Development of a bioavailable Hg(II) sensing system based on MerR-regulated visual pigment biosynthesis

Abstract

Engineered microorganisms have proven to be a highly effective and robust tool to specifically detect heavy metals in the environment. In this study, a highly specific pigment-based whole-cell biosensor has been investigated for the detection of bioavailable Hg(II) based on an artificial heavy metal resistance operon. The basic working principle of biosensors is based on the violacein biosynthesis under the control of mercury resistance (mer) promoter and mercury resistance regulator (MerR). Engineered biosensor cells have been demonstrated to selectively respond to Hg(II), and the specific response was not influenced by interfering metal ions. The response of violacein could be recognized by the naked eye, and the time required for the maximum response of violacein (5 h) was less than that of enhanced green fluorescence protein (eGFP) (8 h) in the single-signal output constructs. The response of violacein was almost unaffected by the eGFP in a double-promoter controlled dual-signals output construct. However, the response strength of eGFP was significantly decreased in this genetic construct. Exponentially growing violacein-based biosensor detected concentrations as low as 0.39 μM Hg(II) in a colorimetric method, and the linear relationship was observed in the concentration range of 0.78–12.5 μM. Non-growing biosensor cells responded to concentrations as low as 0.006 μM Hg(II) in a colorimetric method and in a Hg(II) containing plate sensitive assay, and the linear relationship was demonstrated in a very narrow concentration range. The developed biosensor was finally validated for the detection of spiked bioavailable Hg(II) in environmental water samples.

Detection of environmental pollutant cadmium in water using a visual bacterial biosensor

“…The overnight LB culture of TOP10/pPcad-vio was used to inoculate fresh LB medium (1% v/v inoculum). After 3 h incubation at 37 °C with shaking at 250 rpm, engineered TOP10/pPcad-vio in early exponential phase (OD 600 = 0.17) was induced with 25 μM Cd(II) for 3 h, and the intracellular violacein agglomerates were extracted with butanol as described previously 19 . The butanol extraction phase was placed at 37 °C and sampled at regular time intervals.…”

“…The violacein biosynthetic pathway has been extensively clarified and employed in several recent studies 15 – 17 . Whole-cell biosensors using violacein as a visible readout have been validated in detecting essential micronutrients such as zinc in biological samples 18 , as well as toxic lead and toxic mercury in environmental samples 19 , 20 . In this study, violacein production is performed by the heterologous biosynthetic genes expressed in bacterial biosensor under the control of Cd(II) sensory elements.…”

Differential Detection of Bioavailable Mercury and Cadmium Based on a Robust Dual-Sensing Bacterial Biosensor

“…An 887 bp fragment containing the promoterless egfp gene and rrn B terminator was PCR amplified from the pT-GFP vector and subcloned into the pPmer vector using the Nde I and Hin dIII restriction sites to generate pPmer-G. Similarly, an 878 bp fragment containing the promoterless mcherry gene and rrn B terminator was PCR amplified from the pT-RFP vector and subcloned into the pPcad vector using the Nde I and Xho I restriction sites to produce pPcad-R. A DNA fragment containing a mCherry open reading frame (ORF) was amplified from pT-RFP and fused with the Hg(II) sensory element merR -P mer by an overlapping extension PCR as described previously ( Guo et al, 2021a ) to generate pR-Pmer-G.…”

“…However, the biosensor sensitivity is usually determined by genetic circuit engineering and types of reporter genes ( Gupta et al, 2019 ; Hui et al, 2020 ). Metalloregulator MerR is a Hg(II)-responsive transcriptional factor, which has been employed to develop bacterial whole-cell biosensors using luciferase ( Din et al, 2019 ), β-galactosidase ( Hansen and Sorensen, 2000 ), fluorescence protein ( Zhang et al, 2021 ), and visual pigments ( Guo et al, 2021a ; Hui et al, 2021a ) as the signal outputs. All of these above bacterial biosensors responded to Hg(II) with high selectivity and variable sensitivity.…”

Optimization of Heavy Metal Sensors Based on Transcription Factors and Cell-Free Expression Systems

“…A number of whole-cell and CFE biosensors have been developed to detect heavy metals in water. ,,− However, few of these sensors have been extensively screened against a broad panel of cations in vitro where the absence of a cell membrane and efflux pumps allows for unfettered interactions between the test ions and sensor biorecognition elements. In the present study, we report the development of CFE biosensors designed to detect the toxic heavy metal contaminants arsenic, cadmium, and mercury.…”