Uranous ion oxidation and carbon dioxide fixation byThiobacillus ferrooxidans

The stoichiometric oxidation of uranous-to uranyl-uranium byThiobacllus ferrooxidans is demonstrated. Fixation of¹⁴CO₂ and the effect of inhibitors demonstrate that energy is conserved during the oxidation and used for energy-dependent reverse electron flow and carbon dioxide fixation.Abbreviations HOQNO 2-heptyl-4-hydroxyquinoline-N-oxide - 8-HQ 8-hydroxyquinoline - TTFA thenoyltrifluoroacetone     

Morphology of bacterial leaching patterns by Thiobacillus ferrooxidans on synthetic pyrite

Thiobacillus ferrooxidans has been cultivated on synthetic pyrite (FeS₂) single crystals as the only energy source and the pyrite interface investigated with respect to characteristic morphological changes using scanning electron microscopy. Corrosion patterns of bacterial size were identified in different stages of development and correlated with bacterial activity. It appears that bacterial attack of the sulfide interface starts by secretion of organic substances around the contact area between the bacterial cell and the sulfide energy source. They might either be part of a pseudo capsule which shields the contact area or may form a sulfur absorbing and transporting organic film. Degradation of the sulfide occurs in the contact area below the bacterial cell leading to a corrosion pit which the bacterium may abandon after it has reached a depth of bacterial dimension. Electron spectroscopic (XPS) and X-ray fluorescence studies indicate a layer of organic substances covering the sulfide surface under bacterial leaching conditions, which is sufficiently thick for consideration in interfacial chemical mechanisms.     

Nitrogen fixation activity of a filamentous,nonheterocystous cyanobacterium in the presence and absence of exogenous,organic substrates

Gallionella ferruginea is able to utilize Fe(II) and the reduced sulfur compounds sulfide and thiosulfate as electron donor and energy source. Tetrathionate and elemental sulfur, on the other hand, are not metabolized. In sulfide-O₂ microgradient cultures G. ferruginea grows at the interface between the oxidizing and the reducing zones. Optimal growth depends on low oxygen and sulfide concentrations. Establishing within the gradient protects the bacterium from too high sulfide concentrations. G. ferruginea excretes extracellular polymeric substances (EPS). While in FeS-gradient cultures 2×10⁶ cells/ml were obtained the bacterial mass could be increased to 1–3×10⁸ cells/ml in shaken batch cultures using thiosulfate as substrate. A further increase of bacterial mass by adding an organic carbon source was not possible confirming that G. ferruginea is an obligate autotrophic organism. When growing on sulfide or thiosulfate the otherwise characteristic twisted stalk consisting of ferric hydroxide is lacking. It is thus shown to be a metabolic end product of Fe(II) oxidation rather than metabolically active cellular material.     

The production and utilization of intermediary elemental sulfur during the oxidation of reduced sulfur compounds by Thiobacillus ferrooxidans

The intermediary production of elemental sulfur during the microbial oxidation of reduced sulfur compounds has frequently been reported. Thiobacillus ferrooxidans, an acidophilic chemolithoautotroph, was found to produce an insoluble sulfur compound, primarily elemental sulfur, during the oxidation of thiosulfate, trithionate, tetrathionate and sulfide. This was confirmed by light and electron microscopy. Sulfur was produced from sulfide by an oxidative step, while the production from tetrathionate was initiated by a hydrolytic step, probably followed by a series of chemical reactions. The oxidation of intermediary sulfur was severely inhibited by sulfhydryl-binding reagents such as N-ethylmaleimide, by the addition of uncouplers or after freezing and thawing of the cells, which probably damaged the cell membrane. The mechanisms behind these inhibitions have not yet been clarified. Finally, it was observed that elemental sulfur oxidation by whole cells depended on the medium composition. The absence of sulfate or selenate reduced the sulfur oxidation rate.Non-standard abbreviations NEM N-ethylmaleimide - CCCP carbonyl cyanide m-chlorophenyl hydrazone     

Strain variability and the effects of organic compounds on the growth of the chemolithotrophic bacterium Thiobacillus ferrooxidans

The effects of naturally-occurring organic compounds on ferrous iron oxidation by the bacterium Thiobacillus ferrooxidans were examined with a view to using these compounds to treat or prevent acid mine/rock drainage. The compounds glucose, cellobiose, galacturonic acid, and citric acid were added to the growth medium of five different strains of the bacterium and growth studies were done to determine whether or not strain differences existed with respect to organic compound sensitivity. The effects of these compounds were compared to the effects of sodium dodecyl sulfate (SDS) an anionic detergent. Each of the compounds tested had an inhibitory effect on the strains of the bacterium and sensitivity to these compounds was strain dependent. All strains appeared to be equally susceptible to SDS. Inhibitory concentrations ranged from 70 mM to >280 mM for glucose, 7.5 mM to 150 mM for cellobiose, 20 mM to 230 mM for galacturonic acid, and 50 mM to 130 mM for citric acid. SDS effectively inhibited iron oxidation for all strains at a concentration of 0.3 mM, the lowest concentration tested. Some naturally-occurring organic compounds, therefore, might be candidates for the growth control of T. ferrooxidans.     

Short communication: Adverse effect of surface-active reagents on the bioleaching of pyrite and chalcopyrite by Thiobacillus ferrooxidans

Oxidation of Fe(II) iron and bioleaching of pyrite and chalcopyrite by Thiobacillus ferrooxidans was adversely affected by isopropylxanthate, a flotation agent, and by LIX 984, a solvent-extraction agent, each at 1 g/l. The reagents/l were adsorbed on the bacterial surface, decreasing the bacteria's development and preventing biooxidation. Both reagents inhibited the bioleaching of pyrite and LIX 984 also inhibited the bioleaching of chalcopyrite.     

Utilization of 35S-Thiosulphate and an appraisal of the role of ATP-sulphurylase in chemolithotrophic Thiobacillus ferrooxidans

Differentially labelled ³⁵S-thiosulphate was taken up by washed cells of Thiobacillus ferrooxidans which were previously grown on thiosulphate. The uptake was proportional to the biomass over the range 0.5–4.0 mg dry wt. of bacteria and showed typical saturation kinetics with an estimated K _m value of 0.5 mM for ³⁵S-thiosulphate. Dithionate and Group VI anions inhibited the uptake, which was under pH control and had a temperature optimum of 50°C. In the absence of thiosulphate, the cells bound ³⁵S-sulphate but the binding did not increase on prolonged incubation and the label could be removed completely by washing with dilute sulphuric acid. Increasing amounts of the label were incorporated from [outer-³⁵S]thiosulphate into cellular materials over a 60-min period, whereas little or no assimilation was observed from either the [inner-³⁵S]thiosulphate or ³⁵S-sulphate. The kinetic properties of the sulphate-activating enzyme ATP_sulphurylase enriched from bacteria grown with either thiosulphate or ferrous-iron were similar although this enzyme has an assimilatory function only when the bacterium is grown with ferrous-iron.Abbreviation APS adenosine-5-sulphatophosphate     

Studies on ultrastructure and composition of cell walls of the cyanobacterium Anacystis nidulans

The multilayered cell wall of the cyanobacterium Anacystis nidulans was studied by the freezeetching technique. A characteristic fracture face in the outer cell wall was demonstrated which is densely packed with particles of a diameter of 60–75 Å. This particle layer is comparable with layers which have been described in many cell walls of Gram-negative prokaryotes.The outer membrane of the cell wall was solubilised by extraction with phenol/water or sodium dodecyl sulfate (SDS). In the SDS-extract 31 bands were separated by polyacrylamide gel electrophoresis, among them 3–5 major proteins with molecular weights of approximately 60, 40, and 10 kdaltons, respectively. Several polypeptides of the Anacystis cell wall were comparable in their mobility with polypeptides extracted from cell walls of different Gramnegative bacteria. The analysis of the SDS-unsoluble electron dense layer (sacculi) revealed the typical components of peptidoglycan diaminopimelic acid, muramic acid, glutamic acid, glucosamine and alamine in the molar ratio of 1.0:0.9:1.1:1.5:1.9. In addition, other amino acids (molar ratio from 0.05–0.36), mannosamine (molar ratio 0.54), and lipopolysaccharide components were detected in low concentration.Abbreviations SDS sodium dodecyl sulfate - EDTA ethylene diamine tetraacetate     

Quantification and intracellular distribution of ribulose-1,5-bisphosphate carboxylase in Thiobacillus neapolitanus,as related to possible functions of carboxysomes

Ribulose-1,5-bisphosphate carboxylase (RuBPCase) has been quantified by immunological methods in Thiobacillus neapolitanus cultivated under various growth conditions in the chemostat at a fixed dilution rate of 0.07 h^-1. RuBPCase was a major protein in T. neapolitanus accounting for a maximum of 17% of the total protein during CO₂ limitation and for a minimum of 4% during either ammonium- or thiosulfate limitation in the presence of 5% CO₂ (v/v) in the gasphase. The soluble RuBPCase (i.e. in the cytosol) and the particulate RuBPCase (i.e. in the carboxysomes) were shown to be immunologically identical. The intracellular distribution of RuBPCase protein between carboxysomes and cytosol was quantified by rocket immunoelectrophoresis. The particulate RuBPCase content, which correlated with the volume density of carboxysomes, was minimal during ammonium limitation (1.3% of the total protein) and maximal during CO₂ limitation (6.8% of the total protein). A protein storage function of carboxysomes is doubtful since nitrogen starvation did not result in degradation of particulate RuBPCase within 24 h. Proteolysis of RuBPCase was not detected. Carboxysomes, on the other hand, were degraded rapidly (50% within 1 h) after change-over from CO₂ limitation to thiosulfate limitation with excess CO₂. Particulate RuBPCase protein became soluble during this degradation of carboxysomes, but this did not result in an increase in soluble RuBPCase activity. Modification of RuBPCase resulting in a lower true specific activity was suggested to explain this phenomenon. The true specific activity was very similar for soluble and particulate RuBPCase during various steady state growth conditions (about 700 nmol/min·mg RuBPCase protein), with the exception of CO₂-limited growth when the true specific activity of the soluble RuBPCase was extremely low (260 nmol/min ·mg protein). When chemostat cultures of T. neapolitanus were exposed to different oxygen tensions, neither the intracellular distribution of RuBPCase nor the content of RuBPCase were affected. Short-term labelling experiments showed that during CO₂ limitation, when carboxysomes were most abundant, CO₂ is fixed via the Calvin cycle. The data are assessed in terms of possible functions of carboxysomes.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase - PEP phosphoenolpyruvate - RIE rocket immunoelectrophoresis - CIE crossed immunoelectrophoresis     

Ultrastructure of the synaptic ribbons in photoreceptor cells of Rana catesbeiana revealed by freeze-etching and freeze-substitution

Summary The three-dimensional structure of synaptic ribbons in photoreceptor cells of the frog retina was studied with freeze-etching and freeze-substitution methods, combined with a rapid-freezing technique. Although the synaptic ribbon consisted of two electron-dense plaques bisected by an electron-lucent layer in conventional thin sections, such lamellar nature was not so evident in freeze-etched replicas. The cytoplasmic surfaces of the synaptic ribbon presented an extremely regular arrangements of small particles 4–6 nm in diameter. Fine filaments 8–10 nm in diameter and 30–50 nm in length connected synaptic vesicles and the ribbon surface. These connections were mediated by large particles on both ends of the filaments. Approximately 3–5 filaments attached to one synaptic vesicle. Synaptic ribbons were anchored to a characteristic meshwork underlying the presynaptic membrane via another group of similar fine filaments. The meshwork seemed to be an etched replicated image of the presynaptic archiform density observed in thin sections.     

Kinetics of uranous ion and ferrous iron oxidation byThiobacillus ferrooxidans

Kinetic constants for the oxidation of uranous and ferrous ions byThiobacillus ferrooxidans were estimated. The kinetics indicate a direct biological mechanism for uranium oxidation. The complex interrelations of ferric, uranyl and uranous ion inhibition are considered.     

Microbial desulfurization of coal and oxidation of pure pyrite byThiobacillus ferrooxidans andAcidianus brierleyi

Summary Thiobacillus ferrooxidans andAcidianus brierleyi were capable of oxidizing pure pyrite as well as oxidizing sulfur in coal. First order reactions were assumed in the kinetic analysis performed. For oxidation of pure pyrite the rate constant was higher forA. brierleyi than forT. ferrooxidans. For sulfur removal from coal the values of the rate constants were comparable for the two microorganisms.     

The ultrastructure of cellular division (autosporogenesis) in the coccoid green alga,Trebouxia aggregata,revealed by rapid freeze fixation and freeze substitution

Summary Adequate ultrastructural preservation of cells of the green algaTrebouxia aggregata is achieved by immersion freeze fixation using liquid propane followed by freeze substitution and resin embedding at ambient temperature. Despite differential staining of membranes, using this method we have been able to study plasma membrane biogenesis during cellular division. Daughter protoplasts are separated by an ingrowing septum of plasma membrane that extends into the cell from a particular site at the peripheral plasma membrane marked by centrioles. Septum development involves tip growth followed by lateral growth. This growth seems to involve transfer of membrane from an adjacent partially coated reticulum to the septum plasma membrane. The reticulum which extends from nearby Golgi stacks to the area of septum growth is associated with an extensive array of microtubules. After daughter protoplasts are completely separated, each one becomes surrounded by a cell wall which is distinct from the persisting mother wall. The ultrastructural evidence suggests that cells ofT. aggregata are autospores rather than vegetative cells.Abbreviations C centriole - ER endoplasmic reticulum - G Golgi body - MTOC microtubule organizing center - Mt(s) microtubule(s) - N nucleus - P primary septum - PCR partially coated reticulum - PM plasma membrane - Py pyrenoid - S septum     

Complexation of uranium (VI) by three eco-types of Acidithiobacillus ferrooxidans studied using time-resolved laser-induced fluorescence spectroscopy and infrared spectroscopy

Time-resolved laser-induced fluorescence spectroscopy (TRLFS) was used to study the properties of uranium complexes (emission spectra and fluorescence lifetimes) formed by the cells of the three recently described eco-types of Acidithiobacillus ferrooxidans. The results demonstrated that these complexes have different lifetimes which increase in the same order as the capability of the strains to accumulate uranium. The complexes built by the cells of the eco-type II were the strongest, whereas, those of the eco-types I and III were significantly weaker. The emission spectra of all A. ferrooxidans complexes were almost identical to those of the uranyl organic phosphate compounds. The latter finding was confirmed by infrared spectroscopic analysis.     

The ultrastructure of cell wall formation and of gamma particles during encystment of Allomyces macrogynus zoospores

Structural changes during cell wall formation by populations of semisynchronously germinating zoospores were studied in the water mold Allomyces macrogynus. Fluorescence microscopy using Calcofluor white ST (which binds to -1,4-linked glycans) demonstrated that Calcofluor-specific material was deposited around most cells between 2–10 min after the induction of encystment (beginning when a wall-less zoospore retracts its flagellum and rounds up). During the first 15 min of encystment there was a progressive increase in fluorescence intensity. Ultrastructural analysis of encysting cells showed that within 2–10 min after the induction of encystment small vesicles 35–70 nm diameter were present near the spore surface, and some were in the process of fusing with the plasma membrane. The fusion of vesicles with the zoospore membrane was concomitant with the appearance of electron-opaque fibrillar material outside the plasma membrane. Vesicles similar to those near the spore surface were found within the gamma () particles of encysting cells. These particles had a crystalline inclusion within the electron-opaque matrix. During the period of initial cyst cell wall formation numerous vesicles appeared to arise at the crystal-matrix interface. Approximately 15–20 min was required for the cell wall to be formed. We suggest that the initial response of the zoospore to induction of encystment is the formation of a cell wall mediated by the fusion of cytoplasmic vesicles with the plasma membrane.Non-Standard Abbreviations GlcNac N-Acetylglucosamine - DS sterile dilute salts solution - PYG peptone-yeast extract-glucose broth     

Excitation delocalization in the bacteriochlorophyll c antenna of the green bacterium Chloroflexus aurantiacus as revealed by ultrafast pump-probe spectroscopy

Room temperature absorption difference spectra were measured on the femtosecond through picosecond time scales for chlorosomes isolated from the green bacterium Chloroflexus aurantiacus. Anomalously high values of photoinduced absorption changes were revealed in the BChl c Q_y transition band. Photoinduced absorption changes at the bleaching peak in the BChl c band were found to be 7–8 times greater than those at the bleaching peak in the BChl a band of the chlorosome. This appears to be the first direct experimental proof of excitation delocalization over many BChl c antenna molecules in the chlorosome.     

The ultrastructure of the cell surface and plasma membrane of exponential and stationary phase cells of Schizosaccharomyces pombe,grown in different media

In order to study the ultrastructure of the cell surface and plasma membrane of Schizosaccharomyces pombe as a function of growth conditions we investigated exponential and stationary phase cells grown in rich and minimal medium.Electron microscopic preparation techniques based on rapid cryofixation (without cryoprotectants) were used. The intramembraneous aspects of the plasma membrane were described by freeze fracturing. For the first time the dynamic surface structures could be directly analyzed by freeze drying in the scanning electron microscope and in thin section of freeze substituted samples. This preparation techniques reveal hair-like structures on the surface of yeast cells. The hairs of cells grown in the rich medium are longer than those grown in the minimal medium. A mutant defective in the structure of a cell surface galactomannoprotein (acid phosphatase) reveals (under conditions of maximal acid phosphatase expression) a cell surface structure that differs from the wild type. It is likely that the hairs represent the peripheral galactomannan layer or part of it.On the membrane fracture faces the number, shape, distribution and state of aggregation of the intramembraneous particles are different between membranes of growing and non-growing cells and between cells grown under different physiological conditions. In the minimal medium corresponding periodical structures on the plasmic and exoplasmic fracture faces were observed, which clearly differ between exponential and stationary phase cells. The number, length and depth of plasma membrane invaginations increase as the cells go from the exponential phase to the stationary phase. Short and flattened invaginations are filled with thin periodic structures.     

Composition and ultrastructure of the suberized cell wall of isolated crystal idioblasts from Agave americana L. leaves

Styloid-calcium-oxalate-crystal-containing idioblasts possess an interior cell-wall layer which has a lamellar ultrastructure. Idioblasts were isolated by centrifugation of an Agave americana leaf homogenate through 2M sucrose. The aliphatic monomers of the polymeric material from an idioblast fraction were primarily -hydroxy acids (32%) and dicarboxylic acids (35%), with C_18:1 dicarboxylic acid being the most dominant monomer (25%). Nitrobenzene oxidation of the idioblasts yielded syringaldehyde and vanillin in a ratio of 0.46:1. The major class of wax associated with the idioblasts was free fatty acids (34%). A major homologue of both the fatty acid and fatty alcohol fractions of this wax was C₂₂. The hydrocarbon fraction of the wax had a broad chainlength distribution with a large amount of even-numbered (47%) and shorter-chain homologues. The ultrastructure, the composition of the aliphatic and aromatic components of the polymeric material as well as the composition of the wax show that the idioblast cell wall is suberized. The wax and cutin polymer of the epidermis of A. americana leaves were chemically characterized for comparative purposes.Scientific paper No. 6115, Project 2001, College of Agriculture Research Center, Washington State University, Pullman, WA 99164, USA     

The body wall of Halicryptus spinulosus (Priapulida) — ultrastructure and changes induced by hydrogen sulfide

Halicryptus spinulosus (Priapulida) is extraordinarily resistant to hydrogen sulfide. As described previously, the body wall of specimens from oxic and from hydrogen sulfide-contaminated habitats differs strikingly in colour. In the present paper the ultrastructure of the body wall of specimens kept under oxic conditions is described. These findings serve as a reference for changes induced by exposure to hydrogen sulfide. There are marked ultrastructural differences between epidermal and muscular mitochondria exposed to hydrogen sulfide. Epidermal mitochondria of individuals subjected to hydrogen sulfide are often associated with dense granules of unknown composition. Findings are compared with those from other taxa which may encounter hydrogen sulfide in their environment.Contribution No. 386 of the Alfred-Wegener-Institut für Polar- and Meeresforschung (Awl Bremerhaven).     

Cell types of the endocrine pancreas in the shark Scyliorhinus stellaris as revealed by correlative light and electron microscopy

Summary In the pancreas of Scyliorhinus stellaris large islets are usually found around small ducts, the inner surface of which is covered by elongated epithelial cells; thus the endocrine cells are never exposed directly to the lumen of the duct. Sometimes, single islet cells or small groups of endocrine elements are also incorporated into acini. Using correlative light and electron microscopy, eight islet cell types were identified:Only B-cells (type I) display a positive reaction with pseudoisocyanin and aldehyde-fuchsin staining. This cell type contains numerous small secretory granules (Ø280 nm). Type II- and III-cells possess large granules stainable with orange G and azocarmine and show strong luminescence with dark-field microscopy. Type II-cells have spherical (Ø700 nm), type III-cells spherical to elongated granules (Ø450 × 750 nm). Type II-cells are possibly analogous to A-cells, while type III-cells resemble mammalian enterochromaffin cells. Type IV- cells contain granules (Ø540 nm) of high electron density showing a positive reaction to the Hellman-Hellerström silver impregnation and a negative reaction to Grimelius' silver impregnation; they are most probably analogous to D-cells of other species. Type VI-cells exhibit smaller granules (Ø250 × 500 nm), oval to elongated in shape. Type VI-cells contain small spherical granules (Ø310 nm). Type VII-cells possess two kinds of large granules interspersed in the cytoplasm; one type is spherical and electron dense (Ø650 nm), the other spherical and less electron dense (Ø900 nm). Type VIII-cells have small granules curved in shape and show moderate electron density (Ø100 nm). Grimelius-positive secretory granules were not only found in cell types II and III, but also in types V, VI, and VII. B-cells (type I) and the cell types II to IV were the most frequent cells; types V to VII occurred occasionally, whereas type VIII-cells were very rare.This work was supported by a fellowship from the Ministry of Education of Japan and the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (La 229/8)