The deep-subsurface sulfate reducer Desulfotomaculum kuznetsovii employs two methanol-degrading pathways.

Methanol is generally metabolized through a pathway initiated by a cobalamine-containing methanol methyltransferase by anaerobic methylotrophs (such as methanogens and acetogens), or through oxidation to formaldehyde using a methanol dehydrogenase by aerobes. Methanol is an important substrate in deep-subsurface environments, where thermophilic sulfate-reducing bacteria of the genus Desulfotomaculum have key roles. Here, we study the methanol metabolism of Desulfotomaculum kuznetsovii strain 17T, isolated from a 3000-m deep geothermal water reservoir. We use proteomics to analyze cells grown with methanol and sulfate in the presence and absence of cobalt and vitamin B12. The results indicate the presence of two methanol-degrading pathways in D. kuznetsovii, a cobalt-dependent methanol methyltransferase and a cobalt-independent methanol dehydrogenase, which is further confirmed by stable isotope fractionation. This is the first report of a microorganism utilizing two distinct methanol conversion pathways. We hypothesize that this gives D. kuznetsovii a competitive advantage in its natural environment.

Molecular dissection of a putative iron reductase from Desulfotomaculum reducens MI-1.

Desulfotomaculum reducens MI-1 is a Firmicute strain capable of reducing a variety of heavy metal ions and has a great potential in heavy metal bioremediation. We recently identified Dred_2421 as a potential iron reductase through proteomic study of D. reducens. The current study examines its iron-reduction mechanism. Dred_2421, like its close homolog from Escherichia coli (2, 4-dienoyl-CoA reductase), has an FMN-binding N-terminal domain (NTD), an FAD-binding C-terminal domain (CTD), and a 4Fe-4S cluster between the two domains. To understand the mechanism of the iron-reduction activity and the role of each domain, we generated a series of variants for each domain and investigated their iron-reduction activity. Our results suggest that CTD is the main contributor of the iron-reduction activity, and that NTD and the 4Fe-4S cluster are not directly involved in such activity. This study provides a mechanistic understanding of the iron-reductase activity of Dred_2421 and may also help to elucidate other physiological activities this enzyme may have.

Peroxidase activity in the sulfate-reducing bacterium Desulfotomaculum acetoxidans DSM 771.

Earlier research demonstrated the secretion of benzoate, which must be oxygenated to its 4-hydroxy derivative in order to be included in further sulfate uptake processes. The present study on Desulfotomaculum acetoxidans DSM 771 was designed to determine the activity and catalytic specificity of the enzyme (most probably peroxidase) catalyzing the hydroxylation of secreted benzoate. Peroxidase activity measured with ABTS (2,2'-azino-bis (3-ethylbenzathiazoline-6-sulfonic acid) during cultivation indicated the greatest activity on the third and thirteen days (3.4 and 2.3 nkat per ml sample respectively). The highest (0.7979) correlation coefficient was calculated between peroxidase activity and hydrogen peroxide levels. The cell walls from 3- and 13-day cultures were subjected to an isolation procedure, PIPES (piperazine-N,N'-bis (2-ethane-sulfonic acid) extract followed by preparative electrophoresis. The extracts of a approximately 30 kDa band on the gel were analyzed by Western blotting and the membrane was stained with TMB (3,3',5,5'-tetramethylbenzidine-specific for the presence of peroxidase). This same protein was incubated for 6 h with benzoate, H2O2, Na2SO4. The product formed a complex with Fe3+, whose maximum absorption spectra (501.7 nm) corresponded with a ferric complex of synthetic 4-hydroxy-3-sulfo-benzoate. The H2S level during the cultivation was higher in culture grown with 15.5 mM 4-hydroxy-3-sulfo-benzoate than in culture with lactate supplemented with 15.5 mM sulfate. The role of peroxidase in oxygen utilization and sulfate uptake is discussed.