Dimethylselenide Research Articles

Halitosis associated volatiles in breath of healthy subjects

BackgroundHalitosis can have an intra- or extra-oral origin. In all cases, bad breath is caused by the presence of volatile organic compounds originating from the mouth or the expired air. They can be specific for certain diseases or infections. Study objectiveThis study explored the presence and concentration of these volatile compounds normally associated with halitosis in the breath of healthy symptomless volunteers. MethodsAlveolar and mouth air of 40 healthy volunteers as well as environmental air were analyzed by gas chromatography–mass spectrometry (GC–MS) and by a commercially available GC device (OralChroma®). Results14 compounds, associated with halitosis could be detected. All of them except carbon disulfide, appeared to be (partly) produced endogenously and/or in the mouth. Acetone, 2-butanone, 2-pentanone and 1-propanol were common to all volunteers in both alveolar and mouth air and indole and dimethyl selenide in alveolar air. ConclusionsGC–MS seems a promising tool for differential diagnosis of halitosis, with the possibility to detect extra-oral causes, which often remain undetected unless characterized by a specific smell.

IR laser-induced co-decomposition of dimethyl selenide and trisilane: Gas-phase formation of SiSe and chemical vapor deposition of nanostructured H/Si/Se/C polymers

Chemical changes in IR laser irradiated gaseous mixtures of dimethyl selenide and trisilane have been diagnosed by laser-induced fluorescence (LIF) of transient species and by FTIR, Raman, photoelectron spectroscopy and electron microscopy of the final solid products deposited from the gas phase. It is revealed that decomposition of both compounds leads to gas-phase formation of SiSe and deposition of solid nanostructured polymeric materials. We present indirect evidence on the presence of Si Se bonds in these polymers by revealing that these polymers undergo hydrolysis in air to H 2Se and CH 3SeH, which converts them to solid silicone-based films containing elemental selenium. Plausible reactions taking place in the gas phase and upon exposure of solids to air are suggested.

Consequences of Vapor Enhancement on Selenium Speciation Analysis by HPLC/ICPMS

Recent work has shown the presence of volatile selenium metabolites in human urine and suggested that these compounds could compromise quantitative selenium analyses by ICPMS. We show that with a commonly used sample introduction system (pneumatic nebulizer and spray chamber), two volatile selenium species recently identified in urine, namely, dimethyl selenide and dimethyl diselenide, gave greatly increased ICPMS responses (up to 58-fold) relative to selenite, an effect related to their volatilization in the spray chamber resulting in enhanced transport to the plasma. The quantitative consequences of this effect were demonstrated by measurement of total selenium and selenium species in certified reference material, NIES CRM 18 human urine. Direct flow injection analysis of the urine gave a total selenium concentration more than 2-fold higher than the certified value. These data suggested that NIES CRM 18 may contain part of its selenium as volatile species, and subsequent reversed-phase HPLC/ICPMS showed the presence of dimethyl selenide in addition to selenosugars and trimethylselenonium ion. Although the practice of quantifying unidentified chromatographic peaks against those of known compounds is common in speciation analysis, this approach when applied to NIES CRM 18 gave a value for the sum of selenium species which was twice the certified total selenium concentration. This work shows that the presence of volatile selenium species in urine precludes the use of flow injection analysis for total selenium measurements and imposes severe restrictions on the quantification of urinary selenium metabolites. In addition, it raises broader issues of the validity of the "dilute and shoot" approach to the determination of metals in clinical analysis of biological fluids.

Distribution and genetic diversity of bacterial thiopurine methyltransferases in soils emitting dimethyl selenide

Dimethyl selenide (DMSe) and dimethyl diselenide (DMDSe) emissions by soil samples spiked with selenite or (methyl)selenocysteine, with or without a supplement of nutrient broth and glucose were measured. DMSe was the main form of volatile Se produced, and was observed for both Se-substrates. DMDSe was only emitted from soils spiked with (methyl)selenocysteine. Two bacterial thiopurine methyltransferases (TPMTs), TPMT-I and TPMT-E, have been reported to be involved in DMSe and DMDSe emissions [J. Bacteriol. 184 (2002) 3146; Appl. Environ. Microbiol. 69 (2003) 3784]. To establish if these TPMTs or other members of their gene family could have contributed to the DMSe emissions observed, the diversity of bTPMT gene ( tpm) sequences among the soils of this study was investigated. Total DNAs from these soils were extracted and screened using the tpm PTCF2–PTCR2 consensus primers defined to PCR amplify this gene family. The PCR products obtained from two soils were cloned, analysed by PCR-RFLP, and sequenced. Their analysis showed an important diversity of tpm lineages (around 12) in soils. Phylogenetic analysis of the deduced TPMT sequences of these soils revealed lineages not previously recorded in the databases, sequences closely related or identical to freshwater TPMTs, or sequences encoding TPMTs closely related to those of Pseudomonas fragi TPMT-K, Pseudomonas Hsa.28 TPMT-I, or Colwellia psychrerythraea TPMT-Z. Nested PCRs, allowing detection of about 13 distinct tpm soil and freshwater lineages by PTCF2–PTCR2 PCR screenings, were performed on the soil total DNAs. These PCRs confirmed the sequencing data, and allowed to recover lineages not detected by the cloning strategy. These results indicate that soils, like the freshwater samples, harbour TPMT-I gene sequences but may also have distinct tpm lineages. This study further supports our hypothesis that TPMTs contribute to DMSe soil emissions.

Expression of the ubiE Gene of Geobacillus stearothermophilus V in Escherichia coli K-12 Mediates the Evolution of Selenium Compounds into the Headspace of Selenite- and Selenate-Amended Cultures

The ubiE gene of Geobacillus stearothermophilus V, with its own promoter, was cloned and introduced into Escherichia coli. The cloned gene complemented the ubiE gene deficiency of E. coli AN70. In addition, the expression of this gene in E. coli JM109 resulted in the evolution of volatile selenium compounds when these cells were grown in selenite- or selenate-amended media. These compounds were dimethyl selenide and dimethyl diselenide.

IR laser-induced process for chemical vapor deposition of polyselenocarbosilane films

TEA CO 2 laser irradiation into gaseous mixtures of 1,3-disilacyclobutane (DSCB) and dimethyl selenide (DMS) results in infrared multiple photon-induced, non-interacting homogeneous decompositions of both educts, one yielding silene (H 2Si CH 2) and the other elemental selenium as major products. The reaction between polymerizing silene and agglomerizing Se leads to chemical vapor deposition of novel polyselenocarbosilane films which are unstable in atmosphere. The laser induced co-decomposition represents a new process in allowing (i) reaction between thermally generated transient and element and (ii) chemical vapor deposition of the product of this reaction.

Identification of biogenic dimethyl selenodisulfide in the headspace gases above genetically modified Escherichia coli

Escherichia coli JM109 cells were modified to express the genes encoded in a 3.8-kb chromosomal DNA fragment from a metalloid-resistant thermophile, Geobacillus stearothermophilus V. Manual headspace extraction was used to collect the gases for gas chromatography with fluorine-induced sulfur chemiluminescence analysis while solid-phase microextraction was used for sample collection in gas chromatography/mass spectrometry (GC/MS) analysis. When grown in the presence of selenate or selenite, these bacteria produced both organo-sulfur and organo-selenium in the headspace gases above the cultures. Organo-sulfur compounds detected were methanethiol, dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide. Organo-selenium compounds detected were dimethyl selenide and dimethyl diselenide. Two mixed sulfur-selenium compounds, dimethyl selenenyl sulfide and a chromatographically late-eluting compound, were detected. Dimethyl selenodisulfide, CH 3SeSSCH 3, and dimethyl bis(thio)selenide, CH 3SSeSCH 3, were synthesized and analyzed by GC/MS and fluorine-induced chemiluminescence to determine which corresponded to the late-eluting compound that was bacterially produced. CH 3SeSSCH 3 was positively identified as the compound detected in bacterial headspace above Se-amended cultures. Using GC retention times, the boiling point of CH 3SeSSCH 3 was estimated to be approximately 192 °C. This is the first report of CH 3SeSSCH 3 produced by bacterial cultures.

Gas chromatography with atomic emission detection for dimethylselenide and dimethyldiselenide determination in waters and plant materials using a purge-and-trap preconcentration system

Dimethylselenide (DMSe) and dimethyldiselenide (DMDSe) were determined in plant and water samples by capillary gas chromatography using microwave induced-plasma atomic emission spectrometry for detection. The analytes were leached from the solid samples into methanol by using an ultrasonic probe, and a portion of the extract was preconcentrated by means of a purge-and-trap system before being chromatographed. The analytes were directly purged from the water samples in the presence of 6% (v/v) methanol. Element-specific detection and quantification was carried out by monitoring the selenium (196 nm) emission line. Calibration curves were obtained by plotting peak area versus concentration and the correlation coefficients for linear calibration were 0.9999 for both analytes. Detection limits of 0.8 and 1.1 ng l −1 were obtained for DMSe and DMDSe, respectively, for water samples. For plant materials, the detection limits calculated for 0.5 g samples were 0.3 and 0.4 ng g −1 for DMSe and DMDSe, respectively. Concentration levels of DMSe ranging from 1.2 to 4.2 ng g −1 were found in some of the plant materials analyzed. No DMDSe was found in any of the samples. The accuracy of the method was checked by analyzing different spiked water and plant samples.

GC–ICP–MS determination of dimethylselenide in human breath after ingestion of 77Se-enriched selenite: monitoring of in-vivo methylation of selenium

The amount of volatile dimethylselenide (DMSe) in breath has been monitored after ingestion of sub-toxic amounts of selenium (300 microg (77)Se, as selenite) by a healthy male volunteer. The breath samples were collected in Tedlar bags every hour in the first 12 h and then at longer intervals for the next 10 days. The samples were subjected to speciation analysis for volatile selenium compounds by use of cryotrapping-cryofocussing-GC-ICP-MS. Simultaneously, all urine was collected and subjected to total selenium determination by use of ICP-MS. By monitoring m/z 82 and 77, background or dietary selenium and selenium from the administered selenite were simultaneously determined in the urine and in the breath-dietary selenium only was measured by monitoring m/z 82 whereas the amount of spiked (77)Se (99.1% [enriched spike]) and naturally occurring selenium (7.6% [natural abundance]) were measured by monitoring m/z 77. Quantification of DMSe was performed by using DMSe gas samples prepared in Tedlar bags (linear range 10-300 pg, R (2)=0.996, detection limit of Se as DMSe was 10 pg Se, or 0.02 ng L(-1), when 0.5 L gas was collected). Dimethylselenide was the only selenium species detected in breath samples before and after the ingestion of (77)Se-enriched selenite. Additional DM(77)Se was identified as early as 15 min after ingestion of the isotopically-labelled selenite. Although the maximum concentration of (77)Se in DMSe was recorded 90 min after ingestion, the natural isotope ratio for selenium in DMSe (77/82) was not reached after 20 days. The concentration of DMSe correlated with the total Se concentration in the urine during the experiment (R (2)=0.80). Furthermore, the sub-toxic dose of 300 microg selenium led to a significant increase of DMSe and renal excretion of background selenium, confirming that selenium ingested as selenite is homeostatically controlled by excretion. The maximum concentration of DMSe resulting from the spiked selenite was 1.4 ng Se L(-1) whereas the dietary background level was less than 0.4 ng Se L(-1). Overall excretion as DMSe was calculated to be 11.2% from the ingested selenite within the first 10 days whereas urinary excretion accounts for nearly 18.5%.

Chemical Behavior of the Biradicaloid (HO···ONO) Singlet States of Peroxynitrous Acid. The Oxidation of Hydrocarbons, Sulfides, and Selenides

Various high levels of theory have been applied to the characterization of two higher lying biradicaloid metastable singlet states of peroxynitrous acid. A singlet minimum (cis-2) was located that had an elongated O-O distance (2.17 A) and was only 12.2 kcal/mol [UB3LYP/6-311+G(3df,2p)+ZPVE] higher in energy than its ground-state precursor. A trans-metastable singlet (trans-2) was 10.9 kcal/mol higher in energy than ground-state HO-ONO. CASSCF(12,10)/6-311+G(d,p) calculations predict the optimized geometries of these cis- and trans-metastable singlets to be close to those obtained with DFT. Optimization of cis- and trans-2 within the COSMO solvent model suggests that both exist as energy minima in polar media. Both cis- and trans-2 exist as hydrogen bonded complexes with several water molecules. These collective data suggest that solvated forms of cis-2.3H(2)O and trans-2.3H(2)O represent the elusive higher lying biradicaloid minima that were recently (J. Am. Chem. Soc. 2003, 125, 16204) advocated as the metastable forms of peroxynitrous acid (HOONO). The involvement of metastable trans-2 in the gas phase oxidation of methane and isobutane is firmly established to take place on the unrestricted [UB3LYP/6-311+G(d,p)] potential energy surface (PES) with classical activations barriers for the hydrogen abstraction step that are 15.7 and 5.9 kcal/mol lower than the corresponding activation energies for producing products methanol and tert-butyl alcohol formed on the restricted PES. The oxidation of dimethyl sulfide and dimethyl selenide, two-electron oxidations, proceeds by an S(N)2-like attack of the heteroatom lone pair on the O-O bond of ground-state peroxynitrous acid. No involvement of metastable forms of HO-ONO was discernible.

Tubular Kidney Damage and Centrilobular Liver Injury after Intratracheal Instillation of Dimethyl Selenide

Accidental inhalation of selenium (Se) derivatives, such as dimethyl selenide (DMSe), has been associated with damage of respiratory tissues. However, systemic effects of inhaled Se have not been thoroughly established. We have investigated whether mouse kidney and liver show cellular pathology as a result of a single intratracheal instillation of two different doses of DMSe (0.05 and 0.1 mg Se/kg BW). The animals were sacrificed 1, 7, 14, and 28 days after either 1 of the 2 DMSe treatments; samples were studied by light microscopy. Instillation of the low DMSe dose resulted in acute and transient tubular disease of the kidney expressed by swelling and vacuolation of epithelial cells of proximal tubules; in some mice, tubular necrosis was observed. After 14 days of the DMSe treatment, these lesions were ameliorated and, by day 28, the kidney tubular epithelium depicted a normal morphology. The same low dose of DMSe caused sustained damage to centrilobular hepatocytes characterized by swollen and vacuolized liver cells. After the instillation of the high DMSe dose, the mice presented sustained liver and kidney focal necrosis. Our data suggest that inhalation of DMSe results in: (i) acute tubular injury of the kidney and damage to centrilobular liver cells and (ii) this systemic pathology induced by DMSe is a dose-dependent phenomenon.

Characterization of a novel selenium methyltransferase from freshwater bacteria showing strong similarities with the calicheamicin methyltransferase

A novel group of Se-methyltransferases is presented. The genetic determinant, named mmtA, which revealed this group was isolated from selenite and selenate-resistant freshwater bacteria. E. coli expressing mmtA and grown with a Se supplement emitted dimethyl selenide (DMSe) and dimethyl diselenide (DMDSe). Phylogenetic analysis divided MmtA-like bacterial sequences into two clusters, one grouping MmtA with S- and O-methyltransferases, and one grouping UbiE C-methyltransferases. Se methylation by some of these MmtA phyletic neighbours was investigated.

Determination of dimethylselenide and dimethyldiselenide by gas chromatography–photoionization detection

A simple method for the determination of volatile selenium compounds employing a gas chromatograph equipped with a photoionization detector is described. The method involves the direct injection of dimethylselenide (DMS) or dimethyldiselenide (DMDS) into the gas chromatograph; no derivatization of the sample was required. The photoionization detector was capable of detecting 60 pg (0.55 pmol) of DMS and 150 pg (0.80 pmol) DMDS. Sensitivity was 10–50 times greater with DMS and 4–20 times greater with DMDS when the photoionization detector was employed than when the flame ionization detector was employed.

Changes in bronchoalveolar lavage cells after intratracheal instillation of dimethyl selenide in mice.

CD-1 mice were exposed to a single intratracheal instillation of either 0.025 or 0.075 mg Se/kg wt of dimethyl selenide (DMSe). They were studied over 4 weeks to define the cellular inflammatory response of the airways to DMSe. Bronchoalveolar (BAL) lavage was used to collect the DMSe-induced inflammatory exudates. The DMSe instillation resulted in phlogistic responses that had the neutrophil as the main leukocyte; they were present in BAL samples, mostly at days 1 and 7. Macrophages were also increased during DMSe-induced inflammation. The lower DMSe dose resulted in an inflammatory reaction lasting for 2 weeks. Mice treated with the higher DMSe dose still showed elevated numbers of neutrophils and macrophages 4 weeks after instillation. DMSe did not change the number of lymphocytes harvested from the airways. An early increase in total protein of BAL, and late enhancement in lactate dehydrogenase was observed in mice treated with the high DMSe dose. We conclude that inhalation of DMSe triggers a moderate and dose-dependent inflammatory reaction in the mouse airways, and that this phlogistic reaction is likely to participate in the damage of respiratory epithelia that occurs upon DMSe inhalation.

SPME–multicapillary GC coupled to different detection systems and applied to volatile organo-selenium speciation in yeast

In this work the versatility of solid phase microextraction (SPME) in combination with multicapillary gas chromatography (MC–GC) was evaluated using different common detectors for organo-selenium speciation. The methods compared for detection were inductively coupled plasma mass spectrometry (ICP–MS), microwave induced plasma atomic emission spectroscopy (MIP–AES) and atomic fluorescence spectroscopy (AFS). All detectors were found to be suitable, with highest sensitivity being obtained for MIP–AES detection, with detection limits of 0.57 ng ml−1 for dimethyl selenide, 0.47 ng ml−1 for diethyl selenide and 0.19 ng ml−1 for dimethyl diselenide. The method was applied to the determination of volatile alkyl selenides in selenium enriched yeast samples, which revealed that the presence of inorganic selenium gives rise to at least seven different volatile species after metabolization, with dimethyl diselenide the predominant species. Commercial pasteurized yeast, containing mainly selenomethionine for its use as a food supplement, was found to be still active and produces considerable amounts of organoselenium compounds.

Peroxynitrite Reactions with Dimethylsulfide and Dimethylselenide: An Experimental Study

The kinetics of the peroxynitrite (ONOO- and HOONO, PN for both) reaction with (Me)2X (X = S and Se, dimethyl sulfide (DMS) and dimethylselenide (DMSe)) was studied. The reaction is first order with respect to both PN and (Me)2X concentrations. (Me)2XO and NO2- were formed with 100% yield based on PN in all cases except in the DMS reaction with HOONO, where the yield was ∼80%. DMSe is much more reactive than DMS toward both ONOO- and HOONO, while HOONO reacts much faster than ONOO- with both (Me)2X. The activation entropies (in cal deg-1 mol-1) and enthalpies (given in parentheses, in kcal/mol) for the reactions of DMS and DMSe with HOONO were determined to be −23 ± 3 (5.9 ± 0.5) and −20.5 ± 3 (4.6 ± 0.5), respectively. The corresponding values for the reaction with ONOO- were −29 ± 3 (9.7 ± 0.6) and −28 ± 3 (7.2 ± 0.5). A partial replacement of water with an organic cosolvent resulted in a decrease in the rate of reaction of ONOO- with both DMS and DMSe, while it had no effect on the rates of reaction wi...

Volatile organo-selenium speciation in biological matter by solid phase microextraction–moderate temperature multicapillary gas chromatography with microwave induced plasma atomic emission spectrometry detection

Microwave induced plasma atomic emission spectrometry (MIP-AES) in combination with multicapillary (MC) gas chromatography could be proven to be useful for element specific detection of volatile species. Solid phase microextraction (SPME) was used for preconcentration and sample-matrix separation. The fiber desorption unit as well as the heating control for the MC column were in-house developed and multicapillary column was operated at moderate temperatures (30–100 °C). The method was optimized for organo-selenium species (dimethylselenide (DMSe), diethylselenide (DEtSe) and dimethyldiselenide (DMDSe)), using a chemometric approach. Stationary phases for the separation column were optimized using a conventional GC and contrasted with the results obtained with the MC. Application was focussed on selenium accumulating biological matter, such as lupine, yeast, Indian mustard and garlic. These samples were grown in hydroponic solution containing inorganic selenium (Na 2SeO 3 and Na 2SeO 4). SPME sampling was carried out in fixed volume flow boxes in headspace above the living plants and in vials using treated samples. Results demonstrate inorganic selenium transformation into volatile organic species during metabolism. Separation is fast, a chromatogram can be obtained in less than 3 min and detection limits were at sub-ppb level for all investigated species. The system is independent from the use of a conventional gas chromatographic oven and can be used as a versatile alternative to highly cost intensive methods such as GC–ICP-MS.

Toxic effects of selenium inhalation: acute damage of the respiratory system of mice.

Accidental inhalation of selenium by humans has been associated with damage of respiratory tissues that is lacking a detailed histological definition. We have investigated the natural history of injury to the tracheal epithelium and lungs induced by a single intratracheal instillation of CD-1 mice with two different doses of dimethyl selenide (0.05 and 0.1 mg Se/kg of body weight). The animals were sacrificed 1, 7, 14, and 28 days after the single selenium treatment. Samples of the trachea and lungs were studied by light microscopy. The tracheal epithelium showed loss of cilia and acute necrosis that was followed by metaplastic transformation. Edema and diffuse alveolar damage was observed in the lungs. Our data suggest that: i) severity of respiratory lesions caused by selenium is dose dependent; ii) selenium causes transient metaplastic transformation of the tracheal epithelium; iii) chronic inflammation and increased thickness of alveolar septa occur in the lungs; iv) 4 weeks after selenium treatment, mice recover from the tracheal injury, whereas no amelioration of pulmonary lesions was observed.

Theoretical Studies of the Reaction Mechanisms of Dimethylsulfide and Dimethylselenide with Peroxynitrite

The mechanisms of the reactions of dimethylsulfide (DMS) and dimethylselenide (DMSe) with peroxynitrite anion, ONOO-, and peroxynitrous acid, HOONO, were studied at the B3LYP/6-311+G(d,p) level of theory. It was shown that the gas-phase reactions with ONOO- proceed via an O-atom (O3) transfer (two-electron oxidation) mechanism and produce the corresponding oxides (DMSO and DMSeO, respectively) and NO2- anion. The rate-determining barrier, the O−O bond cleavage, is found to be higher by 6−7 kcal/mol for DMS than for DMSe, indicating that DMSe is more reactive toward ONOO- than DMS. The inclusion of solvent effects decreases the rate-determining barrier and makes it <13.5 kcal/mol in aqueous solution for the DMS reaction. These data indicate that neither DMS nor DMSe catalyze the direct peroxynitrite → nitrate isomerization. It was shown that the reaction of DMS with cis-HOONO might proceed via two distinct pathways, stepwise and concerted, and is much faster than that with ONOO-. The stepwise pathway starts with homolysis of the HO−ONO bond to discrete HO• and ONO• radicals, which then coordinate to DMS and produce the (CH3)2S(OH)(NO2) intermediate product. This product undergoes further transformations via two different pathways: (a) a barrierless and endothermic pathway leading to the (CH3)2S(OH)• and NO2• radicals, and (b) an exothermic H-atom transfer pathway forming DMSO and HONO via a barrier at the TS2(H-transfer) transition state. The ΔH(ΔG) values of the rate-determining steps of these two pathways are 24.3 (11.0) and 11.5 (13.1) kcal/mol, respectively, in the gas phase. The concerted pathway proceeds with an O−O bond cleavage barrier of 6.1 (6.3) kcal/mol and leads to DMSO and HONO. It is predicted that the reaction of DMS with HOONO in the gas phase most likely proceeds via the stepwise (one-electron oxidation) pathway only after including entropy effects. However, in the solution phase the stepwise and concerted pathways will effectively compete with each other.

Freshwater bacteria can methylate selenium through the thiopurine methyltransferase pathway.

Involvement of the bacterial thiopurine methyltransferase (bTPMT) in natural selenium methylation by freshwater was investigated. A freshwater environment that had no known selenium contamination but exhibited reproducible emission of dimethyl selenide (DMSe) or dimethyl diselenide (DMDSe) when it was supplemented with an organic form of selenium [(methyl)selenocysteine] or an inorganic form of selenium (sodium selenite) was used. The distribution of the bTPMT gene (tpm) in the microflora was studied. Freshwater bacteria growing on 10 micro M sodium selenite and 10 micro M sodium selenate were isolated, and 4.5 and 10% of the strains, respectively, were shown by colony blot hybridization to hybridize with a Pseudomonas syringae tpm DNA probe. Ribotyping showed that these strains are closely related. The complete rrs sequence of one of the strains, designated Hsa.28, was obtained and analyzed. Its closest phyletic neighbor was found to be the Pseudomonas anguilliseptica rrs sequence. The Hsa.28 strain grown with sodium selenite or (methyl)selenocysteine produced significant amounts of DMSe and DMDSe. The Hsa.28 tpm gene was isolated by genomic DNA library screening and sequencing. BLASTP comparisons of the deduced Hsa.28 bTPMT sequence with P. syringae, Pseudomonas aeruginosa, Vibrio cholerae, rat, and human thiopurine methyltransferase sequences revealed that the levels of similarity were 52 to 71%. PCR-generated Escherichia coli subclones containing the Hsa.28 tpm open reading frame were constructed. E. coli cells harboring the constructs and grown with sodium selenite or (methyl)selenocysteine produced significant levels of DMSe and DMDSe, confirming that the gene plays a role in selenium methylation. The effect of strain Hsa.28 population levels on freshwater DMSe and DMDSe emission was investigated. An increase in the size of the Hsa.28 population was found to enhance significantly the emission of methyl selenides by freshwater samples supplemented with sodium selenite or (methyl)selenocysteine. These data suggest that bTPMT can play a role in natural freshwater selenium methylation processes.