Organic Carbon Utilization by Obligately and Facultatively Autotrophic Beggiatoa Strains in Homogeneous and Gradient Cultures

Marine Beggiatoa strains MS-81-6 and MS-81-1c are filamentous gliding bacteria that use hydrogen sulfide and thiosulfate as electron donors for chemolithotrophic energy generation. They are known to be capable of chemolithoautotrophic growth in sulfide gradient media; here we report the first successful bulk cultivation of these strains in a defined liquid medium. To investigate their nutritional versatilities, strains MS-81-6 and MS-81-1c were grown in sulfide-oxygen gradient media supplemented with single organic compounds. Respiration rates and biomass production relative to those of controls grown in unsupplemented sulfide-limited media were monitored to determine whether organic compounds were utilized as sources of energy and/or cell carbon. With cells grown in sulfide gradient and liquid media, we showed that strain MS-81-6 strongly regulates two enzymes, the tricarboxylic acid cycle enzyme 2-oxoglutarate dehydrogenase and the Calvin cycle enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, in response to the presence of organic carbon (acetate) in the growth medium. In contrast, strain MS-81-1c lacked 2-oxoglutarate dehydrogenase activity and regulated ribulose-1,5-bisphosphate carboxylase/oxygenase activity only slightly in response to organic substrates. Tracer experiments with radiolabeled acetate showed that strain MS-81-1c did not oxidize acetate to CO(inf2) but could synthesize approximately 20% of its cell carbon from acetate. On the basis of these results, we conclude that Beggiatoa strain MS-81-1c is an obligate chemolithoautotroph, while strain MS-81-6 is a versatile facultative chemolithoautotroph.     

Sulfur metabolism enzymes in thermoacidophilus bacteria Sulfobacillus sibiricus

Sulfur oxygenase, sulfite oxidase, adenylyl sulfate reductase, rhodanase, sulfur:Fe(III) oxidoreductase, and sulfite:Fe(III) oxidoreductase were found in cells of aerobic thermoacidophilic bacteria Sulfobacillus sibiricus strains N1 and SSO. Enzyme activity was revealed in cells grown on the medium with elemental sulfur or in the presence of various sulfide elements and concentrates of sulfide ores. The activity of sulfur-metabolizing enzymes depended little on the degree of aeration during bacterial growth.     

Sulfur-Metabolizing Enzymes in Thermoacidophilic Bacteria Sulfobacillus sibiricus

Sulfur oxygenase, sulfite oxidase, adenylyl sulfate reductase, rhodanase, sulfur : Fe(III) oxidoreductase, and sulfite : Fe(III) oxidoreductase were found in cells of aerobic thermoacidophilic bacteria Sulfobacillus sibiricus, strains N1 and SSO. Enzyme activity was revealed in the cells grown on medium with elemental sulfur or in the presence of various sulfide minerals and concentrates of sulfide ores. The activity of enzymes of sulfur metabolism depended little on the degree of aeration during bacterial growth.     

Enzymatic and genetic characterization of carbon and energy metabolisms by deep-sea hydrothermal chemolithoautotrophic isolates of Epsilonproteobacteria

The carbon and energy metabolisms of a variety of cultured chemolithoautotrophic Epsilonproteobacteria from deep-sea hydrothermal environments were characterized by both enzymatic and genetic analyses. All the Epsilonproteobacteria tested had all three key reductive tricarboxylic acid (rTCA) cycle enzymatic activities--ATP-dependent citrate lyase, pyruvate:ferredoxin oxidoreductase, and 2-oxoglutarate:ferredoxin oxidoreductase--while they had no ribulose 1,5-bisphosphate carboxylase (RubisCO) activity, the key enzyme in the Calvin-Benson cycle. These results paralleled the successful amplification of the key rTCA cycle genes aclB, porAB, and oorAB and the lack of success at amplifying the form I and II RubisCO genes, cbbL and cbbM. The combination of enzymatic and genetic analyses demonstrates that the Epsilonproteobacteria tested use the rTCA cycle for carbon assimilation. The energy metabolisms of deep-sea Epsilonproteobacteria were also well specified by the enzymatic and genetic characterization: hydrogen-oxidizing strains had evident soluble acceptor:methyl viologen hydrogenase activity and hydrogen uptake hydrogenase genes (hyn operon), while sulfur-oxidizing strains lacked both the enzyme activity and the genes. Although the energy metabolism of reduced sulfur compounds was not genetically analyzed and was not fully clarified, sulfur-oxidizing Epsilonproteobacteria showed enzyme activity of a potential sulfite:acceptor oxidoreductase for a direct oxidation pathway to sulfate but no activity of AMP-dependent adenosine 5'-phosphate sulfate reductase for a indirect oxidation pathway. No activity of thiosulfate-oxidizing enzymes was detected. The enzymatic and genetic characteristics described here were consistent with cellular carbon and energy metabolisms and suggest that molecular tools may have great potential for in situ elucidation of the ecophysiological roles of deep-sea Epsilonproteobacteria.     

Existence of a Hydrogen Sulfide:Ferric Ion Oxidoreductase in Iron-Oxidizing Bacteria

The existence of a hydrogen sulfide:ferric ion oxidoreductase, which catalyzes the oxidation of elemental sulfur with ferric ions as an electron acceptor to produce ferrous and sulfite ions, was assayed with washed intact cells and cell extracts of various kinds of iron-oxidizing bacteria, such as Thiobacillus ferrooxidans 13598, 13661, 14119, 19859, 21834, 23270, and 33020 from the American Type Culture Collection, Leptospirillum ferrooxidans 2705 and 2391 from the Deutsche Sammlung von Mikroorganismen, L. ferrooxidans BKM-6-1339 and P3A, and moderately thermophilic iron-oxidizing bacterial strains BC1, TH3, and Alv. It was found that hydrogen sulfide:ferric ion oxidoreductase activity comparable to that of T. ferrooxidans AP19-3 was present in all iron-oxidizing bacteria tested, suggesting a wide distribution of this enzyme in iron-oxidizing bacteria.     

In vivo role of adenosine-5′-phosphosulfate reductase in the purple sulfur bacterium Allochromatium vinosum

Adenosine-5'-phosphosulfate (APS) reductase participates in the oxidation of sulfite to APS in Allochromatium vinosum. Oxidation of sulfite via the APS pathway yields ATP through substrate-level phosphorylation. An alternative enzyme for the oxidation of sulfite to sulfate, sulfite:acceptor oxidoreductase, has also been reported in Ach. vinosum. Oxidation of sulfite through this enzyme does not yield ATP. APS reductase is expressed constitutively in Ach. vinosum, suggesting that it performs an important role in this organism. However, studies carried out with batch cultures of an APS reductase mutant showed little or no differences in growth or in the rates of substrate oxidation when compared to the wild-type, therefore questioning the role of this enzyme. In an attempt to establish whether the ATP gain derived from APS-reductase-mediated oxidation of sulfite is relevant for energy-limited cultures, we compared growth of the wild-type SM50 and the APS-reductase-deficient mutant D3 when grown in continuous culture under different degrees of illumination. Little differences in the specific growth rates of the two strains were observed at light-limiting irradiances, suggesting that the ATP gained during sulfite oxidation through the APS reductase pathway does not constitute a significant energy input. However, at saturating irradiances, wild-type Ach. vinosum grew considerably faster than the mutant. Increasing the irradiance even further resulted in inhibition of the wild-type strain down to the level of the APS reductase mutant. The implications of these results are discussed.     

Enzymatic and Genetic Characterization of Carbon and Energy Metabolisms by Deep-Sea Hydrothermal Chemolithoautotrophic Isolates of Epsilonproteobacteria

The carbon and energy metabolisms of a variety of cultured chemolithoautotrophic Epsilonproteobacteria from deep-sea hydrothermal environments were characterized by both enzymatic and genetic analyses. All the Epsilonproteobacteria tested had all three key reductive tricarboxylic acid (rTCA) cycle enzymatic activities—ATP-dependent citrate lyase, pyruvate:ferredoxin oxidoreductase, and 2-oxoglutarate:ferredoxin oxidoreductase—while they had no ribulose 1,5-bisphosphate carboxylase (RubisCO) activity, the key enzyme in the Calvin-Benson cycle. These results paralleled the successful amplification of the key rTCA cycle genes aclB, porAB, and oorAB and the lack of success at amplifying the form I and II RubisCO genes, cbbL and cbbM. The combination of enzymatic and genetic analyses demonstrates that the Epsilonproteobacteria tested use the rTCA cycle for carbon assimilation. The energy metabolisms of deep-sea Epsilonproteobacteria were also well specified by the enzymatic and genetic characterization: hydrogen-oxidizing strains had evident soluble acceptor:methyl viologen hydrogenase activity and hydrogen uptake hydrogenase genes (hyn operon), while sulfur-oxidizing strains lacked both the enzyme activity and the genes. Although the energy metabolism of reduced sulfur compounds was not genetically analyzed and was not fully clarified, sulfur-oxidizing Epsilonproteobacteria showed enzyme activity of a potential sulfite:acceptor oxidoreductase for a direct oxidation pathway to sulfate but no activity of AMP-dependent adenosine 5′-phosphate sulfate reductase for a indirect oxidation pathway. No activity of thiosulfate-oxidizing enzymes was detected. The enzymatic and genetic characteristics described here were consistent with cellular carbon and energy metabolisms and suggest that molecular tools may have great potential for in situ elucidation of the ecophysiological roles of deep-sea Epsilonproteobacteria.     

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.     

Sulfite-oxido-reductase is involved in the oxidation of sulfite in Desulfocapsa sulfoexigens during disproportionation of thiosulfate and elemental sulfur

The enzymatic pathways of elemental sulfur and thiosulfate disproportionation were investigated using cell-free extract of Desulfocapsa sulfoexigens. Sulfite was observed to be an intermediate in the metabolism of both compounds. Two distinct pathways for the oxidation of sulfite have been identified. One pathway involves APS reductase and ATP sulfurylase and can be described as the reversion of the initial steps of the dissimilatory sulfate reduction pathway. The second pathway is the direct oxidation of sulfite to sulfate by sulfite oxidoreductase. This enzyme has not been reported from sulfate reducers before. Thiosulfate reductase, which cleaves thiosulfate into sulfite and sulfide, was only present in cell-free extract from thiosulfate disproportionating cultures. We propose that this enzyme catalyzes the first step in thiosulfate disproportionation. The initial step in sulfur disproportionation was not identified. Dissimilatory sulfite reductase was present in sulfur and thiosulfate disproportionating cultures. The metabolic function of this enzyme in relation to elemental sulfur or thiosulfate disproportionation was not identified. The presence of the uncouplers HQNO and CCCP in growing cultures had negative effects on both thiosulfate and sulfur disproportionation. CCCP totally inhibited sulfur disproportionation and reduced thiosulfate disproportionation by 80% compared to an unamended control. HQNO reduced thiosulfate disproportionation by 80% and sulfur disproportionation by 90%.     

Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria

Hydrogen sulfide is a potent toxin of aerobic respiration, but also has physiological functions as a signalling molecule and as a substrate for ATP production. A mitochondrial pathway catalyzing sulfide oxidation to thiosulfate in three consecutive reactions has been identified in rat liver as well as in the body-wall tissue of the lugworm, Arenicola marina. A membrane-bound sulfide : quinone oxidoreductase converts sulfide to persulfides and transfers the electrons to the ubiquinone pool. Subsequently, a putative sulfur dioxygenase in the mitochondrial matrix oxidizes one persulfide molecule to sulfite, consuming molecular oxygen. The final reaction is catalyzed by a sulfur transferase, which adds a second persulfide from the sulfide : quinone oxidoreductase to sulfite, resulting in the final product thiosulfate. This role in sulfide oxidation is an additional physiological function of the mitochondrial sulfur transferase, rhodanese.     

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     

嗜酸硫杆菌属硫氧化系统研究进展

硫化矿的酸溶解和化学氧化过程中(H 和Fe3 作用下,金属硫化矿中分解),伴随着硫元素转变成多聚硫S8或硫代硫酸盐的过程。对嗜酸硫杆菌属硫氧化过程的研究表明,胞外环状多聚硫S8可能通过细胞外膜蛋白巯基活化成线状-SnH后,被转运到细胞周质区域,进而被硫加双氧酶氧化成SO32-,活化过程中同时生成少量H2S;这些酶促反应不需要辅助因子参与,不释放电子。胞外硫代硫酸盐通过未知途径进入细胞周质。细胞周质中的SO32-主要经由亚硫酸-受体氧化还原酶氧化成SO42-,S2O32-可能经由硫代硫酸盐-辅酶Q氧化还原酶、硫代硫酸盐脱氢酶、连四硫酸盐水解酶等氧化为硫酸,少量H2S则经由硫化物-辅酶Q氧化还原酶氧化为多聚硫,后者再经由SO32-和S2O32-氧化生成最后产物SO42-。这些生物氧化过程释放的电子进入呼吸链参与产生细菌生长代谢所需的能量。然而,关于A.ferrooxidans硫氧化系统中各种硫化合物的酶催化氧化机制的研究仍很缺乏,胞内外硫化合物的转运机制、是否存在胞外酶催化氧化等仍然有待解决。另外,硫的型态和价态、酶催化反应的细胞微区域以及硫氧化系统中一些关键酶的分离及其表达基因的鉴定等问题都还有待进一步研究。基于对这些事实的分析,提出了一个嗜酸硫杆菌属硫氧化系统的模型。     

Inorganic sulfur oxidizing system in green sulfur bacteria

Green sulfur bacteria use various reduced sulfur compounds such as sulfide, elemental sulfur, and thiosulfate as electron donors for photoautotrophic growth. This article briefly summarizes what is known about the inorganic sulfur oxidizing systems of these bacteria with emphasis on the biochemical aspects. Enzymes that oxidize sulfide in green sulfur bacteria are membrane-bound sulfide-quinone oxidoreductase, periplasmic (sometimes membrane-bound) flavocytochrome c sulfide dehydrogenase, and monomeric flavocytochrome c (SoxF). Some green sulfur bacteria oxidize thiosulfate by the multienzyme system called either the TOMES (thiosulfate oxidizing multi-enzyme system) or Sox (sulfur oxidizing system) composed of the three periplasmic proteins: SoxB, SoxYZ, and SoxAXK with a soluble small molecule cytochrome c as the electron acceptor. The oxidation of sulfide and thiosulfate by these enzymes in vitro is assumed to yield two electrons and result in the transfer of a sulfur atom to persulfides, which are subsequently transformed to elemental sulfur. The elemental sulfur is temporarily stored in the form of globules attached to the extracellular surface of the outer membranes. The oxidation pathway of elemental sulfur to sulfate is currently unclear, although the participation of several proteins including those of the dissimilatory sulfite reductase system etc. is suggested from comparative genomic analyses.     

Sulfate formation via ATP sulfurylase in thiosulfate- and sulfite-disproportionating bacteria

Disproportionation of thiosulfate or sulfite to sulfate plus sulfide was found in several sulfate-reducing bacteria. Out of nineteen strains tested, eight disproportionated thiosulfate, and four sulfite. Growth with thiosulfate or sulfite as the sole energy source was obtained with three strains (Desulfovibrio sulfodismutans and the strains Bra02 and NTA3); additionally, D. desulfuricans strain CSN grew with sulfite but not with thiosulfate, although thiosulfate was disproportionated. Two sulfur-reducing bacteria, four phototrophic sulfur-oxidizing bacteria (incubated in the dark), and Thiobacillus denitrificans did not disproportionate thiosulfate or sulfite. Desulfovibrio sulfodismutans and D. desulfuricans CSN formed sulfate from thiosulfate or sulfite even when simultaneously oxidizing hydrogen or ethanol, or in the presence of 50 mM sulfate. The capacities of sulfate reduction and of thiosulfate and sulfite disproportionation were constitutively present. Enzyme activities required for sulfate reduction (ATP sulfurylase, pyrophosphatase, APS reductase, sulfite reductase, thiosulfate reductase, as well as adenylate kinase and hydrogenase) were detected in sufficient activities to account for the growth rates observed. ADP sulfurylase and sulfite oxidoreductase activities were not detected. Disproportionation was sensitive to the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) but not to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). It is proposed that during thiosulfate and sulfite disproportionation sulfate is formed via APS reductase and ATP sulfurylase, but not by sulfite oxidoreductase. Reversed electron transport must be assumed to explain the reduction of thiosulfate and sulfite by the electrons derived from APS reductase.Abbreviations CCCP Carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - APS adenosine 5-phosphosulfate (adenylylsulfate)     

Purification and characterization of a sulfite:cytochrome c oxidoreductase from Thiobacillus acidophilus

Cell-free extracts of Thiobacillus acidophilus prepared at neutral pH showed oxidation of sulfite to sulfate with ferricyanide as electron acceptor. Horse heart cytochrome c could be used as alternative electron acceptor; however, the observed activity was only 0.1% of that found for ferricyanide. The enzyme responsible for the oxidation of sulfite was purified to homogeneity. The purified enzyme was a monomer of 42 kDa and contained one haem c per monomer. Electron paramagnetic resonance (EPR) spectroscopical analysis of the sulfite:cytochrome c oxidoreductase showed the presence of molybdenum (V), only after reduction of the enzyme with sulfite. The pH optimum for the enzymatic reaction was 7.5 and the temperature optimum 40°C. Enzymatic activity was strongly reduced in the presence of the anions: chloride, phosphate and nitrate. In contrast to other enzymes involved in sulfur metabolism and previously isolated from T. acidophilus, sulfite:cytochrome c oxidoreductase activity is not stimulated by the presence of sulfate ions.     

Organization of the Human Mitochondrial Hydrogen Sulfide Oxidation Pathway

Sulfide oxidation is expected to play an important role in cellular switching between low steady-state intracellular hydrogen sulfide levels and the higher concentrations where the physiological effects are elicited. Yet despite its significance, fundamental questions regarding how the sulfide oxidation pathway is wired remain unanswered, and competing proposals exist that diverge at the very first step catalyzed by sulfide quinone oxidoreductase (SQR). We demonstrate that, in addition to sulfite, glutathione functions as a persulfide acceptor for human SQR and that rhodanese preferentially synthesizes rather than utilizes thiosulfate. The kinetic behavior of these enzymes provides compelling evidence for the flow of sulfide via SQR to glutathione persulfide, which is then partitioned to thiosulfate or sulfite. Kinetic simulations at physiologically relevant metabolite concentrations provide additional support for the organizational logic of the sulfide oxidation pathway in which glutathione persulfide is the first intermediate formed.     

Characterization of two different 2-oxoglutarate:ferredoxin oxidoreductases from Hydrogenobacter thermophilus TK-6

A thermophilic, chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, fixes carbon dioxide via the reductive TCA cycle. 2-Oxoglutarate:ferredoxin oxidoreductase (OGOR) is one of the key enzymes of this cycle. Strain TK-6 has two distinct OGOR enzymes termed For and Kor. These enzymes were purified and characterized following heterologous expression in Escherichia coli. The specific activity of For was approximately one-tenth of that of Kor. Additionally, For showed higher thermo-stability than Kor under both aerobic and anaerobic conditions. Western blot analysis showed that both of For and Kor were expressed when strain TK-6 was grown under aerobic conditions. In contrast, only Kor was expressed when the strain was grown under anaerobic conditions using nitrate as a terminal electron acceptor. These results indicate that For supports the optimal growth of strain TK-6 in the presence of oxygen.     

Dissimilatory Oxidation and Reduction of Elemental Sulfur in Thermophilic Archaea

The oxidation and reduction of elemental sulfur and reduced inorganic sulfur species are some of the most important energy-yielding reactions for microorganisms living in volcanic hot springs, solfataras, and submarine hydrothermal vents, including both heterotrophic, mixotrophic, and chemolithoautotrophic, carbon dioxide-fixing species. Elemental sulfur is the electron donor in aerobic archaea like Acidianus and Sulfolobus. It is oxidized via sulfite and thiosulfate in a pathway involving both soluble and membrane-bound enzymes. This pathway was recently found to be coupled to the aerobic respiratory chain, eliciting a link between sulfur oxidation and oxygen reduction at the level of the respiratory heme copper oxidase. In contrast, elemental sulfur is the electron acceptor in a short electron transport chain consisting of a membrane-bound hydrogenase and a sulfur reductase in (facultatively) anaerobic chemolithotrophic archaea Acidianus and Pyrodictium species. It is also the electron acceptor in organoheterotrophic anaerobic species like Pyrococcus and Thermococcus, however, an electron transport chain has not been described as yet. The current knowledge on the composition and properties of the aerobic and anaerobic pathways of dissimilatory elemental sulfur metabolism in thermophilic archaea is summarized in this contribution.     

Physiological adaptation of a nitrate-storing Beggiatoa sp. to diel cycling in a phototrophic hypersaline mat

The aim of this study was to investigate the supposed vertical diel migration and the accompanying physiology of Beggiatoa bacteria from hypersaline microbial mats. We combined microsensor, stable-isotope, and molecular techniques to clarify the phylogeny and physiology of the most dominant species inhabiting mats of the natural hypersaline Lake Chiprana, Spain. The most dominant morphotype had a filament diameter of 6 to 8 microm and a length varying from 1 to >10 mm. Phylogenetic analysis by 16S rRNA gene comparison revealed that this type appeared to be most closely related (91% sequence identity) to the narrow (4-microm diameter) nonvacuolated marine strain MS-81-6. Stable-isotope analysis showed that the Lake Chiprana species could store nitrate intracellularly to 40 mM. The presence of large intracellular vacuoles was confirmed by fluorescein isothiocyanate staining and subsequent confocal microscopy. In illuminated mats, their highest abundance was found at a depth of 8 mm, where oxygen and sulfide co-occurred. However, in the dark, the highest Beggiatoa densities occurred at 7 mm, and the whole population was present in the anoxic zone of the mat. Our findings suggest that hypersaline Beggiatoa bacteria oxidize sulfide with oxygen under light conditions and with internally stored nitrate under dark conditions. It was concluded that nitrate storage by Beggiatoa is an optimal strategy to both occupy the suboxic zones in sulfidic sediments and survive the dark periods in phototrophic mats.     

Isolation and characterization of Acidithiobacillus ferrooxidans strain D3-2 active in copper bioleaching from a copper mine in Chile

Acidithiobacillus ferrooxidans strain D3-2, which has a high copper bioleaching activity, was isolated from a low-grade sulfide ore dump in Chile. The amounts of Cu(2+) solubilized from 1% chalcopyrite (CuFeS(2)) concentrate medium (pH 2.5) by A. ferrooxidans strains D3-2, D3-6, and ATCC 23270 and 33020 were 1360, 1080, 650, and 600 mg x l(-1) x 30 d(-1). The iron oxidase activities of D3-2, D3-6, and ATCC 23270 were 11.7, 13.2, and 27.9 microl O(2) uptake x mg protein(-1) x min(-1). In contrast, the sulfite oxidase activities of strains D3-2, D3-6, and ATCC 23270 were 5.8, 2.9, and 1.0 mul O(2) uptake.mg protein(-1).min(-1). Both of cell growth and Cu-bioleaching activity of strains D3-6 and ATCC 23270, but not, of D3-2, in the chalcopyrite concentrate medium were completely inhibited in the presence of 5 mM sodium bisulfite. The sulfite oxidase of strain D3-2 was much more resistant to sulfite ion than that of strain ATCC 23270. Since sulfite ion is a highly toxic intermediate produced during sulfur oxidation that strongly inhibits iron oxidase activity, these results confirm that strain D3-2, with a unique sulfite resistant-sulfite oxidase, was able to solubilize more copper from chalcopyrite than strain ATCC 23270, with a sulfite-sensitive sulfite oxidase.