In situ analysis of sulfur in the sulfur globules of phototrophic sulfur bacteria by X-ray absorption near edge spectroscopy.

During the oxidation of sulfide and thiosulfate purple and green sulfur bacteria accumulate globules of 'elemental' sulfur. Although essential for a thorough understanding of sulfur metabolism in these organisms, the exact chemical nature of the stored sulfur is still unclear. We applied sulfur K-edge X-ray absorption near edge spectroscopy (XANES) to probe the forms of sulfur in intact cells. Comparing XANES spectra of Allochromatium vinosum, Thiocapsa roseopersicina, Marichromatium purpuratum, Halorhodospira halophila and Chlorobium vibrioforme grown photolithoautotrophically on sulfide with reference probes (fingerprint method), we found sulfur chains with the structure R-S(n)-R. Evidence for the presence of sulfur rings, polythionates and anionic polysulfides in the sulfur globules of these bacteria was not obtained.     

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.     

Search for polythionates in cultures of Chromatium vinosum after sulfide incubation

Cultures of Chromatium vinosum, devoid of sulfur globules, were supplemented with sulfide and incubated under anoxic conditions in the light. The concentrations of sulfide, polysulfides, thiosulfate, polythionates and elemental sulfur (sulfur rings) were monitored for 3 days by ion-chromatography and reversed-phase HPLC. While sulfide disappeared rapidly, thiosulfate and elemental sulfur (S₆, S₇ S₈ rings) were formed. After sulfide depletion, the concentration of thiosulfate decreased fairly rapidly, but elemental sulfur was oxidized very slowly to sulfate. Neither polysulfides (S _x ^2– ), polythionates (S_nO ₆ ^2– , n=4–6), nor other polysulfur compounds could be detected, which is in accordance with the fact that sulfide-grown cells were able to oxidize polysulfide without lag. The nature of the intracellular sulfur globules is discussed.     

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.     

Interactions between phototrophic bacteria in sediment ecosystems

The environmental conditions in laminated microbial sediment ecosystems on the island of Schiermonnikoog (The Netherlands) were monitoredin situ over 24-hour periods by using micro-electrodes. In the layer of purple sulfur bacteria dramatic diel aerobic/anaerobic shifts occurred, whereas the top layer of cyanobacteria was occasionally confronted with sulfide. Pure cultures of the dominant organisms, being the cyanobacteriumMicrocoleus chthonoplastes and the purple sulfur bacteriumThiocapsa roseopersicina, were subjected to regimes mimicking the natural circumstances. It was demonstrated that both organisms are physiologically very well adapted to the fluctuating environmental conditions. The organisms interact by releasing metabolic end-products, the removal of toxic compounds and by competition for common substrates. It was demonstrated that positive interactions between both organisms are more important than negative interactions.     

Structure and development of a benthic marine microbial mat

Abstract Vertically stratified microbial communities of phototrophic bacteria in the upper intertidal zones of the North Sea island of Mellum were investigated. Growth and population dynamics of the cyanobacterial mat were followed over three successive years. It was concluded that the initial colonization of the sandy sediments was by the cyanobacterium Oscillatoria . In well-established mats, however, the dominant organism was Microcoleus chthonoplastes . The observed succession of cyanobacteria during mat development is correlated with nitrogen fixation. Nitrogen fixation is necessary in this low-nutrient environment to ensure colonization by mat-constructing cyanobacteria. Under certain conditions, a red layer of purple sulfur bacteria developed underneath the cyanobacterial mat in which Chromatium and Thiocapsa spp. dominated, but Thiopedia and Ectothiorhodospira spp. have also been observed. Measurements of light penetrating the cyanobacterial mat indicated that sufficient light is available for the photosynthetic growth of purple sulfur bacteria. Profiles of oxygen, sulfide and redox potential within the microbial mat were measured using microelectrodes. Maximum oxygen concentrations, measured at a depth of 0.7 mm, reached levels more than twice the normal air saturation. Dissolved sulfide was not detected by the microelectrodes. Determination of acid-distilled sulfide, however, revealed appreciable amounts of bound sulfide in the mat. Redox profiles measured in the mat led to the conclusion that the upper 10 mm of the sedimentary sequence is in a relatively oxidized state.     

Coexistence of aerobic chemotrophic and anaerobic phototrophic sulfur bacteria under oxygen limitation

Abstract: The aerobic chemotrophic sulfur bacterium Thiobacillus thioparus T5 and the anaerobic phototrophic sulfur bacterium Thiocapsa roseopersicina M1 were co-cultured in continuously illuminated chemostats at a dilution rate of 0.05 h⁻¹. Sulfide was the only externally supplied electron donor, and oxygen and carbon dioxide served as electron acceptor and carbon source, respectively. Steady states were obtained with oxygen supplies ranging from non-limiting amounts (1.6 mol O₂ per mol sulfide, resulting in sulfide limitation) to severe limitation (0.65 mol O₂ per mol sulfide). Under sulfide limitation Thiocapsa was competitively excluded by Thiobacillus and washed out. Oxygen/sulfide ratios between 0.65 and 1.6 resulted in stable coexistence. It could be deduced that virtually all sulfide was oxidized by Thiobacillus . The present experiments showed that Thiocapsa is able to grow phototrophically on the partially oxidized products of Thiobacillus . In pure Thiobacillus cultures in steady state extracellular zerovalent sulfur accumulated, in contrast to mixed cultures. This suggests that a soluble form of sulfur at the oxidation state of elemental sulfur is formed by Thiobacillus as intermediate. As a result, under oxygen limitation colorless sulfur bacteria and purple sulfur bacteria do not competitively exclude each other but can coexist. It was shown that its ability to use partially oxidized sulfur compounds, formed under oxygen limiting conditions by Thiobacillus , helps explain the bloom formation of Thiocapsa in marine microbial mats.     

Development of mass blooms of Thiocapsa roseopersicina on sheltered beaches on the Orkney Islands

Abstract Mass developments of the purple sulfur bacterium Thiocapsa roseopersicina in the surface layers of sandy beaches on the Orkney Islands were examined with respect to microcolony formation on sand grains, vertical distribution of viable cells and the ability to colonize beach surfaces. It was observed that microcolonies of the non-motile phototrophic bacterium cemented individual sand grains to each other and that the resulting aggregates could withstand severe wave action and may play a decisive role in the stabilization of these sandy beaches.
After removal of the top layer similar population densities of T. roseopersicina were recorded within seven days. It was calculated that the net specific growth rate initially was 0.53 day⁻¹ (0.022 h⁻¹).
Laboratory studies strongly suggest that the populations of T. roseopersicina on sheltered beaches on the Orkney Islands were growing phototrophically in the light even when the microenvironment was oxic. Bacteriochlorophyll a synthesis was repressed by oxygen and occurred during periods with low light intensities when the microenvironment was anoxic and contained sulfide.     

Competition between anoxygenic phototrophic bacteria and colorless sulfur bacteria in a microbial mat

Abstract The populations of chemolithoautotrophic (colorless) sulfur bacteria and anoxygenic phototrophic bacteria were enumerated in a marine microbial mat. The highest population densities were found in the 0–5 mm layer of the mat: 2.0 × 10⁹ cells cm⁻³ sediment, and 4.0 × 10⁷ cells cm⁻³ sediment for the colorless sulfur bacteria and phototrophs, respectively. Kinetic parameters for thiosulfate-limited growth were assessed for Thiobacillus thioparus T5 and Thiocapsa roseopersicina M1, both isolated from microbial mats. For Thiobacillus T5, growing at a constant oxygen concentration of 43 μmol l⁻¹, μ_max was 0.336 h⁻¹ and K _s 0.8 μmol l⁻¹. Phototrophically grown Thiocapsa strain M1 displayed a μ_max of 0.080 h⁻¹ and a K _s of 8 μmol l⁻¹ when anoxically grown under thiosulfate limitation. In a competition experiment with thiosulfate as electron donor, Thiocapsa became dominant during a 10-h oxic/14-h anoxic regimen at continuous illumination, despite the higher affinity for thiosulfate of Thiobacillus .     

嗜酸硫氧化细菌消解元素硫的研究进展

浸矿酸性环境下,金属硫化矿在Fe3+作用下,经过硫代硫酸盐途径或多聚硫化氢途径而分解的过程中导致大量元素硫的累积,进而可能在金属硫化矿表面形成疏水元素硫层,阻碍金属离子的进一步浸出。酸性环境下,惰性元素硫的消解必须借助嗜酸硫氧化细菌来实现。该消解过程包括嗜酸硫氧化细菌对元素硫的吸附、转运以及氧化转化等过程。本文对近年来嗜酸硫氧化细菌消解元素硫过程的相关研究进行了全面评述,认为有关嗜酸硫氧化细菌消解元素硫的分子机制的清晰阐述还有待人们通过对消解过程的各个环节的分子机制进行大量研究来实现。     

Characterization of a new platelet-forming purple sulfur bacterium,Amoebobacter pedioformis sp. nov.

The dominant purple sulfur bacterium of a reddish-colored waste water pond near Taichung, Taiwan, was isolated in pure culture, strain CML2. Individual cells were nearly spherical, nonmotile, and contained in their peripheral parts was vacuoles that appeared like elongated, curved tubes. Four to sixteen cells formed platelet-like aggregates reminiscent of Thiopedia rosea. The intracellular photosynthetic membrane system of the cells was of vesicular type; the photosynthetic pigments consisted of bacteriochlorophyll a and spirilloxanthin as the major carotenoid. The color of cell suspensions was pink to rosered. Under anaerobic conditions photolithoautotrophic growth occurred with sulfide, elemental sulfur or thiosulfate; sulfur globules were stored as an intermediary oxidation product. In the presence of sulfide, acetate, lactate and pyruvate were photoassimilated; strain CML2 lacked assimilatory sulfate reduction. Fastest photoautotrophic growth (11 h doubling time) was obtained at pH 7.5, 35°C and a light intensity of about 1000 lux (tungsten lamp). Chemolithoautotrophic growth in the dark was possible under reduced oxygen partial pressure with reduced sulfur compounds as respiratory substrates. The DNA base composition of strain CML2 was 65.5 mol% G+C. Strain CML2 is described as type strain of a new species, Amoebobacter pedioformis sp. nov., in the family Chromatiaceae.     

Occurrence of Purple Sulfur Bacteria in a Sewage Treatment Lagoon

The ecology of purple sulfur bacteria in a sewage oxidation lagoon was investigated. Chemical changes in the lagoon were investigated by monitoring biochemical oxygen demand (BOD(5)), sulfide, sulfate, phosphate, total carbohydrates, volatile acids, alkalinity, and pH. Lagoon water temperatures were observed daily. Microbial ecological relationships were deduced by enumerating coliforms, total bacteria other than anaerobes [Tryptone Glucose Extract (TGE) agar], methane formers such as Methanobacterium formicicum, sulfate reducers, purple sulfur bacteria, and algae. Finally, two strains of purple sulfur bacteria were characterized. Two populations, purple sulfur bacteria and total bacteria (TGE agar), reached maximal concentrations in the warmest part of the 1967 summer. Purple sulfur bacteria reached maximal numbers as concentrations of sulfide and volatile acids were depleted, whereas carbohydrates and alkalinity remained unchanged. Low sulfate levels, which were not limiting for sulfate reducers, may be attributable to storage of sulfur within purple sulfur bacteria. No biological, chemical, or physical agent was linked to the removal of coliforms. The increase of algae in the late summer of 1967 may have been related to the low organic content of the lagoon during this period. Although lagoon pH (7.7 to 8.2) was favorable for purple sulfur bacterial growth, temperatures and sulfides were not optimal in the lagoon for these organisms. Chromatium vinosum and Thiocapsa floridana (the predominant lagoon purple sulfur organism in 1967 and 1968) utilized certain carbohydrates, amino acids, volatile acids, and Krebs cycle intermediates. Also purple sulfur bacteria lowered BOD levels as demonstrated by the growth of T. floridana in sterilized sewage.     

Photo-autotrophic growth of Thiocapsa roseopersicina on dimethyl sulfide

Abstract: The purple sulfur bacterium Thiocapsa roseopersicina was examined for photo-autotrophic growth on dimethyl sulfide (DMS). The maximum specific growth rate μ _max (0.068 h⁻¹), saturation constant K _s (38 μm l⁻¹), and yield (5.24 mg protein mmol⁻¹ DMS) were determined in chemostat experiments. Dimethyl sulfoxide was the only product of DMS oxidation. Batch experiments revealed the simultaneous oxidation of DMS and hydrogen sulfide.     

Thiosulfate, polythionates and elemental sulfur assimilation and reduction in the bacterial world

Among sulfur compounds, thiosulfate and polythionates are present at least transiently in many environments. These compounds have a similar chemical structure and their metabolism appears closely related. They are commonly used as energy sources for photoautotrophic or chemolithotrophic microorganisms, but their assimilation has been seldom studied and their importance in bacterial physiology is not well understood. Almost all bacterial strains are able to cleave these compounds since they possess thiosulfate sulfur transferase, thiosulfate reductase or S-sulfocysteine synthase activities. However, the role of these enzymes in the assimilation of thiosulfate or polythionates has not always been clearly established. Elemental sulfur is, on the contrary, very common in the environment. It is an energy source for sulfur-reducing eubacteria and archaebacteria and many sulfur-oxidizing archaebacteria. A phenomenon still not well understood is the 'excessive assimilatory sulfur metabolism' as observed in methanogens which perform a sulfur reduction which exceeds their anabolic needs without any apparent benefit. In heterotrophs, assimilation of elemental sulfur is seldom described and it is uncertain whether this process actually has a physiological significance. Thus, reduction of thiosulfate and elemental sulfur is a common but incompletely understood feature among bacteria. These activities could give bacteria a selective advantage, but further investigations are needed to clarify this possibility. Presence of thiosulfate, polythionates and sulfur reductase activities does not imply obligatorily that these activities play a role in thiosulfate, polythionates or sulfur assimilation as these compounds could be merely intermediates in bacterial metabolism. The possibility also exists that the assimilation of these sulfur compounds is just a side effect of an enzymatic activity with a completely different function. As long as these questions remain unanswered, our understanding of sulfur and thiosulfate metabolism will remain incomplete.     

Thiosulfate, polythionates and elemental sulfur assimilation and reduction in the bacterial world

Abstract Among sulfur compounds, thiosulfate and polythionates are present at least transiently in many environments. These compounds have a similar chemical structure and their metabolism appears closely related. They are commonly used as energy sources for photoautotrophic or chemolithotrophic microorganisms, but their assimilation has been seldom studied and their importance in bacterial physiology is not well understood. Almost all bacterial strains are able to cleave these compounds since they possess thiosulfate sulfur transferase, thiosulfate reductace or S -sulfocysteine synthase activities. However, the role of these enzymes in the assimilation of thiosulfate or polythionates has not always been clearly established.
Elemental sulfur is, on the contrary, very common in the environmental. It is an energy source for sulfur-reducing eubacteria and archaebacteria and many sulfur-oxidizing archaebacteria. A phenomenon still not well understood is the 'excessive assimilatory sulfur metabolism' as observed in methanogens which perform a sulfur reduction which exceeds their anabolic needs without any apparent benefit. In heterotrophs, assimilation of elemental sulfur is seldom described and it is uncertain whether this process actually has a physiological significance.
Thus, reduction of thiosulfate and elemental sulfur is a common by incompletely understood feature among bacteria. These activities could give bacteria a selective advantage, but futher investigations are needed to clarify this possibility. Presence of thiosulfate, polythionates and sulfur reductase activities does not imply obligatorily that these activities play a role in thiosulfate, polythionates or sulfur assimilation as these compounds could be merely intermediates in bacterial metabolism. The possibility also exists that the assimilation of these sulfur compounds is just a side effect of an enzymatic activity with a completely different function.     

Sulfide oxidation under oxygen limitation by a thiobacillus thioparus isolated from a marine microbial mat

Abstract The colorless sulfur bacterium Thiobacillus thioparus T5, isolated from a marine microbial mat, was grown in continuous culture under conditions ranging from sulfide limitation to oxygen limitation. Under sulfide-limiting conditions, sulfide was virtually completely oxidized to sulfate. Under oxygen-limiting conditions, sulfide was partially oxidized to zerovalent sulfur (75%) and thiosulfate (17%). In addition, low concentrations of tetrathionate and polysulfide were detected. The finding of in vivo thiosulfate formation supports the discredited observations of thiosulfate formation in cell free extracts in the early sixties. In a microbial mat most sulfide oxidation was shown to take place under oxygen-limiting conditions. It is suggested that zerovalent sulfur formation by thiobacilli is a major process resulting in polysulfide accumulation. Implications for the competition between colorless sulfur bacteria and purple sulfur bacteria are discussed.     

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.     

Rates of sulfate reduction and thiosulfate consumption in a marine microbial mat

Abstract The sulfur cycle in a microbial mat was studied by determining viable counts of sulfate-reducing bacteria, chemolithoautotrophic sulfur bacteria and anoxygenic phototrophic bacteria. All three functional groups of sulfur bacteria revealed a maximum population density in the uppermost 5 mm of the mat: 1.1 × 10⁸ cells of sulfate reducers cm⁻³ sediment, 2.0 × 10⁹ cells of chemolithoautotrophs cm⁻³ sediment, and 4.0 × 10⁷ cells of anoxygenic phototrophs cm⁻³ sediment. Bacterial dynamics were studied by sulfate reduction rate measurements, both under anoxic conditions (dark incubation) and oxic conditions (incubation in the light), and determination of the vertical distribution of the potential rate of thiosulfate consumption under oxic conditions. Sulfate reduction rates in the top 5 mm of the sediment were 566 nmol cm⁻³ d⁻¹ in the absence of oxygen, and 123 nmol cm⁻³ d⁻¹ in the presence of oxygen. In the latter case, the maximum rate was found in the 5–10-mm depth horizon (361 nmol cm⁻³ d⁻¹). Biological consumption of amended thiosulfate was rapid and decreased with depth, while in the presence of molybdate, thiosulfate consumption decreased to 10–30% of the original rate.     

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.     

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