Mechanism of dissimilatory sulfite reduction by Desulfovibrio desulfuricans: purification of a membrane-bound sulfite reductase and coupling iwth cytochrome c 3 and hydrogenase

The localization of the dissimilatory sulfite reductase in Desulfovibrio desulfuricans strain Essex 6 was investigated. After treatment of the cells with lysozyme, 90% of the sulfite reductase activity was found in the membrane fraction, compared to 30% after cell rupture with the French press. Sulfite reductase was purified from the membrane (mSiR) and the soluble (sSiR) fractiion. On SDS-PAGE, both mSiR and sSiR exhibited three bands at 50, 45 and 11 kDa, respectively. From their UV/VIS properties (distinct absorption maxima at 391, 410, 583, 630 nm, enzymes as isolated) and the characteristic red fluorescence in alkaline solution, mSiR and sSiR were identified as desulfoviridin. Sulfite reductase (HSO₃ ^-H₂S) activity was reconstituted by coupling of mSiR to hydrogenase and cytochrome c ₃ from D. desulfuricans. The specific activity of mSiR was 103 nmol H₂ min^-1 mg^-1, and sulfide was the major product (72% of theoretical yield). No coupling was found with sSiR under these conditions. Furthermore, carbon monoxide was used to diferentiate between the membrane-bound and the soluble sulfite reductase. In a colorimetric assay, with photochemically reduced methyl viologen as redox mediator, CO stimulated the activity of sSiR significantly. CO had no effect in the case of mSiR. These studies documented that, as isolated, both forms of sulfite reductase behaved differently in vitro. Clearly, in D. desulfuricans, the six electron conversion HSO₃ ^-H₂S was achieved by a membranebound desulfoviridin without the assistance of artificial redox mediators, such as methyl viologen.Abbreviations SiR sulfite reductase - mSiR sulfite reductase purified from membranes - sSiR sulfite reductase purified from the soluble fraction Enzymes Sulfite reductase, EC 1.8.99.1 Cytochrome c ₃ hydrogenase, EC 1.12.2.1     

Formation of thiosulfate and trithionate during sulfite reduction by washed cells of Desulfovibrio desulfuricans

Deenergized cells of Desulfovibrio desulfuricans strain Essex 6 formed trithionate and thiosulfate during reduction of sulfite with H₂ or formate. The required conditions were pretreatment with the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP), low concentration of the electron donor H₂ or formate (25–200 M) and the presence of sulfite in excess (>250 M). The cells formed up to 20 M thiosulfate, and variable amounts of trithionate (0–9 M) and sulfide (0–62 M). Tetrathionate was not produced. Sulfate could not replace sulfite in these experiments, as deenergized cells cannot activate sulfate. However, up to 5 M thiosulfate was produced by cells growing with H₂ and excess sulfate in a chemostat. Micromolar concentrations of trithionate were incompletely reduced to thiosulfate and sulfide by washed cells in the presence of CCCP. Millimolar trithionate concentrations blocked the formation of sulfide, even in the absence of CCCP, and caused thiosulfate accumulation; sulfide formation from sulfate, sulfite or thiosulfate was stopped, too. Trithionate reduction with H₂ in the presence of thiocyanate was coupled to respiration-driven proton translocation (extrapolated H⁺/H₂ ratios of 1.5±0.6). Up to 150 M trithionate was formed by washed cells during oxidation of sulfite plus thiosulfate with ferricyanide as electron acceptor (reversed trithionate reductase activity). Cell breakage resulted in drastic decrease of sulfide formation. Cell-free extract reduced sulfite incompletely to trithionate, thiosulfate, and sulfide. Thiosulfate was reduced stoichiometrically to sulfite and sulfide (thiosulfate reductase activity). The formation of sulfide from sulfite, thiosulfate or trithionate by cell-free extract was blocked by methyl viologen, leading to increased production of thiosulfate plus trithionate from sulfite, or increased thiosulfate formation from trithionate. Our study demonstrates for the first time the formation of intermediates during sulfite reduction with whole cells of a sulfate-reducing bacterium oxidizing physiological electron donors. All results are in accordance with the trithionate pathway of sulfite reduction.With gratitude dedicated to Prof. Dr. Norbert Pfennig on occasion of his 65th birthday     

A study on electron transport-driven proton translocation in Desulfovibrio desulfuricans

Proton translocation by washed cells of the sulfate-reducing bacterium Desulfovibrio desulfuricans strain Essex 6 was studied by means of pH and sulfide electrodes. Reversible extrusion of protons could be induced either by addition of electron acceptors to cells incubated under hydrogen, or by addition of hydrogen to cells incubated in the presence of an appropriate electron acceptor. Proton translocation was increased in the presence of ionophores that dissipate the membrane potential (thiocyanate, methyl triphenylphosphonium cation, but not valinomycin) and was sensitive to the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). Upon micromolar additions of H₂, usually sulfide was formed in stoichiometric amounts, and extrapolated H⁺/H₂ ratios were 1.8±0.5 with sulfate, 2.3±0.3 with sulfite and 0.5±0.1 with thiosulfate. In several experiments hydrogen pulses caused increased proton extrusion not associated with sulfide production. This was a hint that sulfite might be reduced via intermediates. In the absence of H₂S formation, extrapolated H⁺/H₂ ratios were 3.1±0.8 with sulfate, 3.4±1.1 with sulfite, 4.4±0.8 with thiosulfate and 6.3±1.2 with oxygen. Micromolar pulses of electron acceptors to cells incubated under H₂ caused less proton translocation than H₂ pulses in presence of excess of electron acceptor; extrapolated H⁺/H₂ ratios were 1.3±0.4 with sulfite, 3.3±0.9 with nitrite and 4.2±0.5 with oxygen. No proton translocation was observed after micromolar pulses of sulfate, thiosulfate or nitrate to cells incubated under hydrogen in the presence of thiocyanate. Inhibition experiments with CO and CuCl₂ revealed that the hydrogenase activity was localized in the intracellular space, and that no periplasmic hydrogenase was present. The results indicate that D. desulfuricans can generate a proton gradient by pumping protons across the cytoplasmic membrane.Abbreviations APS adenosine 5-phosphosulfate - CCCP carbonyl cyanide m-chlorophenylhydrazone - MTTP⁺ methyl triphenylphosphonium cation     

Growth yields of Desulfotomaculum orientis with hydrogen in chemostat culture

Desulfotomaculum orientis (strain Singapore 1) was grown autotrophically with H₂+CO₂ and sulfate, thiosulfate or sulfite as electron acceptor in sulfide- and pH-controlled continuous culture. Under sulfate-limiting conditions real growth yields of up to 9.7 g cell dry mass per mol sulfate were obtained. Electron acceptor limitation resulted in the excretion of up to 14.5 mmol acetate per liter, formed by reduction of CO₂ with H₂. Acetate production was not coupled to an increase of growth yields: under hydrogen-limiting conditions only 1.6 mmol acetate per liter was produced, and even higher growth yields of up to 12,4 g cell dry mass per mol sulfate were obtained. With thiosulfate or sulfite as electron acceptor growth yields increased up to 17.9 g cell dry mass per mol electron acceptor. Growth yields were not simply correlated with the growth rate, and did not allow the determination of maintenance coefficients and the extrapolation to maximal yields at infinite growth rate (Y ^max). The maximal growth rates (_max) with sulfate and thiosulfate were 0.090 and 0.109 h^-1, respectively, if cells were grown continuously in sulfidostat culture under nonlimiting conditions.The net energy yield of sulfate reduction and the energy requirement for the activation of sulfate by Desulfotomaculum orientis are discussed.     

Effects of cadmium on gene expression in cadmium-tolerant and cadmium-sensitiveDatura innoxia cells

The effect of Cd on gene expression in suspension cultures of twoDatura innoxia cell lines with differing Cd tolerance was studied.In vivo labeling experiments using [³H] leucine showed that Cd induced the synthesis of a similar range of proteins in both cell lines at a concentration which will kill the sensitive but not the tolerant cells. Corresponding changes in levels of translatable mRNA were also observed. The induction of the synthesis of proteins by Cd was transient since Cd-tolerant cells growing continuously in 250 M CdCl₂ contained a similar set ofin vitro translation products to cells growing in the absence of Cd. Although Cd had a similar effect on gene expression in both cell lines, Cd-tolerant cells possess two abundant mRNAs which are constitutively produced. These mRNAs encode proteins of low molecular weight (about 11 kDa) and are either absent or present at a low level in Cd-sensitive cells. The functions of these proteins are not known but they may be involved in the tolerance mechanism. Two-dimensional gel electrophoresis ofin vitro translation products showed that many of the Cd-induced proteins are also induced by heat shock. A 42°C heat shock resulted in agreater range and more intense induction of translatable mRNAs than 4 h exposure to 250 M CdCl₂. However a subset of mRNAs were induced specifically by Cd while other mRNAs were heat shock-specific. There was no difference in the ability of the two cell lines to tolerate heat shock. This was also reflected by the same pattern of major proteins induced by heat shock in the two cell lines.     

Osmotic stress adaptation, compatible solutes accumulation and biocontrol efficacy of two potential biocontrol agents on Fusarium head blight in wheat

Fusarium head blight (FHB) caused by Gibberella zeae (anamorph = Fusarium graminearum) is a devastating disease that causes extensive yield and quality losses to wheat in humid and semi-humid regions of the world. Biological control has been demonstrated to be effective under laboratory conditions but a few biocontrol products have been effective under field conditions. The improvement in the physiological quality of biocontrol agents may improve survival under field conditions, and therefore, enhance biocontrol activity. Bacillus subtilis RC 218 and Brevibacillus sp. RC 263 were isolated from wheat anthers and showed significant effect on control of FHB under greenhouse assays. This study showed the effect of water availability measured as water activity (a_W) using a growth medium modified with NaCl, glycerol and glucose on: (i) osmotic stress tolerance, (ii) viability in modified liquid medium, (iii) quantitative intracellular accumulation of betaine and ectoine and (iv) the biocontrol efficacy of the physiologically improved agents. Viability of B. subtilis RC 218 in NaCl modified media was similar to the control. Brevibacillus sp. RC 263 showed a limited adaptation to growth in osmotic stress. Betaine was detected in high levels in modified cells but ectoine accumulation was similar to the control cells. Biocontrol activity was studied in greenhouse assays on wheat inoculated at anthesis period with F. graminearum RC 276. Treatments with modified bacteria reduced disease severity from 60% for the control to below 20%. The physiological improvement of biocontrol agents could be an effective strategy to enhance stress tolerance and biocontrol activity under fluctuating environmental conditions.     

Epidermal Growth Factor Induces Proliferation and DNA Synthesis in Ciliate Cells

We studied the effect of murine epidermal growth factor on cell proliferation and DNA synthesis in macronuclei of ciliate Tetrahymena pyriformis Gl. Mitogenic effect of epidermal growth factor on proliferation-induced tetrahymena cells has been revealed. This effect is due to the induced progression of cells at G ₁ and, consequently, their earlier entering DNA synthesis phase of the first cell cycle. Epidermal growth factor had no mitogenic effect on the resting cells in a stationary culture (G ₀ phase) whose development is independent of the growth factors in the medium.     

Elemental sulfur as an intermediate of sulfide oxidation with oxygen byDesulfobulbus propionicus

The sulfate-reducing bacteriumDesulfobulbus propionicus oxidized sulfide, elemental sulfur, and sulfite to sulfate with oxygen as electron acceptor. Thiosulfate was reduced and disproportionated exclusively under anoxic conditions. When small pulses of oxygen were added to washed cells in sulfide-containing assays, up to 3 sulfide molecules per O₂ disappeared transiently. After complete oxygen consumption, part of the sulfide reappeared. The intermediate formed was identified as elemental sulfur by chemical analysis and turbidity measurements. When excess sulfide was present, sulfur dissolved as polysulfide. This process was faster in the presence of cells than in their absence. The formation of sulfide after complete oxygen consumption was due to a disproportionation of elemental sulfur (or polysulfide) to sulfide and sulfate. The uncoupler tetrachlorosalicylanilide (TCS) and the electron transport inhibitor myxothiazol inhibited sulfide oxidation to sulfate and caused accumulation of sulfur. In the presence of the electron transport inhibitor 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), sulfite and thiosulfate were formed. During sulfur oxidation at low oxygen concentrations, intermediary formation of sulfide was observed, indicating disproportionation of sulfur also under these conditions. It is concluded that sulfide oxidation inD. propionicus proceeds via oxidation to elemental sulfur, followed by sulfur disproportionation to sulfide and sulfate. Dedicated to Prof. Dr. Dr. h.c. Norbert Pfennig on the occasion of his 70th birthday     

Comparative aspects of utilization of sulfonate and other sulfur sources by Escherichia coli K12

Selected biochemical features of sulfonate assimilation in Escherichia coli K-12 were studied in detail. Competition between sulfonate-sulfur and sulfur sources with different oxidation states, such as cysteine, sulfite and sulfate, was examined. The ability of the enzyme sulfite reductase to attack the C-S linkage of sulfonates was directly examined. Intact cells formed sulfite from sulfonate-sulfur. In cysteine-grown cells, when cysteine was present with either cysteate or sulfate, assimilation of both of the more oxidized sulfur sources was substantially inhibited. In contrast, none of three sulfonates had a competitive effect on sulfate assimilation. In studies of competition between different sulfonates, the presence of taurine resulted in a decrease in cysteate uptake by one-half, while in the presence of isethionate, cysteate uptake was almost completely inhibited. In sulfite-grown cells, sulfonates had no competitive effect on sulfite utilization. An E. coli mutant lacking sulfite reductase and unable to utilize isethionate as the sole source of sulfur formed significant amounts of sulfite from isethionate. In cell extracts, sulfite reductase itself did not utilize sulfonate-sulfur as an electron acceptor. These findings indicate that sulfonate utilization may share some intermediates (e.g. sulfite) and regulatory features (repression by cysteine) of the assimilatory sulfate reductive pathway, but sulfonates do not exert regulatory effects on sulfate utilization. Other results suggest that unrecognized aspects of sulfonate metabolism, such as specific transport mechanisms for sulfonates and different regulatory features, may exist.     

Geosmin production in the cyanobacterium Oscillatoria brevis

The cyanobacterium Oscillatoria brevis (Kütz.) Gom., strain NIVA CYA 7, was used to investigate how geosmin production is related to the synthesis of chlorophyll a, phycobiliproteins and β-carotene under nitrogen (NH ₄ ⁺ ) and light limiting conditions. Chemostat samples were used to inoculate batch cultures that were treated with inhibitors of isoprenoid synthesis, norflurazon and dimethazone, and gabaculine that inhibits tetrapyrrole synthesis. Dimethazone decreased and norflurazon increased geosmin production under light limited conditions, as was expected due to their sites of action in the isoprenoid pathway. This effect was not so pronounced in nitrogen limited cultures due to the additional effect of increasing nitrogen deficiency during the experimental period. Norflurazon was the only inhibitor that uncoupled geosmin production completely from β-carotene formation which indicates a strikt coupling between geosmin and β-carotene biosynthesis. From the observed increase of geosmin production relative to pigment synthesis after norflurazon treatment it was suggested that isoprenoid precursors are directed to geosmin synthesis when the demand for pigment precursors is very low. Within the framework of this study the data strongly support the hypothesis of geosmin formation via the isoprenoid pathway in Oscillatoria brevis as was found for actinomycetes. This research was performed at the Department of Microbiology, University of Amsterdam, with financial support provided by the royal Norwegian Ministry of Foreign Affairs and the Royal Norwegian Council for Scientific and Industrial Research     

Oxidation of H2, organic compounds and inorganic sulfur compounds coupled to reduction of O2 or nitrate by sulfate-reducing bacteria

All of fourteen sulfate-reducing bacteria tested were able to carry out aerobic respiration with at least one of the following electron donors: H₂, lactate, pyruvate, formate, acetate, butyrate, ethanol, sulfide, thiosulfate, sulfite. Generally, we did not obtain growth with O₂ as electron acceptor. The bacteria were microaerophilic, since the respiration rates increased with decreasing O₂ concentrations or ceased after repeated O₂ additions. The amounts of O₂ consumed indicated that the organic substrates were oxidized incompletely to acetate; only Desulfobacter postgatei oxidized acetate with O₂ completely to CO₂. Many of the strains oxidized sulfite (completely to sulfate) or sulfide (incompletely, except Desulfobulbus propionicus); thiosulfate was oxidized only by strains of Desulfovibrio desulfuricans; trithionate and tetrathionate were not oxidized by any of the strains. With Desulfovibrio desulfuricans CSN and Desulfobulbus propionicus the oxidation of inorganic sulfur compounds was characterized in detail. D. desulfuricans formed sulfate during oxidation of sulfite, thiosulfate or elemental sulfur prepared from polysulfide. D. propionicus oxidized sulfite and sulfide to sulfate, and elemental sulfur mainly to thiosulfate. A novel pathway that couples the sulfur and nitrogen cycles was detected: D. desulfuricans and (only with nitrite) D. propionicus were able to completely oxidize sulfide coupled to the reduction of nitrate or nitrite to ammonia. Cell-free extracts of both strains did not oxidize sulfide or thiosulfate, but formed ATP during oxidation of sulfite (37 nmol per 100 nmol sulfite). This, and the effects of AMP, pyrophosphate and molybdate on sulfite oxidation, suggested that sulfate is formed via the (reversed) sulfate activation pathway (involving APS reductase and ATP sulfurylase). Thiosulfate oxidation with O₂ probably required a reductive first step, since it was obtained only with energized intact cells.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - APS adenosine phosphosulfate or adenylyl sulfate     

Effect of calcium ions on the Hsp104 synthesis and heat tolerance of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Effect of calcium ions on heat tolerance of Saccharomyces cerevisiae and on the induction of Hsp104 synthesis by this microorganism was studied. Short-term (30 min) treatment with CaCl₂ at 30°C enhanced the heat tolerance to the lethal heat shock (50°C); the synthesis of Hsp104 was induced as well. The effect of Ca²⁺ on the heat tolerance and Hsp104 synthesis was shown to be ion-specific and was inhibited by LaCl₃, which is known to block calcium ion channels on the cytoplasmic membrane. The effect of Ca²⁺ depended on the potential of the inner mitochondrial membrane. When the cells were treated with sodium azide, which reduced the electrochemical potential, the effect of calcium both on heat tolerance and Hsp104 synthesis was suppressed. Depending on the concentration of exogenous Ca²⁺ and the ambient conditions, calcium ions may either induce or inhibit the expression of the stress genes and cell viability.     

A model system to study the effect of SO2 on plant cells. III. Effects of sulfite on the ultrastructure of fern protonemal cells

The mechanism of the toxic effects on plant cells of sulfite, a product of the air pollutant sulfur dioxide, is not well understood. Therefore, changes in the fine structure and organization of microtubules and microfibrils induced by sulfite were studied by electron and light microscopy in the protonemata of the fernAdiantum capillusveneris L. Under red-light conditions, growing protonemata fumigated with 0.05 or 0.1 μ1/1 SO₂ for 1 to 4 days showed abnormalities, such as apical swelling, and they sometimes burst at the apex. The incidence of abnormalities seemed to be correlated with the concentration of the sulfite dissolved in the culture medium. At an appropriate concentration (3.3–6.6. mM) of sulfite (applied as K₂SO₃), cell swelling at the apical region of protonema was also induced. When the concentration of sulfite was as high as 6.6 mM, more than 60% of protonemata burst at the tip. During the apical swelling, no distinct changes were observed in the fine structure of organelles, such as the chloroplasts, mitochondria, microbodies, Golgi bodies and nucleus. However, the arrangement of cortical microtubules and that of the innermost layer of microfibrils around the subapical region of protonemata were changed from transverse to the cell axis (i.e., circular) to random and the cell wall was thickened. These observations suggest that sulfite may influence the mechanisms that maintain the transverse orientation of microtubules in the subapical region of a protonema and that the resultant random arrangement of microtubules induces the random arrangement of microfibrils and leads to apical swelling.     

A sulfate-reducing bacterium from the oxic layer of a microbial mat from Solar Lake (Sinai), Desulfovibrio oxyclinae sp. nov.

In an investigation on the oxygen tolerance of sulfate-reducing bacteria, a strain was isolated from a 10⁷-fold dilution of the upper 3-mm layer of a hypersaline cyanobacterial mat (transferred from Solar Lake, Sinai). The isolate, designated P1B, appeared to be well-adapted to the varying concentrations of oxygen and sulfide that occur in this environment. In the presence of oxygen strain P1B respired aerobically with the highest rates [260 nmol O₂ min^–1 (mg protein)^–1] found so far among marine sulfate-reducing bacteria. Besides H₂ and lactate, even sulfide or sulfite could be oxidized with oxygen. The sulfur compounds were completely oxidized to sulfate. Under anoxic conditions, it grew with sulfate, sulfite, or thiosulfate as the electron acceptor using H₂, lactate, pyruvate, ethanol, propanol, or butanol as the electron donor. Furthermore, in the absence of electron donors the isolate grew by disproportionation of sulfite or thiosulfate to sulfate and sulfide. The highest respiration rates with oxygen were obtained with H₂ at low oxygen concentrations. Aerobic growth of homogeneous suspensions was not obtained. Additions of 1% oxygen to the gas phase of a continuous culture resulted in the formation of cell clumps wherein the cells remained viable for at least 200 h. It is concluded that strain P1B is oxygen-tolerant but does not carry out sulfate reduction in the presence of oxygen under the conditions tested. Analysis of the 16S rDNA sequence indicated that strain P1B belongs to the genus Desulfovibrio, with Desulfovibrio halophilus as its closest relative. Based on physiological properties strain P1B could not be assigned to this species. Therefore, a new species, Desulfovibrio oxyclinae, is proposed. Received: 7 August 1996 / Accepted: 29 January 1997     

Influence of sodium orthovanadate on the production of astaxanthin from green algae Haematococcus lacustris

Astaxanthin production is commonly induced under stress conditions such as nutrient deficiency (N or P), high light stress, and variations of temperature, high NaCl concentrations, and other factors. The objective of the present study is the analysis of the effect of oxidative stress by sodium orthovanadate (SOV), a nonspecific inhibitor of protein tyrosine phosphatases, on the cells growth and astaxanthin production of H. lacustris. In the presence of SOV (lower than 5.0 mM), maximum growth of H. lacustris obtained was 2.4 × 10⁵ cells/mL in MBBM medium at 24°C under continuous illumination (40 μE/m²/s) of white fluorescent light, with continuous aeration of CO₂ (0.2 vvm). Total carotenoids accumulated per cell biomass unit treated with 2.5 mM SOV has approximately shown 2.5 folds higher than the control after short period of SOV induction time as 2 days, despite that cells were grown under normal light. Meanwhile, maximal astaxanthin production from H. lacustris was 10.7 mg/g biomass in MBBM with 5 days of continuous illumination at 40 μE/m²/s, which has been established as optimal light intensity for the control culture of H. lacustris. Treating algae H. lacustris with sodium orthovanadate showed promoting the accumulation of astaxanthin by advancing either the inhibition of dephosphorylation or synthesis of ATP. Its potential role of PTPases in microalgae H. lacustris is discussed. The first two authors are equally contributed to this work.     

A light-enhanced metabolism of sulfite in cells of Cucumis sativus L. cotyledons

The effects of light and several photosynthetic inhibitors on the rate of sulfite metabolism in cells obtained from Cucumis sativus L. cotyledons was studied. The cells were treated with 200 M Na₂SO₃ and the disappearance of sulfite was monitored using either dithiobisnitrobenzoic acid or fuchsin. The rate of sulfite disappearance in light was double the dark rate. Disalicylidene propanediamine at 1 mM increased this light-enhanced metabolism approx. 50%; neither 1 M 3,4-dichlorophenyl-N,N-dimethylurea nor 0.1 mM cyanazine, which completely inhibited CO₂-dependent oxygen evolution, affected the rate of sulfite metabolism. Addition of 200 M Na₂SO₃ to the cells partially inhibited ¹⁴CO₂ fixation. The rate of sulfite consumption by the cells did not affect this inhibition. We conclude that light-dependent sulfite metabolism is cucumber cells may utilize reduced ferredoxin generated as a result of photosynthetic electron transport. An injurious interaction between CO₂ fixation and sulfite appears to occur independently of the sulfite-metabolism process.Abbreviations DCMU 3,4-dichlorophenyl-N,N-dimethylurea - DSPD disalicylidene propanediamine - DTNB 5,5-dithiobis-(2-nitrobenzoic acid)     

Sulfite inhibits the F1F0-ATP synthase and activates the F1F0-ATPase of Paracoccus denitrificans

The F₁F₀ complex of Paracoccus denitrificans (PdF₁F₀) is the fastest ATP synthase but the slowest ATPase. Sulfite exerts maximal activation of the PdF₁F₀-ATPase (Pacheco-Moisés, F., García, J. J., Rodríguez-Zavala, J. S., and Moreno-Sánchez, R. (2000). Eur. J. Biochem. 267, 993–1000) but its effect on the PdF₁F₀-ATP synthase activity remains unknown. Therefore, we studied the effect of sulfite on ATP synthesis and ³²Pi ATP exchange reactions of inside-out membrane vesicles of P. denitrificans. Sulfite inhibited both reactions under conditions of maximal pH and normal sensitivity to dicyclohexylcarbodiimide. Sulfite increased by 10- and 5-fold the K _0.5 for Mg²⁺-ADP and Pi during ATP synthesis, respectively, and by 4-fold the IC₅₀ of Mg²⁺-ADP for inhibition of the PdF₁F₀-ATPase activity. Thus, sulfite exerts opposite effects on the forward and reverse functioning of the PdF₁F₀ complex. These effects are not due to membrane or PdF₁F₀ uncoupling. Kinetic and structural modifications that could account for these results are discussed.     

Formation of thionates by freshwater and marine strains of sulfate-reducing bacteria

The formation of thionates (thiosulfate, trithionate and tetrahionate) during the reduction of sulfate or sulfite was studied with four marine and four freshwater strains of sulfate-reducing bacteria. Growing cultures of two strains of the freshwater species Desulfovibrio desulfuricans formed up to 400 M thiosulfate and 100 M trithionate under conditions of electron donor limitation. Tetrathionate was observed in lower concentrations of up to 30 M. Uncoupler-treated washed cells of the four freshwater strains formed thiosulfate and trithionate at low electron donor concentrations with sulfite in excess. In contrast, only one of four marine strains formed thionates. The freshwater strain Desulfobulbus propionicus transformed sulfite almost completely to thiosulfate and trithionate. The amounts produced increased with time, concentration of added sulfite and cell density. Tetrathionate was detected only occasionally and in low concentrations, and was probably formed by chemical oxidation of thiosulfate. The results confirm the diversity of the sulfite reduction pathways in sulfate-reducing bacteria, and suggest that thiosulfate and trithionate are normal by-products of sulfate reduction.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone     

Interaction of benzo[c]phenanthridine and protoberberine alkaloids with animal and yeast cells

We compared the effects of four quaternary benzo[c]phenanthridine alkaloids – chelerythrine, chelilutine, sanguinarine, and sanguilutine – and two quaternary protoberberine alkaloids – berberine and coptisine – on the human cell line HeLa (cervix carcinoma cells) and the yeastsSaccharomyces cerevisiae andSchizosaccharomyces japonicus var. versatilis. The ability of alkaloids to display primary fluorescence, allowed us to record their dynamics and localization in cells. Cytotoxic, anti-microtubular, and anti-actin effects in living cells were studied. In the yeasts, neither microtubules nor cell growth was seriously affected even at the alkaloid concentration of 100 μg/ml. The HeLa cells, however, responded to the toxic effect of alkaloids at concentrations ranging from 1 to 50 μg/ml. IC₅₀ values for individual alkaloids were: sanguinarine IC₅₀ = 0.8 μg/ml, sanguilutine IC₅₀ = 8.3 μg/ml, chelerythrine IC₅₀ = 6.2 μg/ml, chelilutine IC₅₀ = 5.2 μg/ml, coptisine IC₅₀ = 2.6 μg/ml and berberine IC₅₀ >10.0 μg/ml. In living cells, sanguinarine produced a decrease in microtubule numbers, particularly at the cell periphery, at a concentration of 0.1 μg/ml. The other alkaloids showed a similar effect but at higher concentrations (5–50 μg/ml). The strongest effects of sanguinarine were explained as a consequence of its easy penetration through the cell membrane owing to nonpolar pseudobase formation and to a high degree of molecular planarity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Aminooxyacetic acid inhibits antheridiogenesis and development of Anemia phyllitidis gametophytes

Cytomorphological studies of the development of young fern gametophytes (Anemia phyllitidis) have been used to investigate combined effects of gibberellic acid and ethylene on male sex expression. ACC (the key by-product in ethylene biosynthesis pathway) was found to exert a synergetic effect on the gibberellic acid-induced antheridia formation, and this phenomenon could be related with the specific stimulation of cell growth and activity of their differentiation. To complete and verify those observations male sex expression in the fern gametophytes treated with ACC-biosynthesis inhibitor was reinvestigated. Aminooxyacetic acid (AOA) restrained antheridia formation via inhibition of cell divisions. AOA influenced the arrangement and flexibility of cellulose microfibrils in the antheridial zone cells, thus affecting cell expansion. On the other hand, the level of DNA synthesis was not reduced. Transient increase in the number of S-phase cells, followed by the accumulation of G2-phase cells led to the enhancement of cell polyploidization. All these findings correspond with the previous observations and support participation of ethylene in gibberellic acid-induced male sex expression in ferns.Abbreviations AOA Aminooxyacetic acid - CPA Cell profile area - GA Gibberellin - GA₃ Gibberellic acid