Thomas G. Chasteen

Microbial Methylation of Metalloids: Arsenic, Antimony, and Bismuth

SUMMARY A significant 19th century public health problem was that the inhabitants of many houses containing wallpaper decorated with green arsenical pigments experienced illness and death. The problem was caused by certain fungi that grew in the presence of inorganic arsenic to form a toxic, garlic-odored gas. The garlic odor was actually put to use in a very delicate microbiological test for arsenic. In 1933, the gas was shown to be trimethylarsine. It was not until 1971 that arsenic methylation by bacteria was demonstrated. Further research in biomethylation has been facilitated by the development of delicate techniques for the determination of arsenic species. As described in this review, many microorganisms (bacteria, fungi, and yeasts) and animals are now known to biomethylate arsenic, forming both volatile (e.g., methylarsines) and nonvolatile (e.g., methylarsonic acid and dimethylarsinic acid) compounds. The enzymatic mechanisms for this biomethylation are discussed. The microbial conversion of sodium arsenate to trimethylarsine proceeds by alternate reduction and methylation steps, with S-adenosylmethionine as the usual methyl donor. Thiols have important roles in the reductions. In anaerobic bacteria, methylcobalamin may be the donor. The other metalloid elements of the periodic table group 15, antimony and bismuth, also undergo biomethylation to some extent. Trimethylstibine formation by microorganisms is now well established, but this process apparently does not occur in animals. Formation of trimethylbismuth by microorganisms has been reported in a few cases. Microbial methylation plays important roles in the biogeochemical cycling of these metalloid elements and possibly in their detoxification. The wheel has come full circle, and public health considerations are again important.

Tellurite: history, oxidative stress, and molecular mechanisms of resistance

The perceived importance of tellurium (Te) in biological systems has lagged behind selenium (Se), its lighter sister in the Group 16 chalcogens, because of telluriums lower crustal abundance, lower oxyanion solubility and biospheric mobility and the fact that, unlike Se, Te has yet to be found to be an essential trace element. Te applications in electronics, optics, batteries and mining industries have expanded during the last few years, leading to an increase in environmental Te contamination, thus renewing biological interest in Te toxicity. This chalcogen is rarely found in the nontoxic, elemental state (Te(0)), but its soluble oxyanions, tellurite (TeO(3)(2-)) and tellurate (TeO(4)(2-)), are toxic for most forms of life even at very low concentrations. Although a number of Te resistance determinants (Tel) have been identified in plasmids or in the bacterial chromosome of different species of bacteria, the genetic and/or biochemical basis underlying bacterial TeO(3)(2-) toxicity is still poorly understood. This review traces the history of Te in its biological interactions, its enigmatic toxicity, importance in cellular oxidative stress, and interaction in cysteine metabolism.

Confirmation of the biomethylation of antimony compounds

We have evidence that an organic and an inorganic salt of antimony were reduced and methylated biologically by microorganisms in laboratory experiments. The organoantimony compound produced was trimethylstibine [(CH3)3Sb] and was detected in a culture headspace. This was confirmed by matching the compounds retention time in capillary gas chromatography, as detected by fluorine-induced chemiluminescence, with a com- mercial standard and by its mass spectrum determined with gas chromatography/ mass spectrometry (GC–MS). (CH3)3Sb was detected in the headspace of soil samples amended with either potassium antimonyltartrate or potassium hexahydroxyantimonate and augmented with any one of three different nitrate-containing growth media. The identity of the microorganisms in soil that accomplished this are as yet unknown. Of 48 soil samples amended with these two compounds, 24 produced trimethylstibine. Bioreduction of trimethyldibromoantimony was also detected in a liquid monoculture of Pseudomonas fluorescens K27 which also produced tri- methylstibine. This headspace production of (CH3)3Sb was determined to be linked to the cultures cell population as measured by optical density. This microbe, however, did not biomethylate either potassium antimonyltartrate or potassium hexahydroxyantimonate in any experiments we performed.

Fate of Selenate and Selenite Metabolized by Rhodobacter sphaeroides

ABSTRACT Cultures of a purple nonsulfur bacterium, Rhodobacter sphaeroides, amended with ∼1 or ∼100 ppm selenate or selenite, were grown phototrophically to stationary phase. Analyses of culture headspace, separated cells, and filtered culture supernatant were carried out using gas chromatography, X-ray absorption spectroscopy, and inductively coupled plasma spectroscopy-mass spectrometry, respectively. While selenium-amended cultures showed much higher amounts of SeO32− bioconversion than did analogous selenate experiments (94% uptake for SeO32− as compared to 9.6% for SeO42−-amended cultures from 100-ppm solutions), the chemical forms of selenium in the microbial cells were not very different except at exposure to high concentrations of selenite. Volatilization accounted for only a very small portion of the accumulated selenium; most was present in organic forms and the red elemental form.

Fluorine-induced chemiluminescence detection of biologically methylated tellurium, selenium, and sulfur compounds

SummaryThe sensitive detection of volatile, methylated selenium and tellurium compounds based on capillary gas chromatography coupled to fluorine-induced chemiluminescence detection is described. The method requires no sample derivatization, and the detection limits for selenides and tellurides (low pg range) are the lowest reported to date. This technique can resolve and speciate complex mixtures of methylated tellurium, selenium, and sulfur gases and is useful for analysis of selenium and tellurium gases in environmental samples that also contain usually interfering reduced sulfur gases. Applications of the technique for analysis of bacterial and fungal headspace samples are presented.

X-ray absorption spectroscopy of selenium-containing amino acids.

The selenium K-edge X-ray absorption spectra of selenomethionine, selenocysteine, selenocystine, and sulfo-selenocystine in solution are compared with the corresponding sulfur K-edge spectra of the sulfur analogues of these compounds. The selenium and sulfur spectra follow similar trends, although the latter are significantly sharper owing to the longer core hole lifetime at the lower energies where sulfur absorbs. The spectra of the selenium compounds are sufficiently distinct that it is reasonable to expect that curve fitting will allow the speciation of the forms of selenium in complex biological samples.

Cysteine Metabolism-Related Genes and Bacterial Resistance to Potassium Tellurite

Tellurite exerts a deleterious effect on a number of small molecules containing sulfur moieties that have a recognized role in cellular oxidative stress. Because cysteine is involved in the biosynthesis of glutathione and other sulfur-containing compounds, we investigated the expression of Geobacillus stearothermophilus V cysteine-related genes cobA, cysK, and iscS and Escherichia coli cysteine regulon genes under conditions that included the addition of K2TeO3 to the culture medium. Results showed that cell tolerance to tellurite correlates with the expression level of the cysteine metabolic genes and that these genes are up-regulated when tellurite is present in the growth medium.

Biomimetic, mild chemical synthesis of Cdte-GSH quantum dots with improved biocompatibility

Multiple applications of nanotechnology, especially those involving highly fluorescent nanoparticles (NPs) or quantum dots (QDs) have stimulated the research to develop simple, rapid and environmentally friendly protocols for synthesizing NPs exhibiting novel properties and increased biocompatibility. In this study, a simple protocol for the chemical synthesis of glutathione (GSH)-capped CdTe QDs (CdTe-GSH) resembling conditions found in biological systems is described. Using only CdCl2, K2TeO3 and GSH, highly fluorescent QDs were obtained under pH, temperature, buffer and oxygen conditions that allow microorganisms growth. These CdTe-GSH NPs displayed similar size, chemical composition, absorbance and fluorescence spectra and quantum yields as QDs synthesized using more complicated and expensive methods. CdTe QDs were not freely incorporated into eukaryotic cells thus favoring their biocompatibility and potential applications in biomedicine. In addition, NPs entry was facilitated by lipofectamine, resulting in intracellular fluorescence and a slight increase in cell death by necrosis. Toxicity of the as prepared CdTe QDs was lower than that observed with QDs produced by other chemical methods, probably as consequence of decreased levels of Cd+2 and higher amounts of GSH. We present here the simplest, fast and economical method for CdTe QDs synthesis described to date. Also, this biomimetic protocol favors NPs biocompatibility and helps to establish the basis for the development of new, “greener” methods to synthesize cadmium-containing QDs.

Enhanced Glutathione Content Allows the In Vivo Synthesis of Fluorescent CdTe Nanoparticles by Escherichia coli

The vast application of fluorescent semiconductor nanoparticles (NPs) or quantum dots (QDs) has prompted the development of new, cheap and safer methods that allow generating QDs with improved biocompatibility. In this context, green or biological QDs production represents a still unexplored area. This work reports the intracellular CdTe QDs biosynthesis in bacteria. Escherichia coli overexpressing the gshA gene, involved in glutathione (GSH) biosynthesis, was used to produce CdTe QDs. Cells exhibited higher reduced thiols, GSH and Cd/Te contents that allow generating fluorescent intracellular NP-like structures when exposed to CdCl2 and K2TeO3. Fluorescence microscopy revealed that QDs-producing cells accumulate defined structures of various colors, suggesting the production of differently-sized NPs. Purified fluorescent NPs exhibited structural and spectroscopic properties characteristic of CdTe QDs, as size and absorption/emission spectra. Elemental analysis confirmed that biosynthesized QDs were formed by Cd and Te with Cd/Te ratios expected for CdTe QDs. Finally, fluorescent properties of QDs-producing cells, such as color and intensity, were improved by temperature control and the use of reducing buffers.

Using fluorine-induced chemiluminescence to detect organo-metalloids the headspace of phototrophic bacterial cultures amended with selenium and tellurium

Abstract Purple nonsulfur bacteria are resistant to metalloid oxyanions. The key to this resistance seems to lie in their ability to reduce and, in some cases, methylate these toxic chemical species. Six strains of purple nonsulfur bacteria have been grown under photo-heterotrophic conditions and exposed to varied concentrations of inorganic forms of selenium, tellurium and dimethyl selenone, a proposed biological intermediate of the selenium reduction/methylation process. Selenium and tellurium metallic powders have also been added to live cultures. All of the phototrophic bacteria studied here reduce and methylate one or the other oxyanion of Se used, and three strains reduce and methylate Te° and Se°. All strains respond to the addition of dimethyl selenone by producing dimethyl selenide and/or dimethyl diselenide. In all six cases the highest amounts of volatile selenium compounds are found in cultures amended with dimethyl selenone and the lowest amounts in cultures doped with selenate. Some of the phototrophic bacteria studied here, when amended with oxyanions of both Se and Te, increase their release of dimethyl telluride (produced by their bioreduction and methylation of tellurate) in the presence of selenate. Control experiments show that this synergism is biological and not merely caused by the presence of other organo-sulfides or -selenides in culture.