Arsenic (As)-contaminated ground water, occurring from mining or agriculture or natural contamination due to the abundance of arsenic in the Earth’s crust, is a serious global health issue. Human exposing to high As groundwater are at risks of keratosis, hyperpigmentation, dermatological problems, gangrene, and cancer risk. The non-carcinogenic or carcinogenic effects of groundwater As on residents have been reported globally[1]. The serious waterborne As poisoning affects the health of millions of people in many countries, such as America, India, Bangladesh, Hungary, and China. For us living in the Jianghan Plain, it is not only a global but also local problem. More than 73 thousand people, including 20 thousand children, are exposed to the risk of As poisoning in the Jianghan Plain[1,2]. Previous studies found that the depth of high As concentration distributing in groundwater also corresponds well with that of wells where residents obtain for drinking water, which will endanger the safety of residents living in Jianghan Plain. Due to its toxicity and strict arsenic standards for drinking water, cost-effective and sensitive environmental monitoring tools to detect arsenic are needed.
Fig 1. Spatial distribution of arsenic concentration (in ppb) in the Jianghan Plain[1]
Many methods have been reported to detect arsenic at low concentrations, such as chemiluminescent immunoassay, inductively coupled plasma optical emission spectrometry (ICP-OES), and atomic absorption spectrometry (AAS)[2-4]. However, these methods often require complicated and expensive instruments and trained professionals to pretreat and analyze samples, making them hard to use in-situ. To overcome these limitations, biosensors using enzymes, antibodies, and microorganism cells have garnered interest for use in the detection of arsenic in drinking water. Especially, whole-cell biosensors (WCBs) have been studied for the specific and sensitive detection of toxic heavy metal ions. Often, the regulatory elements from a heavy metal resistance operon, including the transcriptional regulator and its cognate promoter, are coupled to a reporter gene such as fluorescence, luminescence, or enzyme assays so that the signal strength from the reporter is correlated to the concentration of the heavy metal to be detected[4,5]. However, low sensitivity and specificity are major issues when using them for arsenic detection.
Recently, extensive research has been carried out for improved sensitivity of electroactive biofilm-based sensor (EAB-sensor), which is recognized as a useful tool in water quality early-warning. EABs are biofilms formed by electroactive bacteria which can interact with electrodes by either transferring electrons to or accepting electrons from the electrodes[6]. EAB enables spontaneous generation of electrical signals without the need of additional chemical mediators. Moreover, biofilms can establish themselves on virtually any accessible surface and exhibit inherent tolerance to harsh conditions. Owing to the self-immobilization, self-sustainability, and high robustness of EABs, EAB-based biosensors have shown promising environmental applications, including pollutant detection and early-warning for water safety[7].
Fig 2. Design and principle of EAB-enbaled biosensors[6]
Compared to the WCBs, electroactive bacteria can colonize on electrodes to form 3-dimensional (3D) structures with cells embedded in a matrix of self-produced extracellular polymeric substances (EPS)[5,8]. The preparation of EAB-based biosensor is a straightforward process which does not require biochemical purification, cell entrapment, and exogenous redox mediators electrically connecting the biocatalyst to the electrode. The natural immobilization of EABs can efficiently retain the metabolic functionality of the bacteria, exhibit self-maintenance, and regenerate, especially when biofilm thickness is in a certain range that allows highly efficient mass transfer. All these features make EAB-enabled biosensors promising for environment applications[6,8]. Increasing number of studies have reported the application of EAB-based biosensors in water quality monitoring.
In 2023, the CUG-China team aims to develop an EABs-enabled biosensors to sense arsenic by using the electroactive bacterium S. oneidensis MR-1 as chassis cells. S. oneidensis MR-1 was among the first identified microorganisms capable of using minerals that contain Fe(Ⅲ), Mn(Ⅲ) or Mn(Ⅳ) as terminal electron acceptors[9,10]. Genetic studies revealed the direct involvement of six multihaem c-type cytochromes— CymA, Fcc3 (also known as FccA), MtrA, MtrC, OmcA and small tetrahaem cytochrome (STC) — and the porin-like outer membrane protein MtrB in the extracellular reduction of minerals that contain Fe(Ⅲ)[11-13]. Functional characterization has confirmed that CymA oxidizes quinol in the cytoplasmic membrane and transfers the released electrons to the periplasmic c-Cyts Fcc3 and STC. MtrA, MtrB and MtrC form a trans-outer membrane protein complex that transfers electrons from the periplasmic proteins to the bacterial surface[17,18]. Finally, on the bacterial surface, MtrC and OmcA can physically interact with each other and transfer electrons directly to minerals that contain Fe(Ⅲ) or electrodes[18-20].
Fig 3. Metal reducing pathways (Mtr) of S. oneidensis[10]
To summarize, CymA, Fcc3, MtrA, MtrB, MtrC, OmcA and STC form a pathway that oxidizes quinol in the cytoplasmic membrane and transfers the released electrons across the entire width of the cell envelope to the surface of minerals[9,10].
S. oneidensis is widely used in bioelectrochemical systems (BESs) for various biotechnological applications, such as bioelectricity generation via microbial fuel cells (MFC)[18,21]. These applications mostly associate with biofilms grown on the surfaces of electrodes. Shewanella biofilms are electrically conductive, which is conferred by matrix-associated electroactive components such as c-type cytochromes and electrically conductive nanowires[6,8,10].
Although EAB-based biosensor has many advantages, it is still limited by relatively low sensitivity, low specificity, and instability[6,8].
Sensitivity is a crucial parameter that evaluates functional characteristics of biosensors. A desirable EAB as a sensitive element should exhibit the following characteristics: efficient mass transfer to facilitate analytes access to cells and efficient electron transfer capable of delivering comparable electrical signals in the presence of analytes[6,22]. The sensitivity of an EAB-enabled biosensor is often restricted by the mass transfer barrier caused by EPS and the low EET efficiency, especially in EABs formed by microbial communities[23]. To improve the sensitivity of sensor, we enhanced EET efficiency in S. oneidensis MR-1 biofilm by engineering biofilm matrix. As a bridge between the microbial cells and terminal electron acceptors, the conductivity of EPS matrix, often occupying 50-90% of biofilm biomass, has gained much research attention[8].
Fig 4. The complex structure of Shewanella biofilms in a bioelectrochemical system[8]
Microcolonies in the mature biofilm are characterized by EPS matrix consisting of polysaccharides, proteins and extracellular DNA (eDNA). Different from that of nonelectrogenic bacteria, the biofilm matrix of exoelectrogens is electrically conductive and contains extracellular electron transfer components, such as cytochromes and other electron mediators in Shewanella EPS matrix[8,10,21]. Previous study showed that Shewanella biofilms with less polysaccharide produced higher electrical outputs of MFCs. We found the ptpA gene in S. oneidensis genome encodes a tyrosine phosphatase involved in the biosynthesis of EPS extracellular polysaccharide[3]. Un-conductive polysaccharide in S. oneidensis MR-1 biofilm matrix attenuate the efficiency of extracellular electron transfer. Thus, we knocked out ptpA from S. oneidensis MR-1 genome to enhance its ability to generate electricity. Our results show that the ability of the mutant strain to form biofilm on both conductive (well plate) and non-conductive (anode) surfaces significantly reduced. Moreover, we found that the engineered strain formed a thinner but more conductive biofilm on the surface of anode in MFCs. We conducted the arsenic shock experiments, and found that the mutant strain exhibited a higher sensitivity than WT, increasing the detection range of arsenic[5,24].
Another major challenge for sensor applications is to specifically detect analytes of interest in complex matrices containing other chemicals. Signal interference might occur when EAB-enabled biosensors are applied to complex aquatic environments[24,25]. The key to developing a sensitive and specific biosensor is to identify the regulatory elements and then optimize performance by engineering the regulatory elements or the genetic circuit. A relatively well-studied arsenic resistance operon is the one found in Escherichia coli, which contains arsR (transcriptional regulator), arsB (arsenite permease), and arsC (arsenate reductase) [26,27]. When arsenic is absent, the transcription regulator ArsR binds to the ArsR-binding site (ABS) within the ars promoter and blocks transcription. Once arsenic is present, it binds to ArsR and activate the transcription of the ars genes and clear arsenic in the cell [5,28,29]. The arsR regulator and the promoter of this operon have been used to construct arsenic whole cell biosensors (WCB) in various microorganism hosts [3,30,31].
Fig 5. Gene circuit we designed for arsenic response
We relied on an mtrC complementation strategy to construct arsenic-responsive genetic circuits in plasmids[3,6]. MtrC plays an essential role in the metal reduction (Mtr) pathway of S. oneidensis MR-1, stabilizing a complex formed with MtrA and MtrB. Because this stable complex is required for electrode reduction, strains deficient in the mtrC coding sequence are unable to produce significant levels of current when inoculated into BESs [6,8,10]. However, when mtrC is re-introduced into a knockout strain, the electrode reduction phenotype is restored. When inoculated into a BES, current production will increase in response to increasing mtrC transcription in such an engineered strain. We exploited this by placing the mtrC coding sequence under the control of an arsenic-inducible promoter region[3]. The arsenic-inducible promoter (Pars) is negatively regulated by ArsR. When arsenic is excluded from a cell with these genetic components, the binding kinetics between ArsR and its associated operator region within Pars are relatively strong, blocking the transcription of downstream genes. In this design, ArsR acts as a negative auto-regulator by limiting its own expression to a low, basal level. The dynamics of the circuit change, however, when arsenic enters the cell. In this condition, arsenic associates with ArsR, inducing a conformational change that leads to its dissociation from the Pars operator. Because the binding kinetics between ArsR and the ABS become more unfavorable with increasing arsenic concentrations, MtrC levels—and hence, electrode reduction capacities—increase with increasing arsenic presence. We also applied to another c-Cyts gene, cymA, as the reporter gene to response arsenic.
In order to improve the performance of our arsenic biosensor, we constructed a modular positive feedback-based amplifier. Positive feedback is a common mechanism used in the regulation of many gene circuits as it can amplify the response to inducers and also generate binary outputs and hysteresis[6,32]. In the context of electrical circuit design, positive feedback is often considered in the design of amplifiers. Similar approaches, therefore, may be used for the design of amplifiers in synthetic gene circuits with applications [9,23,29]. We developed a modular positive feedback circuit that can function as a genetic signal amplifier, heightening the sensitivity to inducer signals without the need for an external cofactor. The design utilizes a constitutively active, autoinducer-independent variant of the quorum sensing regulator LuxR.
In the lux system, LuxI produces 3OC6HSL, which diffuses in and out of the cells. The receptor LuxR and 3OC6HSL form LuxR-3OC6HSL complexes which associate further to polymers[8,32,33]. After binding of the polymer to the lux operon, it positively regulates the gene transcription of luxI. As LuxI (in contrast to the LuxR) is encoded on the lux operon, the system contains a positive feedback. The operon constitutively produces the autoinducer in low amounts. If the cell density increases, the positive feedback loop is induced, resulting in an increased autoinducer production.
Fig 6. Amplifier gene circuit
In order to construct a positive feedback circuit which does not require 3OC6HSL produced by LuxI, we engineered LuxR by deleting 2-262 amino acids in the N-terminal domain (AHL binding domain) and reserving a C-terminal domain with the function of activating transcription, obtaining a resulting regulator LuxR(△2-162). LuxR(△2-162) can active the gene transcription driven by the lux promoter in the absence of AHL. To construct the amplifier, we cloned gfp and LuxR(Δ2-162) behind the lux promoter. In this design, LuxR(Δ2-162) functions in a positive feedback loop as it can bind to the PluxI promoter and activate its own transcription.
Fig 7. LuxR based amplifier for arsenic detection
After the modification of the LuxR(Δ2-162), we applied the amplifier into the arsenic-response system we build. The transcriptional activator, LuxR(Δ2-162), was used to replace mtrC as reporter, and it was regulated by the arsR-Parscircuit, while mtrC together with a second LuxR(Δ2-162) was placed under the promoter PluxI, which was activated by LuxR(Δ2-162). When arsenic is present, it activates the expression of LuxR(Δ2-162) in the first circuit, which turns on the expression of mtrC and LuxR(Δ2-162) from the following circuit. The second LuxR(Δ2-162) activates its own expression as well as that of mtrC and forms a positive feedback loop to enhance the output signal from mtrC. These two parts work together as the arsenic EAS-based sensor with the positive feedback amplifier. Our results suggested that, compared with the sensor without positive feedback, the one with a positive feedback amplifier functions well in enhancing the iron reduction rates and better performance in MFC, increasing the detection range, and improving sensitivity. By introducing the positive feedback amplifier into the arsenic sensor, the output signal was enhanced so much that the specificity of the sensor toward arsenic was also significantly increased.
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