Synthesis,storage and degradation of polyglucose in chlorobium thiosulfatophilum

Cultures of Chlorobium thiosulfatophilum form polyglucose during growth. The polyglucose is laid down within the cells as rosette-like granules, which are made up from smaller grains. The size of each granule appears to be limited to less than 30 nm, since an increase in polyglucose content leads to more granules being formed rather than an increase in granule size.The polyglucose in washed cells is fermented in the dark to acetate, propionate, caproate and succinate, of which acetate by far comprises the largest fraction (68%). During incubation of washed cells without hydrogen donor, the level of polyglucose decreases regardless of whether the cells are incubated in the dark or in the light. Since the products formed from polyglucose under the two different conditions are not the same, it is suggested that polyglucose in the dark serves as an energy source, whereas when in the light the role of polyglucose is mainly to provide the cell with reducing power.     

Occurrence,structure and function of intracellular polyglucose in the obligate chemolithotroph Thiobacillus neapolitanus

Nitrogen-limited cells of the obligate chemolithotroph Thiobacillus neapolitanus formed an intracellular polymer during growth in the chemostat. This polymer was isolated and characterized as a branched polyglucose composed of units joined by -14 and -16 linkages. Polyglucose in T. neapolitanus can be considered a storage compound since formation of this compound took place during excess of energy and CO₂ whilst shortage of CO₂ resulted in rapid breakdown of polyglucose. Moreover the breakdown of polyglucose generated metabolically useful energy as could be demonstrated by polyglucose-dependent protein synthesis. Possession of polyglucose did not influence the viability of T. neapolitanus during prolonged periods of energy starvation. Activities of key enzymes of the oxidative pentose phosphate cycle, glucose-6-phosphatedehydrogenase and 6-phospho-gluconate-dehydrogenase, were demonstrated in cell free extracts of T. neapolitanus and appeared to increase 5- and 3-fold, respectively, during growth on NO ₃ ^- instead of NH ₄ ⁺ as a nitrogen source.     

Effect of light internsity on vesicle formation in Chlorobium

1. Chlorobium limicola forma sp. thiosulfatophilum was cultivated at 22 and 22000 lux. 2. The content of bchl d on a protein basis in the low light intensity cultures was about twice that of the high light intensity cultures. 3. After growth at 22 lux the red bchl d peak was at c. 743 nm, while at the higher intensity this peak was at c. 732 nm. 4. Electron microscopy of thin sections of Chlorobium revealed that vesicle size was greater at the low light intensity than at the high. 5. This was confirmed by sucrose density gradient centrifugation of differentially ¹⁴C-labelled vesicles from cultures grown at the two intensities. 6. The optimum temperature for growth was about 35°C. Incubation at the optimum temperature was particularly beneficial at high light intensity.Abbreviation bchl bacteriochlorophyll     

Frequency and structure of spinae on Chlorobium spp.

Several Chlorobium species have been observed to possess spinae. Spinae are non-prosthecate, helically wound, rigid structures that extend from the outer bacterial cell surface into the external environment. Spinae length was variable within and between Chlorobium species. Spinae width was fairly consistent within species but varied between species (39.4 ± 2.6 nm to 82.6 ± 8.0 nm). The number of spinae per cell varied. The spinae did not penetrate the bacterial cell envelope and were randomly located on the cell surface. Spinae were not geographically restricted. The observation of spinae on pure cultures of Chlorobium spp. maintained for 25–30 years suggests that spinae may be of significant use to the cell.     

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.     

Direct determination of polyglucose metabolites in plasma using anion-exchange chromatography with pulsed amperometric detection

High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE–PAD) was evaluated for the quantitation of polyglucose metabolites (DP2–DP7) in human plasma. The method was investigated for accuracy, precision, specificity, linearity, range and analyte stability. Samples were prepared by dilution into the standard range (0.1–10 μg/ml) followed by deproteinization using a 30?000 molecular mass cut-off filtration device. The limit of detection was 0.05 μg/ml for all metabolites. Method precision for DP2–DP7 varied from approximately 2% R.S.D. in the upper range to approximately 15% R.S.D. at the limit of quantitation. Samples were stable following one or two freeze–thaw cycles and, after preparation, they could be refrigerated for up to 72 h. Application of this method to clinical plasma samples from continuous ambulatory peritoneal dialysis (CAPD) patients administered one daily night-time intraperitoneal exchange of 2 l of 7.5% polyglucose solution for four weeks indicated that plasma levels of DP2, DP3 and DP4 increased from baseline levels of <0.01 g/l to steady-state levels of 1.2±0.3, 1.2±0.3 and 0.4±0.1 g/l (mean±S.D.), respectively. These steady state plasma levels for DP2 and DP3 are comparable to previously reported levels in patients administered daily overnight 7.5% polyglucose dialysis solution.     

Different Sensitivities to Oxygen Between Two Strains of the Photosynthetic Green Sulfur Bacterium Chlorobium vibrioforme NCIB 8327 with Bacteriochlorophyll c and d

Two sub-strains of the anoxygenic photosynthetic green sulfur bacterium Chlorobium vibrioforme NCIB 8327 were derived from the same clone and could be discriminated only by their possession of either bacteriochlorophyll (BChl) c or d as the major pigment in the peripheral light-harvesting antenna system, chlorosome (Saga Y et al. (2003) Anal Sci 19: 1575–1579). In the presence of a proper amount of oxygen in the initial culture medium, the BChl d strain showed longer retardation on its growth initiation than the BChl c strain, indicating that the latter was advantageous for survival under aerobic light conditions which produced reactive oxygen species in vivo. The result would be ascribable to the difference of the midpoint potentials between two kinds of chlorosomes formed by self-aggregates of BChl c and d as measured by their fluorescence quenching.     

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     

Cytochromes of the non-thiosulfate-utilizing green sulfur bacterium Chlorobium limicola

Flavocytochrome c-553 of the non-thiosulfateutilizing green sulfur bacterium Chlorobium limicola strain 6330 was partially purified by ion exchange column chromatography and ammonium sulfate fractionation (highest purity index obtained: A ₂₈₀/A _{417 red}=0.96). It is autoxidizable and located in the soluble fraction. This hemoprotein contains a flavin component and one heme per molecule. The dithionite reduced spectrum reveals the typical maxima of a c-type cytochrome: =553,5 nm; =523 nm; =417 nm, while the oxidized form shows a -band at 410 nm and two shoulders at 440 nm and 480 nm indicating the flavin component. The flavocytochrome is a basic protein with an isoelectric point at pH 9.0 (± 0.5), a redox potential of 65 mV, a molecular weight of 56,000. It participates in sulfide oxidation and shows neither adenylylsulfate reductase nor sulfite reductase activity. C. limicola further contains a soluble cytochrome c-555 (highest purity index obtained: A ₂₈₀/A _{412 ox}=0.13; isoelectric point between pH 9.5 and 10) and the non-heme iron-containing proteins rubredoxin and ferredoxin, but lacks cytochrome c-551. Besides these soluble electron transfer proteins a membrane-bound c-type cytochrome (=554,5 nm) can be detected spectrophotometrically.Non-common abbreviations HIPIP high-potential iron sulfur protein - APS adenylylsulfate     

Species specific release of sulfate from adenylyl sulfate by ATP sulfurylase or ADP sulfurylase in the green sulfur bacteria Chlorobium limicola and Chlorobium vibrioforme

High activities of ATP sulfurylase were found in the soluble protein fraction of two Chlorobium limicola strains, whereas ADP sulfurylase was absent. ATP sulfurylase was partially purified and characterized. It was a stable soluble enzyme with a molecular weight of 230,000, buffer-dependent pH optima at 8.6 and 7.2 and an isoelectric point at pH 4.8. No physiological inhibitor was found. Inhibition was observed with p-CMB and heavy metals. Sulfur compounds had no effect on enzyme activity. The stoichiometry of the reaction was proven. In contrast, an ADP sulfurylase, but no ATP sulfurylase, was found in Chlorobium vibrioforme. This enzyme was very labile with a molecular weight of about 120,000 and buffer-dependent pH optima at 9.0 and 8.5. Under test conditions the apparent K _m value was determined to be 0.28 mM for adenylyl sulfate and 8.0 mM for phosphate.Abbreviations APS adenylyl sulfate - p-CMB parachloromercuribenzoate - PP_i inorganic pyrophosphate     

Identification of the major chlorosomal bacteriochlorophylls of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides; their function in lateral energy transfer

The chlorosomal bacteriochlorophyll (BChl) composition of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides was investigated by means of normal-phase high-performance liquid chromatography. From both species a number of homologues was isolated, which were identified by absorption and ²⁵²Cf-plasma desorption mass spectroscopy. Besides BChl d, C. vibrioforme contained a significant amount of BChl c, which may provide an explanation for the previous observation of at least two spectrally different pools of BChl in the chlorosomes of green sulfur bacteria (Otte et al. 1991). C. phaeovibrioides contained various homologues of BChl e only. Absorption spectra in acetone of BChl c, d and e, as well as bacteriopheophytin e are presented. No systematic differences were found for the various homologues of each pigment. In addition to farnesol, the mass spectra revealed the presence of various minor esterifying alcohols in both species, including phytol, oleol, cetol and 4-undecyl-2-furanmethanol, as well as an alcohol of low molecular mass, which is tentatively assumed to be decenol.Abbreviations BChl bacteriochlorophyll - BPh bacteriopheophytin (used as a general name for the Mg-free compound, irrespective of the esterifying alcohol) - HPLC high-performance liquid chromatography     

The intracellular polyglucose storage granules of Spirochaeta aurantia

Extracts of Spirochaeta aurantia contained granules approximately 36 nm in diameter. These granules were purified by isopycnic centrifugation on CsCl gradients and shown on the basis of chemical and spectroscopic evidence to be glycogen. Electron microscopic cytochemical methods revealed glycogen-like granules in S. aurantia cells.     

The role of polyglucose in oxygen-dependent respiration by a new strain of Desulfovibrio salexigens

Abstract: Desulfovibrio salexigens strain Mastl was isolated from the oxic/anoxic interface of a marine sediment. Growth under sulfate-reducing conditions was accompanied by polyglucose accumulation in the cell with every substrate tested. Highest polyglucose storage was found with glucose (0.8–1.0 g polyglucose (g protein)⁻¹), but the growth rate with this substrate was very low (0.015 h⁻¹). Anaerobically grown cells of strain Mastl exhibited immediate oxygen-dependent respiration. The endogenous oxygen reduction rate was proportional to the polyglucose content. The rate of aerobic respiration of pyruvate was also directly related to the polyglucose content indicating that this organism was only able to respire with oxygen as long as polyglucose was present. Maximum oxygen reduction rates were found at air saturating concentrations and were relatively low (3–50 nmol O₂ min⁻¹ (mg protein)⁻¹). Catalase was constitutively present in anaerobically grown cells. When batch cultures were exposed to oxygen, growth ceased immediately and polyglucose was oxidized to acetate within 40–50 h. Like the oxygen reduction activity, the nitro blue tetrazolium (NBT)-reduction activity in these cells was proportional to the polyglucose content. Under anaerobic starvation conditions there was no correlation between the NBT-reduction activity and polyglucose concentration and polyglucose was degraded slowly within 240 h. The ecological significance of aerobic polyglucose consumption is discussed.     

Aspects of nitrogen fixation in Chlorobium

Four strains of the green sulfur bacterium Chlorobium were studied in respect to nitrogen nutrition and nitrogen fixation. All strains grew on ammonia, N₂, or glutamine as sole nitrogen sources; certain strains also grew on other amino acids. Acetylene-reducing activity was detectable in all strains grown on N₂ or on amino acids (except for glutamine). In N₂ grown Chlorobium thiosulfatophilum strain 8327 1 mM ammonia served to switch-off nitrogenase activity, but the effect of ammonia was much less dramatic in glutamate or limiting ammonia grown cells. The glutamine synthetase inhibitor methionine sulfoximine inhibited ammonia switch-off in all but one strain. Cell extracts of glutamate grown strain 8327 reduced acetylene and required Mg²⁺ and dithionite, but not Mn²⁺, for activity. Partially purified preparations of Rhodospirillum rubrum nitrogenase reductase (iron protein) activating enzyme slightly stimulated acetylene reduction in extracts of strain 8327, but no evidence for an indigenous Chlorobium activating enzyme was obtained. The results suggest that certain Chlorobium strains are fairly versatile in their nitrogen nutrition and that at least in vivo, nitrogenase activity in green bacteria is controlled by ammonia in a fashion similar to that described in nonsulfur purple bacteria and in Chromatium.Non-common abbreviations MSX Methionine sulfoximine - MOPS 3-(N-morpholino) propane sulfonic acid This paper is dedicated to Professor Norbert Pfennig on the occasion of his 60th birthday     

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.     

Biosynthesis of chlorosome proteins is not inhibited in acetylene-treated cultures of Chlorobium vibrioforme

The composition, abundance and apparent molecular masses of chlorosome polypeptides from Chlorobium tepidum and Chlorobium vibrioforme 8327 were compared. The most abundant, low-molecular-mass chlorosome polypeptides of both strains had similar electrophoretic mobilities and abundances, but several of the larger proteins were different in both apparent mass and abundance. Polyclonal antisera raised against recombinant chlorosome proteins of Cb. tepidum recognized the homologous proteins in Cb. vibrioforme, and a one-to-one correspondence between the chlorosome proteins of the two species was confirmed. As previously shown [Ormerod et al. (1990) J Bacteriol 172: 1352–1360], acetylene strongly suppressed the synthesis of bacteriochlorophyll c in Cb. vibrioforme strain 8327. No correlation was found between the bacteriochlorophyll c content of cells and the cellular content of chlorosome proteins. Nine of ten chlorosome proteins were detected in acetylene-treated cultures, and the chlorosome proteins were generally present in similar amounts in control and acetylene-treated cells. These results suggest that the synthesis of chlorosome proteins and the assembly of the chlorosome envelope is constitutive. It remains possible that the synthesis of bacteriochlorophyll c and its insertion into chlorosomes might be regulated by environmental parameters such as light intensity.This revised version was published online in October 2005 with corrections to the Cover Date.     

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.     

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     

Photorespiration in diatoms

Adenylylsulfate reductase was purified from Chlorobium limicola. The most important properties of the enzyme were compared with those of APS reductases from Thiocapsa, thiobacilli and sulfate-reducing bacteria.     

Coexistence of Chlorobium and Chromatium in a sulfide-limited continuous culture

Competition experiments between Chromatium vinosum and Chlorobium limicola in sulfide-limited continuous culture under photolithoautotrophic conditions resulted in the coexistence of both organisms. The ratio between the two bacteria was dilution-rate as well as pH dependent. The observed coexistence can be explained as a hitherto not reported form of dual substrate limitation. The two substrates involved are the electron donors sulfide (growth-limiting substrate in the reservoir vessel) and extracellular elemental sulfur (formed by Chlorobium as a result of sulfide oxidation). It is argued that, although Chlorobium may have the better affinity for both substrates involved, Chromatium can compete successfully on the basis of its intracellular storage of sulfur. Ecological implication of the observed coexistence with respect to natural blooms are discussed.