Use of Tunable Whole-Cell Bioreporters to Assess Bioavailable Cadmium and Remediation Performance in Soils

Abstract

It is important to have tools to measure the bioavailability to assess the risks of pollutants because the bioavailability is defined as the portions of pollutants showing the biological effects on living organisms. This study described the construction of tunable Escherichia coli whole-cell bioreporter (WCB) using the promoter region of zinc-inducible operon and its application on contaminated soils. It was verified that this WCB system showed specific and sensitive responses to cadmium rather than zinc in the experimental conditions. It was inferred that Cd(II) associates stronger with ZntR, a regulatory protein of zinc-inducible operon, than other metal ions. Moreover, the expression of reporter genes, egfp and mcherry, were proportional to the concentration of cadmium, thereby being a quantitative sensor to monitor bioavailable cadmium. The capability to determine bioavailable cadmium was verified with Cd(II) amended LUFA soils, and then the applicability on environmental systems was investigated with field soils collected from smelter area in Korea before and after soil-washing. The total amount of cadmium was decreased after soil washing, while the bioavailability was increased. Consequently, it would be valuable to have tools to assess bioavailability and the effectiveness of soil remediation should be evaluated in the aspect of bioavailability as well as removal efficiency.

Modulating the Properties of Metal-Sensing Whole-Cell Bioreporters by Interfering with Escherichia coli Metal Homeostasis

“…With wild-type WCBs, EGFP induction was observed after cadmium and mercury treatments, consistent with our previous report [12]. However, the metal selectivity was abolished from ∆zntA WCBs.…”

“…Deletion of zntA Abolishes the Metal Selectivity of WCBs Harboring pZntA-EGFP Previously, we used WCBs harboring pZntA-EGFP as a biosensor to quantify bioavailable cadmium in contaminated [12]. The WCBs employed the promoter region of the E. coli znt operon as a sensing domain and egfp as a reporter domain.…”

“…The plasmids pZntA-EGFP and pNik-EGFP, which possessed fusions between the promoter regions of the znt and nik operons, respectively, and egfp, have been previously described [12,15]. The promoter regions were amplified from E. coli chromosomal DNA by PCR and inserted into pET-21(a) with BglII and XbaI; egfp was inserted downstream with BamHI and XhoI.…”

“…However, the selectivity and sensitivity of these biosensors vary depending on the nature of the promoters and regulatory proteins. For example, although the E. coli znt-operon promoter responds to zinc, a WCB harboring znt-promoter::egfp (encoding enhanced green fluorescent protein (EGFP)) responded to cadmium best, followed by mercury [12]. In addition, although it is known to be copper-responsive, an E. coli cop-operon promoterbased WBC had insufficient sensitivity to detect copper.…”

“…In this study, we demonstrate approaches to enhance and modulate the selectivity and sensitivity of WCBs by interfering with the metal homeostasis system and engineering metal-binding regulatory proteins in E. coli. Genes in the znt operon, which are responsive to divalent metal cations such as Pb(II), Zn(II), and Cd(II), were deleted, and their effects on metal-sensing WCBs carrying plasmids harboring zntAp::egfp and nikAp::egfp, which are used to quantify bioavailable cadmium and arsenic in soils, respectively [10,12], were investigated. Interruption of metal homeostasis by gene deletion and regulatory protein bioengineering changed the properties of the WCBs, providing an alternative method to enhance existing WCBs and generate novel WCBs targeting additional materials.…”