Isolation and Characterization of Partial Sequence of merA Gene from Mercury Resistant Bacterium Klebsiella pneumoniae Isolated from Sario River Estuary Manado

The most common bacterial mercury resistance mechanism is based on the reduction of Hg to Hg, which is dependent on the mercuric reductase enzyme (merA) activity. The aims of this research were to isolate and characterize merA gene fragment of mercury resistant bacteria Klebsiella pneumoniae isolate A1.1.1. The gene fragment was amplified by PCR using previously designed primer pairs. Plasmid DNAs were used as template. The result showed that the partial sequence of merA gene has been found on plasmid DNA of mercury resistant bacterium Klebsiella pneumoniae isolates A1.1.1. The nucleotide sequence of the merA gene consists of 285 base pairs (bp) which encodes deduced 94 amino acids of mercury reductase merA protein. The merA protein sequence of isolate A1.1.1 has 99% similarity with some strains of Klebsiella pneumoniae deposited in Gen Bank. There is a gene mutation that causes the deduced amino acid threonine was replaced by serine at position 524 (Thr→Ser) in the merA protein of Klebsiella pneumonia as the accession number: AAR91471.1.


INTRODUCTION
Mercury is a toxic compound that is widely distributed in the global environment and can accumulate in the food chain (Jan et al., 2009).Mercury poisoning has become a problem because of the pollution of mercury in the global environment.Mercury pollution continuously increases from time to time as a result of human activities such as the growth of electronics industry, the increasing use of antimicrobial agents, vaccines, amalgam, cosmetics and the higher activity of gold mines using mercury to extract gold (Jan et al., 2009;Schelert et al., 2004).
Mercuryis accumulated in soil and water as mercury ions (Hg 2+ ) that can be converted into more toxic methyl mercury by microbial activity.Various conventional techniques have been used to dispose toxic metals including preparation and chemical separation, oxidation-reduction reactions, ion exchange, reverse osmosis, filtration, adsorption using activated carbon, electrochemical and evaporation.However, those techniques were considered ineffective, especially for metal concentrations less than 100 mg/L and also quite expensive and their supporting chemicals become secondary pollutants (Habashi, 1978).Therefore the use of microorganisms to remove heavy metal contamination from mining and industrial wastes should be considered (Keramati et al., 2011).Mercury chloride (HgCl 2 ) is often used for research because it is easily soluble but toxic (Schelert et al., 2004).Microbial detoxification of mercury occurs by transforming Hg 2+ to volatile metallic mercury (Hg 0 ).Staphylococcus, Bacillus, Pseudomonas, Citrobacteria, Klebsiella and Rhodococcus are often used in microbial bioremediation for mercury (Adeniji, 2004).
Detoxification of mercury by mercury-resistant bacteria can occur due to the presence of mercury resistance genes located in mer operons unique to each bacterium (Silver and Phung, 1996).Mercury resistance genes are often found in plasmids or transposons (Ravel et al., 2000;Nascimento and Chartone-Souza, 2003) and in chromosome (Wang et al., 1988).The mercury detoxification is mediated by intracellular protein, mercury reductase (merA).Mercury ion is transported from outside the cell by a mercury transporter, merP or merC (Iohara et al., 2001;Sasaki et al., 2005), which is an extracellular protein that binds to mercury ions and merT, which is an inner membrane protein that transports mercury ions into the cells.Inside the cell, Hg 2+ is bound through the process of ligand exchange reactions to the active site of flavine disulfide oxido reductase of mercury reductase merA (Ravel et al., 2000).Mercury reductase catalyzes the reduction of Hg 2+ to volatile and slightly reactive Hg 0 (Nascimento and Chartone-Souza, 2003).Narrow spectrum mercuryresistant bacteria only have protein merA.Broadspectrum mercury resistant bacteria have merA and merB, a lyaseorgano mercury.The later catalyzes the cleavage of mercury-carbon bond to produce organic compounds and Hg 2+ (Barkay et al., 2003;Barkay and Wagner-Döbler, 2005).There are many gold mining in North Sulawesi use mercury to extract gold from rock or ore and mercury waste is discharged into the environment, causing the surrounding water contaminated by mercury.Mercury contaminatedenvironment is a suitable source for the growth of mercury resistant bacteria.In our previous study, we isolated mercury resistant bacterium isolate A1.1.1,identified as Klebsiella pneumoniae from Sario River estuary.It showed a high mercury reduction activity, i.e., 75, 92 and 99.4% in 1, 12 and 24 h of incubation, respectively in nutrient broth (Fatimawali et al., 2011).
This study was aimed to isolate and characterize a merA gene fragment from isolate A1.1.1,as a molecular marker for mercury-resistant bacteria.The results of this research can be used as a basis for further study in mercury detoxification process in mercury waste waters.

MATERIALS AND METHODS
DNA was isolated using Plasmid DNA Isolation Kits (Promega, Madison, USA).The partial merA gene fragment was amplified using a primer pair previously designed by Ni Chadhain et al. (2006) using plasmid DNA as template.The nucleotide sequences of primers were 3'TCCGCAAGTNGCVACBGTNGG5' for A1s-nF and 5'-ACCATCGTAAGRTARGGRAAVA-3' for A5-nR.PCR was done according to previous work with modification (Ni Chadhain et al., 2006) using 1.5 mM MgCl 2 for 35 cycles and annealing temperature at 54°C.PCR products were analyzed using 1.5% agarose gel electrophoresis.

RESULTS
Amplified PCR products from plasmid, shown in Fig. 1, were analyzed using 1.5% agarose gel electrophoresis.Fragments of merA gene with size 285 bp exist in DNA plasmid.Ni Chadhain et al. (2006), which uses the same primer obtained merA gene in genomic DNA of bacterial isolated from marine sediment.Bacteria developed mercury resistance mechanisms depending on the group of genes that are located in the merOperon that can be contained in a plasmid or chromosome (Barkay et al., 2003;Essa et al., 2003).
MerA gene sequencing and blast results: Sequencing was performed to determine the nucleotide sequence of the merA gene of Klebsiella pneumoniae isolate A1.1.1., as shown in Fig. 2.
To study the similarity of nucleotides sequence from merA gene of Klebsiella pneumoniae isolate A1.1.1 with merA gene of Klebsiella pneumoniae deposited in GenBank, blast analyzes was conducted by online at http://blast.ncbi.nlm.nih.gov/Blast.cgi.Blast result shows that merA gene of isolate A1.1.1 has 93% similarities with merA gene of Klebsiella pneumoniae deposited in GenBank.To study nucleotides differences/similarity, blast results were aligned using Clustal 2.1 multiple sequence alignment program.Alignment result was shown in Fig. 3.

Thr →Ser
MerAgene isolate A1.1.1 of Klebsiella pneumoniae has only 93% similarity with those merA of other Klebsiella pneumoniae deposited in GenBank due to many nucleotide bases differences.It indicates that there are number of gene mutations that shown in alignment result (Fig. 3).The mutations consist of transition ofpurinetopyrimidine,namely G6 into A6, A39 into G39, T42 into C42, C48 into T48, T71 into C71, A75 into G75, T84 into C84, A174 into G174, T195 into C195, T225 into C225, T234 into C234 and A261 into G261.Besides that, there are trans-version mutation (purineto pyrimidine or otherwise), namely G81 into C81, G90 into T90, T96 into A96, A237 into T237, C239 into G239and C279 into A279.Number of mutations affects the translation process, so it will result in different deduced amino acid and in turn it will affect the protein structures.
To study the effect of mutation in translation process, protein of MerA isolate A1.1.1 were blasted to MerA proteinof Klebsiella pneumoniae in GenBank.Alignment was further conducted using Clustal 2.1 multiple sequence alignment.MerA protein deduced from the merA geneisolate A1.1.1 has 99% similarity with some strains Klebsiella pneumoniae deposited in GenBank, as shown in Fig. 4.
There is one amino acid difference of merA protein of isolate A.1.1.1 to other Klebsiella pneumonia merA protein in Gen Bank.The amino acid is threonine at position 524, which is replaced with serine (Thr→Ser) in the merA protein of Klebsiella pneumoniae (accession number: AAR91471.1.)

DISCUSSION
The merA gene of Klebsiella pneumoniae isolates A1.1.1 exists in the plasmid.This is in contrast to the result of Ni Chadhain et al. (2006), while using the same primers, they found merA gene in genomic DNA of bacterial isolated from marine sediment.These differences can occur due to mercury resistance mechanisms developed by any bacterium.Depending on the group of genes that are located in the mer Operon, the mer genes can be located in a plasmid or chromosome (Barkay et al., 2003;Adriana et al., 2008).MerA genes previously discovered by Essa et al. (2003), were located in plasmid of mercury-resistant bacterial cultures.
Alignment results of merA gene (Fig. 3) and merA protein of isolate A1.1.1 (Fig. 4) show that most of the mutations do not affect translation products.It is because the most of replaceable bases results in the same amino acid, for example CAG and CAA encode for glutamine, GCA and GCG encode for alanine.CAT and CAC, both encode for histidine, GAC and GAT encodefor asparagine, GGG and GGC encodefor glicine, CTG and CTA encodefor leucine, CTC and CTG encode for leucine and GTG and GTT encode for valin.In contrast with ACC, which encode threonine, is replaced with TCG, which encode for serin.This mutation resultsin different protein product of merA.Threonine (Thr = T) is a polar sidecha in amino acid, because of containing hydroxyl groups that can form strong hydrogen bonds with other amino acid sidecha in group containing atoms O, N and S, where the strength of the bond depends on the pH of the environment.Serine (Ser = S) which share the similar property as threonine, has R group of-CHOH, while threonine has R group of-COHCH 3 .Bothaminoacids, threonine and serine, are not located in active sites, therefore are not involved in the binding process and the reduction of mercury by merA.Therefore, the change of threonine by serine on merA in amino acid chain does not result in structural change, or in mercury detoxification activity.The active site in volved in the reduction process of mercury contain cysteine C207, C212, C628 and C629, which facilitates the binding of Hg 2+ on merA of Y605 and K613, which is described in the crystal structure model of merA of Bacillussp RC607 (Barkay et al., 2003).
The heart of mer on the mechanism of resistance is mercury reductase homodimer (merA), which reduces Hg 2+ to Hg 0 and use the cofactor flavina deninedi nucleotide and electrons from NADPH (Barkay et al., 2003).Catalysis by mer Airport of a sustainable suppresses of nucleotide-disulfideoxido reductase doma in which contains two active cysteine's C207and C212 (Simbahan et al., 2005).Mercury reductase merA is an enzyme that catalyzes changes of thiol-Hg 2+ into volatileHg 0 .NADPH is used as the electron source (Furukawa and Tonomura, 1972) and is located in the cytoplasm (Summers and Sugarman, 1974).High reduction power of Klebsiella pneumoniae isolate A1.1.1 from Fatimawali et al. (2011) reduces 99.4% of HgCl 2 in 24 h.This is related to the presence of mercury reductase merA gene found in this research.MerA gene of isolate A1.1.1isplannedto be isolated and cloned to obtain merA enzyme to be used in detoxification of inorganic Mercury.
Identification and molecular analysis of mercuryresistant bacteria is still widely performed since the use of mercury is still widely used and exploited.MerA enzyme produced by mercury-resistant bacteria can be used in the remediation process or detoxification of mercury.Kargar et al. (2012) analyzed the molecular basis of mercury-resistant bacteria from Kor River Iran.They isolated 12.3 kb plasmid from Pseudomonas sp., Serratia sp. and Escherichia coli, containing merOperon.

CONCLUSION
The partial sequence of merA gene was found on DNA plasmid of mercury resistant bacterium Klebsiella pneumoniae is olate A1.1.1.The nucleotide sequence of the merA gene consist of285 base pairs (bp) which encodes for94deduced amino acids of mercury reductase merA protein.The merA protein sequence has 99% similarity with some strains of Klebsiella pneumonia deposited in GenBank.There is a nucleic acid mutation that causes the deduced amino acid threonine to be replaced by serine at position 524 (Thr→Ser) in the merA protein of Klebsiella pneumonia (accession number: AAR91471.1).