merP Genes Research Articles

MerP/MerT-mediated mechanism: A different approach to mercury resistance and bioaccumulation by marine bacteria

Currently, mechanism underlying mercury resistance and bioaccumulation of marine bacteria remains little understood. A marine bacterium Pseudomonas pseudoalcaligenes S1 is resistant to 120 mg/L Hg2+ with bioaccumulation capacity of 133.33 mg/g. Accordingly, Hg2+ resistance and bioaccumulation mechanism of S1 was investigated at molecular and cellular level. Annotation of S1 transcriptome reveals 772 differentially expressed genes, including Hg2+-relevant genes merT, merP and merA. Both merT and merP gene have three complete copies in S1 genome, while merA gene has only one. In order to evaluate the function of these Hg2+-relevant genes, three recombinant strains were constructed to express MerA (named as A), MerT/MerP (TP) and MerT/MerP/MerA (TPA), respectively. The results show that Hg2+ resistance of strain TP, TPA, and A are improved with minimum inhibition concentration (MIC) being 60 mg/L, 40 mg/L, and 20 mg/L, respectively compared to 2 mg/L of host strain. Strain TP and TPA exhibit enhanced Hg2+ bioaccumulation capacity, while strain A does not differ from the control. Their equilibrium Hg2+ bioaccumulation capacities are 110.48 mg/g, 94.49 mg/g, 83.76 mg/g and 82.29 mg/g, respectively. Summarily, different from most microorganisms that exhibit Hg2+ resistance by MerA-mediated mechanism, marine bacterium S1 achieves Hg2+ resistance and bioaccumulation capability via MerT/MerP-mediated strategy.

Analysis for the presence of determinants involved in the transport of mercury across bacterial membrane from polluted water bodies of India.

Mercury, which is ubiquitous and recalcitrant to biodegradation processes, threatens human health by escaping to the environment via various natural and anthropogenic activities. Non-biodegradability of mercury pollutants has necessitated the development and implementation of economic alternatives with promising potential to remove metals from the environment. Enhancement of microbial based remediation strategies through genetic engineering approaches provides one such alternative with a promising future. In this study, bacterial isolates inhabiting polluted sites were screened for tolerance to varying concentrations of mercuric chloride. Following identification, several Pseudomonas and Klebsiella species were found to exhibit the highest tolerance to both organic and inorganic mercury. Screened bacterial isolates were examined for their genetic make-up in terms of the presence of genes (merP and merT) involved in the transport of mercury across the membrane either alone or in combination to deal with the toxic mercury. Gene sequence analysis revealed that the merP gene showed 86–99% homology, while the merT gene showed >98% homology with previously reported sequences. By exploring the genes involved in imparting metal resistance to bacteria, this study will serve to highlight the credentials that are particularly advantageous for their practical application to remediation of mercury from the environment.

Biosorption of nickel, chromium and zinc by MerP-expressing recombinant Escherichia coli

Escherichia coli hosts able to over-express metal-binding proteins (MerP) originating from Gram-positive ( Bacillus cereus RC607) and Gram-negative ( Pseudomonas sp. K-62) bacterial strains were used to adsorb Ni 2+, Zn 2+ and Cr 3+ in aqueous solutions. The initial adsorption rate and adsorption capacity were determined to evaluate the performance of the biosorbents. With the expression of MerP protein, the metal adsorption capacity of the recombinant strains for Ni 2+, Zn 2+ and Cr 3+ significantly improved. The cells carrying Gram-positive merP gene (GB) adsorbed Zn 2+ and Cr 3+ at a capacity of 22.3 and 0.98 mmol/g biomass, which is 121% and 72% higher, respectively, over that of the MerP-free host cells. Adsorption capacity of the cells carrying Gram-negative merP gene (GP) also increased 144% and 126% for Zn 2+ and Cr 3+, respectively. Both recombinant strains also exhibited 24% and 5% enhancement in adsorption of Ni 2+ for GB and GP, respectively. The initial adsorption rate of the recombinant biosorbents was also higher than that of the MerP-free host, suggesting an increased metal-binding affinity with MerP expression. Severe cell damage on GB biosorbent was observed after Cr 3+ adsorption, probably due to the metal toxicity effect on the cells.

Overexpression of a Single Membrane Component from theBacillus merOperon Enhanced Mercury Resistance in anEscherichia coliHost

Overexpression of a mercuric ion binding protein, MerP, from the mercury resistance operon genes of Gram-positive bacterial strain Bacillus megaterium MB1 and from Gram-negative bacterial strain Pseudomonas aeruginosa K-62 was found to enhance the mercury resistance level of Escherichia coli host cells, even though they share only 27.3% identity. Immunoblot analysis showed that MerP (BMerP) from Bacillus could be expressed on the membrane fraction of E. coli cells. Treated with 10 microM Hg2+, a recombinant strain harboring the BMerP gene significantly improved, showing a 27% increase in mercuric ion adsorption capacity, 16% better than that of a Pseudomonas merP gene (PMerP)-harboring strain. While multiple heavy metals co-existed, the mercuric ion adsorption capacity of the BMerP-harboring E. coli was not affected while that of the PMerP-harboring strain decreased. These results suggest that BMerP can act as a bio-adsorbent compartmentalizing the toxic mercuric ion on the cell membrane and enhancing resistance.

Polypeptides for heavy-metal biosorption: capacity and specificity of two heterogeneous MerP proteins

MerP protein possesses a highly conserved domain with two cysteine residues for Hg 2+ binding. The MerP protein originated from mer operon of Gram-positive bacterium Bacillus cereus RC607 possesses adjoining two cysteines while that of Gram-negative bacterium Pseudomonas sp. K-62 contains two cysteines separated by spacing two other residues. Both heterogeneous merP genes were cloned and over-expressed in Escherichia coli BL21 hosts. The resulting recombinant strains exhibited a six- to eightfold increase in Hg 2+ resistance and an 10% increase in Hg 2+ adsorption capacity. The merP over-expressed strain harboring Gram-positive merP had 142, 84 and 33% increase for Cu 2+, Cd 2+ and Pb 2+ adsorption capability, respectively, over that of merP-free host cells. The strain carrying Gram-negative merP also increased 47, 55 and 12% for Cu 2+, Cd 2+ and Pb 2+ adsorption, respectively. Multi-metal biosorption experiments showed that the affinity of metal biosorption by the recombinant strains decreased in the order of Cu>Pb>Cd. Peptides containing the amino acid sequences of metal-binding motif for both heterogeneous MerP proteins were chemically synthesized and covalently immobilized on Celite carriers to examine their metal-binding ability. For Bacillus MerP-originated peptides, the binding capacity was 0.72, 0.45 and 0.36 mol/mol peptide for Cu, Cd and Pb, respectively, while the capacity was 0.51 0.45 and 0.31 mol/mol peptide for Cu, Cd and Pb, respectively, for the peptide containing Pseudomonas MerP metal-binding motif. These results may show that MerP with adjoining cysteines seems to be more effective in binding metals than that with two separated cysteines.

The role of cysteine residues in the transport of mercuric ions by the Tn501 MerT and MerP mercury-resistance proteins.

Each cysteine residue in the MerT and MerP polypeptides of bacterial transposon Tn501 was replaced by serine, and the mercury-resistance phenotypes of the mutants were determined in Escherichia coli. Cys-24 and Cys-25 in the first transmembrane region of MerT were essential for transport of mercuric ions through the cytoplasmic membrane, and mutations Cys-76-Ser, Cys-82-Ser or Gly-38-Asp in MerT or Cys-36-Ser in MerP all reduced transport and resistance. Deletion of the merP gene slightly reduced mercuric ion resistance and transport, whereas a Cys-33-Ser mutation in MerP appears to block transport of mercuric ions by MerT. The effects of deleting merP on mutations in merT were tested. The 116-amino-acid MerT protein is sufficient for mercuric ion transport across the cytoplasmic membrane.

Lack of involvement of merT and merP in methylmercury transport in mercury resistant Pseudomonas K-62

To clarify whether the merT and merP genes play a role in the transport of methylmercury, we constructed a deletion plasmid, pMRD141 which lacked the genes conferring the organomercurial lyase and the mercuric reductase from plasmid, pMRA17 containing the entire broad-spectrum mercury resistance determinants of the 26 kb-plasmid from Pseudomonas K-62. Plasmid, pMRD141 showed hypersensitivity to Hg 2+ but still expressed a normal sensitivity to methylmercury. The mercury-induced hypersensitive cells carrying pMRD141 took up appreciably more 203Hg 2+ than the induced resistant cells with pMRA17 and the sensitive cells with cloning vector, Bluescript II SK(+) but no difference in the uptake of CH 3 203Hg + among these three strains was found. These results suggested that the merT and merP are only involved in the Hg 2+ transport but do not participate in the transport of methylmercury.

Lack of involvement of merT and merP in methylmercury transport in mercury resistant Pseudomonas K-62.

To clarify whether the merT and merP genes play a role in the transport of methylmercury, we constructed a deletion plasmid, pMRD141 which lacked the genes conferring the organomercurial lyase and the mercuric reductase from plasmid, pMRA17 containing the entire broad-spectrum mercury resistance determinants of the 26 kb-plasmid from Pseudomonas K-62. Plasmid, pMRD141 showed hypersensitivity to Hg2+ but still expressed a normal sensitivity to methylmercury. The mercury-induced hypersensitive cells carrying pMRD141 took up appreciably more 203Hg2+ than the induced resistant cells with pMRA17 and the sensitive cells with cloning vector, Bluescript II SK(+) but no difference in the uptake of CH3(203)Hg2+ among these three strains was found. These results suggested that the merT and merP are only involved in the Hg2+ transport but do not participate in the transport of methylmercury.

Mercury operon regulation by the merR gene of the organomercurial resistance system of plasmid pDU1358.

The structural basis for induction of the mercury resistance operon with inorganic mercury and with the organomercurial compound phenylmercuric acetate was addressed by DNA sequencing analysis and by lac fusion transcription experiments regulated by merR in trans from broad-spectrum-resistance plasmid pDU1358 (Hg2+ and phenylmercury responding). The lac fusion results were compared with those from a narrow-spectrum-resistance (Hg2+ responding but not phenylmercuric responding) operon and the pDU1358 merR deleted at the 3' end. The nucleotide sequence of the beginning region of the broad-spectrum mer operon of plasmid pDU1358 was determined, including that of the merR gene, the operator-promoter region, the merT and merP genes, and the first 60% of the merA gene. Comparison of this sequence with DNA sequences of narrow-spectrum mer operons from transposon Tn501 and plasmid R100 showed that a major difference occurred in the 3' 29 base pairs of the merR gene, resulting in unrelated C-terminal 10 amino acids. A hybrid mer operon consisting of the merR gene from pDU1358, a hybrid merA gene (determining mercuric reductase enzyme), and lacking the merB gene (determining phenylmercury lyase activity) was inducible by both phenylmercury and inorganic Hg2+. This shows that organomercurial lyase is not needed for induction by organomercurial compounds. A mutant form of pDU1358 merR missing the C-terminal 17 amino acids responded to inorganic Hg2+ but not to phenylmercury. Thus, the C-terminal region of the MerR protein of the pDU1358 mer operon is involved in the recognition of phenylmercury.

DNA sequence analysis of bacterial toxic heavy metal resistances

Bacterial plasmids have genes that confer highly specific resistances to As, Bi, Cd, Cu, Cr, Hg, Pb, Te, Zn, and other toxic heavy metals. For each toxic cation or anion, generally a different resistance system exists, and these systems may be "linked" together on multiple resistance plasmids. For Cd2+, AsO2-, AsO4(3)-, Hg2+, and organomercurials, DNA sequence analysis has supplemented direct physiological and biochemical experiments to produce sophisticated understanding. The cadA ATPase of S. aureus plasmids is a 727 amino acid membrane ATPase that pumps Cd2+ from the cells as rapidly as it is accumulated. This polypeptide is related by sequence to other cation translocating ATPases, including the membrane K+ ATPases of Escherichia coli and Streptococcus faecalis, the H+ ATPases of yeast and Neurospora, the Na+/K+ ATPases of vertebrate animals, and the Ca2+ ATPases of rabbit muscle. The conserved residues include the aspartyl residue that is phosphorylated, the lysine involved in ATP binding, and the proline within a membrane translocating region. The arsenate and arsenite translocating ATPase consists of 3 polypeptides (from DNA sequence analysis), including a recognizable ATP binding protein (arsA), an integral membrane protein (arsB gene), and a substrate specificity subunit (arsC gene). Inorganic mercury and organomercurial degradation is carried out by a series of about 6 polypeptides, including 2 soluble intracellular enzymes (organomercurial lyase and mercuric reductase). The latter is related by sequence and function to glutathione reductase and lipoamide dehydrogenase of prokaryotes and eukaryotes. These enzymes are dimeric, FAD-containing, NAD(P)H-dependent oxidoreductases. Other recognizable polypeptides in the mer system include a DNA-binding regulatory protein from the merR gene and a Hg2+ transport system consisting of a periplasmic Hg2(+)-binding protein (merP gene) and a membrane protein (merT gene) in gram negative systems.

Restriction pattern and polypeptide homology among plasmid-borne mercury resistance determinants.

The structural and functional properties of mercury resistance determinants cloned from a series of independently isolated conjugative plasmids were compared with those of the prototype HgR determinants from Tn501 and plasmid R100 (containing Tn21). Restriction endonuclease mapping classified the HgR determinants into at least three different but related structural groups which are distantly related to those from Tn501 and R100. These relationships were confirmed by the functional analysis of sub-clones and gamma delta insertion mutations and from the polypeptides specified by the cloned HgR determinants. Each mercury resistance clone synthesized polypeptides equivalent in size to the merA, merT, and merP gene products. However, those for merA and merT showed considerable size variation. No polypeptide equivalent to merD or merC of R100 was detected.

Role of the merT and merP gene products of transposon Tn 501 in the induction and expression of resistance to mercuric ions

The bacterial transposon Tn 501 carries, in addition to the genetic information for its own transposition, the genes of the mer operon (in the order merRTPAD), which code for resistance to Hg 2+ ions. The basis for the resistance to Hg 2+ is the enzymatic reduction of Hg 2+ to Hg 0 by mercuric reductase, the product of the Tn501 merA gene. We show here that deletion of the merT and merP genes from Tn 501 leads to almost complete loss of the resistance phenotype, even if mercuric reductase is still present in the cytoplasm. Expression of the merT and merP genes in the absence of mercuric reductase gives a mercury-supersensitive phenotype. Mercurydependent induction of transcription of the mer operon can occur in the absence of the merT and merP gene products. However, this induction is reduced by the presence of mercuric reductase in the cell. We conclude that one or both of the merT and merP genes of Tn 501 are required for the expression of the mercury-resistance phenotype. They may in addition have a role in maximising the induction of the mer operon in the presence of Hg 2+ ions. This is consistent with their proposed role in transport of Hg 2+ into the cytoplasm.