Evidence for direct interactions between the mercuric ion transporter (MerT) and mercuric reductase (MerA) from the Tn<Emphasis Type="Italic">501</Emphasis><Emphasis Type="Italic">mer</Emphasis> operon

Mercuric ion resistance in bacteria requires transport of mercuric ions (Hg²⁺) into the cytoplasmic compartment where they are reduced to the less toxic metallic mercury (Hg⁰) by mercuric reductase (MR). The long-established model for the resistance mechanism predicts interactions between the inner membrane mercuric ion transporter, MerT, and the N-terminal domain of cytoplasmic MR, but attempts to demonstrate this interaction have thus far been unsuccessful. A recently developed bacterial two-hybrid protein interaction detection system was used to show that the N-terminal region of MR interacts with the cytoplasmic face of MerT. We also show that the cysteine residues on the cytoplasmic face of the MerT protein are required for maximal mercuric ion transport but not for the interaction with mercuric reductase.     

Overexpression of a single membrane component from the Bacillus mer operon enhanced mercury resistance in an Escherichia coli host

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.     

Overexpression of MerT,the mercuric ion transport protein of transposon Tn501, and genetic selection of mercury hypersensitivity mutations

The small (116 amino acids) inner membrane protein MerT encoded by the transposon Tn501 has been overexpressed under the control of the bacteriophage T7 expression system. Random mutants of MerT were made and screened for loss of mercuric ion hypersensitivity. Several mutantmerT genes were selected and sequenced: Cys24Arg and Cys25Tyr mutations abolish mercury resistance, as do charge-substitution mutations in the first predicted transmembrane helix (Glyl4Arg, Glyl5Arg, Gly27Arg, Ala18Asp), and the termination mutations Trp66Ter and Cys82Ter.     

Overexpression of MerT, the mercuric ion transport protein of transposon Tn501, and genetic selection of mercury hypersensitivity mutations

The small (116 amino acids) inner membrane protein MerT encoded by the transposon Tn501 has been overexpressed under the control of the bacteriophage T7 expression system. Random mutants of MerT were made and screened for loss of mercuric ion hypersensitivity. Several mutantmerT genes were selected and sequenced: Cys24Arg and Cys25Tyr mutations abolish mercury resistance, as do charge-substitution mutations in the first predicted transmembrane helix (Glyl4Arg, Glyl5Arg, Gly27Arg, Ala18Asp), and the termination mutations Trp66Ter and Cys82Ter.     

Transient loss of plasmid-mediated mercuric ion resistance after stress in Pseudomonas aeruginosa

After freezing and thawing, Pseudomonas aeruginosa harboring a drug resistance plasmid (Hg2+r, Strr), became acutely sensitive to mercuric ions but not to streptomycin in the plating medium, whereas its sensitivity to both agents became more pronounced indicating a synergistic effect. This freeze-thaw-induced sensitivity was transient and capable of being repaired to a simple salts medium. Transient outer and cytoplasmic membrane damage was also observed in frozen and thawed preparations. From kinetics studies, repair of cytoplasmic membrane damage superseded repair of outer membrane damage and damage measured by mercuric ions and mercuric ions plus streptomycin. Osmotically shocked cells were also sensitive to mercuric ions, mercuric ions plus streptomycin, and sodium lauryl sulfate, but not to sodium chloride or streptomycin alone. This sensitivity was again transient and capable of repair in the same simple salts medium. Active transport of a non-metabolizable amino acid, alpha-amino isobutyric acid, was sensitive to mercuric ions and became more so after freezing and thawing. A freeze-thaw-resistant mercuric ion-dependent reduced nicotinamide adenine dinucleotide phosphate oxidoreductase was localized in the cytoplasm of this organism. This enzyme and an intact outer membrane appear to be required for mercuric ion resistance in this strain.     

Transient loss of plasmid-mediated mercuric ion resistance after stress in Pseudomonas aeruginosa.

After freezing and thawing, Pseudomonas aeruginosa harboring a drug resistance plasmid (Hg2+r, Strr), became acutely sensitive to mercuric ions but not to streptomycin in the plating medium, whereas its sensitivity to both agents became more pronounced indicating a synergistic effect. This freeze-thaw-induced sensitivity was transient and capable of being repaired to a simple salts medium. Transient outer and cytoplasmic membrane damage was also observed in frozen and thawed preparations. From kinetics studies, repair of cytoplasmic membrane damage superseded repair of outer membrane damage and damage measured by mercuric ions and mercuric ions plus streptomycin. Osmotically shocked cells were also sensitive to mercuric ions, mercuric ions plus streptomycin, and sodium lauryl sulfate, but not to sodium chloride or streptomycin alone. This sensitivity was again transient and capable of repair in the same simple salts medium. Active transport of a non-metabolizable amino acid, alpha-amino isobutyric acid, was sensitive to mercuric ions and became more so after freezing and thawing. A freeze-thaw-resistant mercuric ion-dependent reduced nicotinamide adenine dinucleotide phosphate oxidoreductase was localized in the cytoplasm of this organism. This enzyme and an intact outer membrane appear to be required for mercuric ion resistance in this strain.     

Overexpression of a Single Membrane Component from the Bacillus mer Operon Enhanced Mercury Resistance in an Escherichia coli Host

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 μM 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.     

Tn5044-conferred mercury resistance depends on temperature: the complexity of the character of thermosensitivity

Escherichia coli K12 containing the transposon Tn5044 mer operon (merR, T, P, C, and A genes) is resistant to mercuric chloride at 30°C but sensitive to this compound at 37–41.5°C. We have studied the mechanism underlying the temperature-sensitive nature of this mercury resistance phenotype, and found that the expression of the Tn5044 merA gene coding for mercuric reductase (MerA) is severely inhibited at non-permissive temperatures. Additionally, MerA showed a considerably reduced functional activity in vivo at non-permissive temperatures. However, the temperature-sensitive character of the functioning of this enzyme in cell extracts, where it interacted with one of the low-molecular weight SH compounds rather than with the transport protein MerT (as is the case in vivo), was not apparent. These data suggest that the temperature-sensitive mercury resistance phenotype should stay under control at two stages: when the merA gene is expressed and when its product interacts with MerT to accept the mercuric ion.     

Tn5563, a transposon encoding putative mercuric ion transport proteins located on plasmid pRA2 of Pseudomonas alcaligenes

Sequence analysis of pRA2, an endogenous 33-kb plasmid from Pseudomonas alcaligenes NCIB 9867 (strain P25X), revealed the presence of a 6256-bp transposon of the Tn3 family, designated Tn5563. Tn5563, which is flanked by two 39-bp inverted repeats, encodes a transposase, a resolvase, and two open reading frames which share amino acid sequence similarities with the mercuric ion transport proteins MerT and MerP encoded by several mer operons. However, no other mer operon genes were found on Tn5563. Sequencing of a RP4::Xln hybrid plasmid indicates possible interactions between pRA2 and the P25X chromosome mediated by Tn5563.     

Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg(2+)-contaminated environments.

Escherichia coli strains were genetically engineered to express an Hg2+ transport system and metallothionein. Overexpression of a glutathione S-transferase fusion protein of Saccharomyces cerevisiae or pea metallothionein significantly increased the bioaccumulation of Hg2+ transported by MerT and MerP and protected the cells from the accumulated Hg2+. The recombinant strains have excellent properties for bioremediation of Hg(2+)-contaminated environments.     

1H NMR studies of the mercuric ion binding protein MerP: Sequential assignment,secondary structure and global fold of oxidized MerP

Summary The oxidized form of the mercuric ion binding protein MerP has been studied by two-dimensional NMR. MerP, which is a periplasmic water-soluble protein with 72 amino acids, is involved in the detoxification of mercuric ions in bacteria with resistance against mercury. The mercuric ions in the periplasmic space are first scavenged by the MerP protein, then transported into the cytoplasm by the membrane-bound transport protein MerT, and finally reduced to elementary (nontoxic) mercury by the enzyme mercuric reductase. In this work, the ¹H NMR spectrum of oxidized MerP (closed disulfide bridge) has been assigned by using homonuclear 2D NMR techniques. The secondary structure and global fold have been inferred from the nuclear Overhauser effect (NOE) data. The secondary structure comprises four -strands and two -helices, in the order ₁₁₂₃₂₄. The protein folds into an antiparallel -sheet, ₂₃₁₄, with the two antiparallel helices on one side of the sheet. The folding topology is similar to that of acylphosphatase, the activation domain of porcine pancreatic procarboxypeptidase B, the DNA-binding domain of bovine papillomavirus-1 E2 and the RNA-binding domains of the U1 snRNP A and hnRNP C proteins. However, there is no structural similarity between MerP and other bacterial periplasmic binding proteins.     

Mercury transport and resistance

Resistance to mercuric ions in bacteria is conferred by mercuric reductase, which reduces Hg(II) to Hg(0) in the cytoplasmic compartment. Specific mercuric ion transport systems exist to take up Hg(II) salts and deliver them to the active site of the reductase. This short review discusses the role of transport proteins in resistance and the mechanism of transfer of Hg(II) between the mercury-resistance proteins.     

Crystal structure of the oxidized form of the periplasmic mercury-binding protein MerP from Ralstonia metallidurans CH34

In Ralstonia metallidurans CH34, the gene merP encodes for a periplasmic mercury-binding protein which is capable of binding one mercury atom. The metal-binding site of MerP consists of the highly conserved sequence GMTCXXC found in the family that includes metallochaperones and metal-transporting ATPases. We purified MerP from R.metallidurans CH34 and solved its crystal structure under the oxidized form at 2.0A resolution. Superposition with structures of other metal-binding proteins shows that the global structure of R.metallidurans CH34 oxidized MerP follows the general topology of the whole family. The largest differences are observed with the NMR structure of oxidized Shigella flexneri MerP. Detailed analysis of the metal-binding site suggests a direct role for Y66 in stabilizing the thiolate group of C17 during the mercury-binding reaction. The metal-binding site of oxidized MerP is also similar to the metal-binding sites of oxidized copper chaperone for superoxide dismutase and Atx1, two copper-binding proteins from Saccharomyces cerevisiae. Finally, the packing of the MerP crystals suggests that F38, a well-conserved residue in the MerP family may be important in mercury binding and transfer. We propose a possible mechanism of mercury transfer between two CXXC motifs based on a transient bi-coordinated mercury intermediate.     

Bacterial resistances to mercury and copper.

Heavy metals are toxic to living organisms. Some have no known beneficial biological function, while others have essential roles in physiological reactions. Mechanisms which deal with heavy metal stress must protect against the deleterious effects of heavy metals, yet avoid depleting the cell of a heavy metal which is also an essential nutrient. We describe the mechanisms of resistance in Escherichia coli to two different heavy metals, mercury and copper. Resistance of E. coli to mercury is reasonably well understood and is known to occur by transport of mercuric ions into the cytoplasmic compartment of the bacterial cell and subsequent reductive detoxification of mercuric ions. Recent mutational analysis has started to uncover the mechanistic detail of the mercuric ion transport processes, and has shown the essential nature of cysteine residues in transport of Hg(II). Resistance to copper is much less well understood, but is known to involve the increased export of copper from the bacterial cell and modification of the copper; the details of the process are still being elucidated. Expression of both metal resistance determinants is regulated by the corresponding cation. In each case the response enables the maintenance of cellular homeostasis for the metal. The conclusions drawn allow us to make testable predictions about the regulation of expression of resistance to other heavy metals.     

Growth and Germination Inhibitors in Hog-weed

Properties of a glutathione transport system in T. ferrooxidans strain AP-44 were investigated using a reduced form of ³⁵S-glutathione (³⁵S-GSH). About 71.2% of the total radioactivity taken up into the cells was distributed in the cytosol fraction. The amount of GSH taken up into the cells was in proportion to the amount of ferrous iron oxidized. However, a high concentration of silver ions (50 mm), which completely inhibited an iron-oxidizing activity, did not inhibit the GSH transport. The results suggest that GSH was transported by using a proton electrochemical gradient formed across the cytoplasmic membrane. Since growth inhibition by silver nitrate was decreased by the addition of GSH to both silver ion sensitive-cells and resistant-cells, the GSH transport system may play some role in the silver ion resistance mechanism of the bacterium.