Antimony (Sb) is an emerging contaminant, and its on-site speciation analysis is central to the accurate evaluation of its bioavailability and toxicity. The whole-cell biosensors (WCBs) for Sb(III) are promising but challenging due to the lack of Sb(III)-specific recognition components. Here, we constructed a novel Sb(III)-specific WCB using an Sb(III) transcriptional regulator (antR) and its cognate promoter (Pant). To prevent the promoter leakage of Pant, an additional regulatory gene, antR, was inserted downstream of the Sb(III)-inducible promoter, improving the sensitivity of the WCB by an order of magnitude and reaching the detection limit at 0.009 μM, which is lower than the WHO drinking water standard of Sb. Moreover, the WCB with double antR showed a high specificity toward Sb(III) compared with interfering ions at 3 orders of magnitude higher concentrations. This WCB was capable of measuring Sb(III) bioavailability in natural waters and sediments on-site, and its results were not statistically different from the chemical analysis. The insights gained from this work demonstrate that the addition of regulatory genes prevents promoter leakage and improves the sensitivity of WCBs in field applications. IMPORTANCE Antimony (Sb) is a redox-sensitive pollutant ubiquitous in the environment. Sb(III) is dominant in the subsurface and is readily oxidized to less toxic Sb(V) upon exposure to air, and therefore, on-site Sb speciation analysis is essential to evaluate its bioavailability and toxicity. Dissolved Sb concentration and speciation can be determined accurately using on-site chemical sensors, but chemical sensors have difficulty determining the bioavailable Sb(III) that is taken up by the cells. Here, we constructed an Sb(III)-specific whole-cell biosensor (WCB) using double Sb(III) transcriptional regulators (antR) downstream of its cognate promoter Pant. With an additional antR, the sensitivity of the WCB was improved by approximately 10 times, and the promoter leakage commonly found in WCBs was inhibited. Integrated with a tea-bag design, the WCB is able to measure Sb(III) bioavailability in natural water and sediments on-site. This study demonstrates the importance of inserting one more regulatory gene to improve sensitivity.
Keywords: Sb(III) transcriptional repressors (antR); antimony; bioavailable Sb(III); promoter leakage; whole-cell biosensor (WCB).