"Patrolmen" for the mercury contaminated soil
Soil has always been important to humans, providing a resource that can be used for shelter and food production. However, the problem of soil pollution is very serious. Mercury pollution is one typical type of heavy metal pollution in soil, which will cause serious adverse effects on ecosystem and human health. So prevention of contaminated soil is closely related to health problems in soil-plant-animal-human system. Here, we designed a "patrolmen" for the mercury contaminated soil, which can detect and remedy the mercury contaminated soil. "Patrolmen" includes multifunction engineering bacteria and earthworms. Multifunctional engineering bacteria were cultured, with MerR-dependent hypersensitive switch, mercury monitor, and mercury stabilizer. Once mercury ions are found in the soil, multifunctional engineering bacteria scattered in the soil can be activated, releasing chromogenic protein and stabilizing mercury ions in the form of HgS with low toxicity. Moreover, considering engineering bacteria move slowly in the soil and is greatly affected by the environment, we choose earthworm as the walking carrier. The engineering bacteria will be reproduced in the earthworm body and move through soil followed with the earthworm to monitor and remedy mercury contaminated soil movably.
A.MerR-dependent sensor–hypersensitive switch
A sensor is hoped to regulate the module for mercury detection and remediation, which is not only to improve the efficiency of engineering bacteria to remediation mercury contaminated soil, but also to save the resources of engineering bacteria. MerR proteins are chosen to do this! MerR-dependent sensor is shown to be an activator of the mer genes in the presence of Hg (II) salts and a weak repressor in the absence of Hg (II), as a hypersensitive switch on the module for mercury detection and remediation. MerR also regulated its own synthesis. MerR proteins are located upstream of the mer operons. The N-terminal regions contain predicted helix-turn-helix motifs, which are thought to define the DNA binding regions. The C-terminal regions are originally suggested as the sites of Hg (II) binding by three cysteine residues. The details of MerR-dependent regulation at the merOP region are given in Figure 1. As shown in Figure1, both activation and repression of the mer genes occurred with the MerR regulator bound at the same site on DNA. In the absence of Hg (II), MerR protein is bound to DNA in the repressor conformation and RNA polymerase recruited maintaining repression of the promoter (Figure 1A). In the presence of Hg (II), binding of Hg (II) to one of two binding sites on the MerR causes DNA distortion then gives a unwinding of DNA and straightening of helix backbone to put MerR in the activating conformation (Figure 1B). The reorientation allows them to interact productively with the RNA polymerase subunit to form an open transcriptional complex and transcription is initiated (Figure 1C).
A: MerR bound to DNA and RNA polymerase recruited but not able to form an open complex. B: The conformational change induced by Hg (II) binding to the ternary complex and initiation of transcription. C: The hypothetical repression of the promoter by displacement of Hg–MerR2 with MerD at low Hg. MerR is shown in green and MerD in orange; the −10 and −35 sequences are indicated by red boxes. The separate RNA polymerase holoenzymes are: α=pink, ꞵ,ꞵ= blue, =green. (Ref. Brown NL, Stoyanov JV, Kidd SP, Hobman JL. The MerR family of transcriptional regulators. FEMS Microbiology Reviews, 2003, 27 (2-3): 145–163.) In addition, in order to improve the detection sensitivity of Hg (II) in mercury contaminated soil, Hg (II) signal amplification module is supplemented by reference to the BEAS_China team (a project won the iGEM Gold Award in 2019). Hg ions directly control the expression of RinA_p80α activator, and the activation promoter PRinA_p80α of RinA_p80α activator controls the output BFP (blue fluorescent protein). When Hg ions activate the expression of RinA_p80α activator, RinA_p80α activator will activate BFP expression on high-copy promoters, leading to a higher level of fluorescence values. The principle of signal amplifier is as follows:
B.Mercury ion colorer
Once Hg (II) in soil is detected by the module for mercury detection and remediation, in order to show color change to warn farmers or land owners obviously that the land has been contaminated with mercury, we insert a fluorescent protein followed the structural genes of MerR promoter transcription. After the release of the fluorescent protein, it will combine with Hg (II) in soil to express color rendering. Meanwhile, to avoid confusion with the red or yellow of the soil itself and the green of bryophytes on the soil surface, we choose BFP, a blue fluorescent protein.
C.Mercury ion fixation and remediation
In the module for mercury detection and remediation, the downstream structural genes of MerR promoter transcription also include the proteins which can fix and remedy Hg (II) in mercury contaminated soil. They will enrich and fix Hg (II) firstly then stabilize Hg (II) to HgS with low biological toxicity. They are mainly composed of three proteins: organomercurial lyase (MerB), metallothionein (MT) and Arabidopsis L-cysteine demercase (AtLCD).
C1 Organic mercury dissociation
Mercury is mainly present in soil in two chemical forms–inorganic salts and organomercurial compounds, such as methylmercury (MeHg), the most toxic form. Therefore, the transformation of organomercurials into inorganic mercury ions is particularly important in the fixation and remediation of mercury contaminated soil. Here, MerB is used to catalyze the protonolysis of the carbon-mercury bond in organomercurials, resulting in the formation of a reduced hydrocarbon and Hg (II) (eq 1).
MerB adopts a 3D structure characterized by six β-helices and three separate antiparallel α-sheets (Figure 3A). This unique structure leads to its unusual enzymatic activity. Among them, two conserved cysteines (Cysl59 and Cys96, as shown in Figure 3B) are believed to be critical for the enzymatic activity of MerB. Carbon-mercury bond in organomercurials bound with Cysl59 is broken by the protons provided by Cys96 to yield an aliphatic or aromatic compound and Hg (II).
Figure 3. (A) Stereoview of the ribbon model of one representative structure of MerB from the ensemble of the 20 lowest energy structures. (B) Ribbon model of a representative structure of MerB highlighting the position of the four cysteines. Three (Cys96, Cys159, and Cys160) of the four cysteines in the protein are close in space whereas Cys117 is buried in the hydrophobic core of the protein. (Ref. Paola D L, Gregory C B, Homayoun V K, et al. Biochemistry, 2004, 43: 8322–8332.)
C2 Mercury ion enrichment
In order to improve the efficiency of engineering bacteria for the mercury ions treatment, metallothionein (MT), which can bind with heavy metals efficiently, is chosen to enrich and store the mercury ions in the engineering bacteria.
Metallothioneins (MTs) are low-molecular-weight, metalsequestering proteins rich in cysteines that can bind metal ions in a biologically inactive form. MTs are structurally composed of two globular metal-binding domains (α and β) with differential capacity to associate with metal ions. The C-terminal α domain contains 11 cysteines and the N-terminal β domain contains nine cysteines. The cysteine residues in MTs allow it to bind up to a total of seven mercury ions. Seven metal ions are located in two separate metal-thiolate clusters and form a monomeric dumbbell-shaped protein (Figure 4). For the natural MTs, they have no disulfide bond and no stable free sulfhydryl. All the thiol groups on cysteine can bind with mercury ions, which have significant mercury chelating properties.
C3 Mercury ion fixation
MTs can enrich mercury ion, but it cannot eliminate the pollution. At present, the best way to control mercury pollution is to transform mercury ions into insoluble HgS with low biological toxicity, which is generated by the reaction of mercury ions and H2S (eq 2). Therefore, we add the corresponding proteins which can produce H2S into the module for mercury fixation and remediation.
Here, we choose Arabidopsis L-cysteine demercase (AtLCD) as a key enzyme for the production of H2S. It has been reported that AtLCD can catalyze cysteine to produce free H2S, NH3 and pyruvate in the ratio of 1:1:1.
Considering that the engineering bacteria are susceptible to the environmental changes and can only repair mercury pollution of the limited area where they live due to slow shift in the soil, we choose earthworm, one common ecological engineer in the soil, as the carrier of engineering bacteria to solve the above problems. The engineering bacteria in soil can be ingested by earthworms then propagated extensively in the earthworms gut, a superior environment for engineering bacteria, and return to the soil with the excretion of earthworm. In this way, the engineering bacteria will realize the comprehensive detection and treatment of soil mercury pollution with the earthworms. In addition, earthworms also have the ability to enrich mercury ions by themselves. The engineered bacteria in the earthworms’ body can also play an important role in fixing mercury ions. In conclusion, through the "patrolmen", that is, earthworms as the mobile carriers, the engineering bacteria can complete the real-time detection and remediation of mercury contaminated soil.
In our design, we expect to construct engineering bacteria that can express merR, BFP, atlcd, merB3 and metallothionein, and hope to coordinate the expression of each protein by designing a signal amplification circuit, in order to achieve the most efficient the result of. However, in order to ensure the maximum feasibility of the experiment, we decided to construct engineering bacteria that only express merR, atLCD and merB in the previous experiment, and by using high expression vectors, the steps of designing the signal amplification circuit were simplified. Promoter to achieve signal amplification. In our preliminary experiments, we hope to verify whether the core mercury fixing element composed of merR, merB and atLCD can function as expected. If the results of the experiment are successful, we will add BFP and metallothionein in subsequent experiments to further improve the functions of the engineered bacteria and regulate the protein expression to achieve the synergy of different proteins.
Reference:.Brown NL, Stoyanov JV, Kidd SP, Hobman JL. The MerR family of transcriptional regulators. FEMS Microbiology Reviews, 2003, 27 (2-3): 145–163. .The project from BEAS_China team (iGEM 2019) https://2019.igem.org/Team:BEAS_China /Design. .Paola D L, Gregory C B, Homayoun V K, et al. Biochemistry, 2004, 43: 8322–8332. .Haq F, Mahoney M, Koropatnick J. Signaling events for metallothionein induction. Mutation Research, 2003, 1: 211–226. .Mackay E A, Overnell J, Dunbar B, et al. Complete aminoacid sequences of five dimeric and four monomeric forms of metallothionein from the edible mussel Mytilus edulis. Febs Journal, 2010, 1:183–194. .Jin Z P, Shen J J, Qiao Z J, et a1. Hydrogen sulfide improves drought resistance in Arabidopsis thaliana. Biochemical and Biophysical Research Communications, 2011, 414: 481–486.
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