PCR amplification of the 3'' external transcribed and intergenic spacers of the ribosomal DNA repeat unit in three species of Saccharomyces

Abstract Desulfotomaculum thermobenzoicum TSB converted 4 mol pyruvate to 5 mol acetate in the absence of sulfate. The cells grown on pyruvate without sulfate showed both carbon monoxide dehydrogenase (CODH) and methylmalonyl-CoA: pyruvate transcarboxylase activities. However, considering the fermentation products, the acetogenesis from pyruvate might be conducted by CODH pathway rather than methylmalonyl-CoA pathway. Contrary to this finding, Desulfobulbus propionicus MUD fermented 3 mol pyruvate to 2 mol acetate and 1 mol propionate stoichiometrically via methylmalonyl-CoA pathway. Desulfovibrio vulgaris Marburg, which has neither the CODH pathway nor the methylmalonyl-CoA pathway, converted pyruvate to acetate, H₂ and CO₂ as the main products. These results indicate that the fermentation pattern of pyruvate depends on the metabolic characteristics of each sulfate-reducing bacterium.     

In situ stimulation of bacterial sulfate reduction in sulfate-limited freshwater lake sediments

Abstract By adding sulfate in the form of solid gypsum, it was possible to transform in situ a predominantly methanogenic sediment ecosystem into a sulfate-reducing one. The concentrations of sulfate, sulfide, methane, acetate, propionate, soluble iron, and manganese were determined in the porewater before and after the transition. Although sulfate was no longer limiting, acetate and propionate continued to accumulate and reached much higher concentrations than under sulfate-limited conditions. Metabolic activities of fermenting bacteria and of sulfate reducers, which belong to the group that incompletely oxidizes organic material, might be responsible for the increased production of volatile fatty acids. The elevated concentrations of soluble Fe(II)²⁺ and Mn(II)²⁺ observed in the porewater stem from iron and manganese compounds which may be reduced chemically by hydrogen sulfide and other microbially produced reducing agents or directly through increased activities of the iron and manganese reducing bacteria. In the horizon with high sulfate-reducing activities the methane concentrations in the porewater were lower than in non-stimulated sediment regions. The shape of the concentration depth profile indicates methane consumption through sulfate reducing processes. The in situ experiment demonstrates the response of a natural microbial ecosystem to fluctuations in the environmental conditions.     

Preliminary study of treatment of sulphuric pickling water waste from steelmaking by bio-oxidation with Thiobacillus ferrooxidans

Abstract: This report looks at the laboratory-scale recovery of iron oxides (αFe₂O₃ type) through bio-oxidation with Thiobacillus ferrooxidans of the ferrous sulphate contained in steel industry sulphuric pickling liquors. This is done by calcining iron sulphates and iron and ammonium sulphates obtained from the crystallization of the oxidized solution. The products of the bacterial reaction and the iron oxides are then studied according to calcination temperature. The process carried out produced 50 kg of α Fe₂O₃ per m³ of waste pickling liquor at 700°C with 99.8% weight iron recovery.     

Hydrogenase Activity of Mineral-Associated and Suspended Populations of Desulfovibrio desulfuricans Essex 6

The interactions between sulfate-reducing microorganisms and iron oxides influence a number of important redox-sensitive biogeochemical processes including the formation of iron sulfides. Enzymes, such as hydrogenase which catalyze the reversible oxidation of molecular hydrogen, are known to mediate electron transfer to metals and may contribute to the formation and speciation of ferrous sulfides formed at the cell–mineral interface. In the present study, we compared the whole cell hydrogenase activity of Desulfovibrio desulfuricans strain Essex 6 growing as biofilms on hematite (hematite-associated) or as suspended populations using different metabolic pathways. Hematite-associated cells exhibited significantly greater hydrogenase activity than suspended populations during sulfate respiration but not during pyruvate fermentation. The enhanced activity of the hematite-associated, sulfate-grown cells appears to be dependent on iron availability rather than a general response to surface attachment since the activity of glass-associated cells did not differ from that of suspended populations. Hydrogenase activity of pyruvate-fermenting cells was stimulated by addition of iron as soluble Fe(II)Cl₂ and, in the absence of added iron, both sulfate-reducing and pyruvate-fermenting cells displayed similar rates of hydrogenase activity. These data suggest that iron exerts a stronger influence on whole cell hydrogenase activity than either metabolic pathway or mode of growth. The location of hydrogenase to the cell envelope and the enhanced activity at the hematite surface in sulfate-reducing cells may influence the redox conditions that control the species of iron sulfides on the mineral surface.     

Microbial sulfate reduction in acidic (pH 3) strip-mine lakes

Abstract ³⁵SO₄ reduction was detected in slurries of sediments obtained from Reservoir 29 (pH 3.8) and Lake B (pH 6.2), two acid strip-mine lakes in Indiana. The rates varied seasonally and were higher in summer and fall than in the spring. The optimal pH for sulfate reduction in Reservoir 29 sediments was 5, but samples had increased activity at pH 7 within 24 h after adjusting the pH to this value. In Lake B, the optimal pH for sulfate reduction was the in situ pH (6.2). Sulfate reduction in both lakes was stimulated 2–3-fold by increasing p _H2. High concentrations (5 mM) of organic acids inhibited sulfate reduction at pH 3.8, but stimulation was observed at concentrations of 0.1 mM. Acid-volatile sulfides accounted for about 70% of the products of ³⁵SO₄ reduction.     

The role of reduced iron powder in the fermentative production of tetanus toxin

When tetanus toxin is made by fermentation with Clostridium tetani, the traditional source of iron is an insoluble preparation called reduced iron powder. This material removes oxygen from the system by forming FeO₂ (rust). When inoculated in a newly developed medium lacking animal and dairy products and containing glucose, soy-peptone, and inorganic salts, growth and toxin production were poor without reduced iron powder. The optimum concentration of reduced iron powder for toxin production was found to be 0.5 g/l. Growth was further increased by higher concentrations, but toxin production decreased. Inorganic iron sources failed to replace reduced iron powder for growth or toxin formation. The iron source that came closest was ferrous ammonium sulfate. The organic iron sources ferric citrate and ferrous gluconate were more active than the inorganic compounds but could not replace reduced iron powder. Insoluble iron sources, such as iron wire, iron foil, and activated charcoal, were surprisingly active. Combinations of activated charcoal with soluble iron sources such as ferrous sulfate, ferric citrate, and ferrous gluconate showed increased activity, and the ferrous gluconate combination almost replaced reduced iron powder. It thus appears that the traditional iron source, reduced iron powder, plays a double role in supporting tetanus toxin formation, i.e., releasing soluble sources of iron and providing an insoluble surface.     

Interaction between fish spoilage bacteria Pseudomonas sp. and Shewanella putrefaciens in fish extracts and on fish tissue

The interaction between fish spoilage bacteria, Pseudomonas sp. and Shewanella putrefaciens , was investigated using fish extract and fish tissue as model systems. Isolates of Pseudomonas that produced iron chelators, siderophores, inhibited growth of S. putrefaciens in a fish-extract-agar diffusion assay but no, or only weak, antagonistic activity was seen when the medium was supplemented with iron. Sterile-filtered supernatant fluid from a siderophore-producing Pseudomonas grown in fish extract was inhibitory to S. putrefaciens if the number of Pseudomonas was above 10⁸ cfu ml⁻¹. In contrast, supernatant fluids from siderophore-negative Pseudomonas isolates did not inhibit growth of S. putrefaciens. The inhibitory effect was, except for one strain of Pseudomonas , not seen in supernatant fluids from iron-enriched cultures of Pseudomonas sp. Finally, siderophore-producing Pseudomonas sp. lowered the maximum cell level of S. putrefaciens 1–2 log units from 10⁹ to 10¹⁰ cfu g⁻¹ when the strains were grown on fish muscle blocks at 0°C but the growth rate of S. putrefaciens was not affected.     

Methanogenesis in the hypersaline Solar Lake (Sinai)

Abstract Enrichment studies on microbial mat sediments (potential stromatolites) from the hypersaline Solar Lake (Sinai) indicated high numbers of methanogenic bacteria (up to 10⁵ ml⁻¹ sediment) in spite of the high sulfate reduction rate, sulfate concentration and salinity. Among H₂/CO₂, acetate and monomethylamine, the methylated amine was the preferred substrate. The predominant species enriched was a Methanosarcina sp. The findings indicate that methanogenic bacteria play an important role in hypersaline sulfate-enriched anoxic sediments and stromatolithic microbial mats.     

The toxicity of iron to brown trout and effects on the gills: a comparison of two grades of iron sulphate

The 96-h LC₅₀ on brown trout Salmo trutta of a commercial iron (III) sulphate liquor, used for treating reservoirs to reduce algal growth, was 28 mg total Fe l⁻¹ (0·05 mg soluble Fe l⁻¹). The 96-h LC₅₀ for analar grade iron (III) sulphate was 47 mg total Fe l⁻¹ (0·24 mg soluble Fe l⁻¹). Lethal and sublethal exposure to both grades of iron resulted in accumulation on the gill, which appears to be the main target for iron toxicity. Greater iron accumulation occurred during exposure to commercial iron sulphate liquor. Physical clogging of gills and gill damage was seen during lethal and sublethal exposure to iron. Gill tissue analysis showed no evidence of iron uptake into gill tissues during lethal or sublethal exposure to iron. Iron did not accumulate in plasma of fish exposed to iron compared to controls. Respiratory disruption due to physical clogging of the gills is suggested as a possible mechanism for iron toxicity.     

Inhibition of [3H]MK-801 Binding by Ferrous (II) but Not Ferric (III) Ions in a Manner Different from That by Sodium Nitroprusside (II) in Rat Brain Synaptic Membranes

Abstract: The addition of sodium nitroprusside (SNP) significantly inhibited binding of (+)-5-[³H]methyl-10,11-dihydro-5 H -dibenzo[ a,d ]cyclohepten-5,10-imine ([³H]MK-801) to an ion channel associated with the N -methyl- d -aspartate (NMDA) receptor in a concentration-dependent manner at concentrations of >1 µ M in rat brain synaptic membranes not extensively washed. However, neither S -nitroso- N -acetylpenicillamine nor S -nitroso- l -glutathione inhibited binding even at 100 µ M . Of the two compounds structurally related to SNP (II), similarly potent inhibition was induced by potassium ferrocyanide (II) but not by potassium ferricyanide (III). In addition, ferrous chloride (II) induced much more potent inhibition of binding than ferric chloride (III), at a similar concentration range. In contrast, iron chelators prevented the inhibition by ferrous chloride (II) without markedly affecting that by SNP (II) and potassium ferrocyanide (II). Pretreatment with ferrous chloride (II) also led to potent inhibition of [³H]MK-801 binding in a manner insensitive to subsequent addition of the iron chelators. Pretreatment with Triton X-100 resulted in significant potentiation of the ability of ferrous chloride (II) to inhibit [³H]MK-801 binding irrespective of the addition of agonists, moreover, although binding of other radioligands to the non-NMDA receptors was unaltered after pretreatment first with Triton X-100 and then with ferrous chloride (II). These results suggest that ferrous ions (II) may interfere selectively with opening processes of the NMDA channel through mechanisms entirely different from those underlying the inhibition by both SNP (II) and potassium ferrocyanide (II) in rat brain.     

Role of outer-membrane cytochromes MtrC and OmcA in the biomineralization of ferrihydrite by Shewanella oneidensis MR-1

In an effort to improve the understanding of electron transfer mechanisms at the microbe–mineral interface, Shewanella oneidensis MR-1 mutants with in-frame deletions of outer-membrane cytochromes (OMCs), MtrC and OmcA, were characterized for the ability to reduce ferrihydrite (FH) using a suite of microscopic, spectroscopic, and biochemical techniques. Analysis of purified recombinant proteins demonstrated that both cytochromes undergo rapid electron exchange with FH in vitro with MtrC displaying faster transfer rates than OmcA. Immunomicroscopy with cytochrome-specific antibodies revealed that MtrC co-localizes with iron solids on the cell surface while OmcA exhibits a more diffuse distribution over the cell surface. After 3-day incubation of MR-1 with FH, pronounced reductive transformation mineral products were visible by electron microscopy. Upon further incubation, the predominant phases identified were ferrous phosphates including vivianite [Fe₃(PO₄)₂·8H₂O] and a switzerite-like phase [Mn₃,Fe₃(PO₄)₂·7H₂O] that were heavily colonized by MR-1 cells with surface-exposed outer-membrane cytochromes. In the absence of both MtrC and OmcA, the cells ability to reduce FH was significantly hindered and no mineral transformation products were detected. Collectively, these results highlight the importance of the outer-membrane cytochromes in the reductive transformation of FH and support a role for direct electron transfer from the OMCs at the cell surface to the mineral.     

The effect of different dietary iron levels on growth and hepatic iron concentration in juvenile gibel carp (Carassius auratus gibelio)

An 83-day growth trial was conducted using a flow-through system to examine the effects of different dietary iron levels on growth and hepatic iron concentration in juvenile gibel carp ( Carassius auratus gibelio ). Six purified diets supplemented with different levels of iron (0, 10, 30, 60, 100 and 200 mg kg⁻¹) (as ferrous sulfate) were fed to triplicate groups of fish (initial weight 2.12 ± 0.00 g per fish). The results showed that the addition of iron to the basal diet did not significantly affect the specific growth rate (SGR), feed efficiency (FE), survival, red blood cell amount (RBC), hemoglobin content (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) or mean corpuscular hemoglobin concentration (MCHC). Hepatic iron concentration and hematocrit (Hct) were significantly influenced by dietary iron level (P < 0.05). On the basis of the iron concentration for the maintenance of optimum hepatic iron concentration and Hct, it was concluded that the dietary iron concentration of juvenile gibel carp should be not less than 202 mg Fe kg⁻¹ diet.     

Influence of nicotianamine and iron supply on formation and elongation of adventitious roots in hypocotyl cuttings of the tomato mutant 'chloronerva' (Lycopersicon esculentum)

The influence of nicotianamine (NA) on formation and elongation of adventitious roots in hypocotyls of de-rooted NA-less mutant seedlings of Lycopersicon esculentum Mill, was examined in relation to the iron supply [ferric N-N'-ethylenediaminedi-(2-hydroxyphenylacetate) (FEDDHA), ferric ethylenediaminetetracetate (FeEDTA), ferric N-(2-hydroxyethyl)-ethylenediaminetriacetate (FeHEDTA, Fe-citrate and FeCl₃] in the nutrient solution. The initiation of root primordia in hypocotyl cuttings was independent of NA and occurred with about the same frequency in both, mutant and wild-type. In the mutant the development of primordia to adventitious roots was blocked at all iron sources used, except FeEDTA. Addition of NA (5x 10⁻⁶ to 2 × 10⁻⁵ M ) to the rooting medium resulted in a fast growth of adventitious roots in mutant cuttings with all iron sources tested. Rooting of wild-type cuttings was independent from NA application and iron sources. We suppose that NA is involved in the intracellular transport of iron. Its function is possibly linked with chelation of ferrous iron in the cell.     

Incorporation of [methyl-3H]thymidine by obligate and facultative anaerobic bacteria when grown under defined culture conditions

Abstract Incorporation of [ methyl -³H]thymidine into bacterial DNA was determined for a range of axenic anaerobic bacterial cultures: fermentative heterotrophs, sulphate-reducing bacteria, purple sulphur bacteria, acetogens and methanogens. Anaerobically growing Bacillus sp. and the obligate aerobe Thiobacillus ferrooxidans were also investigated. Actively growing cultures of sulphate-reducing bacteria belonging to the genera Desulfovibrio, Desulfotomaculum, Desulfobacter, Desulfobotulus and Desulfobulbus , purple sulphur bacteria ( Chromatium vinosum OP2 and Thiocapsa roseopersicina OP1), methanogens ( Methanococcus GS16 and Methanosarcina barkeri ) and an acetogen ( Acetobacterium woodii ) did not incorporate [ methyl -³H]thymidine into DNA. The only obligate anaerobes in which thymidine incorporation into DNA could be unequivocally demonstrated were members of the genus Clostridium . Anaerobically growing Bacillus sp. also incorporated thymidine. These data demonstrate that pure culture representatives of major groups of anaerobic bacteria involved in the terminal oxidation of organic carbon and anoxygenic phototrophs within sediments are unable to incorporate [ methyl -³H]thymidine into DNA, although some obligate and facultative anaerobes can. Variability in thymidine incorporation amongst pure culture isolates indicates that unless existing techniques can be calibrated to take this into consideration then productivity estimates in both aerobic and anaerobic environments may be greatly underestimated using the [ methyl -³H]thymidine technique.     

Differential protein expression during growth of Acidithiobacillus ferrooxidans on ferrous iron, sulfur compounds, or metal sulfides

A set of proteins that changed their levels of synthesis during growth of Acidithiobacillus ferrooxidans ATCC 19859 on metal sulfides, thiosulfate, elemental sulfur, and ferrous iron was characterized by using two-dimensional polyacrylamide gel electrophoresis. N-terminal amino acid sequencing and mass spectrometry analysis of these proteins allowed their identification and the localization of the corresponding genes in the available genomic sequence of A. ferrooxidans ATCC 23270. The genomic context around several of these genes suggests their involvement in the energetic metabolism of A. ferrooxidans. Two groups of proteins could be distinguished. The first consisted of proteins highly upregulated by growth on sulfur compounds (and downregulated by growth on ferrous iron): a 44-kDa outer membrane protein, an exported 21-kDa putative thiosulfate sulfur transferase protein, a 33-kDa putative thiosulfate/sulfate binding protein, a 45-kDa putative capsule polysaccharide export protein, and a putative 16-kDa protein of unknown function. The second group of proteins comprised those downregulated by growth on sulfur (and upregulated by growth on ferrous iron): rusticyanin, a cytochrome c(552), a putative phosphate binding protein (PstS), the small and large subunits of ribulose biphosphate carboxylase, and a 30-kDa putative CbbQ protein, among others. The results suggest in general a separation of the iron and sulfur utilization pathways. Rusticyanin, in addition to being highly expressed on ferrous iron, was also newly synthesized, as determined by metabolic labeling, although at lower levels, during growth on sulfur compounds and iron-free metal sulfides. During growth on metal sulfides containing iron, such as pyrite and chalcopyrite, both proteins upregulated on ferrous iron and those upregulated on sulfur compounds were synthesized, indicating that the two energy-generating pathways are induced simultaneously depending on the kind and concentration of oxidizable substrates available.     

Differential Protein Expression during Growth of Acidithiobacillus ferrooxidans on Ferrous Iron, Sulfur Compounds, or Metal Sulfides

A set of proteins that changed their levels of synthesis during growth of Acidithiobacillus ferrooxidans ATCC 19859 on metal sulfides, thiosulfate, elemental sulfur, and ferrous iron was characterized by using two-dimensional polyacrylamide gel electrophoresis. N-terminal amino acid sequencing and mass spectrometry analysis of these proteins allowed their identification and the localization of the corresponding genes in the available genomic sequence of A. ferrooxidans ATCC 23270. The genomic context around several of these genes suggests their involvement in the energetic metabolism of A. ferrooxidans. Two groups of proteins could be distinguished. The first consisted of proteins highly upregulated by growth on sulfur compounds (and downregulated by growth on ferrous iron): a 44-kDa outer membrane protein, an exported 21-kDa putative thiosulfate sulfur transferase protein, a 33-kDa putative thiosulfate/sulfate binding protein, a 45-kDa putative capsule polysaccharide export protein, and a putative 16-kDa protein of unknown function. The second group of proteins comprised those downregulated by growth on sulfur (and upregulated by growth on ferrous iron): rusticyanin, a cytochrome c₅₅₂, a putative phosphate binding protein (PstS), the small and large subunits of ribulose biphosphate carboxylase, and a 30-kDa putative CbbQ protein, among others. The results suggest in general a separation of the iron and sulfur utilization pathways. Rusticyanin, in addition to being highly expressed on ferrous iron, was also newly synthesized, as determined by metabolic labeling, although at lower levels, during growth on sulfur compounds and iron-free metal sulfides. During growth on metal sulfides containing iron, such as pyrite and chalcopyrite, both proteins upregulated on ferrous iron and those upregulated on sulfur compounds were synthesized, indicating that the two energy-generating pathways are induced simultaneously depending on the kind and concentration of oxidizable substrates available.     

Sulfate-reducing bacteria in littoral sediment of Lake Constance

Abstract The viable population of sulfate-reducing bacteria (SRB) in littoral sediments of Lake Constance was investigated using enrichment and enumeration techniques. Enrichment studies established that most types of SRB grew best in media with low salt concentrations (max. 0.4 g Cl⁻/1), consistent with the low salinity of the freshwater habitat. Enumerations were based on an adequate medium with the following electron donors: H₂, lactate, acetate, propionate, butyrate, caprylate, succinate, benzoate, or S₂O₃²⁻ for thiosulfate-disproportionating bacteria. Cultures were incubated for 6 weeks to obtain maximum counts. A maximum cell density of 6.3 × 10⁶ cells per ml sediment was estimated, which is the highest number of SRB ever reported for anoxic sediments. A comparison with measured sulfate reduction rates showed that the enumeration techniques were about 10–100-fold more efficient than those previously used. The population of SRB had a characteristic structure consisting of 87.7% H₂-utilizing SRB (physiologically resembling the classical Desulfovibrio species); 12.0% propionate utilizers (tentatively identified as Desulfobulbus species); 0.3% long chain fatty acid-oxidizing Desulfovibrio sapovorans species. Acetate-utilizing SRB ( Desulfotomaculum acetoxidans ) constituted ≤ 0.05% of the total estimated population. Moreover, the latter species was only present as inactive spores. Benzoate-degrading SRB were not detected.     

Acid-stable cytochromes in ferrous ion oxidizing cell-free preparations from Thiobacillus ferrooxidans

Abstract Cell-free preparations from ferrous ion- and sulfur-grown Thiobacillus ferrooxidans prepared under neutral (pH 7.5) or acidic conditions (pH 2.0) were compared. Under neutral conditions the ferrous ion-oxidizing system of T. ferrooxidans was membrane-bound. At acidic conditions, the enzyme system became partially solubilized. In ferrous ion-oxidizing membranes of ferrous ion-grown cells (neutral conditions) a₁-, c- and b-type cytochromes were present. The acidic preparations contained only cytochrome a₁ and c. At least three acid-stable c-type cytochromes (M_r 60 000, 30 000 and 25 000), an acid-stable protein with non-convalently bound heme group (M_r probably rusticyanin, were detected in ferrous ion oxidizing preparations. Membranes of sulfur-grown cells prepared under neutral conditions still oxidized ferrous ions, and contained a₁-, b-, c- and in addition an aa₃-type cytochrome. Cytochrome b and aa₃ were acid-labile.     

Substrate stabilization of lysophosphatidylcholine-solubilized plasma membrane H+-ATPase from oat roots

Plasma membrane vesicles with H⁺-ATPase activity were purified from 8-day-old oat ( Avena sativa L. cv. Brighton) roots using an aqueous polymer two-phase system. Of several detergents tested, only lysophosphatidylcholine solubilized the H⁺-ATPase in an active form. Solubilization of the H⁺-ATPase with lysophosphatidylcholine was possible in the absence of glycerol, but the ATPase activity decreased about 4–5 times as rapidly in the absence as in the presence of 30% (w/v) glycerol. The solubilized enzyme was further stabilized by ATP and protons. Addition of 1 m M ATP to the plasma membranes halted inactivation of the H⁺-ATPase. Even in the absence of polyol compounds and ATP, the enzyme was stable for hours at relatively low pH with an optimum around pH 6.7 at room temperature. The curve for the stability of soluble H⁺-ATPase as a function of pH closely resembles the pH curve for the activity of the H⁺-ATPase. This suggests that binding of protons to transport sites may stabilize the soluble H⁺-ATPase in an enzymatically active form.     

Pathway of propionate degradation in Desulfobulbus propionicus

Abstract The degradation of [1-¹⁴C]- and [2-¹⁴C] propionate to acetate and bicarbonate by the sulfate- reducing bacterium Desulfobulbus propionicus was studied. When [1-¹⁴C]propionate was used, more than 95% of the label was recovered in the HCO₃⁻ fraction. [2-¹⁴C]Propionate was quantitatively converted into labeled acetate of which the methyl and carboxyl group were equally labeled. These results are in accordance with a randomizing route such as the methylmalonyl-CoA pathway for propionate degradation and support earlier evidence for the functioning of this pathway on the basis of enzyme assays.