Photoassimilation of acetate and metabolism of carbohydrate in Chlorobium thiosulfatophilum

1. Washed cell suspensions of Chlorobium thiosulfatophilum form large amounts of a polyglucose in the light. Addition of acetate to the cells increases the formation of polysaccharide considerably. During incubation in the dark, polysaccharide decreases with time, and organic acids such as succinic and propionic acid are excreted into the medium. 2. Glucose isolated from cells which had photoassimilated 1-, 2-, and U-¹⁴C-acetate had a specific activity which lay between 1 and 2 times that of the acetate substrates. 3. To analyse the distribution of radioactivity in the glucose units formed during photoassimilation of ¹⁴C-acetate, 2 microbial degradations, with bakers' yeast and Zymomonas mobilis respectively, were used. The results show that acetate gives rise to carbon atoms 1+2 and 5+6 of glucose, whereas carbon atomes 3+4 are not labelled. Further, the results indicate that glucose is not formed via the reductive pentose phosphate cycle when acetate is present.     

Growth kinetics of the photosynthetic bacterium Chlorobium thiosulfatophilum in a fed-batch reactor

Hydrogen sulfide dissolved in water can be converted to elementary sulfur or sulfate by the photosynthetic bacterium Chlorobium thiosulfatophilum. Substrate inhibition occurred at sulfide concentrations above 5.7 mM. Light inhibition was found at average light intensities of 40,000 lux in a sulfide concentration of 5 mM, where no substrate inhibition occurred. Light intensity, the most important growth parameter, was attenuated through both scattering by sulfur particles and absorption by the cells. Average cell and sulfur particle sizes were 1.1 and 9.4 mum, respectively. Cells contributed 10 times as much to the turbidity as sulfur particles of the same weight concentration. The light attenuation factor was mathematically modeled, considering both the absorption and scattering effects based on the Beer-Lambert law and the Rayleigh theory, which were introduced to the cell growth model. Optimal operational conditions relating feed rate vs. light intensity were obtained to suppress the accumulation of sulfate and sulfide and save light energy for 2- and 4-L fed-batch reactors. Light intensity should be greater for the same performance (H(2)S removal rate/unit cell concentration) in larger reactors due to the scaleup effect on light transmission. Knowledge of appropriate growth kinetics in photosynthetic fed-batch reactors was essential to increase feed rate and light intensity and therefore cell growth. A mathematical model was developed that describes the cell growth by considering the light attenuation factor due to scattering and absorption and the crowding effect of the cells. This model was in good agreement with the experimental results. (c) 1992 John Wiley & Sons, Inc.     

Ribulose 1,5-diphosphate carboxylase and Chlorobium thiosulfatophilum

1. Cell-free extracts of the photosynthetic bacterium Chlorobium thiosulfatophilum, strains 8327 and Tassajara, were assayed for ribulose 1,5-diphosphate (RuDP) carboxylase and phosphoribulokinase-the two enzymes peculiar to the reductive pentose phosphate cycle. 2. RuDP carboxylase was consistently absent in strain 8327. The Tassajara strain showed a low RuDP-dependent CO₂ fixation activity that was somewhat higher in cells following transatlantic air shipment than in freshly grown cells. The stability and behaviour of this activity in sucrose density gradients were similar to those described by other workers. 3. The radioactive carboxylation products formed in the presence of RuDP by enzyme preparations from the Tassajara strain did not include 3-phosphoglycerate-the known product of the RuDP carboxylase reaction, but instead consisted of the unrelated acids glutamate, aspartate and malate. 4. Phosphoribulokinase was absent in all preparations of the two Chlorobium strains tested. By contrast, phosphoribulokinase as well as RuDP carboxylase were readily demonstrated in preparations from pea chloroplasts and the photosynthetic bacterium Rhodospirillum rubrum. 5. It is concluded that C. thiosulfatophilum appears to lack RuDP carboxylase, phosphoribulokinase, and hence, the reductive pentose phosphate cycle.Support of a J. S. Guggenheim Fellowship is gratefully acknowledged     

A comparative study of the isocitrate dehydrogenases of Chlorobium limicola forma Chlorobium thiosulfatophilum and Rhodopseudomonas palustris

The carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle in the obligate photolithotroph Chlorobium limicola forma thiosulfatophilum and the oxidation of isocitrate in the tricarboxylic acid cycle in the photoheterotroph Rhodopseudomonas palustris are catalyzed by isocitrate dehydrogenases. A comparative study of these enzymes isolated from the two bacteria showed that they virtually do not differ in the enzymatic and kinetic properties.     

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     

Localization and possible role of an adenosine triphosphatase in Chlorobium thiosulfatophilum.

1. Evidence is presented that the ATPase activity detected in cell extracts of Chlorobium thiosulfatophilum is bound to the cytoplasmic membrane rather than to the chlorobium vesicles. 2. The activity of this ATPase is inhibited in vitro by various carbodiimides, phloridzin and sodium azide. 3. The apparent Km for ATP is approximately 0.2 mM and the enzyme shows product inhibition by ADP. 4. Photophosphorylation, characterized in vivo, is inhibited by many of the compounds that inhibit the ATPase.     

Control of Oxidative Sulfur Metabolism of Chlorobium limicola forma thiosulfatophilum

A metered blend of anaerobic-grade N₂, CO₂, and H₂S gases was introduced into an illuminated, 800-ml liquid volume, continuously stirred tank reactor. The system, described as an anaerobic gas-to-liquid phase fed-batch reactor, was used to investigate the effects of H₂S flow rate and light energy on the accumulation of oxidized sulfur compounds formed by the photoautotroph Chlorobium limicola forma thiosulfatophilum during growth. Elemental sulfur was formed and accumulated in stoichiometric quantities when light energy and H₂S molar flow rate levels were optimally adjusted in the presence of nonlimiting CO₂. Deviation from the optimal H₂S and light energy levels resulted in either oxidation of sulfur or complete inhibition of sulfide oxidation. Based on these observations, a model of sulfide and sulfur oxidases electrochemically coupled to the photosynthetic reaction center of Chlorobium spp. is presented. The dynamic deregulation of oxidative pathways may be a mechanism for supplying the photosynthetic reaction center with a continuous source of electrons during periods of varying light and substrate availability, as in pond ecosystems where Chlorobium spp. are found. Possible applications for a sulfide gas removal process are discussed.     

A comparative study on the effect of O2 on photosynthetic carbon metabolism by Chlorobium thiosulfatophilum and Chromatium vinosum

The reductive carboxylic acid cycle appears to be the majorcarbon assimilation pathway in green sulfur bacteria, Chlorobiumthiosulfatophilum. While cyanide was relatively ineffectivein inhibiting the bacterial photosynthetic CO₂ fixation, photosynthesiswas strongly impaired in an O₂-containing atmospheric environment.No glycolate formation was detected in Chlorobium under an O₂atmosphere. In the purple sulfur bacteria, Chromatium vinosum,however, photosynthesis was highly sensitive to cyanide, andin a short-term incubation (up to 10 min) photosynthetic CO₂fixation was found to be relatively indifferent to an O₂-containingatmosphere of up to 100% O₂. Significant formation of glycolatewas demonstrated upon a very brief exposure to O₂, whereas thetotal photosynthetic CO₂ fixation was slightly affected. However,ribulose-1,5-bisphosphate carboxylase activity in Chromatiumextract was competitively inhibited by O₂ in a similar mannerto the higher plant enzyme, K¹(O₂) value being 0.7 mM at pH8.2. The percentage of incorporation of ¹⁴CO₂ into glycolateand glycine under an O₂-containing atmosphere declined withincreasing levels of bicarbonate concentrations in the medium.The Warburg effect and biosynthetic mechanisms involving glycolatein photosynthetic bacteria are discussed. ¹ This is paper XXXIX in the series "Structure and Functionof Chloroplast Proteins". Paper XXXVIII is reference (6) Asamiand Akazawa (1977). This research was supported in part by grantsfrom the Ministry of Education of Japan (111912), the TorayScience Foundation (Tokyo), and the Japan Securities ScholarshipFoundation (Tokyo). (Received January 28, 1977; )     

A novel aminoglycosphingolipid found in Chlorobium limicola f. thiosulfatophilum 6230

An aminolipid from Chlorobium limicola f. thiosulfatophilum has been purified and characterized by thin-layer chromatography, infrared specroscopy, ¹H-NMR, ¹³C-NMR, plasma desorption mass spectrometry, and fast atom bombardment mass spectrometry. The structure is that of an aminosugar (neuraminic acid) attached to a sphingosine backbone with one myristic acid linked to the sphingosine by an amide bond. Related glycosphingolipids and capnoids are found in the Bacterioides/Flavobacteria which are related to the green sulfur bacteria by the criterion of 16S rRNA structure. No aminoglycosphingolipid was found in Chloroflexus aurantiacus.     

ATP-linked citrate lyase activity in the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum

Cell-free extracts of the green sulfur bacterium Chlorobium limicola forma thiosulfatophilum strains 1C and L have been shown to cleave citrate with the formation of oxaloacetate and acetyl-CoA. This capacity was found in autotrophically grown cells as well as in the cells grown on media with acetate or L-glutamate. Citrate lyase activity in cell-free extracts is only measurable in the presence of citrate, adenosine-5-triphosphate, coenzyme A and Mg²⁺ or Mn²⁺. It is concluded on the basis of the obtained data that C. limicola f. thiosulfatophilum contains adenosine-5-triphosphate-linked citrate lyase (E.C.4.1.3.8). In contrast to green bacteria in the purple bacteria Ectothiorhodospira shaposhnikovii, Rhodospirillum rubrum and Thiocapsa roseopersicina citrate lyase activity was not found.     

The lipopolysaccharide of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum

The cell wall lipopolysaccharide of the green sulfur bacterium Chlorobium vibrioforme f. thiosulfatophilum was obtained by the phenol-chloroform-petroleum ether and the hot phenol-water methods, respectively. It contained mannose, glucose, galacturonic acid, glucosamine, glycine, and small amounts of rhamnose, galactose and glucuronic acid. In addition to d-glycero-d-mannoheptose, the corespecific constituents 2-keto-3-deoxyoctonate and l-glycero-d-mannoheptose were found. Polyacrylamide gel-electrophoresis in the presence of sodium deoxycholate gave no indication for the presence of O-specific repeating units. Degradation of the lipopolysaccharide required 10% acetic acid (100° C, 2 h). The lipid A moiety contained the total of glucosamine of the lipopolysaccharide as well as small amounts of 2,3-diamino-2,3-dideoxy-glucose. It was phosphate-free. The fatty acid spectrum comprised 3-OH-14:0, 3-OH-16:0, and iso-3-OH-18:0 besides little 12:0, 14:0 and 16:0. Hydroxylaminolysis and sodium methylate treatment revealed all of the three hydroxy fatty acids to be amidebound.Abbreviations DOC sodium deoxycholate - PAGE polyacrylamide gel-electrophoresis     

The oxidation and reduction of pyridine nucleotides by Rhodopseudomonas spheroides and Chlorobium thiosulfatophilum

Summary The light-induced formation of NADH by whole cells of Rhodopseudomonas spheroides has been followed fluorimetrically and found to lag slightly behind cytochrome c oxidation. The uncoupler, FCCP¹, abolished NADH formation which was also inhibited by HOQNO¹. Electron flow from NADH to oxygen or cytochrome c was inhibited in chromatophores of R. spheroides by HOQNO, antimycin A and rotenone. From the known properties of the inhibitors used it is deduced that NADH formation in the light is dependent upon reversed electron flow. No light-induced formation of NAD(P)H by whole cells or chromatophores of Chlorobium thiosulfatophilum was detected either fluorimetrically or by extraction followed by enzymic assay although cytochrome c oxidation was extensive in whole cells. Extracts of C. thiosulfatophilum catalysed the rapid reduction of endogenous or mammalian cytochrome c; unlike R. spheroides this activity was found almost entirely in the soluble fraction and was insensitive to HOQNO, antimycin A and rotenone. No cytochrome b was detected in C. thiosulfatophilum by difference spectroscopy of pyridine haemochromes of acetone powders. The K _m for NADH of NADH-cytochrome c reductase in both organisms was about 3 mol; the reductase was inhibited by NAD. The rates of NADPH-cytochrome c reductase in R. spheroides particles were too low for K _m determination; for C. thiosulfatophilum particles the K _m for NADPH was about 300 mol. The addition of NADH to soluble extracts of either organism caused the reduction of endogenous flavin that was reoxidised by ferricyanide. The NADH-cytochrome c reductase of C. thiosulfatophilum was not separated from ferredoxin on a DEAE column. It is concluded that in C. thiosulfatophilum the formation of NADH in an energy-linked reaction is unlikely; the possibility of a cyclic electron flow involving chlorophyll, ferredoxin, flavoprotein and cytochrome c is discussed.     

A Comparative Study of the Isocitrate Dehydrogenases of Chlorobium limicola forma thiosulfatophilum and Rhodopseudomonas palustris

The carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle in the obligate photolithotroph Chlorobium limicola forma thiosulfatophilum and the oxidation of isocitrate in the tricarboxylic acid cycle in the photoheterotroph Rhodopseudomonas palustris are catalyzed by isocitrate dehydrogenases. A comparative study of these enzymes isolated from the two bacteria showed that they virtually do not differ in enzymatic and kinetic properties.     

Removal of hydrogen sulfide by Chlorobium thiosulfatophilum in immobilized-cell and sulfur-settling free-cell recycle reactors.

Bioconversion of hydrogen sulfide to elementary sulfur by the photosynthetic bacterium Chlorobium thiosulfatophilum was studied in immobilized-cell and sulfur-settling free-cell recycle reactors. The cells immobilized in strontium alginate beads excreted elementary sulfur and accumulated it as crystal in the bead matrices, which made it possible that the reactor broth remained clear and the light penetrated the reactor deeper than with the free cells. In comparison with the free cells, the immobilized cells required 30% less light energy at a H2S removal rate of 2 mM/(L.h) and showed an activity of 2.4 times that of the free cells. However, in 40 h after the reaction the deterioration of the H2S removal efficiency became significant due to the accumulation of sulfur in the beads. The scanning electron micrograph (SEM) and energy-dispersive X-ray spectrometer (EDS) studies showed that the sulfur in the beads existed within a layer of 0.4 mm from the bead surface. In the sulfur-settling free-cell recycle reactor, about 80% of the sulfur excreted by the free cells could be removed in a settler. The 4-L fed batch reactor with the settler improved the light transmission to result in a H2S removal rate of 3 mumol/(mg of protein.h), 50% higher than that without it. The settling recycle reactor was much better in the removal of H2S than the immobilized-cell reactor because the former was a continuous system with the constant removal of sulfur particles by settling and of spent medium by supplying fresh medium at the same rate as the filtering rate of the reactor broth, while the latter was essentially a batch system where toxic metabolites and produced sulfur could not be removed.