Construction of a bacterial biosensor for zinc and copper and its application to the development of multifunctional heavy metal adsorption bacteria

Abstract

In this study, we designed and applied molecular biosensors for heavy metals, zinc and copper, for use in bioremediation strategies. Bacteria utilize two component systems to sense changes in the environment by multiple signal components including heavy metals and control gene expression in response to changes in signal molecules. zraP and cusC promoters were selected from a genetic circuit of the ZraSR and CusSR two-component system and were fused to a dual-labeling reporter protein as an interactive biological component for zinc and copper to generate a signal from the constructed biosensor. The biosensor efficiently senses zinc and copper with a calculated detection limit of 16 μM and 26 μM, respectively, and was shown to be a sensitive and effective heavy metal monitoring bacterial system. To extend the application of the bacterial biosensor, we assembled a bioadsorption system that can trigger bacteria to sense and adsorb 13 ± 0.3 mg/L of zinc and 11.4 ± 0.42 mg/L of copper per gram of dry cell weight with induction at a concentration of 100 mg/L of the respective metal ion.

Introduction

Environmental pollution by heavy metals is a serious problem in both developing and developed countries worldwide. Therefore, it is necessary to be knowledgeable about the possible effects caused by the increasing levels of heavy metals on human health and the environment. For monitoring pollution, heavy metal concentrations have been quantified by analytical methods, which are expensive and frequently require substantial pretreatment of the samples [1]. Therefore, it is necessary to develop an effective and economical method that can efficiently monitor environmental quality and determine the quantity of hazardous metals. To detect the bioavailable fractions of certain metals, several approaches have been attempted by researchers over the past few decades. One of the best approaches is based on the use of genetically engineered bacteria, which produce measurable signal when the bacterium comes in contact with biocomponents [2], [3]. So far, several metal-specific bacterial sensors for the detection of bioavailable metals have been developed [4], [5], [6], [7], [8] but none of the constructs sense the heavy metal exogenously using bacteria. At present, to remove heavy metals from metal contaminants, a number of expression systems promoting the display of chimeric proteins on the surfaces of microbial cells have emerged for the bioremediation of heavy metals. Cell surface display technology has made a wide range of applications in the biotechnological and industrial fields possible including the recovery of harmful chemicals and heavy metals, live vaccine development, and screening of peptide libraries without causing metabolic abnormalities in the host system [9], [10], [11], [12].

In recent years, zinc and copper biosensor developed on the basis of the bacterial regulatory system namely zntA–zntR and CueO present in the bacterial cytoplasm, which encodes the zinc and copper efflux protein, respectively, leading to detoxification of zinc and copper. At higher concentrations of Zn²⁺ and Cu²⁺, the system fails to efflux the metal and is unable to sense exogenous metal [13], [14]. However, ZraSR and CusSR, a two-component membrane associated sensor kinase system, senses exogenously respective metals and even responds preferentially to even at higher concentrations of metals. Two component systems (TCS) in bacteria serve as a basic stimulus-response coupling mechanism to allow organisms to sense and respond to changes in many different environments [15]. Each TCS consists of a sensor protein-histidine kinase (HK), which senses a specific environmental stimulus, and a corresponding response regulator (RR), which controls the differential expression of target genes [16]. To date, among the various TCS identified, ZraSR and CusSR have been found to sense Zn²⁺ and Cu²⁺, respectively, exogenously for a metal efflux system. In ZraSR and CusSR, a membrane-associated sensor kinase and a response regulator help in regulating the expression of zraP and cusC, which are involved in transcription regulation of the genes for zinc and copper homeostasis, respectively [17], [18].

In the present study, we constructed a novel dual reporter-based two component system sensor plasmid capable of monitoring the heavy metals, zinc and copper in exogenous media. We also extended the application of the biosensor to concomitantly develop a bioadsorption plasmid to express a metal binding peptide onto the cell surface of bacteria using the outer membrane protein C (OmpC) surface anchor, resulting in engineered bacteria that can sense and adsorb exogenous zinc and copper. Escherichia coli OmpC is one of the most abundant and well characterized cell surface display system [19]. For the construction of the biosensor, we integrated two reporters, namely GFP and RFP-based sensor cassettes, that can specifically respond to zinc and copper heavy metal signal molecules. Moreover, the two bi-directionally encoded fluorescent proteins could be coordinately expressed. Here, we demonstrate that the presence of zinc and copper allows visualization of the GFPs and RFPs in living cells. This quantitative analysis at the single cell level indicates that our system represents a novel and useful tool for real-time and multicolor monitoring of heavy metals in exogenous media.