Dimethyl Sulfoxide Reduction Research Articles

The regulatory function of LexA is temperature-dependent in the deep-sea bacterium Shewanella piezotolerans WP3.

The SOS response addresses DNA lesions and is conserved in the bacterial domain. The response is governed by the DNA binding protein LexA, which has been characterized in model microorganisms such as Escherichia coli. However, our understanding of its roles in deep-sea bacteria is limited. Here, the influence of LexA on the phenotype and gene transcription of Shewanella piezotolerans WP3 (WP3) was investigated by constructing a lexA deletion strain (WP3ΔlexA), which was compared with the wild-type strain. No growth defect was observed for WP3ΔlexA. A total of 481 and 108 genes were differentially expressed at 20 and 4°C, respectively, as demonstrated by comparative whole genome microarray analysis. Furthermore, the swarming motility and dimethylsulfoxide reduction assay demonstrated that the function of LexA was related to temperature. The transcription of the lexA gene was up-regulated during cold acclimatization and after cold shock, indicating that the higher expression level of LexA at low temperatures may be responsible for its temperature-dependent functions. The deep-sea microorganism S. piezotolerans WP3 is the only bacterial species whose SOS regulator has been demonstrated to be significantly influenced by environmental temperatures to date. Our data support the hypothesis that SOS is a formidable strategy used by bacteria against various environmental stresses.

Metabolic fate of the 14C-labeled herbicide clodinafop-propargyl in a sediment–water system

The metabolic fate of 14C-phenyl-labeled herbicide clodinafop-propargyl (14C-CfP) was studied for 28 days in lab assays using a sediment–water system derived from a German location. Mineralization was 5.21% of applied 14C after 28 days exhibiting a distinct lag phase until day 14 of incubation. Portions of radioactivity remaining in water phases decreased at moderate rate to 18.48% after 28 days; 62.46% were still detected in water after 14 days. Soxhlet extraction of the sediment using acetonitrile released 35.56% of applied 14C with day 28, while 33.99% remained as non-extractable residues. A remarkable increase of bound 14C was observed between 14 and 28 days correlating with the distinct increase of mineralization. No correlation was found throughout incubation with microbial activity of the sediment as determined by dimethyl sulfoxide reduction. Dissolved oxygen and pH value of water phases remained almost constant for 28 days. Analyses of Soxhlet extracts of the sediment and ethyl acetate extracts of water phases by radio-TLC and radio-HPLC revealed that CfP was rapidly cleaved to free acid clodinafop (Cf), which was further (bio-) transformed. DT50 values (based on radio-HPLC) were below 1 day (CfP) and slightly above 28 days (Cf). Further metabolites were not detected. Fractionation of humic and non-humic components of the sediment demonstrated that CfP's non-extractable residues were predominantly associated with fulvic acids up to 14 days of incubation (3.36%), whereas after 28 days, the majority of radioactivity was found in the humin/mineral fraction (13.30% of applied 14C). Due to high-performance size-exclusion chromatography of the fulvic acids fraction derived from assays incubated for 28 days, this portion of 14C was firmly, possibly covalently bound to fulvic acids and did not consist of CfP or Cf. Using an isolation strategy comprising preincubation of sediment with CfP and mineralization of 14C-CfP as criterion, a microorganism was isolated from the sediment examined. It grew on 14C-CfP as sole carbon source with evolution of 14CO2. The bacterium was characterized by growth on commonly used carbon sources and 16S rDNA sequence analysis. Its sequence exhibited high similarity with that of Nocardioides aromaticivorans strain H-1 (98.85%; DSM 15131, JCM 11674).

Measurement of DMS, DMSO, and DMSP in natural waters by automated sequential chemical analysis

AbstractDimethyl sulfide (DMS), dimethylsulfoniopropionate (DMSP), and dimethyl sulfoxide (DMSO) are key components of the marine sulfur cycle. The concentrations of these compounds exhibit large spatial and temporal variability in the surface ocean, creating a need for high resolution sampling. Existing automated underway measurement systems for DMS do not measure DMSP or DMSO, so their spatial variability is less well‐characterized. We present an accurate and robust method for the automated, high throughput sampling and measurement of DMS, DMSO, and DMSP (DMS/O/P) in a single water sample. The method is based on a three‐step sequence of purge and trap gas chromatography, where DMS analysis is followed by the enzymatic reduction of DMSO to DMS and the alkaline hydrolysis of DMSP to DMS. The system, which we call the Organic Sulfur Sequential Chemical Analysis Robot (OSSCAR), includes automated calibrations and blank determinations. OSSCAR can be used as a front‐end system for any sulfur detector and is suited for continuous underway analysis or the measurement of discrete water samples. The system described here has a minimum detection limit of ∼ 0.1 nM of DMS/O/P in a 2.5 mL sample. Assessment of liquid standards and intercalibration against independent analytical systems demonstrate good precision and accuracy of our method. Shipboard analysis of surface water DMS/O/P concentrations on a transect from Ocean Station Papa (50°N, 145°W) to Vancouver Island demonstrates the utility of OSSCAR for mapping variability in reduced sulfur compounds across dynamic and contrasting oceanographic conditions.

Phenotypic and functional characterization of long-term cryopreserved human adipose-derived stem cells.

Cryopreservation represents an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs) and allows pooling of cells via long-term storage for clinical applications, e.g., cell-based therapies. It is crucial to reduce freezing injury during the cryopreservation process by loading the ASCs with the optimum concentration of suitable cryoprotective agents (CPAs). In this study, human ASCs were preserved for 3 months in different combinations of CPAs, including 1) 0.25 M trehalose; 2) 5% dimethylsulfoxide (DMSO); 3) 10% DMSO; 4) 5% DMSO + 20% fetal bovine serum (FBS); 5) 10% DMSO + 20% FBS; 6) 10% DMSO + 90% FBS. Interestingly, even with a reduction of DMSO to 5% and without FBS, cryopreserved ASCs maintained high cell viability comparable with standard cryomedium (10% DMSO + 90% FBS), with normal cell phenotype and proliferation rate. Cryopreserved ASCs also maintained their differentiation capability (e.g., to adipocytes, osteocytes and chondrocytes) and showed an enhanced expression level of stemness markers (e.g., NANOG, OCT-4, SOX-2 and REX-1). Our findings suggest that 5% DMSO without FBS may be an ideal CPA for an efficient long-term cryopreservation of human ASCs. These results aid in establishing standardized xeno-free long-term cryopreservation of human ASCs for clinical applications.

Electrochemically mediated enantioselective reduction of chiral sulfoxides.

The respiratory DMSO reductase from Rhodobacter capsulatus catalyzes the reduction of dimethyl sulfoxide to dimethyl sulfide. Herein, we have utilized this Mo enzyme as an enantioselective catalyst to generate optically pure sulfoxides (methyl p-tolyl sulfoxide, methyl phenyl sulfoxide and phenyl vinyl sulfoxide) from racemic starting materials. A hexaaminecobalt coordination compound in its divalent oxidation state was employed as the mediator of electron transfer between the working electrode and DMSO reductase to continually reactivate the enzyme after turnover. In all cases, chiral HPLC analysis of the reaction mixture revealed that the S-sulfoxide was reduced more rapidly leading to enrichment or isolation of the R isomer.

Aryl methyl sulfides via SNAr using DMSO as the source of the thiomethyl moiety

A unique synthesis of aryl methyl sulfides is reported proceeding via reduction of dimethylsulfoxide to dimethylsulfide at elevated temperature in the presence of Hunig’s base followed by nucleophilic aromatic substitution and demethylation. Activated aryl fluorides, chlorides, and nitrobenzenes are suitable substrates with 13 examples provided. Dimethylsulfoxide serves as a simple and inexpensive formal source of the thiomethyl moiety.

Dimethylsulfoxide reduction activity is linked to nutrient stress in Thalassiosira pseudonana NCMA 1335

Dimethylsulfoxide reduction activity is linked to nutrient stress in Thalassiosira pseudonana NCMA 1335

Dimethyl Sulfide – Significance, Origins, and Control

Dimethyl sulfide (DMS) is a substantial contributor to the aroma of many lager-style beers. Opinion varies on its desirability. It can be derived in beer from two sources: the thermal decomposition of S-methyl-methionine (SMM) produced in the embryo of barley during germination, or the reduction of dimethyl sulfoxide (DMSO, derived from the breakdown of SMM during the curing of malt) by yeast. The enzyme that effects DMSO reduction is a reductase whose primary function is the reduction of methionine sulfoxide and which competes for reducing power with several other cellular systems. Control of DMS production from SMM is achieved by specifying precursor levels in malt, by attending to the vigor and duration of the boil, and by controlling the length of the whirlpool stand. Control of DMS production from yeast is achieved by specifying yeast strain, wort gravity, free amino nitrogen and pH, the type of fermenter (ergo the extent of volatilization), and the fermentation temperature.

Transposon Mutagenesis Identified Chromosomal and Plasmid Genes Essential for Adaptation of the Marine Bacterium Dinoroseobacter shibae to Anaerobic Conditions

Anaerobic growth and survival are integral parts of the life cycle of many marine bacteria. To identify genes essential for the anoxic life of Dinoroseobacter shibae, a transposon library was screened for strains impaired in anaerobic denitrifying growth. Transposon insertions in 35 chromosomal and 18 plasmid genes were detected. The essential contribution of plasmid genes to anaerobic growth was confirmed with plasmid-cured D. shibae strains. A combined transcriptome and proteome approach identified oxygen tension-regulated genes. Transposon insertion sites of a total of 1,527 mutants without an anaerobic growth phenotype were determined to identify anaerobically induced but not essential genes. A surprisingly small overlap of only three genes (napA, phaA, and the Na(+)/Pi antiporter gene Dshi_0543) between anaerobically essential and induced genes was found. Interestingly, transposon mutations in genes involved in dissimilatory and assimilatory nitrate reduction (napA, nasA) and corresponding cofactor biosynthesis (genomic moaB, moeB, and dsbC and plasmid-carried dsbD and ccmH) were found to cause anaerobic growth defects. In contrast, mutation of anaerobically induced genes encoding proteins required for the later denitrification steps (nirS, nirJ, nosD), dimethyl sulfoxide reduction (dmsA1), and fermentation (pdhB1, arcA, aceE, pta, acs) did not result in decreased anaerobic growth under the conditions tested. Additional essential components (ferredoxin, cccA) of the anaerobic electron transfer chain and central metabolism (pdhB) were identified. Another surprise was the importance of sodium gradient-dependent membrane processes and genomic rearrangements via viruses, transposons, and insertion sequence elements for anaerobic growth. These processes and the observed contributions of cell envelope restructuring (lysM, mipA, fadK), C4-dicarboxylate transport (dctM1, dctM3), and protease functions to anaerobic growth require further investigation to unravel the novel underlying adaptation strategies.

Genomic Plasticity Enables a Secondary Electron Transport Pathway in Shewanella oneidensis

Microbial dissimilatory iron reduction is an important biogeochemical process. It is physiologically challenging because iron occurs in soils and sediments in the form of insoluble minerals such as hematite or ferrihydrite. Shewanella oneidensis MR-1 evolved an extended respiratory chain to the cell surface to reduce iron minerals. Interestingly, the organism evolved a similar strategy for reduction of dimethyl sulfoxide (DMSO), which is reduced at the cell surface as well. It has already been established that electron transfer through the outer membrane is accomplished via a complex in which β-barrel proteins enable interprotein electron transfer between periplasmic oxidoreductases and cell surface-localized terminal reductases. MtrB is the β-barrel protein that is necessary for dissimilatory iron reduction. It forms a complex together with the periplasmic decaheme c-type cytochrome MtrA and the outer membrane decaheme c-type cytochrome MtrC. Consequently, mtrB deletion mutants are unable to reduce ferric iron. The data presented here show that this inability can be overcome by a mobile genomic element with the ability to activate the expression of downstream genes and which is inserted within the SO4362 gene of the SO4362-to-SO4357 gene cluster. This cluster carries genes similar to mtrA and mtrB and encoding a putative cell surface DMSO reductase. Expression of SO4359 and SO4360 alone was sufficient to complement not only an mtrB mutant under ferric citrate-reducing conditions but also a mutant that furthermore lacks any outer membrane cytochromes. Hence, the putative complex formed by the SO4359 and SO4360 gene products is capable not only of membrane-spanning electron transfer but also of reducing extracellular electron acceptors.

Aerobic and anaerobic microbial activities in the foreland of a receding glacier

The foreland soils of a receding glacier were investigated for both aerobic and anaerobic microbial activities. Estimation of microbial biomass via substrate induced respiration, and determination of dehydrogenase activity, dimethylsulfoxide reduction and ammonification were performed to describe soil microflora and its activity. These parameters distinctly increased along the chronosequence from the youngest to the oldest soil. Additionally, these properties showed remarkably higher values in the samples collected at the end of the growing season indicating distinct seasonal changes. The determination of methane emission by the three soils incubated at 10, 37 and 50 °C clearly pointed to higher methanogenic activities within the thermophilic temperature range. Our results also show that water saturation in order to simulate spring snow melt led to increased emissions of methane due to reduced oxygen availability. Methane release was generally low in the primary soil, slightly increased as soil formation proceeded and in all soils was distinctly higher at the end of the growing season. Potential methane consumption also increased along the foreland and during the year. Overall methanotrophic activity greatly exceeded methanogenic activity, and the net CH4 consumption distinctly increased along the chronosequence.

Rapid, Covalent Addition of Phosphine to Dithiolene in a Molybdenum Tris(dithiolene). A New Structural Model for Dimethyl Sulfoxide Reductase

Triphenylphosphine (PPh(3)) rapidly and reversibly adds to the bdt ligand in the molybdenum tris(dithiolene) complex Mo(tfd)(2)(bdt) [tfd = S(2)C(2)(CF(3))(2); bdt = S(2)C(6)H(4)], turning chelating bdt into the monodentate zwitterionic ligand SC(6)H(4)SPPh(3). A second PPh(3) molecule fills the newly created open site in the crystallographically characterized product Mo(tfd)(2)(SC(6)H(4)SPPh(3))(PPh(3)), which is a structural model for dimethyl sulfoxide (DMSO) reductase. While the complex is only a precatalyst for reduction of DMSO by PPh(3) (the initially low catalytic rate increases with time), Mo(tfd)(2)(SMe(2))(2) was found to be catalytically active without an induction period.

High concentrations and turnover rates of DMS, DMSP and DMSO in Antarctic sea ice

[1] The vast Antarctic sea-ice zone (SIZ) is a potentially significant source of the climate-active gas dimethylsulfide (DMS), yet few data are available on the concentrations and turnover rates of DMS and the related compounds dimethylsulfoniopropionate (DMSP) and dimethylsulfoxide (DMSO) in sea ice environments. Here we present new measurements characterizing the spatial variability of DMS, DMSP, and DMSO concentrations across the Antarctic SIZ, and results from tracer experiments quantifying the production rates of DMS from various sources. We observed extremely high concentrations (>200 nM) and turnover rates (>100 nM d−1) of DMS in sea-ice brines, indicating intense cycling of DMS/P/O. Our results demonstrate a previously unrecognized role for DMSO reduction as a major pathway of DMS production in Antarctic sea ice.

Correct Assembly of Iron-Sulfur Cluster FS0 into Escherichia coli Dimethyl Sulfoxide Reductase (DmsABC) Is a Prerequisite for Molybdenum Cofactor Insertion

The FS0 [4Fe-4S] cluster of the catalytic subunit (DmsA) of Escherichia coli dimethyl sulfoxide reductase (DmsABC) plays a key role in the electron transfer relay. We have now established an additional role for the cluster in directing molybdenum cofactor assembly during enzyme maturation. EPR spectroscopy indicates that FS0 has a high spin ground state (S = 3/2) in its reduced form, resulting in an EPR spectrum with a peak at g ∼ 5.0. The cluster is predicted to be in close proximity to the molybdo-bis(pyranopterin guanine dinucleotide) (Mo-bisPGD) cofactor, which provides the site of dimethyl sulfoxide reduction. Comparison with nitrate reductase A (NarGHI) indicates that a sequence of residues ((18)CTVNC(22)) plays a role in both FS0 and Mo-bisPGD coordination. A DmsA(ΔN21) mutant prevented Mo-bisPGD binding and resulted in a degenerate [3Fe-4S] cluster form of FS0 being assembled. DmsA belongs to the Type II subclass of Mo-bisPGD-containing catalytic subunits that is distinguished from the Type I subclass by having three rather than two residues between the first two Cys residues coordinating FS0 and a conserved Arg residue rather than a Lys residue following the fourth cluster coordinating Cys. We introduced a Type I Cys group into DmsA in two stages. We changed its sequence from (18)C(A)TVNC(B)GSRC(C)P(27) to (18)C(A)TYC(B)GVGC(C)G(26) (similar to that of formate dehydrogenase (FdnG)) and demonstrated that this eliminated both Mo-bisPGD binding and EPR-detectable FS0. We then combined this change with a DmsA(R61K) mutation and demonstrated that this additional change partially rescued Mo-bisPGD insertion.

Cobalt hexaamine mediated electrocatalytic voltammetry of dimethyl sulfoxide reductase: driving force effects on catalysis

The bacterial molybdoenzyme dimethyl sulfoxide (DMSO) reductase from Rhodobacter capsulatus catalyzes the reduction of DMSO to dimethyl sulfide in anaerobic respiration. In its native state, DMSO reductase is reduced to its active state by a pentaheme cytochrome (DorC). Alternatively, we show that DMSO reductase catalysis may be driven electrochemically using a series of homologous coordination compounds as mediating synthetic electron donors. All mediators are macrocyclic hexaaminecobalt(II) complexes in their active form, differing principally in their redox potentials over a range of about 250mV. Thus, each complex presents a different reductive driving force to DMSO reductase and this leads to pronounced differences in the electrocatalytic behavior as measured by cyclic voltammetry. Digital simulation of the experimental voltammetry enables the critical features of the catalytic cycle to be extracted.

Influence of Cardiolipin on Lager Beer Dimethyl Sulfide Levels: A Possible Role Involving Mitochondria?

Dimethyl sulfide (DMS) is an important flavor compound in American and European lagers, so brewers strive to control its level in their products. Although the main approach to controlling DMS involves judicious selection of malting and wort production regimes to control one of its precursors, S-methyl methionine (SMM), DMS formation during fermentation by yeast caused by an enzyme coined “dimethyl sulfoxide (DMSO) reductase” cannot be overlooked. During the process of altering the wort carbohydrate spectrum from one predominantly made up of maltose to one much richer in glucose through adjunct changes, a significant reduction in beer DMS levels was noted. An investigation into malt SMM levels demonstrated no significant reduction in this main precursor, suggesting other mechanisms could be involved. Since it is known that glucose inhibits the development of both mitochondria and their unique lipid, cardiolipin, we speculated that this organelle could be involved. Through a series of replicated incubation experiments aimed at altering cardiolipin content in yeast, it was found that beer DMS levels at the end of fermentation were moderately higher (approx. 10 μg/L) under incubation conditions that promote cardiolipin formation, i.e., incubation in glycerol and l-thyroxine. In comparison, conditions that limit cardiolipin formation (control and inositol incubation) resulted in lower DMS levels (P < 0.05). Assuming equal losses due to CO2 stripping during fermentation, these results suggest a possible role involving yeast mitochondria. Two suggestions are provided, of which we believe the latter is most likely based on DMSO- and methionine sulfoxide (MetSO)-addition experiments conducted within this study. First, if a true bacterial-like DMSO reductase protein were responsible, then defects in iron-sulfur cluster (ISC) formation or protein maturation would be likely, because ISCs are catalytic sites for electron transfer of Fe-S proteins. However, due to complete MetSO inhibition of DMSO reduction to DMS in the yeast strain studied, it seems more plausible that this was the result of decreased MetSO reductase activity, specifically an MsrB protein, which is speculated to be localized to the mitochondria, and that somehow the cardiolipin composition of the mitochondrial membrane is involved.

ArcB1, a Homolog of Escherichia coli ArcB, Regulates Dimethyl Sulfoxide Reduction in Shewanella oneidensis MR-1

Shewanella oneidensis is a metal reducer that uses the cyclic AMP receptor protein, CRP, to regulate anaerobic respiration. In addition, ArcA(So) is required for anaerobic growth with dimethyl sulfoxide (DMSO) and plays a role in aerobic respiration. The sensor kinase that activates ArcA(So) in S. oneidensis is not known. ArcB1(So), a homolog of the Escherichia coli sensor kinase ArcB(Ec), was identified and found to be required for DMSO reductase gene expression. In combination with HptA, ArcB1(So) complemented an E. coli arcB(Ec) mutant. ArcA(So), ArcB1(So), and HptA appear to constitute a two-component signal transduction system that regulates DMSO reduction in S. oneidensis.

Inhibition of methionine sulfoxide reduction by dimethyl sulfoxide

Dimethyl sulfoxide (DMSO) is widely used in chemistry and biology as a solvent and as a cryoprotectant. It is also used as a pharmaceutical agent for the treatment of interstitial cystitis and rheumatoid arthritis. Previous reports described DMSO as being reduced by methionine-S-sulfoxide reductase (MsrA). However, little is known about the DMSO reduction capability of methionine-R-sulfoxide reductase (MsrB) or its effect on the catalysis of methionine sulfoxide reduction. We show that mammalian MsrB2 and MsrB3 were unable to reduce DMSO. This compound inhibited MsrB2 activity but did not inhibit MsrB3 activity. We further determined that DMSO functions as an inhibitor of MsrA and MsrB2 in the reduction of methionine sulfoxides via different inhibition mechanisms. DMSO competitively inhibited MsrA activity but acted as a non-competitive inhibitor of MsrB2 activity. Our study also demonstrated that DMSO inhibits in vivo methionine sulfoxide reduction in yeast and mammalian cells. [BMB reports 2009; 42(9): 580-585].

Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.

Control of DMSO in wastewater to prevent DMS nuisance odors

A “canned corn-like” odor was periodically detected at Philadelphia's Northeast Water Pollution Control Plant (NEWPCP) for more than two decades. Previous research concluded that it was caused by dimethyl sulfide (DMS), from the reduction of dimethyl sulfoxide (DMSO) discharged by a local industrial customer. Several process modifications were implemented at the industrial site to eliminate the “canned corn-like” odor. Results showed that enhancing DMSO recovery by 25% and equalizing the aqueous wash discharge over a longer period of time reduced the DMSO source peak discharge from 1124 to 49 kg/h, and the peak concentrations of DMSO and DMS at the NEWPCP by 81 and 88%. Reduction of DMSO discharge by segregating the first wash for off-site disposal further reduced the peak discharge of DMSO from 49 to 18 kg/h at the source, and DMSO and DMS concentrations at the NEWPCP by 48 and 92%. Segregation of the dehydration distillate for off-site disposal reduced DMSO discharge by 3 kg/h. Modifications by concentrating a higher percentage of the DMSO into the first wash and increasing the DMSO solvent recovery by an additional 33% reduced the total DMSO discharge from 522 to 200 kg and peak discharge rate from 15 to 6 kg/h. All of these process modifications collectively reduced the DMSO source discharge by 92% and the DMSO concentration received at NEWPCP by 97%, from 12 mg/L to ∼500 μg/L. At this reduced concentration, the company's wastewater discharge was no longer found to cause the “canned corn” odor at the fence line of NEWPCP, thereby mitigating any further need for odor control.