Anaerobic oxidation of thiosulfate and elemental sulfur in Thiobacillus denitrificans

Thiobacillus denitrificans strain RT could be grown anaerobically in batch culture on thiosulfate but not on other reduced sulfur compounds like sulfide, elemental sulfur, thiocyanate, polythionates or sulfite. During growth on thiosulfate the assimilated cell sulfur was derived totally from the outer or sulfane sulfur. Thiosulfate oxidation started with a rhodanese type cleavage between sulfane and sulfone sulfur leading to elemental sulfur and sulfite. As long as thiosulfate was present elemental sulfur was transiently accumulated within the cells in a form that could be shown to be more reactive than elemental sulfur present in a hydrophilic sulfur sol, however, less reactive than sulfane sulfur of polythionates or organic and inorganic polysulfides. When thiosulfate had been completely consumed, intracellular elemental sulfur was rapidly oxidized to sulfate with a specific rate of 45 natom S°/min·mg protein. Extracellularly offered elemental sulfur was not oxidized under anaerobic conditions.     

硫氧化菌种脱除硫化物生成单质硫限制性因素优化

【目的】针对硫氧化菌种较为特殊的生化特性,优选其氧化硫化物生成单质硫过程的相关限制性因素,以提高该类菌种生成单质硫效率。【方法】采用一株典型脱硫菌Thermithiobacillus tepidarius JNU-2(T.tepidarius JNU-2)氧化硫化物生成单质硫。研究该菌株在以Na2S2O3为能源底物时的培养特性和脱硫性能,并结合单因素实验对菌株氧化硫化物生成单质硫的限制性因素进行优选。【结果】T.tepidarius JNU-2在以Na2S2O3为唯一能源底物培养时的μmax为0.207 h-1,最终生物量为4.0×106 cells/m L。98%的Na2S2O3在24 h时被消耗殆尽,此时单质硫产量达到最大值为0.8 g/L。随后单质硫逐渐被氧化利用,最终稳定在0.2 g/L。经过对该过程主要限制性因素进行单因素实验优化,确定最佳碳氮源、Mg SO4、Fe SO4和能源底物条件分别为:CO2、NH4Cl0.5 g/L、Mg SO4 0.5 g/L、Fe SO4 0.1 g/L和Na2S2O3 15.0 g/L。优化后的氧化Na2S2O3生成单质硫过程的最大生物量可达4.8×106 cells/m L,单质硫产量提升至1.14 g/L。相较于未优化之前,单质硫的产量提高了42.5%。【结论】优化该过程主要限制性因素可有效提高化能自养型T.tepidarius JNU-2氧化硫化物生成单质硫效率。     

Fate of elemental sulfur in an intertidal sediment

Abstract: Sediment from a tidal flat at Wedderwarden, near the mouth of the Weser estuary, northern Germany, was amended with elemental sulfur, and concentrations of metabolic end products were monitored. The production of both sulfate and sulfide was consistent with disproportionation as the most important fate of the added elemental sulfur. A population of bacteria conducting active elemental sulfur disproportionation was also enriched from the sediment. In the enrichments, containing both elemental sulfur and Fe oxides as a sulfide 'scrub', sulfide and sulfate were produced in a ratio of     , somewhat lower than the predicted ratio of     . The mismatch between predicted and observed production ratios is explained by the channelling of electrons into autotrophic or mixotrophic CO₂ fixation rather than sulfide formation. The production of organic carbon, in the correct amount to explain the observed sulfide to sulfate production ratio, was verified by organic carbon analysis. Finally, rates of sulfate reduction were identical in the elemental sulfur amended sediment, and in control sediment with no added sulfur. Hence, the heterotrophic bacterial community was completely unaffected by an active metabolism conducting elemental sulfur disproportionation.     

Genes involved in hydrogen and sulfur metabolism in phototrophic sulfur bacteria

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

Chemolithoautotrophic growth on elemental sulfur (S°) and respiratory oxidation of S° by Thiobacillus versutus and another sulfur-oxidizing bacterium

Abstract Thiobacillus versutus was shown to grow chemolithoautotrophically under microaerophilic conditions, with crystalline elemental sulfur (S°) and thiosulfate as sole electron source. The exponential growth rate on S° ( μ = 0.106 h⁻¹) measured in batch culture was similar to the reported maximum growth rate on thiosulfate in chemostat cultures. The rates of thiosulfate, S° and sulfite oxidation were measured respirometrically using an oxygen electrode. During growth under air on thiosulfate, as well as under low oxygen pressure on S° and thiosulfate, a relatively strong sulfuroxidizing activity (SOA) was measured. The induction of the SOA on cells growing with thiosulfate and the similar growth rates on S° and thiosulfate strongly suggest that S° could be an important intermediate during thiosulfate utilization.     

The role of the sulfur globule proteins of Allochromatium vinosum: mutagenesis of the sulfur globule protein genes and expression studies by real-time RT-PCR

During oxidation of reduced sulfur compounds, the purple sulfur bacterium Allochromatium vinosum stores sulfur in the periplasm in the form of intracellular sulfur globules. The sulfur in the globules is enclosed by a protein envelope that consists of the homologous 10.5-kDa proteins SgpA and SgpB and the smaller 8.5-kDa SgpC. Reporter gene fusions of sgpA and alkaline phosphatase showed the constitutive expression of sgpA in A. vinosum and yielded additional evidence for the periplasmic localization of the sulfur globules. Expression analysis of the wild-type sgp genes by quantitative RT-PCR using the LightCycler system showed the constitutive expression of all three sgp genes. The expression of sgpB and sgpC is significantly enhanced under photolithotrophic conditions. Interestingly, sgpB is expressed ten times less than sgpA and sgpC implying that SgpA and SgpC are the main proteins of the sulfur globule envelope. Mutants with inactivated sgpA or sgpB did not show any differences in comparison with the wild-type, i.e., the encoded proteins can replace each other, whereas inactivation of sgpC leads to the formation of considerably smaller sulfur globules. This indicates a role of SgpC for globule expansion. A sgpBC double mutant was unable to grow on sulfide and could not form sulfur globules, showing that the protein envelope is indispensible for the formation and deposition of intracellular sulfur.The paper is dedicated to Prof. Dr. Dr. h.c. mult. Hans Günter Schlegel, Göttingen, on the occasion of his 80th birthday on October 24th, 2004, with great gratitude, as our interest in microbial sulfur metabolism goes back to the early 1960s, when HGT worked in Prof. Schlegels laboratory and in 1972 established this field in Bonn.     

Physiology of purple sulfur bacteria forming macroscopic aggregates in Great Sippewissett Salt Marsh, Massachusetts

Abstract Purple bacterial aggregates found in tidal pools of Great Sippewissett Salt Marsh (Falmouth, Cape Cod, MA) were investigated in order to elucidate the ecological significance of cell aggregation. Purple sulfur bacteria were the dominant microorganisms in the aggregates which also contained diatoms and a high number of small rod-shaped bacteria. Urea in concentrations of ≥ 1 M caused disintegration of the aggregates while proteolytic enzymes, surfactants or chaotropic agents did not exhibit this effect. This suggests that polysaccharides in the embedding slime matrix stabilize the aggregate structure. In addition cell surface hydrophobicity is involved in aggregate formation. The concentration of dissolved oxygen decreased rapidly below the surface of aggregates while sulfide was not detected. The apparent respiration rate in the aggregates was high when the purple sulfur bacteria contained intracellular sulfur globules. In the presence of DCMU, respiration remained light-inhibited. Light inhibition disappeared in the presence of KCN. These results demonstrated that respiration in the aggregates is due mainly to purple sulfur bacteria. The concentration of bacteriochlorophyll (Bchl) a in the aggregates (0.205 mg Bchl a cm⁻³) was much higher than in the pool sediments but comparable to concentrations in microbial mats of adjacent sand flats. Purple aggregates may therefore originate in the microbial mats rather than in the pools themselves. Rapid sedimentation and high respiration rates of Chromatiaceae in the aggregates would prevent the inhibition of Bchl synthesis if aggregates were lifted off the sediment and up into the oxic pool water by tidal currents.     

Effects of stream phosphorus levels on microbial respiration

SUMMARY 1. We examined microbial respiration among streams in lowland Costa Rica comprising a natural phosphorus gradient (5–350 μg SRP L^?1) resulting from variable inputs of solute‐rich (e.g. P, SO₄ and Cl) groundwater. 2. Microbial respiration rates were determined by measuring oxygen change in situ in nine low‐order streams on three substrate types: mixed leaves collected from the stream bottom, conditioned Ficus leaves and sediments. 3. Respiration rates on both leaf types were positively related to phosphorus and negatively related to N : P ratios. Microbial respiration rates on sediments were not related to any of the variables [i.e. soluble reactive phosphorus (SRP), N‐NO₃ and N : P] measured. 4. Respiration rates on newly colonised Ficus leaves formed an asymptotic curve increasing to a plateau, suggesting that saturation with phosphorus occurred at concentrations <15 μg SRP L^?1. 5. To test the hypothesis that phosphorus was the main solute in solute‐rich water that was driving observed differences in microbial respiration rates, we artificially enriched a small stream with phosphorus and measured changes in respiration before and after enrichment. 6. Experimental phosphorus enrichment produced increases in respiration rates similar in magnitude to those observed in the nine streams forming the natural phosphorus gradient, supporting our hypothesis that phosphorus was the major variable driving interstream differences in microbial respiration rates. Respiration rates were higher in this study than those reported for most other tropical streams and rivers with the exception of those reported for tropical Asian streams. 7. Results indicate that variations in phosphorus concentrations can potentially affect patterns of microbial respiration rates at a landscape level via differential inputs of solute‐rich groundwater into streams.     

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     

N-acetyl-L-cysteine as a source of sulfane sulfur in astrocytoma and astrocyte cultures: correlations with cell proliferation

Summary. N-acetyl-L-cysteine (NAC), a precursor of L-cysteine, not only elevates the level of glutathione in both astrocytoma and astrocyte cultures, but also affects the cellular level of sulfane sulfur. Astrocytoma cells were investigated using the stable U373 human cell line. In the U373 cells, N-acetyl-L-cysteine, depending on the concentration in the culture medium and culture duration, either elevated or diminished the level of sulfane sulfur, and this was respectively accompanied by decreased or increased cellular proliferation. In murine astrocytes, in turn, NAC was capable of lowering the level of sulfane sulfur and in this way decreased cellular proliferation. It seems that normal (astrocyte) and transformed (astrocytoma) cells differed in their reaction to NAC in the culture medium. The effect of N-acetyl-L-cysteine on astrocytoma cells was advantageous in that it inhibited their proliferation through the elevation of the level of sulfane sulfur. Authors’ address: Maria Wróbel, Chair of Medical Biochemistry, Jagiellonian University Medical College, Kopernika 7 St., 31-034 Kraków, Poland     

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。     

Investigations on microbial sulfur respiration. Isolation, purification, and characterization of cellular components from Spirillum 5175.

The sulfur-reducing bacterium Spirillum 5175 was investigated with regard to membrane constituents that might be part of the sulfur oxidoreductase which converts elemental sulfur to hydrogen sulfide. Regardless of the electron acceptor used for cultivation of the bacteria, i.e. elemental sulfur, fumarate, or nitrate (Sp. 5175S,F,N), the qualitative pattern of cytochromes and Fe-S proteins did not change significantly, as documented by ultraviolet/visible and electron paramagnetic resonance spectroscopy of oxidized (as isolated) and reduced (dithionite) samples. With elemental sulfur the prominent cytochrome exhibited absorption maxima at 553, 522.5 and 426 nm in the reduced state. In fumarate-grown cells two prominent cytochromes were found with maxima at 561, 551, 530, 521 and 430 nm. Two b-type cytochromes with Em at -198 mV and -20 mV vs the standard hydrogen electrode were identified in the membrane fraction of Sp. 5175F. A yellow pigment was extracted and identified as a flexirubin-type pigment. Although present in large quantities, it seemed not to be involved in the reduction of elemental sulfur. Menaquinone, MK 6 (Mr 580) was the prominent quinone identified in Sp. 5175. Characterization of a second quinone was not attempted because of its much lower concentration. The membrane constituents of Sp. 5175 were solubilized by a variety of detergents and detergent mixtures. A colorimetric procedure with photochemically reduced phenosafranin as the electron donor and cysteamine trisulfide (RS-S-SR, R = -CH2CH2NH2) as the electron acceptor was used to detect sulfur oxidoreductase activity. Three membrane proteins of Sp. 5175 were purified: (1) an [NiFe] hydrogenase, homogeneous by SDS/polyacrylamide gel electrophoresis, with electron paramagnetic resonance signals as isolated at gx,y,z = 2.01, 2.16, 2.33 (100 K), and a strong signal at g = 2.02 below 20 K; (2) a cytochrome b, Fe-S-dependent fumarate reductase, and (3) a protein apparently linked to the sulfur oxidoreductase activity. In contrast to fumarate reductase, no b-type cytochrome was present in the fractions exhibiting sulfur oxidoreductase activity. The presence of Fe-S centers was demonstrated by electron paramagnetic resonance spectroscopy at 10 K. It is not clear whether the c-type cytochrome in the same fractions is part of the sulfur-reducing apparatus of Sp. 5175.     

Susceptibility of various purple and green sulfur bacteria to different antimicrobial agents

Abstract Several purple and green sulfur bacteria (genera Chromatium, Thiocapsa and Chlorobium ) were tested for their sensitivity to different antimicrobial agents by a disc diffusion assay, using thioacetamide as a source of hydrogen sulfide for plate growth. Chlorobium limicola strains were more sensitive to amoxicillin, erythromycin and nalidixic acid, whereas gentamicin and netilmicin were more active against the purple bacteria tested. None of the organisms were sensitive to oxacillin and trimethoprim + sulfamethoxazole. The critical concentrations at the edge of the inhibition zone were also calculated for three organisms and the antimicrobials colistin, mitomycin C, penicillin G, rifampicin, and streptomycin. The results obtained suggest that colistin, mitomycin C, penicillin G would provide selective conditions against the growth of Chlorobium limicola strains, while streptomycin and other aminoglycoside antibiotics would select against purple bacteria.     

Oxidative metabolism of inorganic sulfur compounds by bacteria

The history of the elucidation of the microbiology and biochemistry of the oxidation of inorganic sulfur compounds in chemolithotrophic bacteria is briefly reviewed, and the contribution of Martinus Beijerinck to the study of sulfur-oxidizing bacteria highlighted. Recent developments in the biochemistry, enzymology and molecular biology of sulfur oxidation in obligately and facultatively lithotrophic bacteria are summarized, and the existence of at least two major pathways of thiosulfate (sulfur and sulfide) oxidation confirmed. These are identified as the Paracoccus sulfur oxidation (or PSO) pathway and the S4intermediate (or S4I) pathway respectively. The former occurs in organisms such as Paracoccus (Thiobacillus) versutus and P. denitrificans, and possibly in Thiobacillus novellus and Xanthobacter spp. The latter pathway is characteristic of the obligate chemolithotrophs (e.g. Thiobacillus tepidarius, T. neapolitanus, T. ferrooxidans, T. thiooxidans) and facultative species such as T. acidophilus and T. aquaesulis, all of which can produce or oxidize tetrathionate when grown on thiosulfate. The central problem, as yet incompletely resolved in all cases, is the enzymology of the conversion of sulfane-sulfur (as in the outer [S-] atom of thiosulfate [-S-SO3-]), or sulfur itself, to sulfate, and whether sulfite is involved as a free intermediate in this process in all, or only some, cases. The study of inorganic sulfur compound oxidation for energetic purposes in bacteria (i.e. chemolithotrophy and sulfur photolithotrophy) poses challenges for comparative biochemistry. It also provides evidence of convergent evolution among diverse bacterial groups to achieve the end of energy-yielding sulfur compound oxidation (to drive autotrophic growth on carbon dioxide) but using a variety of enzymological systems, which share some common features. Some new data are presented on the oxidation of 35S-thiosulfate, and on the effect of other anions (selenate, molybdate, tu ngstate, chromate, vanadate) on sulfur compound oxidation, including observations which relate to the roles of polythionates and elemental sulfur as intermediates.     

Sulfide oxidation in the phototrophic sulfur bacterium Chromatium vinosum

Sulfide oxidation in the phototrophic purple sulfur bacterium Chromatium vinosum D (DSMZ 180^T) was studied by insertional inactivation of the fccAB genes, which encode flavocytochrome c, a protein that exhibits sulfide dehydrogenase activity in vitro. Flavocytochrome c is located in the periplasmic space as shown by a PhoA fusion to the signal peptide of the hemoprotein subunit. The genotype of the flavocytochrome-c-deficient Chr. vinosum strain FD1 was verified by Southern hybridization and PCR, and the absence of flavocytochrome c in the mutant was proven at the protein level. The oxidation of thiosulfate and intracellular sulfur by the flavocytochrome-c-deficient mutant was comparable to that of the wild-type. Disruption of the fccAB genes did not have any significant effect on the sulfide-oxidizing ability of the cells, showing that flavocytochrome c is not essential for oxidation of sulfide to intracellular sulfur and indicating the presence of a distinct sulfide-oxidizing system. In accordance with these results, Chr. vinosum extracts catalyzed electron transfer from sulfide to externally added duroquinone, indicating the presence of the enzyme sulfide:quinone oxidoreductase (EC 1.8.5.-). Further investigations showed that the sulfide:quinone oxidoreductase activity was sensitive to heat and to quinone analogue inhibitors. The enzyme is strictly membrane-bound and is constitutively expressed. The presence of sulfide:quinone oxidoreductase points to a connection of sulfide oxidation to the membrane electron transport system at the level of the quinone pool in Chr. vinosum. Received: 5 November 1997 / Accepted: 30 March 1998     

湖泊硫循环微生物研究进展

湖泊是响应气候和环境变化的关键生态系统,是研究元素(如碳、氮和硫等)生物地球化学循环的热点环境。湖泊(尤其咸盐湖)具有硫酸盐含量高且含硫化合物种类丰富的特点,因而湖泊中硫元素生物地球化学循环过程非常活跃。微生物是驱动湖泊硫循环的重要推手。因此,研究湖泊中微生物参与的硫元素生物地球化学循环过程以及相关微生物类群构成,对于深入探索微生物在湖泊生态系统中的作用具有重要意义。本文综述了湖泊中驱动硫循环的微生物(硫氧化菌和硫酸盐还原菌)种群多样性、功能基因、代谢途径、硫氧化/硫酸盐还原速率及其对环境条件变化响应等方面的研究现状,并对未来湖泊微生物驱动的硫循环研究方向进行了展望。     

Bacteriological studies on the sulfur cycle in the anaerobic part of the hypolimnion and in the surface sediments of rotsee in Switzerland scientific report of the advanced course of microbial ecology, sponsored by FEMS and the Swiss society for Microbiology, held at Kastanienbaum, Switzerland, 12 September–9 October 1982

Abstract The microbial ecology of the sulfur cycle in the anaerobic part of Rotsee (Switzerland) was studied. Almost all the sulfate reduction took place at the sediment surface at a rate of 2 mmol SO²⁻₄ reduced m⁻² day⁻¹. Approx. 10⁴ sulfate reducers per ml were present in the surface sediments. The sulfide produced was phototrophically consumed mainly by Thiopedia rosea, Lamprocystis roseopersicina and ' Pelochromatium roseum ' consortia. Thiopedia rosea migrated diurnally about one meter. Bacterial photosynthesis was limited by light and sulfide rather than by temperature.     

Respiratory oxidation of reduced sulfur compounds by intact cells of Thiobacillus tepidarius (type strain)

Thiobacillus tepidarius (type strain) was grown in microaerophilic conditions, on tetrathionate, thiosulfate or crystalline S^o. The rates of tetrathionate, thiosulfate, elemental sulfur (S^o) and sulfite oxidation of the different cultures were measured respirometrically, using exponentially growing cells, with an oxygen electrode. Cells growing on the three different sulfur compounds retain thiosulfate-, tetrathionate, and S^o-oxidizing activities (SOA), but lack respiratory sulfite-oxidizing activity. The SOA for all the cultures was almost totally inhibited by 50 M myxothiazol, an inhibitor of the quinone-cytochrome b region, and by 10 M of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP). Tetrathionate- and thiosulfate-oxidizing activities were moderately and weakly inhibited by 50 M totally inhibited (>95%) all respiratory activities. This study suggests that electrons released by S^o oxidation enter the respiratory chain in the quinone-cytochrome b region.Abbreviation SOA sulfur-oxidizing activity