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(3)SeSSCH(3), and dimethyl bis(thio)selenide, CH(3)SSeSCH(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(3)SeSSCH(3) was positively identified as the compound detected in bacterial headspace above Se-amended cultures. Using GC retention times, the boiling point of CH(3)SeSSCH(3) was estimated to be approximately 192 degrees C. This is the first report of CH(3)SeSSCH(3) produced by bacterial cultures.

Geobacillus stearothermophilus V ubiE gene product is involved in the evolution of dimethyl telluride in Escherichia coli K-12 cultures amended with potassium tellurate but not with potassium tellurite.

A 3.8-kb fragment of chromosomal DNA of Geobacillus stearothermophilus V cloned in pSP72 (p1VH) confers resistance to potassium tellurite (K(2)TeO(3)) and to potassium tellurate (K(2)TeO(4)) when the encoded genes are expressed in Escherichia coli K-12. The nt sequence of the cloned fragment predicts three ORFs of 780, 399, and 600 bp, whose encoded protein products exhibit about 80% similarity with the SUMT methyltransferase and the BtuR protein of Bacillus megaterium, and with the UbiE methyltransferase of Bacillus anthracis A2012, respectively. In addition, E. coli/p1VH cells evolved dimethyl telluride, which was released into the headspace gas above liquid cultures when amended with K(2)TeO(3) or with K(2)TeO(4). After 48 h of growth in the presence of these compounds, a protein of about 25 kDa was found at a significantly higher level when crude extracts were analyzed by SDS-PAGE. The N-terminal amino acid (aa) sequence of this protein, obtained by Edman degradation, matched the deduced aa sequence predicted by the G. stearothermophilus V ubiE gene. This gene was amplified by PCR, subcloned in pET21b, and transformed into E. coli JM109(DE3). Interestingly, DMTe evolution occurred when these modified cells were grown in K(2)TeO(4) - but not in K(2)TeO(3) - amended media. These results may be indicative that the two Te oxyanions could be detoxified in the cell by different metabolic pathways.

Identification of biogenic organotellurides in Escherichia coli K-12 headspace gases using solid-phase microextraction and gas chromatography.

Escherichia coli JM109 cells, expressing the genes encoded in a 3.8-kb chromosomal DNA fragment from Geobacillus stearothermophilus V, produced volatile organotellurium compounds which were released into the headspace gas above liquid cultures when amended with tellurite anions in micromolar amounts. Headspace sampling was achieved using gas-syringe extraction or solid-phase microextraction using carboxen-polydimethysiloxane fibers. In addition to dimethyl telluride and dimethyl ditelluride, two new organometalloidal compounds were detected using gas chromatograph with mass spectrometric or fluorine-induced chemiluminescence detection. These compounds are methanetellurol and dimethyl tellurenyl sulfide. The significance of these findings with regard to the current knowledge about bacterial tellurite resistance is discussed.