Construction of a sensitive and specific lead biosensor using a genetically engineered bacterial system with a luciferase gene reporter controlled by pbr and cadA promoters

A bacterial biosensor refers to genetically engineered bacteria that produce an assessable signal in the presence of a physical or chemical agent in the environment. We have designed and evaluated a bacterial biosensor expressing a luciferase reporter gene controlled by pbr and cadA promoters in Cupriavidus metallidurans (previously termed Ralstonia metallidurans) containing the CH34 and pI258 plasmids of Staphylococcus aureus, respectively, and that can be used for the detection of heavy metals. In the present study, we have produced and evaluated biosensor plasmids designated pGL3-luc/pbr biosensor and pGL3-luc/cad biosensor, that were based on the expression of luc+ and under the control of the cad promoter and the cadC gene of S. aureus plasmid pI258 and pbr promoter and pbrR gene from plasmid pMOL30 of Cupriavidus metallidurans. We found that the pGL3-luc/pbr biosensor may be used to measure lead concentrations between 1–100 μM in the presence of other metals, including zinc, cadmium, tin and nickel. The latter metals did not result in any significant signal. The pGL3-luc/cad biosensor could detect lead concentrations between 10 nM to 10 μM. This biosensor was found to be specific for measuring lead ions in both environmental and biological samples.

metals in the environment. Lead (Pb) is a toxic heavy metal that is extensively utilized around the world [3,4]. It has been estimated that the world production of lead is more than 3 million tons per year. It causes widespread environmental contamination in the air, water, soil, and food [5]. This element can enter human bodies as well as animals, affecting the integrity of the food chain; in fish it can accumulate in the bone, liver, gills, kidney, ovary, and muscle [6]. Environmental lead may result in high blood concentrations and an increase in vascular endothelial growth factor (VEGF) [7,8], and can lead to neurological and cardiovascular complications [9]. The reproductive system may also lead to developmental disorders in children [10][11][12][13]. Lead can cross the placenta and cause damage to the developing fetal nervous system [14].
The assessment and monitoring of environment heavy metal contamination is therefore very important to prevent harm to human health. Currently, classical analytical methods, such as spectrometry, FIAAS (Flow injection atomic absorption spectrometry), ion chromatography, and electrochemical techniques, are the main methods used for measuring environmental heavy metals pollution. The main disadvantage of these methods is the necessity for sample digestion under high temperature and pressure, or acidic conditions in which metal ions in solution are released [15]. In any case, the specified apparatus is exceptionally expensive, requires appropriately trained analysts, and it may take days or weeks to get results from a specialist laboratory. Therefore, simpler methods for evaluating heavy metals are required. More importantly, heavy metals are found to be present in the biological systems either in bioavailable/toxic or non-available/non-toxic forms, and current measuring methods are unable to distinguish between toxic and non-toxic fractions of these elements [16], and these methods are both timeconsuming and costly [17]. Biosensors have been developed that are an effective alternative to conventional detecting systems. These may be highly sensitive and simple to use [18]. Cell-based biosensors are biological sensors that contain a reporter gene under the control of a promoter that is sensitive to the presence of an agent, such as environmental contaminants that include heavy metals. Biosensors are used in various designs with different reporters and promoters. At low concentration of heavy bioavailable metals, bioluminescence signals are likely to be suitable [19,20]. Hence while classical analytical techniques can detect metal ion contaminants in environmental samples with excellent precision, they are complex and costly and do not differentiate between the unavailable and bioavailable fractions. An approximate of the bioavailable fraction is significant in bioremediation, waste dumping, waste-treatment optimization and the evaluation of environmental impact [21][22][23][24][25][26][27]. Cell-based biosensors can also be applied to monitoring bioavailable concentrations of heavy metals and piezoelectric biosensors as enzymebased electrochemical biosensors [28][29][30]. One of the most obvious advantages of this method is the ability to measure the bioavailable heavy metal at very low concentrations. It is also a cost-effective and time saving method [18]. In these biosensors, the expression of a reporter gene is controlled by a promoter, such as the pbrR promoter in the pMOL30 plasmid of Cupriavidus metallidurans CH34 and cadC promoter in pI258 plasmid of Staphylococcus aureus (S. aureus) that is sensitive to heavy metals. Most of these promoters originate from bacteria that have resistance systems against heavy metals [31,32]. In this study, we have designed and evaluated luciferase reporter gene expression of

Sequencing
In order to ensure the integrity of the sequencing, the promoter region was sequenced in the modified plasmid (Fig. 1c, d). PCR was performed using primers designed for the pbr and cadA promoters, and the promoter sequence and regulatory gene were amplified with 634 bp for pbr and 601 bp for cadA (Fig. 2).

Biosensor activity of pGL3-luc/pbr
The expression of the luciferase gene, in the presence of different concentrations of lead, showed that 1 μM of lead was the lowest concentration that could stimulate the promoter and could be distinguished from the basal expression of luciferase, and the highest measureable expression was seen at 100 μmol/L. A good biosensor should have two characteristics: specificity and sensitivity. According to the data obtained from our experiments, this biosensor had a high specificity, and luciferase gene was only expressed in the presence of lead. The biosensor was cultured in the presence of different concentrations of zinc, tin and cadmium, and did not stimulate the pbr promoter and expression of the reporter gene ( Fig. 3). In Fig. 3, we aimed to show that the pbr promoter is specific to lead, and other heavy metals such as zinc (Zn) (Fig. 3a), tin (Sn) (Fig. 3b) and cadmium (Cd) (Fig. 3c) do not activate the promoter and significant expression of a reporter gene. Data obtained from the expression of the luciferase gene in the presence of various concentrations of tin, zinc and cadmium, indicated that these heavy metals did not stimulate the pbr promoter.

Biosensor activity in the presence of different concentrations of lead (PbCl 3 )
Lead was the only metal that stimulated the pbr promoter. In the absence of lead, the regulator gene prevents the promoter from activation. Lead ions bind to the regulator gene and inhibits its binding to the operator. As a result, the promoter is activated and the luciferase is expressed. The minimum detectable concentration of this biological sensor was approximately 1 µM and a maximum is 100 μmol/L. The expression of luciferase was no longer linear for value of lead from 100 to 200 μmol/L (Fig. 4a).

The expression of pGL3-luc/ pbr biosensor reporter gene at different times
In order to identify the appropriate time for biosensor growth, a biosensor was cultured at different concentrations of lead for different durations (Fig. 4b). The maximum expression of the luciferase gene was at 12 h (Fig. 5a).  The difference in the growth rate of pGL3-luc/ pbr biosensor compared to E. coli strain

DH5α
The sensor bacteria had a recombinant plasmid containing the pbr promoter region and the pbrR regulatory gene. These bacteria have a greater resistance to lead than E. coli DH5α without plasmid. This resistance may be related to the pbrR regulatory gene (Fig. 5b). The resistance genes to heavy metals have heavy metal-binding motifs, they can limit the toxicity of these metals inside the cell, because of these proteins, the relative resistance of the cell to heavy metals.

The activity of pGL3-luc/cad biosensor at different concentrations of lead
The lowest and highest concentrations of lead that could stimulate the expression of the reporter gene were 10 nmol/L and 10 μmol/L, respectively (Figs. 6 and 7a). The sensor bacteria were incubated at 0.2 OD (1 μmol/L concentration) for different times in the incubator. The expression of luciferase was measured at different times (Fig. 7b). As shown in Fig. 7b, the concentration of 1 μM lead can induce luciferase expression. The degree of expression increased with time, with measureable change in luciferase levels by 2 h measure, and in biological sensors pollution is usually measured at low rates, we chose 2 h for culture of the pGL3-luc/cad biosensor.

Discussion
Because of global industrialization and various geochemical processes, heavy metals and metalloids are the natural parts of an ecosystem which approach the food chain. Only a small rise in these non degradable pollutants' concentration creates a serious danger to organisms [33]. Heavy metals, such as organic pollutants, are not degradable but can be transformed to exist in less toxic form. Microbes are the cheap weapon since they change quickly to overcome heavy metal pressure by creating appropriate survival techniques, like sequestration or active metal transport [34]. The key sources of pollutants in water quality are heavy metal ions like Pb 2+ and Cd 2+ . Recently, full-cell detection has been extensively investigated to use genetically modified bacteria to detect the existence of heavy metal ions in water or soil. Whole-cell sensors require simple sample preparation and can continually sense metal contaminants in the cell culture environment in comparison with main cell-free techniques like immunosensor and electrochemical sensor [35]. It was indicated that strain C. metallidurans CH34 is facultative chemolithoautotrophic β-proteobacterium in the Burkholderiaceae/order Burkholderiales family. It was shown to be heavily resistant to Zn 2+ , Cd 2+ , Ni 2+ , AsO 43− CrO 42− , Hg 2+ , Ag + , Cu 1+ / 2+ , Pb 2+ , and Co 2+ [36]. The whole-cell biosensors have been successfully produced using fluorescent and enzymatic reporters as elements of signal-output based on the natural pbr operon [37]. Biosensor is an analytical tool used to detect the targeted compounds easily and quickly. Furthermore, by cadC gene expression and promoter cad of S. aureus plasmid pI258 with GFP gene in E. coli DH5α, a whole-cell biosensor was developed for detecting toxic cadmium metal ions. The response time was 15 min, with a 10 μg/L detection limit. Luciferase reporter gene has also been expressed based on similar promoter and resistance determinant in S. aureus RN 4220 and Bacillus subtilis BR151. Cadmium, lead and zinc were detected by the resultant luminescent sensor [38]. There are several advantages to using bacterial biosensors, including speed, simplicity and cost. Biological sensors containing cadA and pbr promoter regions have been designed by other researchers, the optimization of this cell biological sensor with ability to measure lead comparing the cadA and pbr promoters in a bioassay system was evaluated in this study. The use of biosensors or biological cell sensors containing a reporter gene controlled by promoters susceptible to the heavy metal ions can provide an efficient method to trace particular pollutants in the environment and in a biological solution [39]. The present study assessed a biosensor system for detecting lead ions through construction of a luminescent bacterial sensor containing the luc + regulated by the cad promoter and cadC gene in plasmid pI258 of S. aureus and the pbr promoter and pbrR gene in pMOL30 plasmid of Cupriavidus metallidurans. Pb-specific bacterial biosensors were formerly defined using reporter genes including lacZ, lux, and luc in the transcription fusion constructs [40][41][42]. In our study, the luciferase reporter gene was used. Luciferases, as a set of heterogeneous enzymes, are able to produce light as a byproduct of catalyzing reactions. They are reporter genes extensively used by prokaryotic and eukaryotic organisms due to their high sensitivity and ease of detection.The quantification of the emitted light, i.e., bioluminescence,is of great importance; it can also be measured using a liquid scintillation counter, a luminometer, or even a X-ray film [41]. It was concluded that a pGL3-luc/pbr biosensor can detect Pb 2+ in the range of 1-100 μM using the expression of firefly luciferase as a detector system, and is highly specific, with no expression of reporter in the presence of other metals such as Sn 2+ , Ni 2+ , Cd 2+ are present. Moreover, this biosensor was 50 times more sensitive when compared with the previous biosensors reported by Chakraborty et al. [32]. The R. metallidurans CH34 strain has several resistance systems that can reduce the concentration of toxic substances to their non-toxic levels. A highly specific system for resistance to lead is known in plasmid pMOL30 [43]. It effectively reduced the concentration of lead ions and is equipped with specific mechanisms for the transfer and separation of lead. The pbr operon includes pbrA, pbrB, pbrC and pbrD genes in which pbrD has a role as a chaperone to accumulate lead in the cell and pbrA eliminates lead ions [43].
Our results show that the pGL3-luc/pbr biosensor is not expressed in the presence of cadmium, zinc, or tin, indicating high sensitivity and specificity of the designed system for lead detection. One of the most important heavy metal transfer systems in S. aureus is located in the plasmid pI258. The plasmid has an operon cadA that encodes an ATPas of type P, which causes resistance to metals such as cadmium, lead, zinc, copper, and tin. The expression of the cadA operon is controlled by the cadC homodimeric protein.
This protein is able, in a binary manner, to bind to the promoter and metal ions, such as cadmium, lead, zinc, and tin. The cad belongs to ArsR/SmtB, a regulating protein family [44]. In our study, the luciferase gene was used as a reporter and E. coli strain of DH5α as a host. Our results showed that the pGL3-luc/cad biosensor can detect at least 10 nM of lead and the lead toxicity was not observed until a concentration of 300 μM. However, the maximal expression of the reporter gene was performed at 10 μM. Our results are supported by the report of Liao et al. that showed the regulating role of cad promoter and the cadC gene in plasmid pI258 of S. aureus, the fluorescence emission was intensified with increasing Cd(II), Pb(II), and Sb(III) ions concentrations [45]. For Pb (II), just like our result in pGL3-luc/cad biosensor, to induce GFP expression significantly, 10 nM was the low, and 10 μM was the maximum concentration of lead that induced significantly GFP expression [45]. The metallo-regulatory α 3 N thiolate-rich site in cadC displays a practical selectivity for larger, softer heavy metal like Pb(II), Cd(II), although smaller boundary metal ions such as Zn(II) accommodated [46]. One of the limitations of this method is that bacterial biosensors require the necessary conditions for bacterial growth to operate, and the graphs are based on solving different concentrations of heavy metals in a bacterial culture medium. Therefore, to measure the amount of heavy metals in an unknown environment, it is necessary to optimize the biosensor in the new environment, which would itself require evaluation.

Conclusion
Our results show that the maximum expression of reporter gene was found in the presence of 100 μM of Lead in pGL3-luc/pbr biosensor and 1 μM of lead in pGL3-luc/ cad biosensor. In this study, the specificity and sensitivity of the two heavy metal susceptive probes, pbr and cadA, were investigated. Sensors containing these two promoter regions were able to detect the concentration of lead between 1-100 μM and 10 nM to 10 19:79 was plotted at different lead concentrations. The standard curve was constructed from triplicates values, we evaluated the accuracy of the biosensor with the specific concentrations that we had obtained from lead metals. By developing these sensors, the time required to identify environmental pollution can be minimized.

Construction of biosensor plasmid
pMOL30 (X71400 AJ278984) and PI258 (GQ900378.1) containing the pbrR gene (634 bp) and CadC gene (601 bp) (Accession number: pbrR: WP_003103716.1and CadC: WP_000726009, respectively, were synthesized and supplied by Millegen company. To ensure the accuracy of synthesized plasmid, the promoter region was sequenced. PGl3control as a vector containing the Luciferase gene and E. coli strain DH5α as the host were used in our study. To obtain a large amount of pMA-T plasmid (a synthetic plasmid) which contains p-promoter sequences and the regulatory gene was sent to Mil-liGen, after evaluation at the NCBI site, for the synthesis of sequences. Synthesized sequences consisted of both pbr_pMA-T plasmids containing the promoter sequence of the pRR operon and the pbrR regulator gene including; cadA pMS-RQ-Bs plasmid containing the promoter region of the cadAp and the cadA gene regulating gene), it was cloned to E. coli host. Afterwards, pMA-T was extracted using plasmid extraction kit, and its quantity and quality were both examined by spectrophotometry and agarose gel, respectively, before they got digested by HindIII and NcoI. The promoter regions with the regulator genes were also purified from the gel electrophoresis. The received sequence and pGL3-control vector were cut using the same restriction enzyme (Nco1 and Hind3) and ligation reaction at 37 °C for 3-4 h with ligase enzyme. The firefly luciferase gene was placed under the control of the received promoter sequences and recombinant plasmids of cad and pbr promoters were named pGL3-luc/pbr biosensor and pGL3-luc/Cad biosensor, respectively. Recombinant plasmids pGL3-luc/pbr biosensor (Fig. 1a) and pGL3-luc/Cad biosensor (Fig. 1b) were transferred to the DH5α bacteria using the chemical method of CaCl2 and then were screened using selective plates containing antibiotic ampicillin. After plasmid extraction, PCR was performed to detect colonies containing the promoter region of pbr and cadA using primers designed for the cloned fragments. After these processes, recombinant plasmids were used to evaluating and measuring different concentrations of heavy metals.