A second positive regulatory function in the mer (mercury resistance) operon

Transpositional mutagenesis of the mer operon of the IncFII plasmid, R100, has revealed a second, trans-acting positive regulatory function. Mutants in this function do not synthesize any of the three small mer operon peptides and have no inducible Hg(II) uptake activity. This second regulatory function is part of complementation group B and so depends upon the activity of the previously described trans-acting positive regulatory function merR. All mutants in this new function map in the amino-terminal 20 kDal of the Hg(II) reductase, suggesting either that this enzyme is also a regulatory protein or that there is a distinct protein whose reading frame is superimposed on that of the Hg(II) reductase. While we have only seen the five previously described mer operon peptides of 69, 66, 15.1, 14 and 12 (13) kDal encoded in minicells by single-copy plasmids, we have observed two new HgCl2-inducible polypeptides of approx. 20 kDal in minicells carrying a multicopy derivative of the mer operon of R100. Sequence data for the Hg(II) reductase region of the related mer operon of the transposon, Tn501 [Brown, N.L., Ford, S.J., Pridmore, R.D. and Fritzinger, D.C., Biochemistry 22 (1983) 4089-4095], shows a second reading frame very rich in cysteine and arginine which overlaps the amino-terminal 20 kDal of the Hg(II) reductase structural gene. We believe that this reading frame is the structural gene for this new regulatory function and propose the name merC (for control).     

Cellular and extracellular proteome analysis of Streptococcus mutans grown in a chemostat

The oral pathogen, Streptococcus mutans, was grown under glucose limitation in a chemostat at pH 7.0 and a dilution rate of 0.1 h(-1) to mimic the conditions prevailing in a healthy human oral cavity in between meal times. Solubilized cellular and extracellular proteins were separated by two-dimensional gel electrophoresis (2-DE) and, following tryptic digestion, 421 protein spots analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry or electrospray ionization-tandem mass spectrometry. Analyses of the mass spectral data showed that the proteins matched the translation products of 200 different open reading frames (ORFs) deduced from contigs of the S. mutans UA159 genome and thus represented proteins derived from approximately 11% of the total ORFs of the bacterium. Of the identified proteins, 172 (including one surface protein) were characterized in the cellular fraction, and the remaining 28 (including two surface proteins) were uniquely identified from the culture fluid. The expression and therefore the existence of 30 proteins previously designated as 'hypothetical' or with no known function was confirmed. 2-DE of whole cell lysates revealed only a single intrinsic membrane protein. This is consistent with proteomic analyses of other Gram-positive bacteria where hydrophilic proteins represent the vast majority of those characterized.     

Linkage of a novel mercury resistance operon with streptomycin resistance on a conjugative plasmid in Enterococcus faecium

It has been shown that the mercury in dental amalgam and other environmental sources can select for mercury resistant bacteria and that this can lead to an increase in resistance to antibiotics. To understand more about this linkage we have investigated the genetic basis for mercury and antibiotic resistance in a variety of oral bacteria. In this study we have cloned and sequenced the mer operon from an Enterococcus faecium strain which was resistant to mercury, tetracycline, and streptomycin. This strain was isolated, in a previous investigation, from a cynomolgus monkey post-installation of amalgam fillings. The mer operon was contained within a putative transposon (Tnmer1) of the ISL3 family. This element was located on a streptomycin resistant plasmid, pPPM1000, which shares homology with pRE25.     

Reactivity of Hg(II) with superoxide: evidence for the catalytic dismutation of superoxide by Hg(II).

Mercuric ion, a well-known nephrotoxin, promotes oxidative tissue damage to kidney cells. One principal toxic action of Hg(II) is the disruption of mitochondrial functions, although the exact significance of this effect with regard to Hg(II) toxicity is poorly understood. In studies of the effects of Hg(II) on superoxide (O2-) and hydrogen peroxide (H2O2) production by rat kidney mitochondria, Hg(II) (1-6 microM), in the presence of antimycin A, caused a concentration-dependent increase (up to fivefold) in mitochondrial H2O2 production but an apparent decrease in mitochondrial O2- production. Hg(II) also inhibited O(2-)-dependent cytochrome c reduction (IC50 approximately 2-3 microM) when O2- was produced from xanthine oxidase. In contrast, Hg(I) did not react with O2- in either system, suggesting little involvement of Hg(I) in the apparent dismutation of O2- by Hg(II). Hg(II) also inhibited the reactions of KO2 (i.e., O2-) with hemin or horseradish peroxidase dissolved in dimethyl sulfoxide (DMSO). Finally, a combination of Hg(II) and KO2 in DMSO resulted in a stable UV absorbance spectrum [currently assigned Hg(II)-peroxide] distinct from either Hg(II) or KO2. These results suggest that Hg(II), despite possessing little redox activity, enhances the rate of O2- dismutation, leading to increased production of H2O2 by renal mitochondria. This property of Hg(II) may contribute to the oxidative tissue-damaging properties of mercury compounds.     

Dual-rhodamine urea derivative, a novel chemidosimeter for Hg(II) and its application in imaging Hg(II) in living cells

Abstract  The synthesis and spectral properties of a chemidosimeter 1,4-di[2-(6-ethylamino-3-ethylimino-2,7-dimethyl-3H-xanthen-9-yl) benzoic acid (aminomethyl)-3-phenylthiourea] benzene (1) for Hg(II) ions are reported, and it has been demonstrated that 1 can be used as a fluorescent probe for monitoring Hg(II) ions in living cells. Graphical abstract  A highly sensitive fluorescent probe (1) was developed as a fluorescent and colorimetric chemodosimeter in dimethyl sulfoxide/methanol solution with a broad pH range (pH 5–10) and high selectivity toward Hg²⁺ ions but no significant response toward other competitive cations. Furthermore, by means of confocal laser scanning microscopy experiments, it is demonstrated that 1 can be used as a fluorescent probe for monitoring Hg²⁺ in living cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Sequence and analysis of a plasmid-encoded mercury resistance operon from Mycobacterium marinum identifies MerH, a new mercuric ion transporter

In this study, we report the DNA sequence and biological analysis of a mycobacterial mercury resistance operon encoding a novel Hg²⁺ transporter. MerH was found to transport mercuric ions in Escherichia coli via a pair of essential cysteine residues but only when coexpressed with the mercuric reductase.     

Quantitative proteome analysis of barley seeds using ruthenium(II)-tris-(bathophenanthroline-disulphonate) staining

This paper describes the application of the recently introduced fluorescence stain Ruthenium(II)-tris-(bathophenanthroline-disulphonate) (RuBP) on a comparative proteome analysis of two phenotypically different barley lines. We carried out an analysis of protein patterns from 2-D gels of the parental lines of the Oregon Wolfe Barley mapping population DOM and REC and stained with either the conventional colloidal Coomassie Brilliant Blue (cCBB) or with the novel RuBP solution. We wished to experimentally verify the usefulness of such a stain in evaluating the complex pattern of a seed proteome, in comparison to the previously used cCBB staining technique. To validate the efficiency of visualization by both stains, we first compared the overall number of detected protein spots. On average, 790 spots were visible by cCBB staining and 1200 spots by RuBP staining. Then, the intensity of a set of spots was assessed, and changes in relative abundance were determined using image analysis software. As expected, staining with RuBP performed better in quantitation in terms of sensitivity and dynamic range. Furthermore, spots from a cultivar-specific region in the protein map were chosen for identification to asses the gain of biological information due to the staining procedure. From this particular region, eight spots were visualized exclusively by RuBP and identification was successful for all spots, proving the ability to identify even very low abundant proteins. Performance in MS analysis was comparable for both protein stains. Proteins were identified by MALDI-TOF MS peptide mass fingerprinting. This approach was not successful for all spots, due to the restricted entry number for barley in the database. Therefore, we subsequently used LC-ESI-Q-TOF MS/MS and de novo sequencing for identification. Because only an insufficient number of proteins from barley is annotated, an EST-based identification strategy was chosen for our experiment. We wished to test whether under these limitations the application of a more sensitive stain would lead to a more advanced proteome approach. In summary, we demonstrate here that the application of RuBP as an economical but reliable and sensitive fluorescence stain is highly suitable for quantitative proteome analysis of plant seeds.     

Dithiocarbamates in heavy metal poisoning: complexes of N,N-di(2-hidroxyethyl)dithiocarbamate with Zn(II), Cd(II), Hg(II), CH3Hg(II), and C6H5Hg(II)

The complexes M(DHDC)2, CH3Hg(DHDC), and C6H5Hg(DHDC) (M = Zn, Cd, Hg; DHDC = N,N-di(2-hydroxyethyl)dithiocarbamate) were prepared and investigated in solution and in the solid state by using 1H and 13C NMR, ir, and Raman spectroscopy. The dithiocarbamate group is anisobidentate and the complexes are associated in solution and the solid state via hydrogen bonding. The possible relation of these structural properties to the behavior of DHDC in the treatment of cadmium poisoning is discussed.     

A proteome analysis of the cadmium and mercury response in Corynebacterium glutamicum

Cadmium and mercury are well-known toxic heavy metals, but the basis of their toxicity is not well understood. In this study, we analyzed the cellular response of Corynebacterium glutamicum to sublethal concentrations of cadmium and mercury ions using 2-DE and MS. Mercury induced the over-expression of 13 C. glutamicum proteins, whereas 35 proteins were induced, and 8 proteins were repressed, respectively, under cadmium stress. The principal response to these metals was protection against oxidative stress, as demonstrated by upregulation of, e.g., Mn/Zn superoxide dismutase. Thioredoxin and oxidoreductase responded most strongly to cadmium and mercury. The increased level of heat-shock proteins, enzymes involved in energy metabolism, as well as in lipoic acid and terpenoid biosynthesis after the treatment of cells with cadmium was also registered. Identification of these proteins and their mapping into specific cellular processes enable a global understanding of the way in which C. glutamicum adapts to heavy-metal stress and may help to gain deeper insight into the toxic mechanism of these metals.     

Linear free-energy analysis of mercury(II) and cadmium(II) binding to three-stranded coiled coils

Investigators have studied how proteins enforce nonstandard geometries on metal centers to assess the question of how protein structures can define the coordination geometry and binding affinity of an active-site metal cofactor. We have shown that cysteine-substituted versions of the TRI peptide series [AcG-(LKALEEK)(4)G-NH(2)] bind Hg(II) and Cd(II) in geometries that are different from what is normally found with thiol ligands in aqueous solution. A fundamental question has been whether this structural perturbation is due to protein influence or a change in the metal geometry preference. To address this question, we have completed linear free-energy analyses that correlate the association of three-stranded coiled coils in the absence of a metal with the binding affinity of the peptides to the heavy metals, Hg(II) and Cd(II). In this paper, six new members of this family have been synthesized, replacing core leucine residues with smaller and less hydrophobic residues, consequently leading to varying degrees of self-association affinities. At the same time, studies with some smaller and longer sequenced peptides have also been examined. All of these peptides are seen to sequester Hg(II) and Cd(II) in an uncommon trigonal environment. For both metals, the binding is strong with micromolar dissociation constants. For binding of Hg(II) to the peptides, the dissociation constants range from 2.4 x 10(-)(5) M for Baby L12C to 2.5 x 10(-)(9) M for Grand L9C for binding of the third thiolate to a linear Hg(II)(pep)(2) species. The binding of Hg(II) to the peptide Grand L9C is similar in energetics for metal binding in the metalloregulatory protein, mercury responsive (merR), displaying approximately 50% trigonal Hg(II) formation at nanomolar metal concentrations. Approximately, 11 kcal/mol of the Hg(II)(Grand L9C)(3)(-) stability is due to peptide interactions, whereas only 1-4 kcal/mol stabilization results from Hg(II)(RS)(2) binding the third thiolate ligand. This further validates the hypothesis that the favorable tertiary interactions in protein systems such as merR go a long way in stabilizing nonnatural coordination environments in biological systems. Similarly, for the binding of Cd(II) to the TRI family, the dissociation constants range from 1.3 x 10(-)(6) M for Baby L9C to 8.3 x 10(-)(9) M for TRI L9C, showing a similar nature of stable aggregate formation.     

Hg(II)-coordination by sugar-acids: role of the hydroxy groups

A solution study on the ability of some derivatised sugars [glucuronic acid (GluA), galacturonic acid (GalA) and glucosaminic acid (GlNA)] to complex the Hg(II) ion is reported. The stability constants of the complex species were determined by potentiometric measurements while (1)H NMR experiments allow to define the coordination sites of sugar molecules. GluA coordinates the metal ion through the carboxylic oxygen and the O-4 hydroxyl group and is found to form more stable complexes with respect to GalA in which metal ligation is from the carboxylic oxygen and the O-5 ring oxygen. GlNA forms stable complexes chelating Hg(II) ion through carboxylic oxygen and the alpha-amino group. The ternary 2,2'-bipyridine containing systems were also investigated by means of potentiometric studies. The ML(2) complexes were also isolated in the solid state and characterised by IR spectroscopy.     

Synthesis and x-ray structure of a Hg(II) complex with adenine N(1)-oxide. A model for mercury binding to DNA

The synthesis and crystal structure of the adenine N(1)-oxide complex with mercury(II) chloride, (C₅H₅N₅O)HgCl₂ are reported. Crystals of the coordination compound belong to the monoclinic system, space group P2₁/n with the following primary crystallographic data: a = 6.685(1) Å, b = 11.798(2) Å, c = 10.155(1) Å, β = 100.22(1)°, V = 906.04 ų, Z = 4. The structure was elucidated by conventional Patterson and Fourier methods and refined by the full matrix least-squares technique on the basis of 1977 observed reflections to an R value of 0.074. The basic unit of the structure is a dimer, with a centre of symmetry, consisting of two HgCl₂ moieties and two adenine N(1)-oxide ligands. A polymeric structure results from the bridging interactions of chloride ions. Adenine N(1)-oxide acts as a bidentate bridging ligand, coordinating through N(7) and O(1). The coordination geometry around the mercury ion is a distorted square pyramid with N(7) and three chlorines (two of which are centro-symmetrically related) forming the square plane and O(1) occupying the axial position. Hg also interacts indirectly with N(6) through a Cl

HN hydrogen bond. Principal intracomplex geometrical parameters are as follows: HgN(7) = 2.61(1) Å, HgO(1) = 2.55(1) Å, HgCl(1) = 2.330(3) Å, HgCl(2) = 2.318(3) Å, HgCl(2′) = 3.347(3) Å. The cis angles range from 77.5° to 107.9° and the two trans angles are 155.5° and 163.1°. The centro-symmetrically related bases overlap partially and pack at a distance of 3.2 Å. The glide-related bases are linked by a hydrogen bond, N(9)H

O(1) and are inclined to one another by 109.7°. The results are compared with those derived from spectroscopic and other physicochemical studies on metal interaction with adenine N(1)-oxide. Based on the present structural observations and earlier experimental results a possible mechanism is proposed for mercury interaction with DNA.     

Reactivity of Hg(II) with superoxide: Evidence for the catalytic dismutation of superoxide by HG(II)

Mercuric ion, a well-known nephrotoxin, promotes oxidative tissue damage to kidney cells. One principal toxic action of Hg(II) is the disruption of mitochondrial functions, although the exact significance of this effect with regard to Hg(II) toxicity is poorly understood. In studies of the effects of Hg(II) on superoxide (O) and hydrogen peroxide (H₂O₂) production by rat kidney mitochondria, Hg(II) (1–6 μM), in the presence of antimycin A, caused a concentration-dependent increase (up to fivefold) in mitochondrial H₂O₂ production but an apparent decrease in mitochondrial O production. Hg(II) also inhibited O-dependent cytochrome c reduction (IC₅₀ ≈?2–3 μM) when O was produced from xanthine oxidase. In contrast, Hg(I) did not react with O in either system, suggesting little involvement of Hg(I) in the apparent dismutation of O by Hg(II). Hg(II) also inhibited the reactions of KO₂ (i.e., O) with hemin or horseradish peroxidase dissolved in dimethyl sulfoxide (DMSO). Finally, a combination of Hg(II) and KO₂ in DMSO resulted in a stable UV absorbance spectrum [currently assigned Hg(II)-peroxide] distinct from either Hg(II) or KO₂. These results suggest that Hg(II), despite possessing little redox activity, enhances the rate of O dismutation, leading to increased production of H₂O₂ by renal mitochondria. This property of Hg(II) may contribute to the oxidative tissue-damaging properties of mercury compounds.     

Prevalence of the multiple antibiotic resistance operon (marRAB) in the genus Salmonella

The multiple antibiotic resistance operon (marRAB) is a member of the multidrug resistance (mdr) systems. Similar to other mdr systems, this operon when induced encodes resistance to structurally and functionally unrelated antibiotics. marRAB has been shown to be conserved in the family Enterobacteriaceae, but within the genus Salmonella certain species appeared to be lacking this operon. To investigate how conserved the marRAB operon was in Salmonella, 30 veterinary isolates were examined by PCR, Southern blot, and dot blot analysis. Using DNA primers based on the marRAB operon of S. typhimurium, a predicted 2.3-kb amplicon resulted after PCR in 16 of the 30 organisms. The 2.3-kb DNA band from S. enteritidis was cloned and sequenced and shown to possess 99% sequence homology to marRAB from S. typhimurium. Using a labeled marRAB gene probe from S. enteritidis, Southern blot and dot blot analysis confirmed the presence of the operon in all 30 Salmonella species examined. Furthermore, when these isolates were induced with low levels of either tetracycline or chloramphenicol, increased antimicrobial resistance was observed to structurally and functionally unrelated antibiotics. Thus, the widespread occurrence of the marRAB locus in this genus prescribes judicious use of antimicrobials to avoid induction of a mdr phenotype.     

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.     

Zn(II) and Hg(II) complexes of naphthalene based thiosemicarbazone: Structure and spectroscopic studies

Synthesis, structure and spectroscopic characterization of 2-thiophenealdehyde-N(4)-napthylthiosemicarbazone and its complexes with biologically important Zn(II) and toxic Hg(II) metal ions have been reported. The crystal structure of the complexes reveals that both are distorted tetrahedral. In the Hg(II) complex the ligand is neutral and mondented where as in Zn(II) complex the ligand is bidented and anionic. Whereas conductivity measurement study shows both the complexes are molecular species. The beautiful changes in absorption spectra along with isosbestic points upon addition of respective metal salts to the ligand solution convincingly support the formation of metal complexes in solution phase. The other spectroscopic studies also show good correlation with their solid state structures.     

福建漳江口红树林沉积物重金属汞(Hg)的分布特征

对福建漳江口红树林沉积物中重金属汞(Hg)含量的时空分布进行了研究。结果表明漳江口红树林湿地汞含量为0.0815 mg•kg–1—0.3431 mg•kg–1。在潮间带水平分布上, 从光滩到高潮位汞含量显著上升(P<0.05), 相对光滩, 红树林沉积物更易富集汞, 其中高潮位红树林底泥中汞含量最高。在垂直方向上, 大多数的汞富集在表层及亚表层, 随着深度的加深, 汞含量呈现下降趋势。汞在红树林湿地沉积物中的积累主要与有机碳(TOC), 含水率, 盐度相关。从计算的单一污染指数(Pi)来看, 漳江口地区汞污染等级为Ⅲ级以下, 大多数采样点属于自由污染或轻度污染。就该地区汞污染的时空变化趋势以及和其他红树林区汞污染状况的对比来看, 漳江口地区存在一定程度的汞污染威胁加剧的现象, 值得引起注意。