Plasmid-mediated resistance to tellurite: expressed and cryptic.

The ability of some bacteria to grow in the presence of high concentrations of tellurium compounds has been recognized for almost 100 years. Since then, interest in this phenomenon has generated a slow but steady trickle of literature. In the past few years, the use of modern techniques in molecular biology has led to a dramatic increase in our understanding of the genetics of several bacterial determinants for resistance to tellurium compounds. These determinants are frequently found to be encoded by plasmids which carry multiple antibiotic resistance determinants. Our understanding of the biochemistry of these systems remains limited. In this article, the history of the study of bacterial resistance to tellurium compounds is briefly reviewed. This is followed by an analysis of the recent developments in the study of plasmid-mediated resistance determinants. Finally, preliminary investigations on the possible mechanisms of bacterial resistance to tellurium compounds are presented.     

The Te-Assay: a black and white method for environmental sample pre-screening exploiting tellurite reduction

We present the tellurite bioassay (Te-Assay) as an alternative approach for quantification of cell viability. The Te-Assay was developed to pre-screen environmental samples for potential bacterial toxicants in which the reduction of tellurite to tellurium is used as a metabolic marker; black phenotype development only occurs in metabolically competent bacteria capable of reducing tellurite (TeO(3)(2-)) to elemental tellurium. The black and white phenotypes equate to nonsignificant or significant impediment of normal metabolic processes, thus permitting the rapid visual assessment of the relative toxicity of environmental samples. Bacterial inocula were exposed in 96-well plates to arrays of diluted analytes or environmental samples before addition of a tellurite to assess cell health/viability. Toxicity was quantified as the analyte concentration at which a 50% reduction in blackness occurred (IC(50)) compared to control wells containing no added analyte. No proprietary strains or reagents are required for Te-Assay, in which characterised strains or recent environmental isolates performed equally well. Strain selection was independent of tellurite-resistance provided that tellurite was reduced intracellularly by active non-growing cells.     

Comparative study of the cytotoxicity of the nanosized and microsized tellurium powders on HeLa cells

To compare the cytotoxicity on HeLa cells induced by nanosized and microsized tellurium powders, HeLa cells were exposed to different concentrations of tellurium powders (0, 50, 100, 150 and 200 μg/mL) for 12 h. In this study, detection of a series of biomarkers, including reactive oxygen species (ROS), glutathione (GSH), 8-hydroxy-2′-deoxyguanosine (8-OHdG), in addition to DNA and protein crosslink (DPC) and MTTassay, were conducted to evaluate the cytotoxicity. It is indicated that compared with the control group, there was no significant difference in the induced cytotoxicity at concentrations lower than 50 μg/mL for both nanosized and microsized tellurium powders. While there appears a significant difference in the induced cytotoxicity for nanosized tellurium powders when the concentration is higher than 100 μg/mL as well as for microsized tellurium powders when the concentration is higher than 200 μg/mL. Moreover, it is found that the cytotoxicity induced on HeLa cells exhibits a certain dose-effect relationship with the concentration of tellurium powders. A conclusion has been reached that the toxicity on HeLa cells can be induced by both nanosized and microsized tellurium powders, and the toxicity of the nanosized tellurium powders is significantly greater than the microsized one.     

Pyridine-2,6-bis(thiocarboxylic acid) produced by Pseudomonas stutzeri KC reduces and precipitates selenium and tellurium oxyanions

The siderophore of Pseudomonas stutzeri KC, pyridine-2,6-bis(thiocarboxylic acid) (pdtc), is shown to detoxify selenium and tellurium oxyanions in bacterial cultures. A mechanism for pdtc's detoxification of tellurite and selenite is proposed. The mechanism is based upon determination using mass spectrometry and energy-dispersive X-ray spectrometry of the chemical structures of compounds formed during initial reactions of tellurite and selenite with pdtc. Selenite and tellurite are reduced by pdtc or its hydrolysis product H(2)S, forming zero-valent pdtc selenides and pdtc tellurides that precipitate from solution. These insoluble compounds then hydrolyze, releasing nanometer-sized particles of elemental selenium or tellurium. Electron microscopy studies showed both extracellular precipitation and internal deposition of these metalloids by bacterial cells. The precipitates formed with synthetic pdtc were similar to those formed in pdtc-producing cultures of P. stutzeri KC. Culture filtrates of P. stutzeri KC containing pdtc were also active in removing selenite and precipitating elemental selenium and tellurium. The pdtc-producing wild-type strain KC conferred higher tolerance against selenite and tellurite toxicity than a pdtc-negative mutant strain, CTN1. These observations support the hypothesis that pdtc not only functions as a siderophore but also is involved in an initial line of defense against toxicity from various metals and metalloids.     

Pyridine-2,6-Bis(Thiocarboxylic Acid) Produced by Pseudomonas stutzeri KC Reduces and Precipitates Selenium and Tellurium Oxyanions

The siderophore of Pseudomonas stutzeri KC, pyridine-2,6-bis(thiocarboxylic acid) (pdtc), is shown to detoxify selenium and tellurium oxyanions in bacterial cultures. A mechanism for pdtc's detoxification of tellurite and selenite is proposed. The mechanism is based upon determination using mass spectrometry and energy-dispersive X-ray spectrometry of the chemical structures of compounds formed during initial reactions of tellurite and selenite with pdtc. Selenite and tellurite are reduced by pdtc or its hydrolysis product H₂S, forming zero-valent pdtc selenides and pdtc tellurides that precipitate from solution. These insoluble compounds then hydrolyze, releasing nanometer-sized particles of elemental selenium or tellurium. Electron microscopy studies showed both extracellular precipitation and internal deposition of these metalloids by bacterial cells. The precipitates formed with synthetic pdtc were similar to those formed in pdtc-producing cultures of P. stutzeri KC. Culture filtrates of P. stutzeri KC containing pdtc were also active in removing selenite and precipitating elemental selenium and tellurium. The pdtc-producing wild-type strain KC conferred higher tolerance against selenite and tellurite toxicity than a pdtc-negative mutant strain, CTN1. These observations support the hypothesis that pdtc not only functions as a siderophore but also is involved in an initial line of defense against toxicity from various metals and metalloids.     

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.     

Binding of tellurium to hepatocellular selenoproteins during incubation with inorganic tellurite: consequences for the activity of selenium-dependent glutathione peroxidase

The metallic group XVIa elements selenium and tellurium possess remarkably similar chemical properties. However, unlike selenium, tellurium is not an essential micronutrient and, indeed, induces both acute and chronic toxicity in a variety of species. Despite this, very little is known of the molecular mechanisms of toxicity of tellurium, particularly with respect to potential chemical interactions with selenium-containing components in the cell. In this work we describe a novel interaction of inorganic tellurite with hepatocellular selenoproteins, particularly with selenium-dependent glutathione peroxidase. The accumulation of (121Te)-tellurite into cultured primary rat liver hepatocytes was shown to be much more rapid than that of (75Se)-selenite on a molar basis. Neither the uptake of (121Te)-tellurite nor of (75Se)-selenite was affected by a large molar excess of the unlabelled counterpart, respectively. Interestingly, separation of the hepatocellular proteins on continuous pH denaturing gels demonstrated clear binding of radiolabelled tellurium to a number of protein bands, including one at 23 and one at 58 kDa, which corresponded to proteins readily labelled in cells treated with (75Se)-selenite. The binding of (121Te) to these proteins was insensitive to reduction with mercaptoethanol and not affected by pre-treatment of the cells with cycloheximide. When purified selenium-dependent glutathione peroxidase was treated directly with (121Te)-tellurite, the protein became labelled in an analogous manner to that achieved in intact cells. This was not affected by coincubation of the enzyme with (121Te)-tellurite and one or both of its substrates. Additionally, incubation of the peroxidase with tellurite effectively inhibited its ability to catalyse glutathione-dependent reduction of hydrogen peroxide. These data suggest that inorganic tellurite delivers tellurium to the intracellular milieu in a form capable of binding to some intracellular selenoproteins and at least in the case of glutathione peroxidase, cause inhibition of catalytic activity. The nature of the binding seems not to be due to the insertion of tellurocysteine into the protein and the insensitivity to reductive cleavage with mercaptoethanol seems to preclude the formation of stable telluro-selenides in the proteins. These data may offer alternative explanations for the established toxicity of tellurium via disruption of selenoprotein function, particularly by the induction of intracellular oxidative stress by the inhibition of Se-dependent glutathione peroxidase.     

Isolation and initial characterization of the tellurite reducing moderately halophilic bacterium, Salinicoccus sp. strain QW6

Among the 49 strains of moderately halophilic bacteria isolated from the salty environments of Iran, a Gram-positive coccus designated as strain QW6 showed high capacity in the removal of toxic oxyanions of tellurium in a wide range of culture medium factors including pH (5.5-10.5), temperature (25-45 degrees C), various salts including NaCl, KCl, and Na(2)SO(4) (0.5-4M), selenooxyanions (2-10mM), and at different concentrations of potassium tellurite (0.5-1mM) under aerobic condition. Phenotypic characterization and phylogenetic analyses based on 16S rDNA sequence comparisons indicated that this strain was a member of the genus Salinicoccus. The maximum tellurite removal was exhibited in 1.5M NaCl at 35 degrees C, while the activity reduced by 53% and 47% at 25 and 45 degrees C, respectively. The optimum pH for removal activity was shown to be 7.5, with 90% and 83% reduced removal capacities at the two extreme values of 5.5 and 10, respectively. The impact of different concentrations of selenooxyanions (2-10mM) on tellurite removal by strain QW6 was evaluated. The ability of strain QW6 in the removal of tellurite in the presence of 6mM selenite increased by 25%. The concentration of toxic potassium tellurite in the supernatant of the bacterial culture medium decreased by 99% (from 0.5 to 0.005mM) after 6 days and the color of the medium changed to black due to the formation of less toxic elemental tellurium.     

Characterization of moderately halotolerant selenate- and tellurite-reducing bacteria isolated from brackish areas in Osaka

Moderately halotolerant selenate- and tellurite-reducing bacteria were characterized for wastewater treatment applications. A selenate-reducing strain 9a was isolated from the biofilm of a leachate treatment plant at a sea-based waste disposal site. A tellurite-reducing strain Taa was isolated from an enrichment culture derived from brackish sediment. Both bacterial strains were Shewanella species. Strain 9a could anaerobically remove 45–70% of 1.0 mM selenate and selenite from water containing up to 3% NaCl within 4 days, while strain Taa could anaerobically and aerobically remove 70–90% of 0.4 mM tellurite from water containing up to 6% NaCl within 3 days. Globular particles of insoluble selenium were observed both outside and inside the cells of strain 9a. The insoluble tellurium formed by strain Taa was globular under microaerobic conditions but nanorod under aerobic conditions. These bacteria will yield a range of useful selenium and tellurium nanomaterials as well as wastewater treatment applications.     

Enantioselective reduction of ortho-substituted acetophenones by bacterial strains isolated from medium enriched with biphenyl or diesel fuel

Application of 21 new bacterial strains from natural environments (coastal plain of Santos and Atlantic Rain Forest, São Paulo, Brazil) in the asymmetric reduction of acetophenone derivatives is described. The bioreduction was carried out with whole bacterial cells leading to (S)-chiral alcohols in up to ≥99% e.e. The (S)-(−)-1-(2-bromo-phenyl)-ethanol was employed in the preparation of chiral tellurium derivatives.     

Determination of tellurium in the cells of Gram-negative bacteria

Several methods of determining tellurium reduced by and deposited in the cells of Gram-negative bacteria are analysed and evaluated. In a series of techniques elaborated by the author, the most satisfactory was found to be the lysis of cells containing tellurium by boiling in sodium laurylsulphate. The resultant colloidal tellurium solution is black-brown, with maximum absorption at wavelength 280 mμ. The method is simple, rapid and sensitive.     

Synthesis of Polynucleotides by Microorganisms

Polynucleotides could be synthesized from nucleoside diphosphates by microorganisms belonging to genera Pseudotnonas, Serratia, Xatuhonwnas, Proteus, Aerobacter, Bacillus, and Brevibacterium. These strains were rich in polynucleotide phosphorylase easily extractable from cells and poor in both nuclease and nucleoside-diphosphate-degrading enzymes. Polynucleotide phosphorylase was effectively extracted from the bacterial cells, that had been once soaked in saturated saline solution, with hypotonic solution. Synthesis of polynucleotides was observed not only when the substrates were incubated with polynucleotide phosphorylase preparation isolated from the bacterial cells, but also when the substrates were added directly to the bacterial cultures.     

Ubiquitination of intracellular bacteria: a new bacteria-sensing system?

Ubiquitination is a protein modification generally used by cells to tag proteins that are destined for proteasomal degradation. In a recent article, Perrin et al. reported that the ubiquitination system has a role in the recognition of bacterial pathogens in the cytosol of mammalian cells. They showed that polyubiquitinated proteins accumulate on the surface of cytosolic Salmonella typhimurium. In macrophages, but not epithelial cells, proteasomes become associated with the surface of cytosolic bacteria. The authors proposed that the ubiquitin-proteasome machinery might be implicated indirectly in bacterial clearance.     

Synoptic determination of living/dead and active/dormant bacterial fractions in marine sediments

The most widely used methods for the estimation of the living/dead fractions of bacterial cells involve specific stains that are able to reveal membrane integrity. Here, we have compared two different probes (propidium iodide and ethidium homodimer-2) that have different molecular weights and steric hindrance effects. We have also combined this method with the staining/destaining procedure that is currently used in the identification of potentially active cells. The procedure for marine sediments described here allows the synoptic (i.e. from the same filter) identification of: (i) the number of living bacteria; (ii) the number of active vs. dormant cells within this living fraction; (iii) the bacterial fraction with an intact nucleoid region without membrane integrity; and (iv) dead cells (devoid of the nucleoid region and without membrane integrity). Our results demonstrate that the concentration of propidium is crucial for the correct estimation of the dead bacterial fraction, ethidium homodimer-2 allows efficient and accurate estimates that are independent of the concentrations used and the sample storage. The active bacterial fraction represented c. 40% of the total bacterial abundance, the inactive/dormant fraction c. 30%, and the dead fraction was, on average, c. 30%. This method allows the processing of a large number of samples with high precision and at relatively low cost, and thus it provides additional synoptic insights into the metabolic state of bacteria in marine sediments.     

Chemical and biological mobilization of Fe(III) in marsh sediments

Iron reduction in marine sulfitic environments may occur via a mechanism involving direct bacterial reduction with the use of hydrogen as an electron donor, direct bacterial reduction involving carbon turnover, or by indirect reduction where sulfide acts to reduce iron. In the presented experiments, the relative importance of direct and indirect mechanisms of iron reduction, and the contribution of these two mechanisms to overall carbon turnover has been evaluated in two marsh environments. Sediments collected from two Northeastern US salt marshes each having different Fe (III) histories were incubated with the addition of reactive iron (as amorphous oxyhydroxide). These sediments were either incubated alone or in conjunction with sodium molybdate. Production of both inorganic and organic pore water constituents and a calculation of net carbon production were used as measures to compare the relative importance of direct bacterial reduction and indirect bacterial reduction. Results indicate that in the environments tested, the majority of the reduced iron found results from indirect reduction mediated by hydrogen sulfide, a result of dissolution and precipitation phenomena, or is a result of direct bacterial reduction using hydrogen as an electron donor. Direct iron reduction plays a minor role in carbon turnover in these environments.     

Inhibition of human squalene monooxygenase by tellurium compounds: evidence of interaction with vicinal sulfhydryls

Squalene monooxygenase is a flavin adenine dinucleotide-containing, microsomal enzyme that catalyzes the second step in the committed pathway for cholesterol biosynthesis. Feeding weanling rats a diet containing 1% elemental tellurium causes a transient, peripheral demyelination due to the disruption of cholesterol synthesis in Schwann cells secondary to inhibition of squalene monooxygenase. The tellurium species responsible for the inhibition is unknown, as is the mechanism of inhibition. To study the potential mechanisms of tellurium toxicity in humans, three likely in vivo metabolites of tellurium (tellurite, dimethyltellurium dichloride, and dimethyltelluride) were tested as inhibitors of purified human squalene monooxygenase. All three inhibitors reacted with the enzyme slowly and the resulting interaction was not freely reversible. The 50% inhibitory concentration for the methyltellurium compounds (approximately 100 nM) after a 30-min preincubation was 100-fold lower than that of tellurite, indicating a role for hydrophobicity in the enzyme-inhibitor interaction. The ability of glutathione and 2,3-dimercaptopropanol to prevent and reverse the inhibition indicated that the tellurium compounds were reacting with sulfhydryls on squalene monooxygenase, and the ability of phenylarsine oxide, which reacts specifically with vicinal sulfhydryls, to inhibit the enzyme indicated that these sulfhydryls are located proximal to one another on the enzyme. These results suggest that the unusual sensitivity of squalene monooxygenase to tellurium compounds is due to the binding of these compounds to vicinal cysteines, and that methylation of tellurium in vivo may enhance the toxicity of tellurium for this enzyme.     

Role of bacterial polysaccharides in the derepression of ex-planta nitrogenase activity with rhizobia

Abstract Since bacterial polysaccharides may limit the availability of oxygen to the cells, we have investigated the role of rhizobial extracellular polysaccharides (EPS) and the non-rhizobial polyscharide, xanthan, in the depression of ex-planta nitrogenase activity with rhizobia in liquid medium. Two rhizobial strains known to exhibit ex-planta nitrogenase activity on solid media were used; the slow-growing Bradyrhizobium japonicum USDA 110 and the arctic Rhizobium strain N31, both being prolific EPS producers. In low nitrogen mannitol (LNM) liquid medium strain N31 exhibited nitrogenase activity only after 15 days, when sufficient EPS had accumulated in the medium, and activity was correlated with EPS production. When rhizobial EPS from an old culture was added to the LNM medium, nitrogenase activity was detected after 48 h incubation, indicating that EPS of the medium decreased oxygen diffusion to cells to a level that depressed nitrogenase activity. In modified LNM medium with xanthan nitrogenase activity was readily depressed. In both strains activity increased with increased xanthan concentration, but decreased sharply at higher concentrations. Strain N31 exhibited a narrower range of polysaccharide concentration for nitrogenase activity than the slow strain USDA 110. Thus, the condition for derepression of nitrogenase might be a careful balancing of the oxygen concentration surrounding the cells, and this condition is met when a balancing of polsaccharide, either synthesized by the rhizobia or added to the medium, can permit oxygen diffusion to within the narrow range required for the depression and expression of nitrogenase.     

Sulfur,selenium and tellurium pseudopeptides: Synthesis and biological evaluation

A new series of sulfur, selenium and tellurium peptidomimetic compounds was prepared employing the Passerini and Ugi isocyanide based multicomponent reactions (IMCRs). These reactions were clearly superior to conventional methods traditionally used for organoselenium and organotellurium synthesis, such as classical nucleophilic substitution and coupling methods. From the biological point of view, these compounds are of considerable interest because of suspected anticancer and antimicrobial activities. While the sulfur and selenium containing compounds generally did not show either anticancer or antimicrobial activities, their tellurium based counterparts frequently exhibited antimicrobial activity and were also cytotoxic. Some of the compounds synthesized even showed selective activity against certain cancer cells in cell culture. These compounds induced a cell cycle delay in the G0/G1 phase. At closer inspection, the ER and the actin cytoskeleton appeared to be the primary cellular targets of these tellurium compounds, in line with some of our previous studies. As most of these peptidomimetic compounds also comply with Lipinski’s Rule of Five, they promise good bioavailability, which needs to be studied as part of future investigations.