Inorganic Sulfur Oxidation by Aureobasidium pullulans

Aureobasidium pullulans (de Bary) Arnaud isolated from the phylloplane of sycamore exposed to heavy atmospheric pollution oxidized S⁰ to S₂O₃²⁻, S₄O₆²⁻, and SO₄²⁻ in vitro. The intermediates S₂O₃²⁻ and S₄O₆²⁻ were also oxidized to SO₄²⁻. Cell-free extracts of A. pullulans also oxidized reduced forms of S, the oxidation increasing linearly with increasing protein concentration, showing that the process is enzymatic. The possible role of fungi in S oxidation in soils is discussed.     

Rate Equations and Kinetic Parameters of the Reactions Involved in Pyrite Oxidation by Thiobacillus ferrooxidans

Rate equations and kinetic parameters were obtained for various reactions involved in the bacterial oxidation of pyrite. The rate constants were 3.5 μM Fe²⁺ per min per FeS₂ percent pulp density for the spontaneous pyrite dissolution, 10 μM Fe²⁺ per min per mM Fe³⁺ for the indirect leaching with Fe³⁺, 90 μM O₂ per min per mg of wet cells per ml for the Thiobacillus ferrooxidans oxidation of washed pyrite, and 250 μM O₂ per min per mg of wet cells per ml for the T. ferrooxidans oxidation of unwashed pyrite. The K_m values for pyrite concentration were similar and were 1.9, 2.5, and 2.75% pulp density for indirect leaching, washed pyrite oxidation by T. ferrooxidans, and unwashed pyrite oxidation by T. ferrooxidans, respectively. The last reaction was competitively inhibited by increasing concentrations of cells, with a K_i value of 0.13 mg of wet cells per ml. T. ferrooxidans cells also increased the rate of Fe²⁺ production from Fe³⁺ plus pyrite.     

Pyrite oxidation by Thiobacillus ferrooxidans with special reference to the sulphur moiety of the mineral

Available cultures of Thiobacillus ferrooxidans were found to be contaminated with bacteria very similar to Thiobacillus acidophilus. The experiments described were performed with a homogeneous culture of Thiobacillus ferrooxidans.Pyrite (FeS₂) was oxidized by Thiobacillus ferrooxidans grown on iron (Fe²⁺), elemental sulphur (S^o) or FeS₂.Evidence for the direct utilization of the sulphur moiety of pyrite by Thiobacillus ferrooxidans was derived from the following observations: a. Known inhibitors of Fe²⁺ and S^o oxidation, NaN₃ and NEM, respectively, partially abolished FeS₂ oxidation. b. A b-type cytochrome was detectable in FeS₂-and S^o-grown cells but not in Fe²⁺-grown cells. c. FeS₂ and S^o reduced b-type cytochromes in whole cells grown on S^o. d. CO₂ fixation at pH 4.0 per mole of oxygen consumed was the highest with S^o, lowest with Fe²⁺ and medium with FeS₂ as substrate. e. Bacterial Fe²⁺ oxidation was found to be negligible at pH 5.0 whereas both FeS₂ and S^o oxidation was still appreciable above this pH. f. Separation of pyrite and bacteria by means of a dialysis bag caused a pronounced drop of the oxidation rate which was similar to the reduction of pyrite oxidation by NEM; indirect oxidation of the sulphur moiety by Fe³⁺ was not affected by separation of pyrite and bacteria.Bacterial oxidation and utilization of the sulphur moiety of pyrite were relatively more important with increasing pH.     

Microbial Manganese and Sulfate Reduction in Black Sea Shelf Sediments

The microbial ecology of anaerobic carbon oxidation processes was investigated in Black Sea shelf sediments from mid-shelf with well-oxygenated bottom water to the oxic-anoxic chemocline at the shelf-break. At all stations, organic carbon (C_org) oxidation rates were rapidly attenuated with depth in anoxically incubated sediment. Dissimilatory Mn reduction was the most important terminal electron-accepting process in the active surface layer to a depth of ~1 cm, while SO₄²⁻ reduction accounted for the entire C_org oxidation below. Manganese reduction was supported by moderately high Mn oxide concentrations. A contribution from microbial Fe reduction could not be discerned, and the process was not stimulated by addition of ferrihydrite. Manganese reduction resulted in carbonate precipitation, which complicated the quantification of C_org oxidation rates. The relative contribution of Mn reduction to C_org oxidation in the anaerobic incubations was 25 to 73% at the stations with oxic bottom water. In situ, where Mn reduction must compete with oxygen respiration, the contribution of the process will vary in response to fluctuations in bottom water oxygen concentrations. Total bacterial numbers as well as the detection frequency of bacteria with fluorescent in situ hybridization scaled to the mineralization rates. Most-probable-number enumerations yielded up to 10⁵ cells of acetate-oxidizing Mn-reducing bacteria (MnRB) cm⁻³, while counts of Fe reducers were <10² cm⁻³. At two stations, organisms affiliated with Arcobacter were the only types identified from 16S rRNA clone libraries from the highest positive MPN dilutions for MnRB. At the third station, a clone type affiliated with Pelobacter was also observed. Our results delineate a niche for dissimilatory Mn-reducing bacteria in sediments with Mn oxide concentrations greater than ~10 μmol cm⁻³ and indicate that bacteria that are specialized in Mn reduction, rather than known Mn and Fe reducers, are important in this niche.     

Impact of Bacterial NO3? Transport on Sediment Biogeochemistry

Experiments demonstrated that Beggiatoa could induce a H₂S-depleted suboxic zone of more than 10 mm in marine sediments and cause a divergence in sediment NO₃⁻ reduction from denitrification to dissimilatory NO₃⁻ reduction to ammonium. pH, O₂, and H₂S profiles indicated that the bacteria oxidized H₂S with NO₃⁻ and transported S⁰ to the sediment surface for aerobic oxidation.     

Anaerobic Sulfide Oxidation with Nitrate by a Freshwater Beggiatoa Enrichment Culture

A lithotrophic freshwater Beggiatoa strain was enriched in O₂-H₂S gradient tubes to investigate its ability to oxidize sulfide with NO₃⁻ as an alternative electron acceptor. The gradient tubes contained different NO₃⁻ concentrations, and the chemotactic response of the Beggiatoa mats was observed. The effects of the Beggiatoa sp. on vertical gradients of O₂, H₂S, pH, and NO₃⁻ were determined with microsensors. The more NO₃⁻ that was added to the agar, the deeper the Beggiatoa filaments glided into anoxic agar layers, suggesting that the Beggiatoa sp. used NO₃⁻ to oxidize sulfide at depths below the depth that O₂ penetrated. In the presence of NO₃⁻ Beggiatoa formed thick mats (>8 mm), compared to the thin mats (ca. 0.4 mm) that were formed when no NO₃⁻ was added. These thick mats spatially separated O₂ and sulfide but not NO₃⁻ and sulfide, and therefore NO₃⁻ must have served as the electron acceptor for sulfide oxidation. This interpretation is consistent with a fourfold-lower O₂ flux and a twofold-higher sulfide flux into the NO₃⁻-exposed mats compared to the fluxes for controls without NO₃⁻. Additionally, a pronounced pH maximum was observed within the Beggiatoa mat; such a pH maximum is known to occur when sulfide is oxidized to S⁰ with NO₃⁻ as the electron acceptor.     

Defluorination of Aqueous Perfluorooctanesulfonate by Activated Persulfate Oxidation

Activated persulfate oxidation technologies based on sulfate radicals were first evaluated for defluorination of aqueous perfluorooctanesulfonate (PFOS). The influences of catalytic method, time, pH and K₂S₂O₈ amounts on PFOS defluorination were investigated. The intermediate products during PFOS defluorination were detected by using LC/MS/MS. The results showed that the S₂O₈ ²⁻ had weak effect on the defluorination of PFOS, while the PFOS was oxidatively defluorinated by sulfate radicals in water. The defluorination efficiency of PFOS under various treatment was followed the order: HT (hydrothermal)/K₂S₂O₈ > UV (ultraviolet)/K₂S₂O₈ > Fe²⁺/K₂S₂O₈ > US (ultrasound)/K₂S₂O₈. Low pH was favorable for the PFOS defluorination with sulfate radicals. Increase in the amount of S₂O₈ ²⁻ had positive effect on PFOS defluorination. However, further increase in amounts of S₂O₈ ²⁻ caused insignificant improvement in PFOS defluorination due to elimination of sulfate radicals under high concentration of S₂O₈ ²⁻. CF₃(CF₂)_nCOOH (n = 0–6) were detected as intermediates during PFOS defluorination. Sulfate radicals oxidation and hydrolysis were the main mechanisms involved in defluorination process of PFOS.     

Transition from Anoxygenic to Oxygenic Photosynthesis in a Microcoleus chthonoplastes Cyanobacterial Mat

Benthic cyanobacterial mats with the filamentous Microcoleus chthonoplastes as the dominant phototroph grow in oxic hypersaline environments such as Solar Lake, Sinai. The cyanobacteria are in situ exposed to chemical variations between 200 μmol of sulfide liter⁻¹ at night and 1 atm pO₂ during the day. During experimental H₂S to O₂ transitions the microbial community was shown to shift from anoxygenic photosynthesis, with H₂S as the electron donor, to oxygenic photosynthesis. Microcoleus filaments could carry out both types of photosynthesis concurrently. Anoxygenic photosynthesis dominated at high sulfide levels, 500 μmol liter⁻¹, while the oxygenic reaction became dominant when the sulfide level was reduced below 100 to 300 μmol liter⁻¹ (25 to 75 μmol of H₂S liter⁻¹). An increasing inhibition of the oxygenic photosynthesis was observed upon transition to oxic conditions from increasing sulfide concentrations. Oxygen built up within the Microcoleus layer of the mat even under 5 mmol of sulfide liter⁻¹ (500 μmol of H₂S liter⁻¹) in the overlying water. The implications of such a localized O₂ production in a highly reducing environment are discussed in relation to the evolution of oxygenic photosynthesis during the Proterozoic era.     

Motility of Marichromatium gracile in Response to Light, Oxygen, and Sulfide

The motility of the purple sulfur bacterium Marichromatium gracile was investigated under different light regimes in a gradient capillary setup with opposing oxygen and sulfide gradients. The gradients were quantified with microsensors, while the behavior of swimming cells was studied by video microscopy in combination with a computerized cell tracking system. M. gracile exhibited photokinesis, photophobic responses, and phobic responses toward oxygen and sulfide. The observed migration patterns could be explained solely by the various phobic responses. In the dark, M. gracile formed an ~500-μm-thick band at the oxic-anoxic interface, with a sharp border toward the oxic zone always positioned at ~10 μM O₂. Flux calculations yielded a molar conversion ratio S_tot/O₂ of 2.03:1 (S_tot = [H₂S] + [HS⁻] + [S²⁻]) for the sulfide oxidation within the band, indicating that in darkness the bacteria oxidized sulfide incompletely to sulfur stored in intracellular sulfur globules. In the light, M. gracile spread into the anoxic zone while still avoiding regions with >10 μM O₂. The cells also preferred low sulfide concentrations if the oxygen was replaced by nitrogen. A light-dark transition experiment demonstrated a dynamic interaction between the chemical gradients and the cell's metabolism. In darkness and anoxia, M. gracile lost its motility after ca. 1 h. In contrast, at oxygen concentrations of >100 μM with no sulfide present the cells remained viable and motile for ca. 3 days both in light and darkness. Oxygen was respired also in the light, but respiration rates were lower than in the dark. Observed aggregation patterns are interpreted as effective protection strategies against high oxygen concentrations and might represent first stages of biofilm formation.     

Chemical weathering and solute export by meltwater in a maritime Antarctic glacier basin

Solute yields, laboratory dissolution data and both chemical and isotopic markers of rock weathering reactions are used to characterise the biogeochemistry of glacial meltwaters draining a maritime Antarctic glacier. We find that delayed flowpaths through ice-marginal talus and moraine sediments are critical for the acquisition of solute from rock minerals because delayed flowpaths through subglacial sediments are absent beneath this small, cold-based glacier. Here the mechanisms of weathering are similar to those reported in subglacial environments, and include sub-oxic conditions in the early summer and increasingly oxic conditions thereafter. Up to 85% of the NO₃ ^? and 65% of the SO₄ ^2? are most likely produced by bacterially mediated reactions in these ice marginal sediments. However, reactive pyrite phases are sparse in the host rocks, limiting the export of Fe, SO₄ ^2? and cations that may be removed by weathering once pyrite oxidation has taken place. This means that dissolution of Ca²⁺ and Na⁺ from carbonate and silicate minerals dominate, producing moderate cationic denudation yields from Tuva Glacier (163 Σ*meq⁺ m^?2 a^?1) compared to a global range of values (94–4,200 Σ*meq⁺ km^?2 a^?1). Overall, crustally derived cations represent 42% of the total cationic flux, the rest being accounted for by snowpack sources.     

Regulation of Endothelial Nitric-oxide Synthase (NOS) S-Glutathionylation by Neuronal NOS: EVIDENCE OF A FUNCTIONAL INTERACTION BETWEEN MYOCARDIAL CONSTITUTIVE NOS ISOFORMS*

Myocardial constitutive No production depends on the activity of both endothelial and neuronal NOS (eNOS and nNOS, respectively). Stimulation of myocardial β₃-adrenergic receptor (β₃-AR) produces a negative inotropic effect that is dependent on eNOS. We evaluated whether nNOS also plays a role in β₃-AR signaling and found that the β₃-AR-mediated reduction in cell shortening and [Ca²⁺]_i transient amplitude was abolished both in eNOS^−/− and nNOS^−/− left ventricular (LV) myocytes and in wild type LV myocytes after nNOS inhibition with S-methyl-l-thiocitrulline. LV superoxide (O₂^˙̄) production was increased in nNOS^−/− mice and reduced by l-N^ω-nitroarginine methyl ester (l-NAME), indicating uncoupling of eNOS activity. eNOS S-glutathionylation and Ser-1177 phosphorylation were significantly increased in nNOS^−/− myocytes, whereas myocardial tetrahydrobiopterin, eNOS Thr-495 phosphorylation, and arginase activity did not differ between genotypes. Although inhibitors of xanthine oxidoreductase (XOR) or NOX2 NADPH oxidase caused a similar reduction in myocardial O₂^˙̄, only XOR inhibition reduced eNOS S-glutathionylation and Ser-1177 phosphorylation and restored both eNOS coupled activity and the negative inotropic and [Ca²⁺]_i transient response to β₃-AR stimulation in nNOS^−/− mice. In summary, our data show that increased O₂^˙̄ production by XOR selectively uncouples eNOS activity and abolishes the negative inotropic effect of β₃-AR stimulation in nNOS^−/− myocytes. These findings provide unequivocal evidence of a functional interaction between the myocardial constitutive NOS isoforms and indicate that aspects of the myocardial phenotype of nNOS^−/− mice result from disruption of eNOS signaling.     

Isolation, Characterization, and In Situ Detection of a Novel Chemolithoautotrophic Sulfur-Oxidizing Bacterium in Wastewater Biofilms Growing under Microaerophilic Conditions

We successfully isolated a novel aerobic chemolithotrophic sulfur-oxidizing bacterium, designated strain SO07, from wastewater biofilms growing under microaerophilic conditions. For isolation, the use of elemental sulfur (S⁰), which is the most abundant sulfur pool in the wastewater biofilms, as the electron donor was an effective measure to establish an enrichment culture of strain SO07 and further isolation. 16S rRNA gene sequence analysis revealed that newly isolated strain SO07 was affiliated with members of the genus Halothiobacillus, but it was only distantly related to previously isolated species (89% identity). Strain SO07 oxidized elemental sulfur, thiosulfate, and sulfide to sulfate under oxic conditions. Strain SO07 could not grow on nitrate. Organic carbons, including acetate, propionate, and formate, could not serve as carbon and energy sources. Unlike other aerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 150 mM was negligible. In situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells hybridized with a probe specific for strain SO07 were mainly present in the oxic biofilm strata (ca. 0 to 100 μm) and that they often coexisted with sulfate-reducing bacteria in this zone. These results demonstrated that strain SO07 was one of the important sulfur-oxidizing populations involved in the sulfur cycle occurring in the wastewater biofilm and was primarily responsible for the oxidation of H₂S and S⁰ to SO₄²⁻ under oxic conditions.     

Succession of Internal Sulfur Cycles and Sulfur-Oxidizing Bacterial Communities in Microaerophilic Wastewater Biofilms

The succession of sulfur-oxidizing bacterial (SOB) community structure and the complex internal sulfur cycle occurring in wastewater biofilms growing under microaerophilic conditions was analyzed by using a polyphasic approach that employed 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization, microelectrode measurements, and standard batch and reactor experiments. A complete sulfur cycle was established via S⁰ accumulation within 80 days in the biofilms in replicate. This development was generally split into two phases, (i) a sulfur-accumulating phase and (ii) a sulfate-producing phase. In the first phase (until about 40 days), since the sulfide production rate (sulfate-reducing activity) exceeded the maximum sulfide-oxidizing capacity of SOB in the biofilms, H₂S was only partially oxidized to S⁰ by mainly Thiomicrospira denitirificans with NO₃⁻ as an electron acceptor, leading to significant accumulation of S⁰ in the biofilms. In the second phase, the SOB populations developed further and diversified with time. In particular, S⁰ accumulation promoted the growth of a novel strain, strain SO07, which predominantly carried out the oxidation of S⁰ to SO₄²⁻ under oxic conditions, and Thiothrix sp. strain CT3. In situ hybridization analysis revealed that the dense populations of Thiothrix (ca. 10⁹ cells cm⁻³) and strain SO07 (ca. 10⁸ cells cm⁻³) were found at the sulfur-rich surface (100 μm), while the population of Thiomicrospira denitirificans was distributed throughout the biofilms with a density of ca. 10⁷ to 10⁸ cells cm⁻³. Microelectrode measurements revealed that active sulfide-oxidizing zones overlapped the spatial distributions of different phylogenetic SOB groups in the biofilms. As a consequence, the sulfide-oxidizing capacities of the biofilms became high enough to completely oxidize all H₂S produced by SRB to SO₄²⁻ in the second phase, indicating establishment of the complete sulfur cycle in the biofilms.     

Polysulfides as Intermediates in the Oxidation of Sulfide to Sulfate by Beggiatoa spp.

Zero-valent sulfur is a key intermediate in the microbial oxidation of sulfide to sulfate. Many sulfide-oxidizing bacteria produce and store large amounts of sulfur intra- or extracellularly. It is still not understood how the stored sulfur is metabolized, as the most stable form of S⁰ under standard biological conditions, orthorhombic α-sulfur, is most likely inaccessible to bacterial enzymes. Here we analyzed the speciation of sulfur in single cells of living sulfide-oxidizing bacteria via Raman spectroscopy. Our results showed that under various ecological and physiological conditions, all three investigated Beggiatoa strains stored sulfur as a combination of cyclooctasulfur (S₈) and inorganic polysulfides (S_n²⁻). Linear sulfur chains were detected during both the oxidation and reduction of stored sulfur, suggesting that S_n²⁻ species represent a universal pool of bioavailable sulfur. Formation of polysulfides due to the cleavage of sulfur rings could occur biologically by thiol-containing enzymes or chemically by the strong nucleophile HS⁻ as Beggiatoa migrates vertically between oxic and sulfidic zones in the environment. Most Beggiatoa spp. thus far studied can oxidize sulfur further to sulfate. Our results suggest that the ratio of produced sulfur and sulfate varies depending on the sulfide flux. Almost all of the sulfide was oxidized directly to sulfate under low-sulfide-flux conditions, whereas only 50% was oxidized to sulfate under high-sulfide-flux conditions leading to S⁰ deposition. With Raman spectroscopy we could show that sulfate accumulated in Beggiatoa filaments, reaching intracellular concentrations of 0.72 to 1.73 M.     

Anaerobic Respiration of Elemental Sulfur and Thiosulfate by Shewanella oneidensis MR-1 Requires psrA,a Homolog of the phsA Gene of Salmonella enterica Serovar Typhimurium LT2

Shewanella oneidensis MR-1, a facultatively anaerobic gammaproteobacterium, respires a variety of anaerobic terminal electron acceptors, including the inorganic sulfur compounds sulfite (SO₃²⁻), thiosulfate (S₂O₃²⁻), tetrathionate (S₄O₆²⁻), and elemental sulfur (S⁰). The molecular mechanism of anaerobic respiration of inorganic sulfur compounds by S. oneidensis, however, is poorly understood. In the present study, we identified a three-gene cluster in the S. oneidensis genome whose translated products displayed 59 to 73% amino acid similarity to the products of phsABC, a gene cluster required for S⁰ and S₂O₃²⁻ respiration by Salmonella enterica serovar Typhimurium LT2. Homologs of phsA (annotated as psrA) were identified in the genomes of Shewanella strains that reduce S⁰ and S₂O₃²⁻ yet were missing from the genomes of Shewanella strains unable to reduce these electron acceptors. A new suicide vector was constructed and used to generate a markerless, in-frame deletion of psrA, the gene encoding the putative thiosulfate reductase. The psrA deletion mutant (PSRA1) retained expression of downstream genes psrB and psrC but was unable to respire S⁰ or S₂O₃²⁻ as the terminal electron acceptor. Based on these results, we postulate that PsrA functions as the main subunit of the S. oneidensis S₂O₃²⁻ terminal reductase whose end products (sulfide [HS⁻] or SO₃²⁻) participate in an intraspecies sulfur cycle that drives S⁰ respiration.Microbial reduction of inorganic sulfur compounds is central to the biogeochemical cycling of sulfur and other elements such as carbon and metals (29). The ability to reduce elemental sulfur (S⁰) is found in members of both prokaryotic domains (20), including mesophilic deltaproteobacteria (Desulfovibrio vulgaris, Pelobacter carbinolicus, Geobacter sulfurreducens) (6, 9, 36, 51), thermophilic deltaproteobacteria (Desulfurella acetivorans) (39), gammaproteobacteria (Shewanella putrefaciens) (41), epsilonproteobacteria (Wolinella succinogenes) (49), cyanobacteria (“Oscillatoria limnetica”) (45), and hyperthermophilic archaea (1, 53). Partially reduced inorganic sulfur compounds such as tetrathionate (S₄O₆²⁻), thiosulfate (S₂O₃²⁻), and sulfite (SO₃²⁻) are also important electron acceptors in the biogeochemical cycling of sulfur (29, 51). S₄O₆²⁻-reducing bacteria, for example, may produce S₂O₃²⁻ as a metabolic end product of S₄O₆²⁻ reduction, while S₂O₃²⁻ disproportionation is a key reaction catalyzed by sulfate-reducing bacteria, resulting in the formation of sulfate (SO₄²⁻) and sulfide (S²⁻) (26).Shewanella oneidensis MR-1, a facultatively anaerobic gammaproteobacterium, respires a variety of compounds as an anaerobic electron acceptor, including the inorganic sulfur compounds S⁰, SO₃²⁻, S₂O₃²⁻, and S₄O₆²⁻; transition metals [e.g., Fe(III) and Mn(IV)]; and radionuclides [e.g., U(VI) and Tc(VII)] (8, 21, 41, 44, 50, 55, 56). The majority of studies of anaerobic respiration by S. oneidensis have focused on the mechanism of electron transport to transition metals and radionuclides (11, 14, 34, 46, 58, 59), while the mechanism of electron transport to inorganic sulfur compounds has not been thoroughly examined.Microbial S⁰ respiration is postulated to occur via two pathways, both of which are based on an intraspecies sulfur cycle. In the first pathway (catalyzed by members of the genus Salmonella [20]), S₂O₃²⁻ is reduced, yielding HS⁻ and SO₃²⁻ (24). SO₃²⁻ diffuses from the cell and reacts chemically with extracellular S⁰ to form S₂O₃²⁻, which reenters the periplasm and is rereduced, thereby sustaining an intraspecies sulfur cycle. In the second pathway (catalyzed by W. succinogenes [24]), water-soluble polysulfides (S_n²⁻; n > 2), formed by chemical interactions of S⁰ at pHs >7 (52), are reduced stepwise in the periplasm to S_{n − 1}²⁻ and HS⁻. Similarly to what occurs with the first pathway, microbially produced HS⁻ diffuses from the cell and reacts chemically with S⁰ to produce additional S_n²⁻, which reenters the periplasm and is rereduced to sustain an analogous intraspecies sulfur cycle (24).Genetic analyses of S₂O₃²⁻ reduction-deficient mutants of Salmonella enterica serovar Typhimurium have demonstrated that phsA (denoting production of hydrogen sulfide) is required for HS⁻ production during S₂O₃²⁻ respiration (10, 17, 22). In addition, phsA-deficient mutants are unable to reduce S⁰ as an electron acceptor (24). The phsA homolog of W. succinogenes (annotated as psrA, for polysulfide reduction) is required for S⁰ respiration (32, 37). W. succinogenes psrA is the first gene of a three-gene cluster (including psrA, psrB, and psrC) whose products encode a polysulfide reductase, a quinol oxidase, and a membrane anchor, respectively (15). In addition, the structure of the polysulfide reductase complex (PsrABC) from Thermus thermophilus has recently been solved, and results indicate that PsrC acts as a quinol oxidase that transfers electrons stepwise via PsrB and PsrA to S_n²⁻ during anaerobic S⁰ respiration (27). The main objectives of the present study were to (i) identify the S. Typhimurium phsA homolog in the S. oneidensis genome, (ii) employ a newly constructed suicide cloning vector for in-frame gene deletion mutagenesis in S. oneidensis to delete the S. Typhimurium phsA homolog of S. oneidensis, and (iii) test the S. oneidensis psrA deletion mutant for respiratory activity on a combination of two electron donors and 11 electron acceptors, including the inorganic sulfur compounds S₄O₆²⁻, S₂O₃²⁻, and S⁰.     

Influence of the sulfur species reactivity on biofilm conformation during pyrite colonization by Acidithiobacillus thiooxidans

Massive pyrite (FeS₂) electrodes were potentiostatically modified by means of variable oxidation pulse to induce formation of diverse surface sulfur species (S _n ^2?, S⁰). The evolution of reactivity of the resulting surfaces considers transition from passive (e.g., Fe_1?x S₂) to active sulfur species (e.g., Fe_1?x S_2?y , S⁰). Selected modified pyrite surfaces were incubated with cells of sulfur-oxidizing Acidithiobacillus thiooxidans for 24 h in a specific culture medium (pH 2). Abiotic control experiments were also performed to compare chemical and biological oxidation. After incubation, the attached cells density and their exopolysaccharides were analyzed by confocal laser scanning microscopy (CLMS) and atomic force microscopy (AFM) on bio-oxidized surfaces; additionally, S _n ^2?/S⁰ speciation was carried out on bio-oxidized and abiotic pyrite surfaces using Raman spectroscopy. Our results indicate an important correlation between the evolution of S _n ^2?/S⁰ surface species ratio and biofilm formation. Hence, pyrite surfaces with mainly passive-sulfur species were less colonized by A. thiooxidans as compared to surfaces with active sulfur species. These results provide knowledge that may contribute to establishing interfacial conditions that enhance or delay metal sulfide (MS) dissolution, as a function of the biofilm formed by sulfur-oxidizing bacteria.     

Oxygen Activation during Oxidation of Methoxyhydroquinones by Laccase from Pleurotus eryngii

Oxygen activation during oxidation of the lignin-derived hydroquinones 2-methoxy-1,4-benzohydroquinone (MBQH₂) and 2,6-dimethoxy-1,4-benzohydroquinone (DBQH₂) by laccase from Pleurotus eryngii was examined. Laccase oxidized DBQH₂ more efficiently than it oxidized MBQH₂; both the affinity and maximal velocity of oxidation were higher for DBQH₂ than for MBQH₂. Autoxidation of the semiquinones produced by laccase led to the activation of oxygen, producing superoxide anion radicals (Q^·− + O₂ ↔ Q + O₂^·−). As this reaction is reversible, its existence was first noted in studies of the effect of systems consuming and producing O₂^·− on quinone formation rates. Then, the production of H₂O₂ in laccase reactions, as a consequence of O₂^·− dismutation, confirmed that semiquinones autoxidized. The highest H₂O₂ levels were obtained with DBQH₂, indicating that DBQ^·− autoxidized to a greater extent than did MBQ^·−. Besides undergoing autoxidation, semiquinones were found to be transformed into quinones via dismutation and laccase oxidation. Two ways of favoring semiquinone autoxidation over dismutation and laccase oxidation were increasing the rate of O₂^·− consumption with superoxide dismutase (SOD) and recycling of quinones with diaphorase (a reductase catalyzing the divalent reduction of quinones). These two strategies made the laccase reaction conditions more natural, since O₂^·−, besides undergoing dismutation, reacts with Mn²⁺, Fe³⁺, and aromatic radicals. In addition, quinones are continuously reduced by the mycelium of white-rot fungi. The presence of SOD in laccase reactions increased the extent of autoxidation of 100 μM concentrations of MBQ^·− and DBQ^·− from 4.5 to 30.6% and from 19.6 to 40.0%, respectively. With diaphorase, the extent of MBQ^·− autoxidation rose to 13.8% and that of DBQ^·− increased to 39.9%.     

Redox and Chemical Activities of the Hemes in the Sulfur Oxidation Pathway Enzyme SoxAX

SoxAX enzymes couple disulfide bond formation to the reduction of cytochrome c in the first step of the phylogenetically widespread Sox microbial sulfur oxidation pathway. Rhodovulum sulfidophilum SoxAX contains three hemes. An electrochemical cell compatible with magnetic circular dichroism at near infrared wavelengths has been developed to resolve redox and chemical properties of the SoxAX hemes. In combination with potentiometric titrations monitored by electronic absorbance and EPR, this method defines midpoint potentials (E_m) at pH 7.0 of approximately +210, −340, and −400 mV for the His/Met, His/Cys⁻, and active site His/CysS⁻-ligated heme, respectively. Exposing SoxAX to S₂O₄²⁻, a substrate analog with E_m ∼−450 mV, but not Eu(II) complexed with diethylene triamine pentaacetic acid (E_m ∼−1140 mV), allows cyanide to displace the cysteine persulfide (CysS⁻) ligand to the active site heme. This provides the first evidence for the dissociation of CysS⁻ that has been proposed as a key event in SoxAX catalysis.     

Inhibition of Chemoautotrophic Nitrification by Sodium Chlorate and Sodium Chlorite: a Reexamination

The oxidation of NH₄⁺ by Nitrosomonas europaea was insensitive to 10 mM NaClO₃ (sodium chlorate) but was strongly inhibited by NaClO₂ (sodium chlorite; K_i, 2 μM). The oxidation of NO₂⁻ by Nitrobacter winogradskyi was inhibited by both ClO₃⁻ and ClO₂⁻ (K_i for ClO₂⁻, 100 μM). N. winogradskyi reduced ClO₃⁻ to ClO₂⁻ under both aerobic and anaerobic conditions, and as much as 0.25 mM ClO₂⁻ was detected in the culture filtrate. In mixed N. europaea-N. winogradskyi cell suspensions, the oxidation of both NH₄⁺ and NO₂⁻ was inhibited in the presence of 10 mM ClO₃⁻ after a 2-h lag period, despite the fact that, under these conditions, ClO₂⁻ was not detected in the filtrate. The data are consistent with the hypothesis that, in mixed culture, NH₄⁺ oxidation is inhibited by ClO₂⁻ produced by reduction of ClO₃⁻ by the NO₂⁻ oxidizer. The use of ClO₃⁻ inhibition of NO₂⁻ oxidation in assays of nitrification by mixed populations necessitates cautious interpretation unless it can be shown that the oxidation of NH₄⁺ is not affected.