E. coli cells. The three surface displayed E. coli cells (OmpA–CadR, OmpA–TC21, OmpA–TC68) are labelled as D, the three controlled E. coli cells without OmpA engineering (CadR, TC21, TC68) are labelled as U. (b) Adsorption of other metal ions with OmpA–TC68 cells in the same condition. (c) Plate assays of Cd²⁺ tolerance with the E. coli cells same as in (a). E. coli cells were spotted on solid medium containing 0.05% arabinose and 50 μM Cd²⁺. The plate was incubated at 37 °C overnight before being read. (d) Cd²⁺ tolerance of surface displayed E. coli cells (OmpA–CadR, OmpA–TC21, OmpA–TC68) under the treatment with different concentrations of Cd²⁺. Cd²⁺ solution was added on the solid medium, containing the induced E. coli cells and 0.05% arabinose. The plate was incubated at 37 °C overnight before being read. Dotted boxes indicate the suppression plaques. Smaller dotted boxes indicated more bacteria survival.

Notably, the surface engineered E. coli cells showed tremendously improved Cd²⁺ tolerance. Plate assays showed more than 1000 fold Cd²⁺ tolerance improvement for all three surface engineered E. coli cells (Fig. 4c). The TC68 construct could even survive and grow without any visible defect at a Cd²⁺ concentration at 1 mmol L⁻¹ (Fig. 4d). This finding suggests that the surface displayed CadR motif could adsorb Cd²⁺ and protect the cells from cadmium intoxication, which is crucial for its potential application in cadmium pollution remediation.

Conclusions

In summary, CadR, a Cd²⁺ selective metalloprotein, was displayed on the surface of E. coli cells and the surface engineered E. coli cells showed high tolerance to Cd²⁺ intoxication and high Cd²⁺ adsorption capability. This method may have potential application as a selective Cd²⁺ biosorbent and this concept could be applied to other selective metal binding motifs and other organisms.

Acknowledgements

This work is supported by Natural Science Foundation of China Grant 21102007 and 21372023, the Shenzhen Science and Technology Innovation Committee SW201110018, SGLH20120928095602764, ZDSY20130331145112855, Pfizer-Peking University platform to Z. G. L.; the Shenzhen Peacock Program (KQTD201103) to Z. G. L.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra07805e

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