Team:UFAM-UEA Brazil/Project/MerOperon
Mer Operon Improvement
Description
In a contaminated environment, microorganisms such as bacteria are able to use its contaminated inorganic matter for oxi-reduction reaction, metabolizing it. 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. Through this, bioremediation arose: environmental problems solved by biological organisms.
Mer operon is a unique adaptattie strategy to environments contaminated by heavy metals, characterized by conversion of methylmercury to its volatile and less toxical form, elemental mercury, through a catalytic pathway, orchestrated by diverse enzymes and proteins.
Mer Operon is composed by MerA, MerB, MerP, MerT, MerD, MerF, MerC, and others. Wwe selected five genes to construct a functional and efficient Operon, codifying the proteins schematizated and described below:
(1) MerP and (2) MerT transporter proteins which carry mercury ion from periplasm to inner membrane and, then, to cytoplasm, respectively.
(3) MerA, mercuric ion reductase, responsible for reducing mercury from Hg(II) to Hg(0), its volatile and less toxical form.
(4) MerB, organomercurial lyase, catalyzes the break of binding between organical radicals and mercury, releasing Hg(II).
(5) MerR, regulatory protein. In absence of mercury, it binds at Operon’s promoter (MerO) and blocks RNA polymerase recognition, so there is no transcription of operon genes.
Want to know more about each protein? Click on it and have fun!
MerA integrates Mer Operon, a molecular mechanism that confers mercury resistance to bacteria and has potential to become an applicable and efficient tool to bioremediate mercury polluted environments. MerA codifies the cytosolic enzyme mercuric ion reductase, and it is the core of mercury resistance mechanism: this enzyme catalyses the reduction of ionic mercury [Hg(II)] to it’s volatile, non-toxical and less reactive elemental form (Hg0), which flows passively through membrane to outer side.
Figure 01: Global mechanism of mercury resistance and bioremediation by microorganisms. Each component of the mer operon orchestrates meticulously with each other, and MerA is a key component on it. Source: Bioremediation of mercury and importance of mer genes.
Mercuric ion reductase needs contribution of a NADPH molecule, able to offer the reductive power that turns Hg(II) into Hg0. MerA also may work alongside MerB, another gene from the operon which codifies the enzyme organomercurial liase, capable of break the bonds between mercury and organic radicals (organomercury), leaving Hg(II) available to the following mercuric ion reductase reduction. Since organomercury ties itself in biological tissues, it can be accumulated in living organisms and biomagnified through food chain, till it reaches humans. So, the cooperation of MerA and MerB is essential to the effectiveness of the bioremediation system.
Structure and mechanism:
Mercuric ion reductase presents itself as an homodimer. It has two terminal sites, each containing a pair of cysteines: a short and mobile C-terminal and a long N-terminal. The C-terminal cysteines of one monomer would lie next to the redox-active cysteines of the other. Together, these four cysteines engage in Hg(II) binding at the active site but, alone, don’t take part in Hg(II) reduction. C- terminal cysteines are responsible for catching Hg(II) ions from solution and delivering them to the active site, where the pair of inner cysteines would establish chemical bonds with mercury. C-terminal cysteines would also provide a pathway to remove basic thiolates at the active site before mercury binding, since the presence of negative charge would inhibit reduction.
Figure 02: General structure of MerA homodimer. Source: Xray Structure of a Hg2+ Complex of Mercuric Reductase (MerA) and Quantum Mechanical/Molecular Mechanical Study of Hg2+ Transfer between the CTerminal and Buried Catalytic Site Cysteine Pairs.
Since N-terminal site has several similarities with MerP periplasmatic mercury transporter protein it was suggested that this domain would be part of mercury capture mechanism, but mutagenesis studies have ruled out this hypothesis. The role of N-terminal in general enzyme function was unclear until very recently. It facilitates the delivery of Hg(II) into the active site through its pair of cysteines. The reduction process is the enzyme’s main mechanism, and it uses a NADPH molecule bound to mercuric ion reductase as ion source. At the core site, FAD mediates the electron transfer between NADPH and Hg(II); primarily NADPH is oxidized to NADP + while FAD is reduced to FADH - , then FADH - reduces ionic mercury Hg(II) to Hg0. Finally, as a volatile vapor, mercury leaves the core of mercuric ion reductase and flows to the outside through the cell membrane.
Figure 03: Three-dimensional crystallized structure of Tn501 MerA interacting with FAD nucleotides, sulfate ions and glycerin; from Pseudomonas aeruginosa. Source: NCBI Database.
Sources:
BARKAY, T.; MILLER, S. M.; SUMMERS, A. O. Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiology Reviews 27 (2003) 355-384.
DASH, H. K.; DAS, S. Bioremediation of mercury and importance of mer genes. International Biodegradation & Biodeterioration 75 (2012) 207-213.
LIAN, P.; GUO, H. B.; RICCARDI, D.; DONG, A.; PARKS, J. M.; XU, Q.; PAI, E. F.; MILLER, S. M.; WEI, D. Q.; SMITH, J. C.; GUO, H. Xray Structure of a Hg2+ Complex of Mercuric Reductase (MerA) and Quantum Mechanical/Molecular Mechanical Study of Hg2+ Transfer between the CTerminal and Buried Catalytic Site Cysteine Pairs. Biochemistry 2014 (53) 7211-7222.
The three major naturally occurring forms of mercury- elemental, ionic, and organic are widely distributed throughout the biosphere. All three forms are constantly interconverted by a variety of abiotic and biological mechanisms. Microbial mechanisms dominate the conversion of ionic mercury to organic mercury compounds, especially methyl mercury. Some microorganisms are also capable of transforming ionic or organic mercury into elemental mercury. This capability constitutes a detoxification mechanism due to the lower toxicity of elemental mercury relative to ionic or organic mercury. Organic and ionic mercury resistance is due to a dedicated set of plasmid-encoded mercury resistance genes (the mer operon.) merR is a regulatory gene. merP and merT are involved in mercury binding and transport. merA encodes for a reductase which reduces ionic mercury to elemental mercury using NADPH. merB encodes for an organomercurial lyase, which converts organomercurials into ionic mercury and a reduced organic product.
Figure 01: Model of carbon-mercury lyase operon. The symbol indicates a cysteine residue. RSH indicates cytosolic thiol redox buffers such as glutathione. Shows the interactions of MerB, in green, with mercury compounds and other gene products of mer operon. (Figure adapted from "Bacterial mercury resistance from atoms to ecosystems.")
The enzymatic reduction mechanism from Mer Operon is headed by two main enzymes codified by MerA and MerB genes, organomercurial lyase and mercuric ion reductase, respectively. MerB breaks the bond between Hg and a carbon atom from an organic radical. In this complex, mercury has the ability to bind itself to biological tissues and propagate through food chain until it reaches humans, a phenomenon called biomagnifications. MerB orchestrates alongside MerA by releasing Hg(II) from the organic complex, so MerA can reduce it to the volatile, less toxic Hg0.
Structure and mechanism:
MerB gene (0.6Kb) was first described by Kenzo Tonomura and is an integrated Mer Operon gene, a functional cluster of genes that can be used for mercury bioremediation. It catalyzes the bond breaking of methylmercury, releasing Hg(II).
The purified protein required a minimum two-fold molar excess of thiol over organomercurial substrate to exhibit any activity and preferred the physiological thiol cysteine to non-physiological mercaptans. The enzyme has a very broad substrate tolerance, handling both alkyl and aryl mercurials, with a slight preference for the latter. As is often the case with enzymes of low substrate specificity, MerB has very slow turnover rates ranging from 0.7 to 240 min−1 on various substrates. Although relatively slow for an enzyme, these rates do represent a 106–107-fold acceleration over chemical protonolysis rates of organomercurials. Paradoxically for a protonolysis catalyst, MerB's pH optimum was >9. Mechanistic studies revealed retention of the skeletal configuration of the substrate consistent with the rare SE2 mechanism rather than a radical-based mechanism.
Figura 2: Structure from Mer B protein, organomercurial lyase, involved in the bacterial mercury resistance system; from Bacillus megaterium. Source: NCBI Database The enzyme has a very broad substrate tolerance, handling both alkyl and aryl mercurials, with a slight preference for the latter.
References
Bacterial mercury resistance from atoms to ecosystems. Barkay T , Miller SM , Summers AO FEMS Microbiology Reviews 27 (2003) 355-384
Tomura, K., Kanzaki, F. (1969) The reductive decomposition of organic mercurials by cell-free extracts of a mercury resistant pseudomonad. Biochim. Biophys. Acta 184, 227–229.
Gregory C. Benison ,‡ Paola Di Lello ,§ Jacob E. Shokes ,‡ Nathaniel J. Cosper ,‡‖ Robert A. Scott ,‡§‖ Pascale Legault ,*§ and James G. Omichinski Department of Chemistry, department of Biochemistry and Molecular Biology, and Center for Metalloenzyme Studies, University of Georgia, Athens, Georgia 30602. Biochemistry, 2004, 43 (26), pp 8333–8345 DOI: 10.1021/bi049662h.Publication Date (Web): June 12, 2004
Di Lello P, Benison GC, Valafar H, Pitts KE, Summers AO, Legault P, Omichinski JG. Biochemistry (2004) 43 p.8322
Results
The first step to characterize this part was testing its Hg resistance and bioremediation with and without MerB gene, as represented below, through an inhibition zone.
It has been use a 10 times concentration variation (20mg/mL, 200µg/mL and 20µg/mL) of HgCl2 in LM (Luria-Bertani variation with half salt) solid media, adding 10µL of mercury chloride solution on its paper disks. The samples were inoculated in triplicate and incubate in BOD at 37°C for 2 days. The results are shown below.
As we can analyze in the figure above, our construction with MerB gene, increasing mer operon spectrum, had a smaller inhibition zone (nearest to the disk), growing better in Hg conditions, with clear difference from other samples (control and mer operon without MerB). As we can see in the graph, measuring inhibition zone length, our construction with MerB had 30% reduced it!
On the next mercury chloride concentration, as shown on the figure below, our construction with MerB gene continued with a smaller inhibition zone, growing even more nearest to the disk!
In 200µg/mL of HgCl2, our construction with MerB gene reached approximately 60% of inhibition zone reduction, one more time enhanced in contrast to genetic circuits only with MerA. Now… the “Grand Finale” experiment in 20µg/mL, presented below!
In 20ppm of HgCl2, our construction with MerB was totally resistant and don’t had any inhibition zone, showing its potential in bioremediation process, metabolizing all the available mercury!
To continue our characterization, we used this part in an improved Mer Operon, with new strongers regulated promoters, to increase mercury bioremediation, as you can see in the synthetic genetic circuits below.
We used two new regulated promoters to compare with natural one. The first test was validated the growth curve in 7.5ppm of HgCl2 in liquid LM media. The result are shown below.
In the growth tests we used 7,5 µg/mL of mercury, comparing with the device K1355004 (used in 2014) and the new construction. At the best take, the new construction showed 4,6 times better result than the previous device.
The curve from the new construction under our bioreactor conditions had an incredible maintenance of its growth rhythm under 5µg/mL mercury concentration in a 42 hours period. In posterior analysis, mercury was reduced in 97% from its initial concentration, proving the efficiency of this new construction and its potential to depollute contaminated waters.
Notebook
Parts origin;
-Based on the team’s 2014 construction, known as essential Biobrick (BBa_K1355004), the sequences of RBS+MerR+Terminator (MerR) and RBS+MerT+RBS+MerP (MerTP) were amplified by PCR and then purified.
The purified amplicons were bound and transformed in the cloning plasmidial vector, pGEM T easy, to obtain essential restriction sites in further Operon Mer construction and cloning processes.
After being transformed, some clones were selected and went through colony PCR to confirm the bound insert. Then, clones were inoculated and its plasmids extracted with a Kit to subsequent digestion by using only Eco RI (knowing that there were two restriction sites for Eco RI upstream and downstream to the cloned sequence, and the confirm the insert.
MerR amplification in pGEM T easy;
For the digestion test, were expected two bands at the electrophoresis gel: the first of approximately 3015 base pairs(for pGEM T easy) and other from cloned inserted of about 534 base pairs, representing Mer R band.
MerTP amplification in pGEM T easy;
For the digestion test, was expected two bands at the electrophoresis gel: the first of approximately 3015 base pairs(for pGEM T easy) and other from cloned inserted, about 674 base pairs, representing MerTP band.
Mer BA construction
For this construction, we utilized a part from last year containing RBS+MerA+Terminator B0015 (MerA) and another specially synthesized for this construction, RBS+MerB.
The parts were transformed by electroporation, and its clones were selected for later cloned plasmid extraction.
As soon as the plasmids were confirmed through electrophoresis, a digestion test was performed with EcoRI and PstI, prove inserted clones.
For the MerA digestion, the expectation was of two bands: one from MerA insert and the other from plasmidial vector pBSK, with the insert length of approximately 1823 base pairs and the vector about 2958 base pairs.
For the MerB digestion, two bands were expected: one from MerB insert (700 base pairs) and the other from plasmidial vector (2958 base pairs).
After confirm the sizes, the samples went through another digestion, at bigger volume, so we could obtain a huge amount of samples for cloning. For Mer A sample, a double digestion was proceeded expecting that the plasmid would linearize, and for MerB, a single digestion, expecting the Mer B insert to be free.
The samples underwent purification and were linked, with Mer B being embedded upstream to Mer. Then, the linking was transformed, and the transforming clones were selected and its plasmids, extracted. A digestion was proceeded with extraction and the inserted was confirmed, with the length of 2523 base pairs. It was named Mer BA.
Mer TPBA construction
For this one, the already done samples of MerTP (cloned in pGEM T easy) and Mer BA (cloned recently in pBSK) were used to this construction. Samples underwent a previous digestion to ascertain the parts, in digestion test. After this process, the samples went through a new digestion for purifying and linking of the parts. In this digestion, the Mer BA sample was linearized and the MerTP had its insert freed. Then, samples were purified, linked, transformed and the clones were selected and inoculated to extract the plasmids. The plasmids were digested as a test, intending to free the insert (~3197 base pairs) and the plasmid vactor (~2958 base pairs) This part was named Mer TPBA.
Changing the plasmidial vector pBSK for vector pSB1C3 attached to sample Mer TPBA
As in the plannings from Mer Operon construction, the samples would be added upstream to Mer A, as it is seen. Therefore, the plasmidial vector which would prevail in the construction might be the vector from Mer A, by then in pBSK. So, to construct MerTPBA we had to change vectors.
To this changing, any part from iGEM parts kit, which were at pSB1C3, was used to give the plasmidial vector. This sample was digested in test and its vector, confirmed (~2070 base pairs).
After confirming the vector, Mer TPBA sample was digested to release the insert (~3197 base pairs) and other digestion was performed on a random sample, to obtain the vector at equal length as the one cited before. The samples were purified, linked and transformed; the transforming clones were selected, inoculated and the plasmids were extracted and digested, to confirm the cloned construction.
CMer R construction with constitutive promoters
For this one, we chose a biobrick containing promoter J23100 from Anderson Promoters catalogue (Berkeley University, 2006). Beyond this promoter, other one was utilized, with average expression, I14033 (Penn State University). These promoters were transformed and the clones were selected and inoculated to, later, be extracted and digested in test. After confirming, a new digesting reaction was performed with bigger volume of sample for purification. Alongside this sample, MerR was digested with greater volume as well, to release the insert, while promoters were linearized, along with its plasmidial vectors.
Constitutive promoter construction + Mer R + regulated promoter + RFP
This construction used the previous one and a part from the laboratory parts bank. This laboratory part contained a regulated promoter with strong expression and RFP sequence (Red Fluorescent Protein – Bba_E1010), which already were in pSB1C3. This sequence was digested in test to proof the insert. Next to confirming, Mer R samples with constitutive promoter were digested alongside regulated promoter sample (BBa_K2123104 or PJK26) with RFP. Samples were purified, linked, transformed and the clones were selected, digested in test and, alongside the confirmed insert, the test kept going.
Mer R construction (containing constitutive promoter) with regulated promoters in RFP;
The already purified Mer R samples were stored and new regulated promoters (besides PJK26) were used. The promoters were BBa_K2123101 (TacMer3) and BBa_K2123102 (TacMer2), which were already linked to RFP. These promoters were linearized upstream and purified, for further linking in the already purified Mer R. After the linking, the sample was transformed and the clones were selected, extracted and digested to confirm the samples.
Mer R construction (containing constitutive promoter) with regulated promoters and Mer TPBA in pSB1C3
The previous samples were linearized downstream and the RFP sequence was replaced by Mer TPBA. The samples containing Mer R were digested to released the constitutive promoter sequence + Mer R + regulated promoter. The Mer TPBA sample was linearized upstream with insertion of all previous sequences.
The samples were digested, purified, linked and transformed, the clones were selected, extracted and digested to confirm the linked insert and end the construction.
Fluorescent promoters test, to analyze Mer R repression
For this test, we made a growth curve with fluorescence analysis, in spectrofluorometer. The chosen sample was the more repressed one along the curve, considering that Mer R was regulating RFP expression. The sample which the repression stood out was the sample with J23100 promoter. This sample prevailed in Mer Operon construction.
Fluorescent promoters test, inducting promoters with low-level of Mercury
In this test, the Mer R sample with RFP, at different concentrations of mercury (0 µg/mL; 0,5 µg/mL and 1µg/mL; in triplicate) were analyzed for 30 hours. The concentration level which better inducted RFP expression was 1µg/mL at last analysis
Test with mercury under concentration of 5µg/mL, 7,5µg/mL, 10µg/mL and 20µg/mL with former RTPA construction
The samples alongside BBa_K1355004 construction (made by the team in 2014) were evaluated on bacterial resistance, considering the growing of each sample under 5ug/mL of mercury. In the beginning, the construction with regulated promoter PJK26 stood out but at the end it fell over.
The tests were repeated under the following concentrations: 20 ug/mL, 10 ug/mL and 7,5 ug/mL, where the PJK26 regulated promoter stood out again.
