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.     

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.     

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.     

Sulfide quinone reductase (SQR) activity in Chlorobium.

Membranes of the green sulfur bacterium, Chlorobium limicola f. thiosulfatophilum, catalyze the reduction of externally added isoprenoid quinones by sulfide. This activity is highly sensitive to stigmatellin and aurachins. It is also inhibited by 2-n-nonyl-4-hydroxyquinoline-N-oxide, antimycin, myxothiazol and cyanide. It is concluded that in sulfide oxidizing bacteria like Chlorobium, sulfide oxidation involves a sulfide-quinone reductase (SQR) similar to the one found in Oscilatoria limnetica [Arieli, B., Padan, E. and Shahak, Y. (1991) J. Biol. Chem. 266, 104-111].     

Red shift of absorption maxima in chlorobiineae through enzymic methylation of their antenna bacteriochlorophylls

The bacteriochlorophyll d producing photosynthetic green sulfur bacteria Chlorobium vibrioforme forma thiosulfatophilum strain NCIB 8327 and C. vibrioforme strain B1-20 respond to reduced light conditions in culture by performing methylations at the 4- and 5-substituents, for example, converting the 4-Et into 4-n-Pr, 4-i-Bu, and even 4-neoPn. During this process, the absorption maximum in living cells of C. vibrioforme strain B1-20 red shifts from 714 to about 728 nm. Eventually, the C. vibrioforme forma thiosulfatophilum strain NCIB 8327 culture carries out a delta-methylation to produce the bacteriochlorophylls c (lambda max ca. 750 nm); the new UC Davis bacteriochlorophyll c culture is named C. vibrioforme forma thiosulfatophilum strain D. It is possible that the homologation process increases hydrophobic interactions between individual BChl molecules, giving rise to larger aggregates in the antenna system. Alternatively, the additional methyl units attached to the 4-position shift the absolute configuration of the 2-(1-hydroxyethyl) group from pure R in the case of 4-Et to pure S in the case of 4-neoPn, which in turn might determine the size of the in vivo aggregates due to the intrinsic nature of the pigment protein system. It is suggested that the bacteriochlorophylls c from Chloroflexus aurantiacus strain J-10-fl and the bacteriochlorophylls e from Chlorobium phaeovibrioides might have undergone similar meso methylation as a response to external environmental pressure such as low light intensity.     

Green sulfur bacteria from hypersaline Chiprana Lake (Monegros,Spain): habitat description and phylogenetic relationship of isolated strains

The 'Salada de Chiprana' (Chiprana Lake) is a hypersaline (30-73 per thousand), permanent and shallow lake of endorheic origin in a semi-arid region of the Ebro depression (Aragon, Spain). Magnesium sulfate and sodium chloride represent the main salts of this athalassohaline environment. Anoxic conditions occurred periodically in the bottom layers of the lake during the study period. When stratified, high sulfide concentrations (up to 7 mM) were measured in the hypolimnion. Physical and chemical conditions gave rise to the development of very dense green sulfur bacteria blooms (10.7 mg l(-1) of BChl c and 16.7 mg l(-1) of BChl d) at 0.5-1 m from the bottom. Microscopic observations revealed that cells morphologically similar to Chlorobium vibrioforme were dominant in the phototrophic bacterial community, but Prosthecochloris aestuarii was also found sometimes at lower concentrations, as revealed by both microscopic observation and flow cytometric analyses. Deep agar dilution series allowed to obtain several axenic cultures of phototrophic bacteria. They were identified according to their morphology, pigment composition and phylogenetic relationships (16S rDNA sequence analysis). Two of the sequenced strains (CHP3401 and CHP3402) belonged to the green sulfur bacteria and were related to Prosthecochloris aestuarii SK413(T) and Chlorobium vibrioforme DSM260(T), respectively. HPLC analyses of both natural samples and Chlorobium vibrioforme isolates indicated that these strains contained both BChl c and BChl d. Phylogenetic results suggested that Chlorobium vibrioforme strains DSM260(T) and CHP3402, all sequenced strains of Prosthecochloris aestuarii and strain CIB2401 constitute a separate cluster of green sulfur bacteria, all of them isolated from marine to hypersaline habitats.     

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.     

Thiosulfate sulfur transferases (Rhodaneses) ofChlorobium vibrioforme f.thiosulfatophilum

Two enzymes containing thiosulfate sulfur transferase activity were purified fromChlorobium vibrioforme f.thiosulfatophilum by ion exchange chromatography, gel filtration and isoelectrofocusing. Enzyme I is a basic protein with an isoelectric point at pH 9.2 and has a molecular weight of 39,000. TheK _m-values for thiosulfate and cyanide of the purified basic protein were 0.25 mM (thiosulfate) and 5 mM (cyanide). Enzyme II is an acidic protein. The enzyme has an isoelectric point at pH 4.6–4.7 and a molecular weight of 34,000. TheK _m-values of the acidic protein were found to be 5 mM for thiosulfate and 125 mM for cyanide.In addition to thiosulfate sulfur transferase activity, cellfree extracts ofChlorobium vibrioforme f.thiosulfatophilum also contained low thiosulfate oxidase activity and negligible thiosulfate reductase activity. The percent distribution of thiosulfate sulfur transferase and thiosulfate oxidase activities in the organism was independent of the offered sulfur compound (thiosulfate, sulfide or both) in the medium.Abbreviations C Chlorobium - SDS sodium dodecylsulfate Dedicated to Prof. Dr. Norbert Pfennig on the occasion of his 60th birthday     

Cytochromes of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum. Purification,characterization and sulfur metabolism

Three cytochromes of the thiosulfate-utilizing green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum were highly purified by ion exchange column chromatography and ammonium sulfate fractionation. All three cytochromes are located in the soluble fraction. Cytochrome c-551 (highest purity index obtained: A₂₈₀/A₄₁₆=0.39) shows maxima at 551 nm (-band), 521 nm (-band), and 416 nm (-band) for the reduced form. This cytochrome is an acidic protein with a molecular weight of 32,000, a redox potential of 150 mV, and an isoelectric point at pH 6.0. Cytochrome c-553 (highest purity index obtained: A₂₈₀/A₄₁₇=0.8) is also an acidic protein with maxima at 553,5 nm, 523,5 nm and 417 nm for the reduced form, a molecular weight of 63,000, a redox potential of 90 mV, an isoelectric point at pH 6.3, and it contains FAD as flavin component. It is autoxidizable and participates in sulfide oxidation, but cannot catalyze the reverse reaction. The cytochrome c-555 (highest purity index obtained: A₂₈₀/A₄₁₈=0.16) is a small basic protein with maxima at 555 nm, 523 nm and 418 nm (reduced form), a molecular weight of 12,500, an isoelectric point between pH 10 and 10.5, and a redox potential of 155 mV. The ratio of the cytochrome contents to each other is constant and does not change when the organism has only thiosulfate or sulfide as the main electron donor in the medium.The soluble fraction further contains the non-heme ironcontaining proteins rubredoxin and ferredoxin. The anaerobic sulfide oxidation in a growing culture of Chlorobium vibrioforme f. thiosulfatophilum is accompanied by a rapid formation of thiosulfate, which is only utilized when sulfide is no longer available, while the elemental sulfur concentration increases constantly until thiosulfate is consumed.Non-common abbreviations C Chlorobium - SDS sodium dodecylsulfate - HIPIP high-potential-iron-sulfur-protein     

Identification of a thiosulfate utilization gene cluster from the green phototrophic bacterium Chlorobium limicola

Chlorobium is an autotrophic, green phototrophic bacterium which uses reduced sulfur compounds to fix carbon dioxide in the light. The pathways for the oxidation of sulfide, sulfur, and thiosulfate have not been characterized with certainty for any species of bacteria. However, soluble cytochrome c-551 and flavocytochrome c (FCSD) have previously been implicated in the oxidation of thiosulfate and sulfide on the basis of enzyme assays in Chlorobium. We have now made a number of observations relating to the oxidation of reduced sulfur compounds. (1) Western analysis shows that soluble cytochrome c-551 in Chlorobium limicola is regulated by thiosulfate, consistent with a role in the utilization of thiosulfate. (2) A membrane-bound flavocytochrome c-sulfide dehydrogenase (which is normally a soluble protein in other species) is constitutive and not regulated by sulfide as expected for an obligately autotrophic species dependent upon sulfide. (3) We have cloned the cytochrome c-551 gene from C. limicola and have found seven other genes, which are also presumably involved in sulfur metabolism and located near that for cytochrome c-551 (SoxA). These include genes for a flavocytochrome c flavoprotein homologue (SoxF2), a nucleotidase homologue (SoxB), four small proteins (including SoxX, SoxY, and SoxZ), and a thiol-disulfide interchange protein homologue (SoxW). (4) We have established that the constitutively expressed FCSD genes (soxEF1) are located elsewhere in the genome. (5) Through a database search, we have found that the eight thiosulfate utilization genes are clustered in the same order in the Chlorobium tepidum genome (www.tigr.org). Similar thiosulfate utilization gene clusters occur in at least six other bacterial species but may additionally include genes for rhodanese and sulfite dehydrogenase.     

Evaluating the Potential of Halothiobacillus Bacteria for Sulfur Oxidation and Biomass Production under Saline Soil

Abstract

Salinity negatively affects growth of sulfur-oxidizing bacteria (SOB) and their sulfate production ability, meanwhile decreases the available sulfate for plants in soil. The aim of this study was to isolate and characterize the bacteria of genus Halothiobacillus, as a salt-tolerant SOB, from saline and sulfidic habitats of Iran for the first time and evaluating the effect of salinity on their biomass and sulfate production during the oxidation of different sulfur sources. Isolation process and surveying the morphological, biochemical and 16S rRNA gene analysis resulted into identification of three species (eight strains) of Halothiobacillus genus including H. neapolitanus, H. hydrothermalis and H. halophilus. Salinity (0, 0.5, 1, 2 and 4?M NaCl) had a significant impact (p?≤?0.01) on bacterial biomass and sulfate production during the oxidation of thiosulfate and elemental sulfur. Biomass and sulfate production by strains was accompanied by a decrease in residual content of thiosulfate (RCT) in medium. The amount of produced biomass and sulfate in medium supplemented by thiosulfate was much higher than elemental sulfur. The highest amount of biomass and sulfate was produced by H. neapolitanus strain I19 at 0.5 and 1?M NaCl concentration. The results of this study could be the first step to focus on the application of these bacteria to increase sulfate storage of saline soils and crop production.     

Oxidation of Inorganic Sulfur Compounds by Obligately 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 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 which are 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.     

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     

Model-based control of feed rate and illuminance in a photosynthetic fed-batch reactor for H2S removal

Traditional application of computer to fermentation processes has focused on the measurement and control of parameters such as temperature, pH, vessel pressure, sparge rate, dissolved oxygen, substrate concentration, and product concentration. In a fed-batch reactor with the photosynthetic green sulfur bacterium Chlorobium thiosulfatophilum which converts hydrogen sulfide to elementary sulfur or sulfate, separate measurement of cell mass concentration and sulfur particle concentration turbidimetrically was difficult due to their combined contributions to the total turbidity. Instead of on-line measurement of many process variables, a model-based control of feed rate and illuminance was designed. Optimal operation condition relating feed rate vs. light intensity was obtained to suppress the accumulation of sulfate and sulfide, and to save light energy in a 4-1 photosynthetic fed-batch reactor. This relation was correlated with the inreasing cell mass concentration. A model which describes the cell growth by considering the light attenuation effects due to scattering and absorption, and to crowding effect of the cells, was established beforehand with the results from the experiments. Based on these optimal operating conditions and the cell growth model, automatic controls of feed rate and illuminance were carried out alternatively to the traditional application of computer to fermentation with on-line measurement, realtime response and adjustment of process variables.List of Symbols F ml/min Flow rate of gas mixture - hV lux Average illuminance - Q mmol/(l h) Removal rate of hydrogen sulfide - X mg protein/l Cell mass concentration as protein - X ₀ mg protein/l Initial cell mass concentration - X _m mg protein/l Maximum cell mass concentration - _a h^–1 Apparent specific growth rate     

The bchU gene of Chlorobium tepidum encodes the c-20 methyltransferase in bacteriochlorophyll c biosynthesis

Bacteriochlorophylls (BChls) c and d, two of the major light-harvesting pigments in photosynthetic green sulfur bacteria, differ only by the presence of a methyl group at the C-20 methine bridge position in BChl c. A gene potentially encoding the C-20 methyltransferase, bchU, was identified by comparative analysis of the Chlorobium tepidum and Chloroflexus aurantiacus genome sequences. Homologs of this gene were amplified and sequenced from Chlorobium phaeobacteroides strain 1549, Chlorobium vibrioforme strain 8327d, and C. vibrioforme strain 8327c, which produce BChls e, d, and c, respectively. A single nucleotide insertion in the bchU gene of C. vibrioforme strain 8327d was found to cause a premature, in-frame stop codon and thus the formation of a truncated, nonfunctional gene product. The spontaneous mutant of this strain that produces BChl c (strain 8327c) has a second frameshift mutation that restores the correct reading frame in bchU. The bchU gene was inactivated in C. tepidum, a BChl c-producing species, and the resulting mutant produced only BChl d. Growth rate measurements showed that BChl c- and d-producing strains of the same organism (C. tepidum or C. vibrioforme) have similar growth rates at high and intermediate light intensities but that strains producing BChl c grow faster than those with BChl d at low light intensities. Thus, the bchU gene encodes the C-20 methyltransferase for BChl c biosynthesis in Chlorobium species, and methylation at the C-20 position to produce BChl c rather than BChl d confers a significant competitive advantage to green sulfur bacteria living at limiting red and near-infrared light intensities.     

Sulfur oxidation in mutants of the photosynthetic green sulfur bacterium Chlorobium tepidum devoid of cytochrome c-554 and SoxB

A mutant devoid of cytochrome c-554 (CT0075) in Chlorobium tepidum (syn. Chlorobaculum tepidum) exhibited a decreased growth rate but normal growth yield when compared to the wild type. From quantitative determinations of sulfur compounds in media, the mutant was found to oxidize thiosulfate more slowly than the wild type but completely to sulfate as the wild type. This indicates that cytochrome c-554 would increase the rate of thiosulfate oxidation by serving as an efficient electron carrier but is not indispensable for thiosulfate oxidation itself. On the other hand, mutants in which a portion of the soxB gene (CT1021) was replaced with the aacC1 cassette did not grow at all in a medium containing only thiosulfate as an electron source. They exhibited partial growth yields in media containing only sulfide when compared to the wild type. This indicates that SoxB is not only essential for thiosulfate oxidation but also responsible for sulfide oxidation. An alternative electron carrier or electron transfer path would thus be operating between the Sox system and the reaction center in the mutant devoid of cytochrome c-554. Cytochrome c-554 might function in any other pathway(s) as well as the thiosulfate oxidation one, since even green sulfur bacteria that cannot oxidize thiosulfate contain a cycA gene encoding this electron carrier.     

A thermophilic green sulfur bacterium from New Zealand hot springs,Chlorobium tepidum sp. nov.

Thermophilic green sulfur bacteria of the genus Chlorobium were isolated from certain acidic high sulfide New Zealand hot springs. Cells were Gram-negative nonmotile rods of variable length and contained bacteriochlorophyll c and chlorosomes. Cultures of thermophilic chlorobia grew only under anaerobic, phototrophic conditions, either photoautotrophically or photoheterotrophically. The optimum growth temperature for the strains of thermophilic green sulfur bacteria isolated was 47–48°C with generation times of about 2 h being observed. The upper temperature limit for growth was about 52°C. Thiosulfate was a major electron donor for photoautotrophic growth while sulfide alone was only poorly used. N₂ fixation was observed at 48°C and cell suspensions readily reduced acetylene to ethylene. The G+C content of DNA from strains of thermophilic chlorobia was 56.5–58.2 mol% and the organisms positioned phylogenetically within the green sulfur bacterial branch of the domain Bacteria. The new phototrophs are described as a new species of the genus Chlorobium, Chlorobium tepidum.This paper is dedicated to Professor Norbert Pfennig on the occasion of his 65th birthday     

Orinithine as a constituent of the peptidoglycan of Chloroflexus aurantiacus,diaminopimelic acid in that of Chlorobium vibrioforme f. thiosulfatophilum

L-Ornithine is the only diamino acid of the peptidoglycan of the gliding phototrophic Chloroflexus aurantiacus. The other constituents are L- and D-alanine, D-glutamic acid, N-acetyl-glucosamine and N-acetyl-muramic acid (in part as muramic acid-6-phosphate), all in approximate equimolar ratios to L-ornithine, aside from small amounts of glycine and histidine. Furthermore unlike typical Gram-negative bacteria, protein is not bound to this peptidoglycan. Instead, the rigid layer (sodium dodecyl sulfate insoluble cell wall fraction) contained large amounts of a complex polysaccharide consisting of sugar O-methyl ethers, hexoses and pentoses. Its binding site is presumably muramic acid-6-phosphate of the peptidoglycan.In contrast, in Chlorobium vibrioforme f. thiosulfatophilium, meso-diaminopimelic acid was found as the only diamino acid of this peptidoglycan. As with other Gramnegative bacteria, L- and D-alanine, D-glutamic acid, N-acetyl-glucosamine and N-acetyl-muramic acid (no muramic acid-6-phosphate) were observed in approximate equimolar ratios to meso-diaminopimelic acid, except a lower D-alanine content. The rigid layer of Chlorobium vibrioforme f. thiosulfatophilum contained protein, and there were no indications for a complex polysaccharide comparable to that of Chloroflexus aurantiacus.Abbreviations Ala alanine - A₂pm diaminopimelic acid - GC/MS combined gas-liquid chromatography/mass spectrometry - GlcNAc N-acetyl-glucosamine - Glu glutamic acid - Gly glycine - HF hydrofluoric acid - Lys lysine - MurNAc N-acetyl-muramic acid - Orn ornithine - SDS sodium dodecyl sulfate     

Photoproduction of H2 from acetate by syntrophic cocultures of green sulfur bacteria and sulfur-reducing bacteria

The marine green sulfur bacterium Chlorobium vibrioforme strain 1930 produced H₂ and elemental sulfur from sulfide or thiosulfate under N limitation in the light. H₂ production depended on nitrogenase and occurred only in the absence of ammonia. Methionine sulfoximine, an inhibitor of glutamine synthetase, prevented the switch-off by ammonia. In defined syntrophic cocultures of the acetate-oxidizing, sulfur-reducing bacterium Desulfuromonas acetoxidans with green sulfur bacteria, H₂ was produced from acetate via a light-driven sulfur cycle. The sulfur-reducing bacterium could not be replaced by sulfate-reducing bacteria in these experiments. In a coculture of the marine Chlorobium vibrioforme strain 1930 and the sulfur-reducing bacterium Desulfuromonas acetoxidans strain 5071, optimum long-term H₂ production from acetate was obtained with molecular nitrogen as N source, at low light intensity (110 mol · m^-2 · s^-1), in sulfide-reduced mineral medium (2 mM Na₂S) at pH 6.8. Traces of sulfide (10 M) were sufficient to keep the sulfur cycle running. The coculture formed no poly--hydroxyalkanoates (PHA), but 20%–40% polysaccharide per cell dry mass. Per mol acetate added, the coculture formed 3.1 mol of H₂ (78% of the theoretical maximum). Only 8% of the reducing equivalents was incorporated into biomass. The maximum rate of H₂ production was 1300 ml H₂ per day and g cell dry mass.Non-standard abbrevations MOPS 2-(N-morpholino) propane sulfonic acid - MSX Methionine sulfoximine - PHA poly--hydroxyalkanoates     

X-ray crystallographic analysis of the sulfur carrier protein SoxY from Chlorobium limicola f. thiosulfatophilum reveals a tetrameric structure

Dissimilatory oxidation of thiosulfate in the green sulfur bacterium Chlorobium limicola f. thiosulfatophilum is carried out by the ubiquitous sulfur-oxidizing (Sox) multi-enzyme system. In this system, SoxY plays a key role, functioning as the sulfur substrate-binding protein that offers its sulfur substrate, which is covalently bound to a conserved C-terminal cysteine, to another oxidizing Sox enzyme. Here, we report the crystal structures of a stand-alone SoxY protein of C. limicola f. thiosulfatophilum, solved at 2.15 A and 2.40 A resolution using X-ray diffraction data collected at 100 K and room temperature, respectively. The structure reveals a monomeric Ig-like protein, with an N-terminal alpha-helix, that oligomerizes into a tetramer via conserved contact regions between the monomers. The tetramer can be described as a dimer of dimers that exhibits one large hydrophobic contact region in each dimer and two small hydrophilic interface patches in the tetramer. At the tetramer interface patch, two conserved redox-active C-terminal cysteines form an intersubunit disulfide bridge. Intriguingly, SoxY exhibits a dimer/tetramer equilibrium that is dependent on the redox state of the cysteines and on the type of sulfur substrate component bound to them. Taken together, the dimer/tetramer equilibrium, the specific interactions between the subunits in the tetramer, and the significant conservation level of the interfaces strongly indicate that these SoxY oligomers are biologically relevant.