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.     

硫氧化细菌源单质硫的生成、转运和回收

单质硫(硫粒)是硫化物生物氧化的中间产物.按化学计量式精准调控O/S比(溶解氧与硫化物的摩尔比),单质硫可成为硫氧化细菌(Sulfur-oxidizing bacteria,SOB)的主要代谢产物.根据单质硫的分布,单质硫可分为胞内硫粒和胞外硫粒.单质硫由胞内向胞外的跨膜转运过程是泌硫型SOB的重要生理特征.从生物脱硫...     

Analysis of sulfur biochemistry of sulfur bacteria using X-ray absorption spectroscopy.

Many sulfide-oxidizing organisms, including the photosynthetic sulfur bacteria, store sulfur in "sulfur globules" that are readily detected microscopically. The chemical form of sulfur in these globules is currently the focus of a debate, because they have been described as "liquid" by some observers, although no known allotrope of sulfur is liquid at physiological temperatures. In the present work we have used sulfur K-edge X-ray absorption spectroscopy to identify and quantify the chemical forms of sulfur in a variety of bacterial cells, including photosynthetic sulfur bacteria. We have also taken advantage of X-ray fluorescence self-absorption to derive estimates of the size and density of the sulfur globules in photosynthetic bacteria. We find that the form of sulfur that most resembles the globule sulfur is simply solid S(8), rather than more exotic forms previously proposed.     

Filamentous sulfur bacteria preserved in modern and ancient phosphatic sediments: implications for the role of oxygen and bacteria in phosphogenesis

Marine phosphate‐rich sedimentary deposits (phosphorites) are important geological reservoirs for the biologically essential nutrient phosphorous. Phosphorites first appear in abundance approximately 600 million years ago, but their proliferation at that time is poorly understood. Recent marine phosphorites spatially correlate with the habitats of vacuolated sulfide‐oxidizing bacteria that store polyphosphates under oxic conditions to be utilized under sulfidic conditions. Hydrolysis of the stored polyphosphate results in the rapid precipitation of the phosphate‐rich mineral apatite—providing a mechanism to explain the association between modern phosphorites and these bacteria. Whether sulfur bacteria were important to the formation of ancient phosphorites has been unresolved. Here, we present the remains of modern sulfide‐oxidizing bacteria that are partially encrusted in apatite, providing evidence that bacterially mediated phosphogenesis can rapidly permineralize sulfide‐oxidizing bacteria and perhaps other types of organic remains. We also describe filamentous microfossils that resemble modern sulfide‐oxidizing bacteria from two major phosphogenic episodes in the geologic record. These microfossils contain sulfur‐rich inclusions that may represent relict sulfur globules, a diagnostic feature of modern sulfide‐oxidizing bacteria. These findings suggest that sulfur bacteria, which are known to mediate the precipitation of apatite in modern sediments, were also present in certain phosphogenic settings for at least the last 600 million years. If polyphosphate‐utilizing sulfide‐oxidizing bacteria also played a role in the formation of ancient phosphorites, their requirements for oxygen, or oxygen‐requiring metabolites such as nitrate, might explain the temporal correlation between the first appearance of globally distributed marine phosphorites and increasing oxygenation of Neoproterozoic oceans.     

Sulfide removal and elemental sulfur recycling from a sulfide-polluted medium by Allochromatium vinosum strain 21D.

Phototrophic purple sulfur bacteria oxidize sulfide to elemental sulfur, which is stored as intracellular sulfur globules. The mutant Allochromatium vinosum strain 21D, containing an inactivated dsrB gene, is unable to further oxidize intracellularly stored sulfur to sulfate. This mutant was used as a biocatalyst in a biotechnological process to eliminate sulfide from synthetic wastewater and to recycle elemental sulfur as a raw material. For this purpose, the mutant was grown in an illuminated 5-liter bioreactor (30 microE/m2/s PAR) at 30 degrees C for 61 days in anoxic phototrophic medium. The process of sulfide removal was semi-continuous and consisted of three consecutive fed-batch sections. Sulfide was repeatedly added into the bioreactor and oxidized by the cells to sulfur. In the presence of the mutant, no unwanted sulfate was produced during sulfide removal. A maximum sulfide removal rate of 49.3 microM/h, a maximum sulfide removal efficiency of 98.7%, and 60.4% sulfur recycling were achieved.     

Insights into the sulfur metabolism of Chlorobaculum tepidum by label-free quantitative proteomics

Chlorobaculum tepidum is an anaerobic green sulfur bacterium which oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. It can also oxidize sulfide to produce extracellular S⁰ globules, which can be further oxidized to sulfate and used as an electron donor. Here, we performed label-free quantitative proteomics on total cell lysates prepared from different metabolic states, including a sulfur production state (10 h post-incubation [PI]), the beginning of sulfur consumption (20 h PI), and the end of sulfur consumption (40 h PI), respectively. We observed an increased abundance of the sulfide:quinone oxidoreductase (Sqr) proteins in 10 h PI indicating a sulfur production state. The periplasmic thiosulfate-oxidizing Sox enzymes and the dissimilatory sulfite reductase (Dsr) subunits showed an increased abundance in 20 h PI, corresponding to the sulfur-consuming state. In addition, we found that the abundance of the heterodisulfide-reductase and the sulfhydrogenase operons was influenced by electron donor availability and may be associated with sulfur metabolism. Further, we isolated and analyzed the extracellular sulfur globules in the different metabolic states to study their morphology and the sulfur cluster composition, yielding 58 previously uncharacterized proteins in purified globules. Our results show that C. tepidum regulates the cellular levels of enzymes involved in sulfur metabolism in response to the availability of reduced sulfur compounds.     

Flow cytometric identification and enumeration of photosynthetic sulfur bacteria and potential for ecophysiological studies at the single-cell level

We show the potential of flow cytometry as a fast tool for population identification and enumeration of photosynthetic sulfur bacteria. Purple (PSB) and green sulfur bacteria (GSB) oxidize hydrogen sulfide to elemental sulfur that can act as storage compound to be further oxidized to sulfate generating the reducing power required for growth. Both groups have different elemental sulfur allocation strategies: whereas PSB store elemental sulfur as intracellular inclusions, GSB allocate sulfur globules externally. We used well-characterized laboratory strains and complex natural photosynthetic populations developing in a sharply stratified meromictic lake to show that PSB and GSB could be detected, differentiated and enumerated in unstained samples using a blue laser-based flow cytometer. Variations in cell-specific pigment content and the dynamics of sulfur accumulation, both intra- and extracellularly, were also detected in flow cytometric plots as sulfur accumulation changed the light scatter characteristics of the cells. These data were used to show the potential for studies on the metabolic status and the rate of activity at the single-cell level. Flow cytometric identification and enumeration resulted in faster and more precise analyses than previous approaches, and may open the door to more complex ecophysiological experiments with photosynthetic sulfur bacteria in mixed cultures and natural environments.     

含奥氏酮嗜盐紫色硫细菌的分离鉴定及系统发育分析

[目的]为挖掘我国紫色硫细菌物种和光合蛋白基因资源.[方法]采用Pfennig紫色硫细菌无机选择性培养基和琼脂稀释法.[结果]从青岛东风盐场分离获得一株含奥氏酮、耐高浓度硫化物、嗜盐耐碱紫色硫细菌菌株283-1.该菌株能氧化硫化物产生硫粒储存在细胞内、嗜盐、细胞含有奥氏酮类胡萝卜素、细菌叶绿素a强吸收峰位于830 nm处、运动、不产生气囊,表明属于Marichromatium属.16S rDNA序列同源性比较和系统发育分析也表明这一点.但该菌株能在1%～15%NaCl、7.5 mmol/L 高浓度硫化物、45℃、5000lux、pH9.0条件下生长良好,能很好的光同化C3和C4有机酸和葡萄糖酸钠等特性,与Marichromatium属4个种有明显不同.[结论]菌株283-1是Marichromatium属一个新分离物,编号 Marichromatium sp.283-1.     

In situ analysis of sulfur species in sulfur globules produced from thiosulfate by Thermoanaerobacter sulfurigignens and Thermoanaerobacterium thermosulfurigenes

The Firmicutes Thermoanaerobacter sulfurigignens and Thermoanaerobacterium thermosulfurigenes convert thiosulfate, forming sulfur globules inside and outside cells. X-ray absorption near-edge structure analysis revealed that the sulfur consisted mainly of sulfur chains with organic end groups similar to sulfur formed in purple sulfur bacteria, suggesting the possibility that the process of sulfur globule formation by bacteria is an ancient feature.     

Investigation of Elemental Sulfur Speciation Transformation Mediated by Acidithiobacillus ferrooxidans

The speciation transformation of elemental sulfur mediated by the leaching bacterium Acidithiobacillus ferrooxidans was investigated using an integrated approach including scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, energy dispersive X-ray spectroscopy, and X-ray absorption near edge spectroscopy (XANES). Our results showed that when grown on elemental sulfur powder, At. ferrooxidans ATCC23270 cells were first attached to sulfur particles and modified the surface sulfur with some amphiphilic compounds. In addition, part of the elemental sulfur powder might be converted to polysulfides. Furthermore, sulfur globules were accumulated inside the cells. XANES spectra of these cells suggested that these globules consisted of elemental sulfur bound to thiol groups of protein. Huan He and Cheng-Gui Zhang made equal contributions to this paper.     

Quantitative proteomics of Chlorobaculum tepidum: insights into the sulfur metabolism of a phototrophic green sulfur bacterium

Chlorobaculum (Cba.) tepidum is a green sulfur bacterium that oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. To gain insight into the sulfur metabolism, the proteome of Cba. tepidum cells sampled under different growth conditions has been quantified using a rapid gel-free, filter-aided sample preparation (FASP) protocol with an in-solution isotopic labeling strategy. Among the 2245 proteins predicted from the Cba. tepidum genome, approximately 970 proteins were detected in unlabeled samples, whereas approximately 630-640 proteins were detected in labeled samples comparing two different growth conditions. Wild-type cells growing on thiosulfate had an increased abundance of periplasmic cytochrome c-555 and proteins of the periplasmic thiosulfate-oxidizing SOX enzyme system when compared with cells growing on sulfide. A dsrM mutant of Cba. tepidum, which lacks the dissimilatory sulfite reductase DsrM protein and therefore is unable to oxidize sulfur globules to sulfite, was also investigated. When compared with wild type, the dsrM cells exhibited an increased abundance of DSR enzymes involved in the initial steps of sulfur globule oxidation (DsrABCL) and a decreased abundance of enzymes putatively involved in sulfite oxidation (Sat-AprAB-QmoABC). The results show that Cba. tepidum regulates the cellular levels of enzymes involved in sulfur metabolism and other electron-transferring processes in response to the availability of reduced sulfur compounds.     

X-ray absorption spectroscopy as a probe of microbial sulfur biochemistry: the nature of bacterial sulfur globules revisited

The chemical nature of the sulfur in bacterial sulfur globules has been the subject of controversy for a number of years. Sulfur K-edge X-ray absorption spectroscopy (XAS) is a powerful technique for probing the chemical forms of sulfur in situ, but two groups have used it with very different conclusions. The root of the controversy lies with the different detection strategies used by the two groups, which result in very different spectra. This paper seeks to resolve the controversy. We experimentally demonstrate that the use of transmittance detection for sulfur K-edge XAS measurements is highly prone to spectroscopic distortions and that much of the published work on sulfur bacteria is very likely based on distorted data. We also demonstrate that all three detection methods used for X-ray absorption experiments yield essentially identical spectra when the measurements are carried out under conditions where no experimental distortions are expected. Finally, we turn to the original question—the chemical nature of bacterial sulfur. We examine isolated sulfur globules of Allochromatium vinosum and intact cells of a strain of magnetotactic coccus and show that XAS indicates the presence of a chemical form of sulfur resembling S₈.     

Speciation of sulfur from filamentous microbial mats from sulfidic cave springs using X-ray absorption near-edge spectroscopy

Most transformations within the sulfur cycle are controlled by the biosphere, and deciphering the abiotic and biotic nature and turnover of sulfur is critical to understand the geochemical and ecological changes that have occurred throughout the Earth's history. Here, synchrotron radiation-based sulfur K-edge X-ray absorption near-edge structure (XANES) spectroscopy is used to examine sulfur speciation in natural microbial mats from two aphotic (cave) settings. Habitat geochemistry, microbial community compositions, and sulfur isotope systematics were also evaluated. Microorganisms associated with sulfur metabolism dominated the mats, including members of the Epsilonproteobacteria and Gammaproteobacteria. These groups have not been examined previously by sulfur K-edge XANES. All of the mats consisted of elemental sulfur, with greater contributions of cyclo-octasulfur (S8) compared with polymeric sulfur (Smicro). While this could be a biological fingerprint for some bacteria, the signature may also indicate preferential oxidation of Smicro and S8 accumulation. Higher sulfate content correlated to less S8 in the presence of Epsilonproteobacteria. Sulfur isotope compositions confirmed that sulfur content and sulfur speciation may not correlate to microbial metabolic processes in natural samples, thereby complicating the interpretation of modern and ancient sulfur records.     

Sulfur Species Investigation in Extra- and Intracellular Sulfur Globules of Acidithiobacillus ferrooxidans and Acidithiobacillus caldus

The sulfur chemical speciation in extracellular and intracellular sulfur globules of Acidithiobacillus ferrooxidans and Acidithiobacillus caldus were investigated with an integrated approach including scanning electron microscopy, transmission electron microscopy, energy dispersive spectroscopy and sulfur K-edge X-ray absorption near edge structure spectroscopy (XANES). The results indicated that both strains can accumulate extracellular sulfur globules when grown on thiosulfate, and the major sulfur chemical speciation of which were S₈ for A. ferrooxidans and mixture of ring sulfur and polythionate for A. caldus, respectively. In contrast, A. ferrooxidans can accumulate both linear sulfur and S₈ internally when grown with sulfur powder and thiosulfate, whereas A. caldus did not accumulate intracellular sulfur globules. In addition, the fitted results of sulfur K-edge XANES spectra indicated that the reduced glutathione (containing thiols groups) were involved in sulfur bio-oxidation of both strains and the tetrathionate were the intermediate products during thiosulfate metabolism by two strains.     

Colourless sulfur bacteria and their role in the sulfur cycle

Summary The bacteria belonging to the families of the Thiobacteriaceae, Beggiatoaceae and Achromatiaceae are commonly called the colourless sulfur bacteria. While their ability to oxidize reduced inorganic sulfur compounds has clearly been established, it is still not known whether all these organisms can derive metabolically useful energy from these oxidations. During the last decades research has mainly focussed on the genus Thiobacillus. Bacteria belonging to this genus can oxidize a variety of reduced inorganic sulfur compounds and detailed information is available on the biochemistry and physiology of these energy-yielding reactions. The thiobacilli, most of which can synthesize all cell material from CO₂, possess a well-regulated metabolic machinery with high biosynthetic capacities, which is essentially similar to that of other procaryotic organisms. Although the qualitative role of colourless sulfur bacteria in the sulfur cycle is well documented, quantitative data are virtually absent. Activities of colourless sulfur bacteria in nature must be related to direct and indirect parameters, such as: the rate of oxidation of (S³⁵) sulfur compounds, the rate of C¹⁴O₂-fixation, the rate of acid production and numbers and growth rates of the bacteria. However, chemical reactions and similar activities of heterotrophic organisms mask the activities of the colourless sulfur bacteria to various extents, depending on the condition of the natural environment. This interference is minimal in regions where high temperature and/or low pH allow the development of a dominant population of colourless sulfur bacteria, such as hot acid sulfur springs, sulfide ores, sulfur deposits and some acid soils. The oxidation of inorganic sulfur compounds is carried out by a spectrum of sulfur-oxidizing organisms which includes: 1) obligately chemolithotrophic organisms 2) mixotrophs 3) chemolithotrophic heterotrophs 4) heterotrophs which do not gain energy from the oxidation of sulfur compounds but benefit in other ways from this reaction, and 5) heterotrophs which do not benefit from the oxidation of sulfur compounds. The spectrum is completed by a hypothetical group of heterotrophic organisms, which may have a symbiotic relationship with thiobacilli and related bacteria. Such heterotrophs may stimulate the growth of colourless sulfur bacteria and thereby contribute to the oxidation of sulfur compounds. Future research should focus in the first place on obtaining and studying pure cultures of many of the colourless sulfur bacteria. In the second place, studies on the physiological and ecological aspects of mixed cultures of colourless sulfur bacteria and heterotrophs may add to a better understanding of the role of the colourless sulfur bacteria in the sulfur cycle. Paper read at the Symposium on the Sulphur Cycle, Wageningen, May 1974.     

Oxidation of inorganic sulfur compounds by obligatory organotrophic bacteria

New data obtained by the author and other researchers on two different groups of obligately heterotrophic bacteria capable of inorganic sulfur oxidation are reviewed. Among culturable marine and (halo)alkaliphilic heterotrophs oxidizing sulfur compounds (thiosulfate and, much less actively, elemental sulfur and sulfide) incompletely to tetrathionate, representatives of the gammaproteobacteria, especially from the Halomonas group, dominate. Some of denitrifying species from this group are able to carry out anaerobic oxidation of thiosulfate and sulfide using nitrogen oxides as electron acceptors. Despite the low energy output of the reaction of thiosulfate oxidation to tetrathionate, it can be utilized for ATP synthesis by some tetrathionate-producing heterotrophs; however, this potential is not always realized during their growth. Another group of marine and (halo)alkaliphilic heterotrophic bacteria capable of complete oxidation of sulfur compounds to sulfate mostly includes representatives of the alphaproteobacteria most closely related to nonsulfur purple bacteria. They can oxidize sulfide (polysulfide), thiosulfate, and elemental sulfur via sulfite to sulfate but neither produce nor oxidize tetrathionate. All of the investigated sulfate-forming heterotrophic bacteria belong to lithoheterotrophs, being able to gain additional energy from the oxidation of sulfur compounds during heterotrophic growth on organic substrates. Some doubtful cases of heterotrophic sulfur oxidation described in the literature are also discussed.     

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.     

34S/32S fractionation in sulfur cycles catalyzed by anaerobic bacteria

Stable isotopic distributions in the sulfur cycle were studied with pure and mixed cultures of the anaerobic bacteria, Chlorobium vibrioforme and Desulfovibrio vulgaris. D. vulgaris and C. vibrioforme can catalyze three reactions constituting a complete anaerobic sulfur cycle: reduction of sulfate to sulfide (D. vulgaris), oxidation of sulfide to elemental sulfur (C. vibrioforme), and oxidation of sulfur to sulfate (C. vibrioforme). In all experiments, the first and last reactions favored concentration of the light 32S isotope in products (isotopic fractionation factor epsilon = -7.2 and -1.7%, respectively), whereas oxidation of sulfide favored concentration of the heavy 34S isotope in products (epsilon = +1.7%). Experimental results and model calculations suggest that elemental sulfur enriched in 34S versus sulfide may be a biogeochemical marker for the presence of sulfide-oxidizing bacteria in modern and ancient environments.     

A new ecological adaptation to high sulfide by a Hydrogenobacter sp. growing on sulfur compounds but not on hydrogen

Thermophilic bacteria were isolated from a sulfide-rich, neutral hot spring in Iceland on gelrite minimal medium with 16 mM thiosulfate. The isolates were aerobic, obligate chemolithoautotrophs and used thiosulfate and sulfur as electron donors, producing sulfate from both substrates. No growth was observed with hydrogen as the sole electron donor, and no hydrogenase activity was detected. The cells were gram-negative and usually single, 4-5 microm long and 0.7 microm in diameter and formed sulfur globules after a few days of incubation. By SSU rRNA sequence comparisons, the bacterium was placed in the genus Hydrogenobacter with the closest relative to be Calderobacterium hydrogenophilum with 98.3% sequence similarity. This novel bacterium shows an ecological adaptation to high sulfide springs and is differentiated from its closest known relatives by lack of H2 oxidation, deposition of sulfur and lower growth temperature.     

34S/32S fractionation in sulfur cycles catalyzed by anaerobic bacteria.

Stable isotopic distributions in the sulfur cycle were studied with pure and mixed cultures of the anaerobic bacteria, Chlorobium vibrioforme and Desulfovibrio vulgaris. D. vulgaris and C. vibrioforme can catalyze three reactions constituting a complete anaerobic sulfur cycle: reduction of sulfate to sulfide (D. vulgaris), oxidation of sulfide to elemental sulfur (C. vibrioforme), and oxidation of sulfur to sulfate (C. vibrioforme). In all experiments, the first and last reactions favored concentration of the light 32S isotope in products (isotopic fractionation factor epsilon = -7.2 and -1.7%, respectively), whereas oxidation of sulfide favored concentration of the heavy 34S isotope in products (epsilon = +1.7%). Experimental results and model calculations suggest that elemental sulfur enriched in 34S versus sulfide may be a biogeochemical marker for the presence of sulfide-oxidizing bacteria in modern and ancient environments.