Store Sulfur Research Articles

Structural and Molecular Genetic Insight into a Widespread Sulfur Oxidation Pathway

Many environmentally important photo- and chemolithoautotrophic bacteria accumulate globules of polymeric, water-insoluble sulfur as a transient product during oxidation of reduced sulfur compounds. Oxidation of this sulfur requires the concerted action of Dsr proteins. However, individual functions and interplay of these proteins are largely unclear. We proved with a ΔdsrE mutant experiment that the cytoplasmic α2β2γ2-structured protein DsrEFH is absolutely essential for the oxidation of sulfur stored in the intracellular sulfur globules of the purple sulfur bacterial model organism Allochromatium vinosum. The ability to degrade stored sulfur was fully regained upon complementation with dsrEFH in trans. The crystal structure of DsrEFH was determined at 2.5 Å resolution to assist functional assignment in detail. In conjunction with phylogenetic analyses, two different types of putative active sites were identified in DsrE and DsrH and shown to be characteristic for sulfur-oxidizing bacteria. Conserved Cys78 of A. vinosum DsrE corresponds to the active cysteines of Escherichia coli YchN and TusD. TusBCD and the protein TusE are parts of sulfur relay system involved in thiouridine biosynthesis. DsrEFH interacts with DsrC, a TusE homologue encoded in the same operon. The conserved penultimate cysteine residue in the carboxy-terminus of DsrC is essential for the interaction. Here, we show that Cys78 of DsrE is strictly required for interaction with DsrC while Cys20 in the putative active site of DsrH is dispensable for that reaction. In summary, our findings point at the occurrence of sulfur transfer reactions during sulfur oxidation via the Dsr proteins.

Importance of the DsrMKJOP complex for sulfur oxidation in Allochromatium vinosum and phylogenetic analysis of related complexes in other prokaryotes

In the phototrophic sulfur bacterium Allochromatium vinosum, sulfur of oxidation state zero stored in intracellular sulfur globules is an obligate intermediate during the oxidation of sulfide and thiosulfate. The proteins encoded in the dissimilatory sulfite reductase (dsr) locus are essential for the oxidation of the stored sulfur. DsrMKJOP form a membrane-spanning complex proposed to accept electrons from or to deliver electrons to cytoplasmic sulfur-oxidizing proteins. In frame deletion mutagenesis showed that each individual of the complex-encoding genes is an absolute requirement for the oxidation of the stored sulfur in Alc. vinosum. Complementation of the DeltadsrJ mutant using the conjugative broad host range plasmid pBBR1-MCS2 and the dsr promoter was successful. The importance of the DsrMKJOP complex is underlined by the fact that the respective genes occur in all currently sequenced genomes of sulfur-forming bacteria such as Thiobacillus denitrificans and Chlorobaculum tepidum. Furthermore, closely related genes are present in the genomes of sulfate- and sulfite-reducing prokaryotes. A phylogenetic analysis showed that most dsr genes from sulfide oxidizers are clearly separated of those from sulfate reducers. Surprisingly, the dsrMKJOP genes of the Chlorobiaceae all cluster together with those of the sulfate/sulfite-reducing prokaryotes, indicating a lateral gene transfer at the base of the Chlorobiaceae.

Evaluating the bulk state of granulated sulfur in stores at oil refineries

The temperature pattern in oxidizing sulfur under natural climatic conditions has been determined. A working equation is proposed for the temperatures in the bulk of stored sulfur.

Estimating historical in-lake alkalinity generation from sulfate reduction and its relationship to lake chemistry as inferred from algal microfossils

Sediment cores were used to estimate in-lake alkalinity generation resulting from sulfate reduction relative to inferred changes in lakewater pH and trophic status over the last century in three Connecticut lakes. Despite being situated in geological settings with crystalline bedrock and thin, poorly buffered soils, and being impacted with high rates of acidic precipitation, none of the study lakes have declined in inferred pH based on scaled chrysophyte and diatom remains. In fact, the pH of one of the lakes, Coventry Lake, has significantly increased over the last century. Over the last 44 to 69 years the amount of sulfur stored in the sediments from each lake increased from ~two to three times resulting in mean rates of alkalinity generation ranging from 78 to 145 meq m−2 yr−1, significantly higher than the 45 to 48 meq m−2 yr−1 of hydrogen ions falling directly on the lake surfaces. In-lake alkalinity generation resulting from sulfate reduction has been sufficient to neutralize all of the acid falling directly onto the lake surfaces, as well as between 9% and 25% of the acid deposited onto the surrounding watersheds. Despite the increased importance of in-lake alkalinity generation, our findings support the hypothesis that significant amounts of alkalinity are also being generated in the catchments of the study lakes. The bulk of the increases in stored sulfur in all three lakes were as Fe sulfides and not in the form of organic sulfur, suggesting that the increases were the result of dissimilatory bacterial reduction of sulfate. As a result of the large increases in storage of Fe sulfides the ratio of total iron to chromium reducible sulfur (Fe:CRS) has declined in all cores over time. Despite the overall decline in Fe:CRS in recent sediments, values are still largely above 3 in more recent sediments of two of the lakes. However, values of Fe:CRS have dropped below 1 in surface sediments of Uncas Lake, suggesting that in-lake loading of phosphorus may be responsible for a recent shift in the algal flora towards a slightly more eutrophic condition.

Analysis of sulfur biochemistry of sulfur bacteria using X-ray absorption spectroscopy.

Many sulfide-oxidizing organisms, including the photosynthetic sulfur bacteria, store sulfur in "sulfur globules" that are readily detected microscopically. The chemical form of sulfur in these globules is currently the focus of a debate, because they have been described as "liquid" by some observers, although no known allotrope of sulfur is liquid at physiological temperatures. In the present work we have used sulfur K-edge X-ray absorption spectroscopy to identify and quantify the chemical forms of sulfur in a variety of bacterial cells, including photosynthetic sulfur bacteria. We have also taken advantage of X-ray fluorescence self-absorption to derive estimates of the size and density of the sulfur globules in photosynthetic bacteria. We find that the form of sulfur that most resembles the globule sulfur is simply solid S(8), rather than more exotic forms previously proposed.

Genes involved in hydrogen and sulfur metabolism in phototrophic sulfur bacteria

The dsr genes and the hydSL operon are present as separate entities in phototrophic sulfur oxidizers of the genera Allochromatium, Marichromatium, Thiocapsa and Thiocystis and are organized similarly as in Allochromatium vinosum and Thiocapsa roseopersicina, respectively. The dsrA gene, encoding the α subunit of ‘reverse’ siroheme sulfite reductase, is also present in two species of green sulfur bacteria pointing to an important and universal role of this enzyme and probably other proteins encoded in the dsr locus in the oxidation of stored sulfur by phototrophic bacteria. The hupSL genes are uniformly present in the members of the Chromatiaceae family tested. The two genes between hydS and hydL encode a membrane-bound b-type cytochrome and a soluble iron-sulfur protein, respectively, resembling subunits of heterodisulfide reductase from methanogenic archaea. These genes are similar but not identical to dsrM and dsrK, indicating that the derived proteins have distinct functions, the former in hydrogen metabolism and the latter in oxidative sulfur metabolism.

Genes involved in hydrogen and sulfur metabolism in phototrophic sulfur bacteria.

The dsr genes and the hydSL operon are present as separate entities in phototrophic sulfur oxidizers of the genera Allochromatium, Marichromatium, Thiocapsa and Thiocystis and are organized similarly as in Allochromatium vinosum and Thiocapsa roseopersicina, respectively. The dsrA gene, encoding the alpha subunit of 'reverse' siroheme sulfite reductase, is also present in two species of green sulfur bacteria pointing to an important and universal role of this enzyme and probably other proteins encoded in the dsr locus in the oxidation of stored sulfur by phototrophic bacteria. The hupSL genes are uniformly present in the members of the Chromatiaceae family tested. The two genes between hydS and hydL encode a membrane-bound b-type cytochrome and a soluble iron-sulfur protein, respectively, resembling subunits of heterodisulfide reductase from methanogenic archaea. These genes are similar but not identical to dsrM and dsrK, indicating that the derived proteins have distinct functions, the former in hydrogen metabolism and the latter in oxidative sulfur metabolism.

Molecular genetic evidence for extracytoplasmic localization of sulfur globules in Chromatium vinosum.

Purple sulfur bacteria store sulfur as intracellular globules enclosed by a protein envelope. We cloned the genes sgpA, sgpB, and sgpC, which encode the three different proteins that constitute the sulfur globule envelope of Chromatium vinosum D (DSMZ 180(T)). Southern hybridization analyses and nucleotide sequencing showed that these three genes are not clustered in the same operon. All three genes are preceded by sequences resembling sigma70-dependent promoters, and hairpin structures typical for rho-independent terminators are found immediately downstream of the translational stop codons of sgpA, sgpB, and sgpC. Insertional inactivation of sgpA in Chr. vinosum showed that the presence of only one of the homologous proteins SgpA and SgpB suffices for formation of intact sulfur globules. All three sgp genes encode translation products which - when compared to the isolated proteins - carry amino-terminal extensions. These extensions meet all requirements for typical signal peptides indicating an extracytoplasmic localization of the sulfur globule proteins. A fusion of the phoA gene to the sequence encoding the proposed signal peptide of sgpA led to high specific alkaline phosphatase activities in Escherichia coli, further supporting the envisaged targeting process. Together with electron microscopic evidence these results provide strong indication for an extracytoplasmic localization of the sulfur globules in Chr. vinosum and probably in other Chromatiaceae. Extracytoplasmic formation of stored sulfur could contribute to the transmembranous Deltap that drives ATP synthesis and reverse electron flow in Chr. vinosum.

Isolation and characterization of sulfur globule proteins from Chromatium vinosum and Thiocapsa roseopersicina.

Purple sulfur bacteria store sulfur as intracellular globules enclosed by a protein envelope. The proteins associated with sulfur globules of Chromatium vinosum and Thiocapsa roseopersicina were isolated by extraction into 50% aqueous acetonitrile containing 1% trifluoroacetic acid and 10 mM dithiothreitol. The extracted proteins were separated by reversed-phase HPLC, revealing three major proteins from C. vinosum and two from T. roseopersicina. All of these proteins have similar, rather unusual amino acid compositions, being rich in glycine and aromatic amino acids, particularly tyrosine. The molecular masses of the C. vinosum proteins were determined to be 10,498, 10,651, and 8,479 Da, while those from T. roseopersicina were found to be 10,661 and 8,759 Da by laser desorption time-of-flight mass spectrometry. The larger T. roseopersicina protein is N-terminally blocked, probably by acetylation, but small amounts of the unblocked form (mass = 10,619) were also isolated by HPLC. Protein sequencing showed that the two larger C. vinosum proteins are homologous to each other and to the large T. roseopersicina protein. The 8,479 Da C. vinosum and 8,759 Da T. roseopersicina proteins are also homologous, indicating that sulfur globule proteins are conserved between different species of purple sulfur bacteria.

Continuous chemotrophic growth and respiration of Chromatiaceae species at low oxygen concentrations

Endogenous and maximum respiration rates of nine purple sulfur bacterial strains were determined. Endogenous rates were below 10 nmol O2 · (mg protein · min)-1 for sulfur-free cells and 15–35 nmol O2 · (mg protein · min)-1 for cells containg intracellular sulfur globules. With sulfide as electron-donating substrate respiration rates were considerably higher than with thiosulfate. Maximum respiration rates of Thiocystis violacea 2711 and Thiorhodovibrio winogradskyi SSP1 (254.8 and 264.2 nmol O2 · (mg protein · min)-1, respectively) are similar to those of aerobic bacteria. Biphasic respiration curves were obtained for sulfur-free cells of Thiocystis violacea 2711 and Chromatium vinosum 2811. In Thiocystis violacea the rapid and incomplete oxidation of thiosulfate was five times faster than the oxidation of stored sulfur. A high affinity of the respiratoty system for oxygen (K m =0.3–0.9 μM O2, V max=260 nmol O2 · (mg protein · min)-1 with sulfide as substrate, K m =0.6–2.4 μM O2, V max=14–40 nmol O2 · (mg protein · min)-1 with thiosulfate as substrate), for sulfide (K m =0.47 μM, V max=650 nmol H2S · (mg protein × min)-1, and for thiosulfate (K m =5–6 μM, V max =24–72 nmol S2O 2-3 · (mg protein · min)-1 was obtained for different strains. Respiration of Thiocystis violacea was inhibited by very low concentrations of NaCN (K i =1.7 μM) while CO concentrations of up to 300 μM were not inhibitory. The capacity for chemotrophic growth of six species was studied in continuous culture at oxygen concentrations of 11 to 67 μM. Thiocystis violacea 2711, Amoebobacter roseus 6611, Thiocapsa roseopersicina 6311 and Thiorhodovibrio winogradskyi SSP1 were able to grow chemotrophically with thiosulfate/acetate or sulfide/acetate. Chromatium vinosum 2811 and Amoebobacter purpureus ML1 failed to grow under these conditions. During shift from phototrophic to chemotrophic conditions intracellular sulfur and carbohydrate accumulated transiently inside the cells. During chemotrophic growth bacteriochlorophyll a was below the detection limit.

Sulfur pool sizes and stable isotope ratios in HUMEX peat before and immediately after the onset of acidification

Total nonsulfate sulfur (TS), reduced inorganic sulfur (RIS), ester sulfate (ES) and carbon bonded sulfur (CBS) pool sizes, and TS and RIS stable sulfur isotopes were examined in peat cores from control and experimental sites within the Skjervatjern catchment before and 8.5 months after the onset of acidification. The total amount of excess sulfate added to the catchment up to this point was less than expected due to mechanical problems. There have been no observable effects on sulfur cycling within the peat soils. The results from this study, however, provide insight into the natural factors controlling sulfur cycling in this system and suggest that dissimilatory sulfate reduction plays a key role. For instance, the control site, which apparently receives the highest input of sulfate because of its position within the catchment drainage system, has the highest TS, RIS, and CBS pool sizes, the lowest C:S ratios, and the most negative δ 34S TS ratios. All of these characteristics are consistent with a dissimilatory origin for a substantial portion of the peat sulfur. Thus, a mechanism that can potentially store and neutralize at least some of the acidic sulfate to be added to this system is already in operation. The ultimate ability of dissimilatory reduction to store sulfur will be tested as the acidification of the Skjervatjern catchment proceeds and will provide important information on how other wetland ecosystems respond to excessive acidic sulfate inputs.

Effects of sulphur on noble metal automotive catalysts

The small amount of sulfur, ca. 300 ppm, in gasoline which is equivalent to ca. 20 ppm SO 2 in exhaust, has multiple effects on the operation of automotive catalysts. These effects are not widely appreciated. Firstly, there are large differences in the capacity of different noble metals to chemisorb sulfur dioxide and secondly, the dispersion of the noble metal influences the activity in the oxidation of sulfur dioxide to sulfur trioxide. The ability of the catalyst to oxidize SO 2 determines the resistance to poisoning by residual amounts of lead in the fuel and whether the surface of the catalyst support will store sulfur, as sulfate, under oxidizing conditions. The surface sulfate groups on the support enhance the oxidation of saturated hydrocarbons, which is very desirable for the attainment of the stringent regulatory limits on hydrocarbon emissions. On the other hand, the stored sulfur can be emitted as a transient spike of hydrogen sulfide if the engine operating conditions shift momentarily to the rich side of the stoichiometric air/fuel ratio, which is an undesirable phenomenon. The presence of sulfur species in the exhaust influences the partitioning of the products of nitric oxide reduction between dinitrogen and ammonia strongly suppressing the latter. The optimization of the composition of the three-way catalyst has to take into account the effect of sulfur compounds present in the exhaust.

Electrochemical identification of microbially mediated hydrogen sulfide oxidation

The process of H2S oxidation by the phototrophic bacteriaThiocapsa roseopersicina andChlorobium phaeobacteroides, respectively, was monitored using a Pt-glass-Ag0, Ag2S electrode combination without liquid junction. Due to the resulting pe(pH) and pH2S plottings three steps can be distinguished: oxidation of H2S to an S(0) state, oxidation of S (0) to SO42−, and oxidation of the remaining H2S directly to SO42−. Differences between the investigated bacteria exist with respect to their individual oxidation strategies.Thiocapsa apparently stops oxidizing H2S at pH2S 7.5 (e.g. 10−7.5M H2S) and shifts to the utilization of the intracellularly stored S (0). In contrastChlorobium utilizes its extracellularly stored sulfur parallel to the extracellular H2S fraction. The corresponding Pt-sensor responses (pe7 values) were found to be similar to the corresponding partial redox equilibria (pe7 values) of H2S oxidation stoichiometries as proposed by Van Niel (1931) and Truper (1964). It is concluded that the recording of pe enables investigators to understand (and control) in situ redox processes, independent of their thermodynamic equilibration, only bound to changes of electroactivity vs. sensor.

Diel cycle of metabolism of phototrophic purple sulfur bacteria in Lake Cisó (Spain)1

During a bloom of purple sulfur bacteria in Lake Cisó (Spain) data were collected on diel changes in the concentrations of sulfide, sulfur, glycogen, and poly‐β‐hydroxybutyrate (PHB), the rates of CO2 fixation and H2S oxidation, and the viability of cells along the vertical profile. During the day, sulfide oxidation resulted in the intracellular accumulation of sulfur and glycogen. At night, the concentrations of glycogen and sulfur decreased concomitant with the production of sulfide and PHB. Throughout the day, CO2 fixation and H2S oxidation were maximal in the zone of maximal population density (peak of the layer); however, in the top of the layer the specific rates were 2–6 times higher. The ratio of CO2 fixation to H2S oxidation, the sulfur content of the cells, and their specific density all indicated that sulfide was predominantly oxidized to sulfur. In the top and the peak of the layer cell viability was high but decreased rapidly with increasing depth. It was concluded that the existence of the bloom is the combined result of slow growth at the peak of the layer and a much faster rate of reproduction at the top of the layer. The cells at the top stored sulfur extensively and then sank to deeper layers where they could not oxidize the sulfur because of insufficient light.

Oxidation of Sulfide and Thiosulfate and Storage of Sulfur Granules in Thiothrix from Activated Sludge

Thiothrix from activated sludge is, in the presence of oxygen, able to oxidize sulfide and thiosulfate and store many clear sulfur granules at sulfide concentrations less than about 1 mM and thiosulfate concentrations of 0.1-20 mM. On this basis, a sulfur storage test for the identification of Thiothrix in activated sludge was developed. It is shown that the filamentous bacteria, Type 021N (commonly causing bulking in sewage plants) , is able to store sulfur granules and thus is a Thiothrix. In enrichment cultures with thiosulfate typical Thiothrix rosettes and motile gonidia are developed, and the presence of a sheath is demonstrated. Filamental diameter varies between 0.8-2.4 µm, the youngest filaments being thinnest. Attempts to isolate Thiothrix have been unsuccesful, but Thiothrix filaments with only a few contaminants are able to grow on solid media with acetate as the only carbon and energy source, indicating the presence of heterotrophic strains. The filamentous organisms of 14 Danish sewage treatment plants are identified, Thiothrix being the most common in 8, and responsible for bulking in 4 plants.