A prodigiosin-based whole-cell biosensor for sensitive colorimetric detection of Hg(II)

Abstract

A whole-cell biosensor based on prodigiosin was developed for Hg(II) detection by screening four PigC homologs. The optimized sensor exhibited a detection limit of 0.41 nM, absolute selectivity against competing metal ions, and robust performance in tap, lake, and seawater matrices, providing a sensitive platform for environmental mercury monitoring.

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Persistent mercury contamination poses severe risks to aquatic ecosystems and public health, necessitating rapid, on-site monitoring tools that circumvent the cost and complexity of instrumental analysis such as cold-vapour atomic fluorescence spectrometry.¹ While whole-cell biosensors offer a promising low-cost alternative, conventional fluorescent or luminescent reporters require specialized excitation equipment and dedicated optics, limiting their deployment in resource-limited settings.² Existing chromogenic systems—such as violacein,³ indigoidine,⁴ carotene,⁵ or pyomelanin⁶ – suffer from either the need for organic solvent extraction, insufficient color contrast, or rapid oxidative fading under environmental conditions. Here, we establish prodigiosin, a bright red, lipophilic pigment with intense visible absorbance, as a robust reporter for mercury(II) detection by screening four homologous prodiginine condensation enzymes (PigC, HapC, TreaP, and TamQ) and coupling the optimized candidates to a redesigned MerR-Pmer regulatory circuit in Escherichia coli (E. coli). This engineered whole-cell sensor leverages enzymatic signal amplification—where each expressed enzyme catalyzes multiple turnover cycles to generate intensely colored product—while circumventing organic extraction requirements through direct recovery of the pigment from cellular biomass using acidified ethanol,^7,8 enabling naked-eye quantification of Hg(II) across diverse environmental water matrices with nanomolar sensitivity and absolute metal selectivity.

To identify an optimal reporter enzyme, we screened four homologous prodiginine condensation enzymes (PigC from Serratia marcescens, HapC from Hahella chejuensis, TreaP from Pseudoalteromonas citrea, and TamQ from Pseudoalteromonas tunicata),^7,9–11 all codon-optimized for E. coli. We cloned into the pET-21a vector under T7lac control. SDS-PAGE analysis confirmed soluble expression of PigC, HapC, and TreaP at their predicted molecular masses (99.2, 102.2, and 100.3 kDa, respectively). In contrast, TamQ failed to accumulate detectable protein under standard induction conditions (Fig. 1B).

Fig. 1 Heterologous expression and catalytic activity screening of four prodiginine condensation enzymes. (A) Schematic of pT7lac expression vectors for IPTG-inducible production of codon-optimized PigC, HapC, TreaP, and TamQ. (B) and (C) SDS-PAGE analysis of total protein from E. coli BL21(DE3) strains: PigC and HapC (B), TreaP and TamQ (C). M, protein marker; (−), uninduced; (+), IPTG-induced. (D)–(G) Absorption spectra (400–750 nm) of acidified ethanol extracts from induced cultures supplemented with MBC and MAP precursors: (D) PigC, (E) HapC, (F) TreaP, (G) TamQ. Insets show magnified views of the 535 nm peak and representative extract colors. Data are representative of three independent experiments.

The synthetic precursors 4-methoxy-2,2′-bipyrrole-5-carbaldehyde (MBC) and 2-methyl-3-amylpyrrole (MAP) were purchased from Sigma-Aldrich (Cat. No. 10476-41-2 and 18320-91-7, purity ≥98%) and dissolved in DMSO as 10 mM stock solutions. Recombinant strains were cultivated in LB medium containing 50 µg mL⁻¹ ampicillin at 37 °C with constant shaking at 250 rpm until the early exponential phase. Induction was initiated by direct addition of IPTG (1 mM) and simultaneous supplementation with MBC and MAP precursors (100 µM each),¹⁰ with cultures maintained at 37 °C and 250 rpm throughout the 4-hour induction period. Only PigC and HapC generated intense red pigment with characteristic absorbance at 535 nm, corresponding to prodigiosin formation.⁸ Time-course analysis revealed that pigment accumulation peaked within 1 h of substrate addition, followed by a gradual decline in absorbance over prolonged incubation, suggesting intracellular degradation or conversion of the product (Fig. S3, SI). Notably, PigC exhibited superior catalytic turnover and higher maximal absorbance (A₅₃₅ ≈ 0.65). At the same time, HapC displayed significantly lower background leakage under non-induced conditions (A₅₃₅ ≈ 0.07 vs 0.15 for PigC), indicating that host–enzyme compatibility critically determines signal-to-noise ratios in pigment-based sensing systems.

We therefore selected both PigC and HapC for subsequent biosensor construction by coupling their coding sequences downstream of a redesigned MerR-Pmer regulatory element,^12,13 creating “OFF–ON” sensor strains (TOP10/pHg-PigC and TOP10/pHg-HapC) where Hg(II) binding to the MerR transcription factor derepresses the Pmer promoter, initiating prodigiosin biosynthesis in a dose-dependent manner.

The biosensors exhibited a characteristic bell-shaped dose–response profile over 0–2000 nM Hg(II), with maximal pigment accumulation observed at approximately 250 nM for TOP10/pHg-PigC and 500 nM for TOP10/pHg-HapC, followed by attenuation at supra-optimal concentrations likely reflecting metabolic burden or oxidative stress (Fig. 2 and Fig. S8). Under optimized induction conditions, TOP10/pHg-PigC achieved a detection limit of 0.41 nM (P < 0.05 vs. blank, n = 3)—well below the WHO drinking-water guideline of 30 nM and superior to most fluorescent whole-cell counterparts^2,14,15 – with a quantitative range of 1–250 nM (R² = 0.966). TOP10/pHg-HapC exhibited a slightly higher detection threshold of approximately 3 nM (P < 0.05) but maintained a broader dynamic range up to 250 nM (R² = 0.993).

Fig. 2 Characterization of Hg(II)-responsive prodigiosin biosensors. (A) Schematic of the MerR-Pmer-regulated sensor mechanism. (B) Absorbance at 535 nm of acidified ethanol extracts from TOP10/pHg-PigC and TOP10/pHg-HapC after exposure to varying Hg(II) concentrations. (C) Dose–response curves with non-linear regression analysis. (D) Representative photographs of extracts showing colorimetric response to Hg(II). Data are mean ± standard deviation from three independent biological replicates (n = 3). Statistical significance was determined by one-way ANOVA with Tukey's post hoc test (P < 0.05).

Notably, the sensor demonstrated absolute selectivity for Hg(II) over competing metal ions; when challenged with Mg(II), Ca(II), Cd(II), Mn(II), Cu(II), Pb(II), or Zn(II) at concentrations up to 25 µM (1000-fold excess), only Hg(II) triggered significant prodigiosin production (P < 0.001). In contrast, other metals produced responses indistinguishable from the metal-free control (Fig. 3A–C). Furthermore, binary mixture experiments confirmed that the presence of 5–10 µM competing ions did not interfere with the detection of 50–100 nM Hg(II), with no significant difference observed between Hg(II)-only and mixed-metal treatments (P > 0.05), validating the high specificity of the MerR regulatory element (Fig. 3D–F). The red pigment remained chromogenically stable in acidified ethanol extracts (4% v/v 1 M HCl) for over 120 hours without spectral shifts or fading (Fig. S2), demonstrating robust stability under the employed extraction conditions. This prolonged chromogenic persistence offers practical advantages for field-deployable colorimetric analysis compared to water-soluble chromophores that are susceptible to oxidative fading during storage.^16–19 Notably, the cell pellets appeared white after acidified ethanol extraction, and a second extraction yielded A₅₃₅ values <5% of the first, confirming >95% recovery efficiency and ruling out systematic error from incomplete pigment recovery.

Fig. 3 Metal selectivity and anti-interference performance of the biosensors. (A), (B) Prodigiosin production by TOP10/pHg-PigC (A) and TOP10/pHg-HapC (B) exposed to individual metal ions (horizontal lines indicate metal-free control A₅₃₅ values). (C) Corresponding photographs of acidified ethanol extracts. (D) and (E) Co-exposure to 50 nM or 100 nM Hg(II) with 5–10 µM interfering metal ions of TOP10/pHg-PigC (D) and TOP10/pHg-HapC (E). (F) Representative photographs of extracts. Data are mean ± standard deviation from three independent biological replicates (n = 3). Statistical significance was determined by one-way ANOVA with Tukey's post hoc test (P < 0.05).

While the biosensor demonstrated absolute selectivity for Hg(II) over competing metal ions, with no cross-reactivity observed even at a 1000-fold excess (Fig. 3), recent advances in green-synthesized abiotic nanosensors entail distinct trade-offs in specificity and sensitivity. Sunlight-assisted synthesis routes—exemplified by Equisetum diffusum-mediated AgNPs (LOD: 70 nM)²⁰ and Carissa macrocarpa extract-stabilized AgNPs (LOD: 18.45 µM)²¹ – enable rapid field-deployable colorimetry within seconds to minutes, yet rely on non-specific redox or aggregation mechanisms that cannot discriminate between Hg(II) and other transition metals without additional masking strategies.

This limitation is particularly evident in multifunctional nanosystems such as sulfanilamide-stabilized AgNPs, which achieve dual-colorimetric sensing of Hg(II) and Fe(III) (LODs: 22 nM and 35 nM, respectively),²² and Causonis trifolia-based AgNPs that integrate mercury/ferric sensing with photocatalysis and antimicrobial activity.²³ At the same time, these platforms offer “sense-and-treat” capabilities (e.g., 77–92% photodegradation of organic dyes within 80–150 min),²⁰ their cross-reactivity with Fe(III) and dependence on colloidal stability (susceptible to high ionic strength and pH variations),²⁴ compromise quantitative accuracy in complex environmental matrices such as seawater or iron-rich groundwaters.

In contrast, the genetically encoded MerR-Pmer regulatory circuit employed herein confers single-analyte specificity through molecular recognition rather than physicochemical interaction, eliminating the need for spectral deconvolution or surface charge maintenance. Compared to AgNPs-modified electrodes, which offer alternative trade-offs between sensitivity and portability²⁵ but require sophisticated instrumentation and are prone to electrode fouling, the prodigiosin-based biosensor combines the visual simplicity of nanoparticle colorimetry with biological specificity and enzymatic signal amplification (0.41 nM LOD). Although currently configured as a single-use endpoint assay, the biological chassis offers inherent self-replication via simple reinoculation, effectively circumventing the reusability limitations of disposable abiotic nanosensors. Future work may integrate orthogonal regulatory elements (e.g., Fur for Fe(III)) into the pigment expression chassis to achieve multiplexed diagnostics without sacrificing the enzymatic signal-to-noise advantage observed in the present single-analyte system.

Real-world applicability was validated by challenging the sensors with Hg(II) in complex environmental matrices without prior sample digestion. Unlike conventional assays that dilute environmental samples to 10–20% to reduce matrix interference,^26–28 we employed high-volume ratios (90% v/v for freshwater matrices, including tap water and lake surface water, and 50% v/v for coastal seawater) to minimize elevation of the detection limit. Both TOP10/pHg-PigC and TOP10/pHg-HapC retained quantitative accuracy across all tested matrices, exhibiting consistent dose–response relationships for 0–100 nM Hg(II) with high coefficients of determination (R² ≥ 0.96) (Fig. 4). Notably, the sensors maintained robust performance even in high-ionic-strength seawater, where dissolved organic matter and competing salts might otherwise interfere with protein function.²⁹ The MerR regulatory element confers selective Hg(II) recognition through specific metal–protein coordination rather than surface charge-dependent interactions, as evidenced by the consistent dose–response performance in high-ionic-strength seawater matrices (Fig. 4), indicating that colloidal stability of the cellular surface does not compromise the genetically encoded sensing mechanism. The progressive darkening of acidified ethanol extraction from colorless to deep red enabled naked-eye discrimination of Hg(II) concentrations at nanomolecular levels, eliminating the need for spectrophotometric analysis in preliminary field screening.

Fig. 4 Hg(II) detection in environmental water matrices. (A) Schematic of the workflow. Prodigiosin production by TOP10/pHg-PigC (B) and TOP10/pHg-HapC (E) in different water matrices (deionized water, tap water, surface water, and seawater) at varying Hg(II) concentrations. (C), (F) Representative photographs of acidified ethanol extracts. (D), (G) Non-linear regression analysis of A₅₃₅versus Hg(II) concentration. Data are mean ± standard deviation (n = 3).

In summary, this work establishes prodigiosin as a high-performance chromogenic reporter for heavy-metal biosensing, representing the first application of this pigment for Hg(II) detection in whole-cell sensors. By leveraging enzymatic signal amplification, inherent photostability, and the high contrast of the red pigment against bacterial cultures, the sensor achieves nanomolar sensitivity with acidified ethanol extraction from the biomass. The modular MerR-Pmer regulatory architecture further enables adaptation to other analytes by swapping the sensory element, expanding the toolkit for visible-wavelength whole-cell diagnostics suitable for on-site environmental monitoring in resource-limited settings.

Conflicts of interest

There are no conflicts to declare.

Data availability

Supporting data are available in the supplementary information (SI). Supplementary information: detailed experimental procedures, optimization of pigment synthesis kinetics, cell growth characteristics, prodigiosin stability assessment, cytotoxicity and cell aggregation analysis, methylmercury selectivity assay, and colony-forming unit viability data. See DOI: https://doi.org/10.1039/d6cc01432a.

Acknowledgements

This work was supported by the Natural Science Foundation of Shenzhen Municipality (JCYJ20230807151400002) and the Natural Science Foundation of Guangdong Province (2025A1515011667).

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Footnote

† Contributed equally.

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