Mercury resistance in aromatic compound degrading Pseudomonas strains

Abstract: Six of ten Pseudomonas strains selected from environmental samples for their ability to degrade aromatic compounds were found to be mercury resistant. Mercury detoxification proceeded through Hg²⁺ volatilization and the genes involved were chromosomally located. All the mercury resistant strains proved able to degrade aromatic compounds in the presence of Hg²⁺.     

Volatilisation of metals and metalloids by the microbial population of an alluvial soil

In order to assess the microbial contribution to the volatilisation of metal(loid)s by methylation and hydridisation in the environment, we focused on soils of different origin. Here, we describe the biogenic production of volatile metal(loid) species of an alluvial soil with rather low metal(loid) contamination. The production of volatile metal(loid) compounds was monitored in soil suspensions kept under anaerobic conditions over an incubation time of 3 months. In the headspace of the samples, we detected mainly hydrids and methylated derivatives of a broad variety of elements such as arsenic, antimony, bismuth, selenium, tellurium, mercury, tin and lead, with the volatile products of arsenic, antimony and selenium representing the highest portions. Classical cultivation-dependent procedures resulted in the isolation of a strictly anaerobic Gram-positive strain (ASI-1), which shows a high versatility in transforming metal(loid) ions to volatile derivatives. Strain ASI-1 is affiliated to the species Clostridium glycolicum due to its high 16S rDNA sequence similarity with members of that species. As shown by fluorescence in situ hybridisation, strain ASI-1 amounts to approximately 2% of the total microbial flora of the alluvial soil. Since the spectrum of volatile metal(loid) compounds produced by this strain is very similar to that obtained by the whole population regarding both the broad variety of metal(loid)s converted and the preference for volatilising arsenic, antimony and selenium, we suggest that this strain may represent a dominant member of the metal(loid) volatilisating population in this habitat.     

Influence of 1,10-phenanthroline and its analogues, other chelators and transition metal ions on dipeptidase activity of the rumen bacterium, Prevotella ruminicola

Prevotella ruminicola plays a prominent role in the breakdown of peptides in the rumen, a process which contributes to excessive ammonia production and inefficient nitrogen retention in ruminants. Various metal ions and chelators were examined to assess how the metal ion-dependent dipeptidase activity of P. ruminicola M384 might be inhibited. Using sonicated extracts, Cu²⁺, Cr²⁺ and Hg²⁺ were most inhibitory, decreasing Ala₂ breakdown to 15, 15 and 5% of control activity, whereas Co²⁺, Mn²⁺ and Zn²⁺ stimulated activity by 189, 30 and 26%, respectively. The chelators, EDTA, EGTA, TPEN and 1,10-phenanthroline, were inhibitory, as were several phenanthroline analogues. Among the stereoisomers of 1,10-phenanthroline tested, derivatives methylated on C-2 and C-9 were less effective than the parent molecule, but 3,4,7,8-tetramethyl,10-phenanthroline (TMP) was more inhibitory. Titration of the most effective inhibitors showed that EDTA, TPEN and TMP had similar potency and were effective at 0.1 mmol l⁻¹ and above. Thus some metal ions and chelators are potent inhibitors of P. ruminicola dipeptidase, although they are unlikely to be sufficiently specific to peptide metabolism to be useful in vivo.     

Volatilisation of metals and metalloids: An inherent feature of methanoarchaea?

As shown by recent studies, anaerobic members of Archaea and Bacteria are involved in processes that transform ionic species of metals and metalloids (arsenic, antimony, bismuth, selenium, tellurium and mercury) into volatile and mostly toxic derivatives (mainly methyl derivatives or hydrides). Since the fact that these transformations proceed in both environmental settings and in parts of the human body, we have to consider that these processes also interfere directly with human health. The diversity of the volatile derivatives produced and their emission rates were significantly higher in methanoarchaeal than in bacterial strains, which supports the pivotal role of methanoarchaea in transforming metals and metalloids (metal(loid)s) into their volatile derivatives. Compared with methanoarchaea, 14 anaerobic bacterial strains showed a significantly restricted spectrum of volatilised derivatives and mostly lower production rates of volatile bismuth and selenium derivatives. Since methanoarchaea isolated from the human gut (Methanosphaera stadtmanae, Methanobrevibacter smithii) showed a higher potential for metal(loid) derivatisation compared to bacterial gut isolates, we assume that methanoarchaea in the human gut are mainly responsible for the production of these volatile derivatives. The observation that trimethylbismuth ((CH(3))(3)Bi), the main volatile derivative of bismuth produced in human feces, inhibited growing cultures of Bacteroides thetaiotaomicron, a representative member of the human physiological gut flora, suggests that these volatiles exert their toxic effects on human health not only by direct interaction with host cells but also by disturbing the physiological gut microflora.     

Cations Affect [3H]Mazindol and [3H]WIN 35,428 Binding to the Human Dopamine Transporter in a Similar Fashion

Abstract: The present study addresses the possibility that there are different cocaine-related and mazindol-related binding domains on the dopamine transporter (DAT) that show differential sensitivity to cations. The effects of Zn²⁺, Mg²⁺, Hg²⁺, Li⁺, K⁺, and Na⁺ were assessed on the binding of [³H]mazindol and [³H]WIN 35,428 to the human (h) DAT expressed in C6 glioma cells under identical conditions for intact cell and membrane assays. The latter were performed at both 0 and 21°C. Zn²⁺ (30–100 µ M ) stimulated binding of both radioligands to membranes, with a relatively smaller effect for [³H]mazindol; Mg²⁺ (0.1–100 µ M ) had no effect; Hg²⁺ at ∼3 µ M stimulated binding to membranes, with a relatively smaller effect for [³H]mazindol than [³H]WIN 35,428 at 0°C, and at 30–100 µ M inhibited both intact cell and membrane binding; Li⁺ and K⁺ substitution (30–100 m M ) inhibited binding to membranes more severely than to intact cells; and Na⁺ substitution was strongly stimulatory. With only a few exceptions, the patterns of ion effects were remarkably similar for both radioligands at both 0 and 21°C, suggesting the involvement of common binding domains on the hDAT impacted similarly by cations. Therefore, if there are different binding domains for WIN 35,428 and mazindol, these are not affected differentially by the cations studied in the present experiments, except for the stimulatory effect of Zn²⁺ at 0 and 21°C and Hg²⁺ at 0°C.     

Arsenic metabolism in freshwater and terrestrial plants

Freshwater and terrestrial plants differ markedly in their ability to metabolize arsenate. In experiments with higher terrestrial plants, e.g. tomato, Lycopersicon esculentum Mill. cv. Better boy, ⁷⁴As-arsenate was readily taken up and reduced to arsenite. Methylation and reduction to methanearsonic acid, methanearsinic acid (indicated for the first time) and dimethylarsinic acid were apparent only in phosphate deficient plants. Lower and higher freshwater plants, e.g. Nitella tenuissima Kütz. and Lemna minima Phill., not only methylated arsenic but also produced considerable amounts of an arsoniumphospholipid previously identified in marine algae. These differences indicate that freshwater but not terrestrial plants have evolved mechanisms for rapid detoxication of arsenate, arsenite and other toxic arsenic species.     

Possible involvement of red pigments in defense against mercury in Pseudomonas K-62

Abstract To study the physiological role of the red pigments in soil strain Pseudomonas K-62, we isolated a red pigment-deficient white mutant from the soil strain by treatment with mitomycin C and compared the phenotypic properties of the mutant and parent strain. The red pigments, which were classified as one of carotenoids based on their physicochemical properties, were separated into two groups, designated pigment A and B respectively on NH-Chromatorex HPLC.The crude pigments and pigment B which could react with Hg²⁺ in the wild-type Pseudomonas K-62 and its mercury-resistant plasmid-deficient strain were enhanced by the addition of Hg²⁺. The white mutant thus obtained showed a greater sensitivity to Hg²⁺ than the wild-type reddish strain despite containing the resistant plasmids. The major component in pigment B was identified by mass spectrometric analysis as 1-hydroxy-1-methoxy-1,2, 1',2',7',8'-hexahydro-ψ,ψ-caroten-4-one, a carotenoid monoketone. These results suggested that red pigments, especially pigment B, may account, at least partially, for defense against Hg²⁺ in the bacterial environments.     

Endogenously Occurring β-Carboline Induces Parkinsonism in Nonprimate Animals: A Possible Causative Protoxin in Idiopathic Parkinson's Disease

Abstract: To examine whether simple β-carbolines induce parkinsonian-like symptoms in vivo via N -methylation, the simple β-carbolines norharman (NH), 2-mono- N -methylated norharmanium cation (2-MeNH⁺), and 9-mono- N '-methylnorharman (9-MeNH) were systematically administered to C57BL/6 mice for 7 days. These substances induced bradykinesia with reduction of locomotion activity. NH or 2-MeNH⁺ decreased dopamine (DA) contents to 50–70% of values in controls in the striatum and midbrain. 9-MeNH potently decreased not only DA but also serotonin content in various regions. Immunohistochemical examination revealed that the numbers of tyrosine hydroxylase (TH)-positive cells in the substantia nigra pars compacta of NH- and 9-MeNH-treated mice were diminished to 76 and 66% of values in control mice, respectively. The formation of a toxic metabolite, 2,9-di- N , N '-methylated norharmanium cation (2,9-Me₂NH⁺), was 14 and eight times higher in the brain of mice receiving 9-MeNH than that in NH- and 2-MeNH⁺-treated mice, respectively. In cultured mesencephalic cells from rat embryo, 2,9-Me₂NH⁺ selectively killed TH-positive neurons only at a lower dose but was toxic to all neurons at higher doses. Thus, the excess formation of 2,9-Me₂NH⁺ would induce nonspecific neurotoxicity. These results indicated that 9-indole nitrogen methylation should be the limiting step in the development of the toxicity. NH, a selective dopaminergic toxin precursor, is sequentially methylated to form 2,9-Me₂NH⁺, which could be an underlying factor in idiopathic Parkinson's disease.     

Partial purification and some biochemical properties of acid phosphatase in germinating chickpea (Cicer arietinum) seeds

An acid phosphatase (EC 3.1.3.2.) from the embryonic axes of chickpea seeds ( Cicer arietinum L. cv. Castellana) was purified by ammonium sulphate precipitation, chromatography on Sephacryl S-200 and polyacrylamide gel electrophoresis. The preparation has an apparent molecular weight of 39 kDa, pH optimum for p -nitrophenylphosphate hydrolysis of 5.25, and K _m of 0.57 m M . The enzyme hydrolyzed all the mono- and di-phosphorylated sugars tested, but had no effect on ATP, ADP, AMP and phosphoenolpyruvate. Phosphate was a competitive inhibitor. Mg²⁺. Ca²⁺, Hg²⁺, Fe³⁺, arsenate, K⁺ and Zn²⁺ were inhibitory. Mn²⁺, dithiothreitol and EDTA had no effect, and polyamines were activators.     

Purification and properties of mercuric reductase from Azotobacter chroococcum

Mercury resistance determinants in bacteria are often plasmid-borne or transposon-mediated. Mercuric reductase, one of the proteins encoded by the mercury resistance operon, catalyses a unique reaction in which mercuric ions, Hg (II), are reduced to mercury metal Hg(O) using NADPH as a source of reducing power. Mercuric reductase was purified from Azotobacter chroococcum SS₂ using Red A dye matrix affinity chromatography. Freshly purified preparations of the enzyme showed a single band on polyacrylamide gel electrophoresis under non-denaturing conditions. After SDS-polyacrylamide gel electrophoresis of the freshly prepared enzyme, two protein bands, a major and a minor one, were observed with molecular weight 69 000 and 54 000, respectively. The molecular weight of the native enzyme as determined by gel filtration in Sephacryl S-300 was 142 000. The Km of Hg²⁺-reductase for HgCl₂ was 11·11 μmol l⁻¹. Titration with 5,5'-dithiobis (2-nitrobenzoate) demonstrated that two enzyme–SH groups become kinetically accessible on reduction with NADPH.     

Influence of pulsing and postpulsing media tonicity on electrotransformation of intact yeast cells

Abstract The carboxylesterases from Proteus vulgaris, Salmonella enterica and Citrobacter amalonaticus were purified 104-, 95- and 120-fold, respectively by chromatography. The enzymes had similar catalytic activities but differed considerably in their inactivation by heat, di-isopropyl fluorophosphate and Cd²⁺, Zn²⁺, Hg²⁺ and Cu²⁺. Quantitative neutralization of hydrolytic activity with specific immunoglobulins indicated that the three enzymes were antigenically distinct.     

The Pmr1 protein, the major yeast Ca2+-ATPase in the Golgi, regulates intracellular levels of the cadmium ion

Cadmium is a nonessential, highly toxic heavy metal that shows ionic properties similar to calcium. These ionic similarities imply that the cadmium ion, Cd²⁺, is a calcium ion, Ca²⁺, receptor-agonist, affecting the same biochemical pathways involved in Ca²⁺ homeostasis. In the yeast Saccharomyces cerevisiae , the PMC1 and PMR1 genes encode vacuolar and Golgi Ca²⁺-ATPases, respectively. The PMR1 protein product Pmr1p is involved in both Ca²⁺ and Mn²⁺ homeostasis. This study investigated the importance of Pmc1p and Pmr1p for Cd²⁺ cellular detoxification. Using the standard techniques of yeast molecular research and a multielemental procedure named particle-induced X-ray emission, Pmr1p was identified as a protein that directly participates in the detoxification of Cd²⁺, possibly through the secretory pathway. The results allow us to posit a model of Cd²⁺ detoxification where Pmr1p has a central role in cell survival in a Cd²⁺-rich environment.     

Inhibition of anthocyanin synthesis and phenylalanine ammonialyase activity by Co2+ in leaf disks of Terminalia catappa

Treatment with sucrose induced anthocyanin synthesis and phenylalanine ammonialyase (PAL, EC 4. 3. 1. 5) activity in leaf disks of Indian almond ( Terminalia catappa L. Duthie). Co²⁺, an inhibitor of ethylene biosynthesis, inhibited anthocyanin synthesis and PAL activity when given together with sucrose. Ethephon (an exogenous source of ethylene) given along with sucrose, promoted anthocyanin synthesis and PAL activity, but in the presence of Co²⁺ its effectiveness decreased. In an attempt to understand the inhibitory action of Co²⁺ in the presence of ethephon, the effect of Co²⁺ on PAL activity was studied in vitro. A kinetic study showed an uncompetitive type of inhibition of PAL by Co²⁺, which was not time dependent. Addition of 2-mercaptoethanol, cysteine or glutathione overcame the in vitro effect of Co²⁺, and 2-mercaptoethanol also restored the activity of PAL extracted from Co²⁺-treated leaf disks. It is suggested that sulfhydryl group(s) might be involved in the inactivation of PAL by Co²⁺. The effects of N-ethylmaleimide (NEM) and HgCl₂ (other sulfhydryl reagents) were also studied. Both NEM and Hg²⁺ competitively inhibited PAL activity in vitro, and the inhibition could be reversed by sulfhydryl compounds.     

Species of Anguilla as indicators of mercury in the coastal rivers and lakes of Victoria, Australia

Mercury concentrations in the axial muscle tissue of most (243) of the 254 Anguilla australis and most (20) of the 27 A. reinhardtii collected from 30 sites in coastal rivers and lakes in Victoria, Australia, during 1975–78 were well below the Australian statutory health limit (0.5 μg g⁻¹ wet weight). For A. australis the mean mercury concentration was 0.17 μg g⁻¹ (±0.16 s.d. , range 0.01–1.60 μg g⁻¹); for A. reinhardtii the values were 0.37 ± 0.23 μg g⁻¹ (range 0.12–1.10 μg g⁻¹). Statistical analyses showed that variation in mercury concentration due to total length accounted for only 13% of the total variation in A. australis and 2% in A. reinhardtii whereas locality accounted for 54 and 68%, respectively. Both species are thus considered suitable as indicators of mercury pollution.     

Toxicity versus avoidance response of golden shiner, Notemigonus crysoleucas, to five metals

Avoidance thresholds and 96-h LC50 values were determined for golden shiners, Noiemigonus crysoleucas , for five individual elements: chromium, Cr; copper, Cu; cadmium, Cd; arsenic, As; selenium, Se. The avoidance concentrations were 73, 26 and 28 μgl^-1 for Cr-VI, Cu and As-III, respectively. Cadmium and Se were not avoided at experimental concentrations up to 68 and 3489 μg1^-1, respectively. Acute flow-through 96-h LC50 values were Cr-VI 55·0, Cu 84·6, and As-III 12·5 mg 1^-1, which were more than two orders of magnitude above avoidance concentrations. The acute flow-through 96-h LC50 values for Cd and Se were 2·8 and 11·2 mg 1^-1, respectively. These concentrations are 31 and 2·2 times the highest concentration employed in the avoidance tests, neither of which were avoided by the test organisms. Thus, simple toxicity tests do not identify the environmental hazard of some elements, and the most toxic elements may not elicit a behavioural response. When used in concert with tests of organism function, more realistic indicators of environmental hazard or safety may be determined.     

Effect of low growth temperature on coupling between electron transport and proton flux in Vicia faba thylakoids

Coupling between electron transport and proton flux has been compared in chloroplasts from Vicia faba (cv. Windsor) plants grown at 20 and 5°C. Proton uptake by warm-grown thylakoids was sensitive to external pH and stimulated by micromolar adenine nucleotide above pH 7.0. Electron transport was modulated by pH, adenine nucleotide and energy transfer inhibitors (triphenyltin and Hg²⁺). By contrast, proton uptake by cold-grown thylakoids was generally lower and was insensitive to micromolar ATP. The rate of non-phosphorylating electron flow in cold-grown thylakoids was relatively insensitive to pH and Hg²⁺ and was not modulated by adenine nucleotides or triphenyltin. Stimulation of electron transport by phosphorylating conditions in cold-grown thylakoids was generally lower and insensitive to pH. It is concluded that the control of proton efflux through CF₀-CF₁ differs in thylakoids of V. faba grown at warm and cold temperatures.     

Synergistic toxicity between arsenic and methylated selenium compounds

Arsenite has been known for half a century to have a protective effect against selenium poisoning. Paradoxically, arsenite inhibits the conversion of inorganic selenium salts to methylated excretory products, although methylation has long been regarded as a detoxification mechanism for selenium. Moreover, there is evidence for a pronounced synergistic toxicity between arsenite and methylated selenium metabolites. We investigated the effect of arsenite on the acute toxicity of a variety of methylated or nonmethylated selenium compounds, as well as methylated forms of sulfur and tellurium. Adult male rats were injected with sodium arsenite (4 mg As/kg bw, s.c.) 10 min prior to injection of the test compounds; at the doses employed, none of the test compounds caused mortality, nor did arsenite, when given alone. When given with arsenite, the following methylated compounds produced toxic signs and high morality at the indicated dosages (mg Se/kg): Methylseleninic acid (2), dimethylselenoxide (2), trimethylselenonium chloride (3), selenobetaine (2), selenobetaine methylester (2, also 1 and 0.5), and Se-methylselenocysteine (2). Toxic signs but not mortality occurred when arsenite was given with selenomethionine (2 mg Se/kg). No enhancement of toxic signs or mortality occurred when arsenite was given with sulfobetaine (0.8 mg S/kg), dimethylsulfide (320 mg S/kg), or the following (nonmethylated) forms of selenium: sodium selenite (2), selenocystine (2), and phenylselenol (2). Arsenite also increased the toxicity of trimethyltelluronium chloride (4.8 mg Te/kg). Like arsenite, periodate-oxidized adenosine (100 mumoles/kg), which is known to inhibit the formation of dimethylselenide and trimethylselenonium ion in vivo, caused increased 24 h mortality when given with various methylated selenium compounds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of sperm motility in sea bass: the effect of heavy metals and physicochemical variables on sperm motility

Computer assisted sperm analysis (CASA) was used to characterize the motility of sea bass Dicentrarchus labrax spermatozoa and to study the effect of several physicochemical variables and heavy metals on sperm swimming performance. Duration of sperm motility in sea bass was very short (<50 s). During the first 20 s all the motility variables measured remained approximately constant, the velocity and linearity of the movement being maximum during this period, while both variables decreased sharply later. While slight variations in pH did not significantly modify sperm swimming performance, changes in osmolality affected all the measured motility variables. Two of the heavy metals tested, Cu²⁺ and Pb²⁺, did not affect sperm motility when the activating media contained up to 100 ppm of the metal salts. In contrast, Hg²⁺ modified the morphology of post-swimming spermatozoa at 0·4–1 ppm (sperm dilution rate 1:39) and completely arrested sperm motility at concentrations as low as 0·1 ppm (sperm dilution rate 1:2500). Assuming a covalent binding to sperm cells, this revealed a finite number of c. 10 million Hg²⁺ binding sites per spermatozoon. Complementary results using demembranated spermatozoa suggested that the main target of HgCl₂ would be located in the plasma membrane and that HgCl₂ would inhibit water channels, hence preventing sperm motility.