Introduction
In an uncontaminated environment, bacteria and other microorganisms are constantly using and sharing the same organic matter as source of energy for metabolism. However, what happens when an inorganic pollutant, as a metal is present on the environment? How could microorganisms survive? The answer is that some would die and others would be able to utilize the inorganic matter to survive without being affected by it. Through this, bioremediation arose: environmental problems solved by biological organisms. During bioremediation, microorganisms such as bacteria are able to use inorganic matter for oxi-reduction reaction, metabolizing the target inorganic pollutant. This specific and complex system is part of bacteria survival mechanism that became resistant to contamination. The generated products released by bacterial metabolism, are typically less toxic form of the initial pollutant.
One of the principal pollutant in Amazon region is mercury, known worldwide for its toxicity. Conventional methods have been used to remove Hg from contaminated environments. In general, laborious treatment systems, with high cost. In order to solve the environmental problem regarding to mercury, and make its bioremediation, we built a bioremediator using Escherichia coli DH5α through the protein mercury reductase (MerA).
Why Mercury reductase?
Mercuric ion reductase (MerA) is a cytosolic enzyme, responsible to convert thiol-avid Hg2+ to its volatile form H0, using NADPH as electrons’ source. Mer A is an homodimeric enzyme, and has similar properties to pyridine nucleotide disulfide oxidoreductase family, by the presence of active site region with two cysteines, which confers the ability of redox-active disulfide/dithiol.
However, differently from other members of this protein family, MerA has two unique regions that prevent inhibition by Hg. The C-terminal region of one monomer, containing two cysteines is close to the redox-active cysteines of the other monomer, and might be important to bind Hg to the active site. Its N-terminal region could be involved in capturing Hg and handing it to the catalytic nucleus, and then reduced by electrons provided by the bound NADPH. MerA is the key for mercury remediation for its characteristic to reduce Hg2+ into H0, making it a prospective protein to use in bioreactors to polluted water treatment stations.
Our construction (How it works?)
To develop a bioremediator, we designed a biobrick device to express MerA protein in mercury’s occurrence. The Mercury ions’ bioremediator device biobrick (BBa_K1355004) is composed by mer bidirectional promoter (BBa_K1355001) attached to the MerA translational unit (BBa_K1355000). It has dual function: A) In reverse: MerR protein regulator transcription; and B) In forward: transcription of MerP - MerT - MerA proteins, as represented below:
In absence of mercury, MerR forms a MerR-promoter-operator complex, preventing RNA polymerase to recognize the promoter, consequently, mRNA for MerPT and MerA will not be transcript. In presence of Hg2+, MerR protein binds to this element and dissociates from the promoter-operator complex, allowing MerPT and MerA expression, as represented below:
Experiments and Results
The experiment for Hg bioremediation was made according to the protocol “Cell growth quantification of mercury resistant Escherichia coli DH5α at differents Hg concentrations by Optical Density”.
DH5-alpha transformed with BBa_K1355004 was inoculated in LM (LB with low concentration of NaCl) liquid medium with 0.05μg/ml of mercury chloride and 34μg/ml of chloramphenicol. The inoculum was incubated in shaker overnight at 37°C. After cell growth, we measured Optical Density (O.D.) of the cell culture in spectrophotometer at 600 nm wavelength. A standard quantity aliquot of bacterial suspension was taken in six falcons (50ml) with 10 ml of LM liquid medium and then added mercury chloride in order to achieve the concentrations: 0μg/ml, 1 μg/ml; 2.5 μg/ml; 5 μg/ml; 10μg/ml; 20 μg/ml. The samples were incubated at 37°C on shaker. We collected each sample at the given times, 1 (02:30 hours of incubation), time 2 (04:30 hours of incubation), time 3 (06:30 hours of incubation), time 4 (17 hours of incubation), time 5 (24 hours of incubation) to measure O.D. on spectrophotometer. As control, we used DH5-alpha transformed with BBa_K1355002 (Hg bio detector device) which does not present coding regions for the mercuric ion reductase. We also measured Optical Density of each sample. After cell growth measurement, the samples were frozen to subsequently quantification of Hg remediated by the Mercury Bacter.
The graph represented on Figure 1 and Figure 2 shows Cell growth of Mercury Bacter and of the control bacteria in different Hg concentrations in function of time, respectively;
Figure 1: Cell growth of Mercury Bacter in different Hg concentrations in function of time;
Figure 2: Cell growth of control bacteria in different Hg concentrations in function of time;
Comparing the graphs shown on figure 1 and 2, it can be observed that the biobrick BBa_K135504 provided resistance to high Hg concentrations. Control bacteria did not present resistance to Hg concentration equal or higher to 1 ppm. On the contrary, Mercury Bacter grew up even in 10 ppm Hg concentrations. Interestingly, at the concentration of 10 ppm Mercury Bacter needed 24 hours to exhibit increase in cell growth, and after 48 hours it maintained the same OD, unlike the others. At 1 ppm, 2.5 ppm and 5 ppm Mercury Bacter performed an overwhelming cellular growth after 24 hours of exposure to Hg, reaching approximately the same cellular growth as the non-exposed bacteria!!
The graph represented on Figure 3 compares Hg concentration in LM medium after 48 hours of experiment, between medium with bacteria used as control and engineered Mercury Bacter.
Figure 3: Hg concentration in LM medium after 48 hours of cell growth in control bacteria and in the genetically engineered Mercury Bacter.
In the graph above it can be observed that our super Mercury Bacter bioremediated! In action, Mercury Bacter reduced 72% of the mercury levels, in comparison to control at the sample 5 ppm! In 1 ppm, Even though on 10 ppm concentration, where bacteria had a slow growth rate only after 24 hours, it bioremediated 48.02%! These results demonstrate our constructions’ efficiency, both for biobrick BBa_K1355001 as a promoter regulated by MerR protein and to BBa_K1355000 as MerA encoding gene!
References
Cobbett, C. S. (2001). Heavy metal detoxification in plants: Phytochelatin biosynthesis and function. Iubmb Life, 51(3), 183-188.
Mathema, V. B., Thakuri, B. C., & Sillanpää, M. (2011). Bacterial mer operon-mediated detoxification of mercurial compounds: a short review. Archives of microbiology, 193(12), 837-844.
Kumar, S. (2013). Studies on mercury transporter merT and mercury reductase merA proteins.
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