Environmental risks continue to grow due to heavy metal contamination caused by anthropogenic activities. Accumulation of harmful quantities of lead poses a threat to aquatic organisms, plants, and human beings. Whole-cell biosensors, which can proliferate independently, can detect the bioavailable fraction to assess the effect of target heavy metal on the environmental ecosystem. In this study, the biosynthesis pathway of violacein was heterogeneously constructed under the control of the T7 lac promoter in E. coli. A dose–response relationship existed between the inducer and the production of violacein. The biosynthesis pathway of violacein was finally engineered under the regulation of Pb(II)-dependent metalloregulator PbrR to assemble Pb(II)-inducible whole-cell biosensor. It permitted specific biosensing of Pb(II) with extraordinary selectivity, and could resist the interferences from various metal ions. Color change by the intracellular accumulation of violacein could be recognized with the naked eye directly with high concentration of lead exposure, and quantified by determining the absorbance at 490 nm after butanol extraction. A good linear range for Pb(II) concentrations of 0.1875–1.5 μM was obtained. The novel pigment-based whole-cell biosensor could detect concentrations as low as 0.1875 μM Pb(II) based on in vitro quantification of violacein extracted by butanol, which is significantly lower than reported fluorescent protein-based PbrR-regulated biosensors based on direct measurement of whole cell fluorescence. These results indicate that genetically controlled violacein biosynthesis can enable a sensitive, visual, and qualitative biosensor for monitoring the presence of bioavailable Pb(II) in lead-contaminated water.

Cadmium (Cd) contamination in water and soil is considered an environmental pollutant. Food crops can absorb and accumulate bioavailable Cd. Continuous monitoring of Cd levels in the environment can minimize exposure and harm to humans. Visual pigments have been demonstrated to have great potential in the development of minimal-equipment biosensors. In the present study, a metabolically engineered bacterium was employed to produce blue-purple pigment violacein responsive to toxic Cd(II). The high stability of the bisindole pigment contributed to determining the violacein at wavelengths of 578 nm. Visual and quantifiable signals could be captured after a 1.5-h Cd(II) exposure. This novel biosensor showed significantly stronger responses to Cd(II) than to other heavy metals including Pb(II), Zn(II), and Hg(II). A significant increase in pigment signal was found to respond to as low as 0.049 μM Cd(II). The naked eye can detect the color change when violacein-based biosensor is exposed to 25 μM Cd(II). A high-throughput method for rapid determination of soluble Cd(II) in environmental water was developed using a colorimetric microplate.

Human exposure to lead mainly occurs by ingestion of contaminated food, water and soil. Blocking lead uptake in the gastrointestinal tract is a novel prevention strategy. Whole-cell biosorbent for lead was constructed with PbrR genetically engineered on the cell surface of Escherichia coli (E. coli), a predominant strain among intestinal microflora, using lipoprotein (Lpp)-OmpA as the anchoring protein. In vitro, the PbrR displayed cells had an enhanced ability for immobilizing toxic lead(II) ions from the external media at both acidic and neutral pH, and exhibited a higher specific adsorption for lead compared to other physiological two valence metal ions. In vivo, the persistence of recombinant E. coli in the murine intestinal tract and the integrity of surface displayed PbrR were confirmed. In addition, oral administration of surface-engineered E. coli was safe in mice, in which the concentrations of physiological metal ions in blood were not affected. More importantly, lead associated with PbrR-displayed E. coli was demonstrated to be less bioavailable in the experimental mouse model with exposure to oral lead. This is reflected by significantly lower blood and femur lead concentrations in PbrR-displayed E. coli groups compared to the control. These results open up the possibility for the removal of toxic metal ions in vivo using engineered microorganisms as adsorbents.

Cadmium (Cd) is carcinogenic to humans and can accumulate in the liver, kidneys, and bones. There is widespread presence of cadmium in the environment as a consequence of anthropogenic activities. It is important to detect cadmium in the environment to prevent further exposure to humans. Previous whole-cell biosensor designs were focused on single-sensing constructs but have had difficulty in distinguishing cadmium from other metal ions such as lead (Pb) and mercury (Hg). We developed a dual-sensing bacterial bioreporter system to detect bioavailable cadmium by employing CadC and CadR as separate metal sensory elements and eGFP and mCherry as fluorescent reporters in one genetic construct. The capability of this dual-sensing biosensor was proved to simultaneously detect bioavailable cadmium and its toxic effects using two sets of sensing systems while still maintaining similar specificity and sensitivity of respective signal-sensing biosensors. The productions of double-color fluorescence were directly proportional to the exposure concentration of cadmium, thereby serving as an effective quantitative biosensor to detect bioavailable cadmium. This novel dual-sensing biosensor was then validated to respond to Cd(II) spiked in environmental water samples. This is the first report of the development of a novel dual-sensing, whole-cell biosensor for simultaneous detection of bioavailable cadmium. The application of two biosensing modules provides versatile biosensing signals and improved performance that can make a significant impact on monitoring high concentration of bioavailable Cd(II) in environmental water to reduce human exposure to the harmful effects of cadmium.