Background

Human actions derived from industrial activities, agriculture and domestic sewage are among those responsible for the pollution of the environment by toxic metals (Biondo, 2008). Mercury occurs in the environment combined with other elements and naturally in different chemical species with distinct solubility, reactivity and toxicity. Occurring in volcanoes and mercury mines, which are responsible for emissions of mercury in the order of 2700-6000 tons/year (Micaroni et al. 2000). In the Amazon, it is also used in mines in the amalgamation of gold (Au-Hg) (Figura 1), where a quantity of metallic mercury (Hg0) is released upon rivers and soils by the handling in insecure situations, and through the outdoor burning process, which releases element's vapor to the atmosphere, that leads to the aquatic ecosystem contamination.

Figure 1. Direct contact with mercury in the Amalgamation of gold process.

In Amazon rivers, about 3000 tons of mercury are available and can be bioconverted in its most toxic forms for living beings, through methylation - the addition of the radical CH3 – of mercury, bioaccumulative properties are acquired by its high affinity to animal tissues (Souza and Barbosa, 2010). The mercury methylation is a crucial step to its entry in the aquatic food chain organisms, because of it riverside communitiesA traditional pupulation that resides in the rivers proximities and they have the artisanal fishing as main food supplying activity. are the main affected by contaminated fish ingestion.

Figure 2. Death of fish caused by dumping Mercury in the river.

Worldwide, there are cases of accidents with mercury as occurred in 1908 with the chemical industry Chisso Corporation that produced fertilizers settled in Minamata Bay in Japan. In 1932 the factory started to produce acetaldehyde, mercury sulfate in production, reaching 6,000 tons in 1951 and 42,245 tons in 1960.

Chisso's factory did not properly dispose their liquid waste, which were released into Minamata Bay since 1946, being continuously disposed residue, eventually affect sea Shiranui and others around it. In April 1956 the first signs of poisoning in people and changes in the ecosystem of the bay were observed. After humans and cats had the same symptoms and marine creatures are constantly found dead, the condition was named Minamata disease. The level of mercury contamination of the Minamata disease patients in the 50`s, reaching up to 705 ppm.

The second major accident is recorded in Iraq (1972), where epidemic proportions of mercury contamination were observed after wheat seeds were treated with alkyl mercury and were inappropriately consumed as food. It is not known how long it takes to develop symptoms of mercury poisoning or not, Because it is directly dependent on the form, concentration and time of exposure to the metal. After these accidents occurred, conventions and legislation in countries were determined individually, in addition, this type of action demonstrates the awareness and concern by government part.

The use of mercury has diversified into areas such as industrial, metallurgy, chemical industry, medical equipment, dentistry, among others. Particularly, the use of mercury has become high in the Amazon, this metal is used in prospecting for gold extraction. The misuse in the region generates not only human intoxication, but serious environmental problems.

Bacteria are able to live in different environments, including the contaminated growing under the pressure of these pollutants many groups become resistant to these contaminants developing systems of homeostasis to protect against damage and maintenance of survival (Biondo, 2008). Considering the metal mercury (Hg) as a main contaminant toxic non-degradable, remaining indefinitely in the environment, the ability of the bacteria is related to the processing mechanism of this substance. It is known that some gram-positive and gram-negative bacteria have this mechanism, including Serratia marcescens, Pseudomonas putida, Staphylococcus aureus, Bacillus sp, Cupriavidus metallidurans, and Enterobacter (Giovanella et al. 2010).

Figure 3. Genes responsible for mercury metabolism identified.

The mechanism is structured to change from a toxic oxidative state to a less toxic to the bacterium by methylation, chelating agents, efflux pumps and enzymatic reduction. This one is related to the mer operon. The resistance of the operon is associated with the activity of regulated proteins and expressed by a group of genes called mer operon, which is present in the region of transposon plasmids and in some cases, on the chromosome. The mechanism of resistance is similar and the genes are conserved for any group of bacteria (Trevors, 1986; Pinto, 2004). In terms of organization and sequence, the plasmids containing the operon present themselves so well preserved homologous genes, and its basic structure known as merRTPAD, in some cases there is occurrence of merRTPA(B) and bacteria that behave megaplasmids with merRTP(C/F)A(B)D(E)(F) (Osborn et al, 1997;. Mergeay et al, 2003;. Nascimento and Chartone-Souza, 2003; Champier et al, 2004).

Figure 4. Working system from Mer operon.

This operon has great bioremediator capacity for presenting carrier function protein (merP and merT) displacing Hg from the periplasm to the cytoplasm: merT encoding a hydrophobic polypeptide of approximately 12.4 kDa comprising 116 amino acid residues. merP is a protein located in the periplasmic space and has size of about 9.4 kDa to about 91 amino acid residues (Belliveau & Trevorst, 1989; Miller, 1999, Brown et al. 2002). There catalytic function of proteins (Mera and Merb) that transform Hg²⁺ and CH₃Hg on Hg0.

MerR is the gene encoding cytoplasmic protein that controls transcription of the structural genes merTP(C)A(B)D(E). Its product is a homodimer of about 15.9 kDa with 144 amino acid residues (Belliveau & Trevorst, 1989; Miller, 1999, Brown et al. 2002). According Shewchuk et al (1989) merR dimer binds to a single atom of Hg.

The regulation of expression of the mer operon is negative inducible, being performed by merR. In the absence of mercury, merR forms a promoter-operator complex, so the RNA polymerase cannot recognize the promoter and messenger RNA from other genes involved in resistance to mercury is not produced (repressor). In the presence of Hg2+ merR binds to this element and is displaced from the promoter-operator, which allows the expression of the genes of the operon (inducible) region.

Figure 5. Mercury’s action to activate the genes on the operon.

The regulatory merR gene is transcribed from a promoter, which is oriented divergently from the promoter of the other mer genes, and also down-regulate their own expression (Mukhopadhyay et al. 1991). When the operon is being expressed, the structural genes merD, MerA, merP and merT are also activated.

Figure 6. Genes orientation involved in the Hg metabolism process. metabolismo Hg.

The other merC, merA, merB, merD, merE and merF genes are involved in bacteria broad-spectrum and narrow-spectrum. Bacteria narrow- spectrum have resistance to mercury inorganic compounds that besides include merA, mercury reductase that converts Hg²⁺ to Hg⁰, and merD, regulatory protein, may have the merC and merF genes, which are responsible for the transport of Hg in the cell. The broad spectrum possess beyond resistance to inorganic compounds, genes involved in resistance activities to organic mercury compounds, namely: merB, organomercurial lyase protein that breaks bonds C^-Hg⁺; and merE carrier protein of methylmercury that captures organo-mercury compounds to the cytoplasm.

Genes of mer operon were made by bioinformatics tools. From this, we construct the merRTP and merA genes, thinking such as essential genes for the transport and breakdown of mercury in the cell, acting as a bioremediador. From iGEM parts of the kit 2014, we used a GFP to construct a biosensor in which genes merRTP were connected with the fluorescent protein. Besides this part, we used another part called MBP, this would be linked to merRTP to allow bioaccumulation of mercury present in the medium, linking this protein to mercury.

The future goals of the team is to make the recombinant bacteria will be cultured in medium with mercury, and total mercury detection is analyzed before and after cultivation of bacteria by means of atomic absorption spectrometry. The biosensor system will be done through the construction of tanks in series, where the first bacterium will be able to detect mercury (merRTP + GFP), the second bacteria with the construction merRTP + MerA will be able to vaporize the mercury, which is captured and subsequently precipitated, the third tank will make the bacteria capable of bioaccumulation of mercury (merRTP + MBP) remaining in the effluent and in the fourth tank will be again a new detection of mercury by biosensoring bacteria to prove the efficiency of the system, if there is still mercury the effluent is sent to the merRTP+merA bacteria, so that all mercury is removed.

Figure 7. Our future goal is to make the system work in tanks.

The cases of exposure to high doses involving intoxication by MeHg gived the birth to offspring with severe neurological disorders such as mental retardation, deafness, blindness, cerebral palsy, cerebellar ataxia, strabismus, dysarthria, among others. Poisoning by high levels of MeHg is rare nowadays. Although quantities of mercury above the national average has been found in the hair of the local population, which indicates that the risk of contamination by this metal is real and needs to be fought. The reason of that is the accumulation of mercury through the food chain, especially in its methylated form in fish, which has a direct effect on the lives of the local population.

The growth of gold extraction in the Amazon caused a seriously problem in the environment, the caracteristic of mercury to accumulate in organic organisms makes it last for years. It is most commom to find mines in the nascente of rivers, because of that, the mercury flows with the river and can get to the metropolitan area. Also, the fish is getting mercury by eating particles of this metal and by the water contamination. Fish is the main font of protein to the local population, people are eating mercury without knowing!!

A way to know more or less the quantity of Mercury in the organism is by screening the hair composition, and, doing that, was discovered that the concentration of these metals in the hair of children in Manaus is higher than average of other regions in the country. After ingestion of contaminated food, about 95% of MeHg are absorbed quite rapidly in gastro-intestinal tract through alternative forms linked to a tiol residues in aminoacids formed. Once adsorbed and into the bloodstream just over 90% of MeHg is inside the red blood cells in a ratio of approximately 10-20:1. Then it goes to the liver, where it can spread to all tissues of the body. The MeHg can overcome the blood-brain barrier by gaining a form of thiol conjugate that is similar to the amino acid methionine then it is easily accumulated in nervous tissue. The slow rates of biotransformation into Hg2+ explain the higher levels of MeHg in the CNS (Central Nervous System) than in the rest of the body. The MeHg can also cause damage to the kidney tissue quite rapidly, reaching stable levels within 3 hours after intoxication.

The damage and cell loss, such as neurons and glia cells, due to the toxic effect of MeHg are represented macroscopically with loss of cortical areas of the CNS. Besides pure neurological symptoms, many neuropsychiatric symptoms were seen on children that were conceived by mothers who were exposed to large amounts of MeHg during their prenatal lives. All due to acute injuries in the encephalon, causing loss of brain mass in determined cortex areas. Besides pure neurological symptoms, many neuropsychiatric symptoms were seen on children that were conceived by mothers who were exposed to large amounts of MeHg such as Akinetic mutism, Extreme shame, Communication difficulties, Lack of initiative and Lack of facial expression.

Poisoning by chronic exposure to low doses of MeHg also has the ability to cause harm to adults, even if not to the same severity in relation to fetal intoxication. Ekino et al. (2007), have reported cases in the distal regions of paresthesia of extremities, and around the lip region, in patients that had time exposure to MeHg prolonged at low doses even if exposure to the toxic agent has already ended 30 years. Regions such as the tongue, lips, fingers and thumb represent areas with the greatest loss of sensitivity due to these regions have large areas in the cortex devoted to their sensitivity.