Combined use of flow cytometry and microscopy to study the interactions between the gram-negative betaproteobacterium Acidovorax facilis and uranium(VI).

The former uranium mine Königstein (Saxony, Germany) is currently in the process of remediation by means of controlled underground flooding. Nevertheless, the flooding water has to be cleaned up by a conventional wastewater treatment plant. In this study, the uranium(VI) removal and tolerance mechanisms of the gram-negative betaproteobacterium Acidovorax facilis were investigated by a multidisciplinary approach combining wet chemistry, flow cytometry, and microscopy. The kinetics of uranium removal and the corresponding mechanisms were investigated. The results showed a biphasic process of uranium removal characterized by a first phase where 95% of uranium was removed within the first 8h followed by a second phase that reached equilibrium after 24h. The bacterial cells displayed a total uranium removal capacity of 130mgU/g dry biomass. The removal of uranium was also temperature-dependent, indicating that metabolic activity heavily influenced bacterial interactions with uranium. TEM analyses showed biosorption on the cell surface and intracellular accumulation of uranium. Uranium tolerance tests showed that A. facilis was able to withstand concentrations up to 0.1mM. This work demonstrates that A. facilis is a suitable candidate for in situ bioremediation of flooding water in Königstein as well as for other contaminated waste waters.

The influence of biofilms on the migration of uranium in acid mine drainage (AMD) waters.

The uranium mine in Königstein (Germany) is currently in the process of being flooded. Huge mass of Ferrovum myxofaciens dominated biofilms are growing in the acid mine drainage (AMD) water as macroscopic streamers and as stalactite-like snottites hanging from the ceiling of the galleries. Microsensor measurements were performed in the AMD water as well as in the biofilms from the drainage channel on-site and in the laboratory. The analytical data of the AMD water was used for the thermodynamic calculation of the predominance fields of the aquatic uranium sulfate (UO(2)SO(4)) and UO(2)(++) speciation as well as of the solid uranium species Uranophane [Ca(UO(2))(2)(SiO(3)OH)(2)∙5H(2)O] and Coffinite [U(SiO(4))(1-x)(OH)(4x)], which are defined in the stability field of pH>4.8 and Eh<960 mV and pH>0 and Eh<300 mV, respectively. The plotting of the measured redox potential and pH of the AMD water and the biofilm into the calculated pH-Eh diagram showed that an aqueous uranium(VI) sulfate complex exists under the ambient conditions. According to thermodynamic calculations a retention of uranium from the AMD water by forming solid uranium(VI) or uranium(IV) species will be inhibited until the pH will increase to >4.8. Even analysis by Energy-filtered Transmission Electron Microscopy (EF-TEM) and electron energy loss spectroscopy (EELS) within the biofilms did not provide any microscopic or spectroscopic evidence for the presence of uranium immobilization. In laboratory experiments the first phase of the flooding process was simulated by increasing the pH of the AMD water. The results of the experiments indicated that the F. myxofaciens dominated biofilms may have a substantial impact on the migration of uranium. The AMD water remained acid although it was permanently neutralized with the consequence that the retention of uranium from the aqueous solution by the formation of solid uranium species will be inhibited.

GARP: a key receptor controlling FOXP3 in human regulatory T cells.

Recent evidence suggests that regulatory pathways might control sustained high levels of FOXP3 in regulatory CD4(+)CD25(hi) T (T(reg)) cells. Based on transcriptional profiling of ex vivo activated T(reg) and helper CD4(+)CD25(-) T (T(h)) cells we have identified GARP (glycoprotein-A repetitions predominant), LGALS3 (lectin, galactoside-binding, soluble, 3) and LGMN (legumain) as novel genes implicated in human T(reg) cell function, which are induced upon T-cell receptor stimulation. Retroviral overexpression of GARP in antigen-specific T(h) cells leads to an efficient and stable re-programming of an effector T cell towards a regulatory T cell, which involves up-regulation of FOXP3, LGALS3, LGMN and other T(reg)-associated markers. In contrast, overexpression of LGALS3 and LGMN enhance FOXP3 and GARP expression, but only partially induced a regulatory phenotype. Lentiviral down-regulation of GARP in T(reg) cells significantly impaired the suppressor function and was associated with down-regulation of FOXP3. Moreover, down-regulation of FOXP3 resulted in similar phenotypic changes and down-regulation of GARP. This provides compelling evidence for a GARP-FOXP3 positive feedback loop and provides a rational molecular basis for the known difference between natural and transforming growth factor-beta induced T(reg) cells as we show here that the latter do not up-regulate GARP. In summary, we have identified GARP as a key receptor controlling FOXP3 in T(reg) cells following T-cell activation in a positive feedback loop assisted by LGALS3 and LGMN, which represents a promising new system for the therapeutic manipulation of T cells in human disease.

Polyphosphate at the Streptomyces lividans cytoplasmic membrane is enhanced in the presence of the potassium channel KcsA.

The distribution of polyphosphate (polyP) within the cytoplasmic membrane of Streptomyces lividans hyphae or protoplasts has been determined at high spatial resolution by elemental mapping using energy-filtered electron microscopy (EFTEM). The results revealed that polyP was best traceable after its interaction with lead ions followed by their precipitation as lead sulphide. Concomitant studies of the S.lividans wildtype (WT) strain and its co-embedded mutant DeltaK (lacking a functional kcsA gene) were conducted by labelling as the surface matrix of either one was labelled by cationic colloidal thorium dioxide. Within the WT strain, additional polyP was found to accumulate distinctly at the inner face of the cytoplasmic membrane. After removal of the cell wall (within protoplasts), the polyP-derived lead-sulphide (PbS) precipitate formed clusters of fibrillar material extending up to 50 nm into the cytoplasm. This feature was absent in the DeltaK mutant strain. Together the results revealed that the presence of the KcsA channel and the structured polyP coincide.

Impact of biofilm matrix components on interaction of commensal Escherichia coli with the gastrointestinal cell line HT-29.

Commensal Escherichia coli form biofilms at body temperature by expressing the extracellular matrix components curli fimbriae and cellulose. The role of curli fimbriae and cellulose in the interaction of commensal E. coli with the intestinal epithelial cell line HT-29 was investigated. Expression of curli fimbriae by the typical commensal isolate E. coli TOB1 caused adherence and internalization of the bacteria and triggered IL-8 production in HT-29 cells. In particular, induction of IL-8 production was complex and involved curli-bound flagellin. While cellulose alone had no effect on the interaction of TOB1 with HT-29 cells, co-expression of cellulose with curli fimbriae decreased adherence to, internalization and IL-8 induction of HT-29 cells. Investigation of a panel of commensal isolates showed a partial correlation between expression of curli fimbriae and enhanced internalization and IL-8 production. In addition, a high immunostimulatory flagellin was identified. Thus, the consequences of expression of extracellular matrix components on commensal bacterial-host interactions are complex.

Genome sequence completed of Alcanivorax borkumensis, a hydrocarbon-degrading bacterium that plays a global role in oil removal from marine systems.

In this paper, we provide background to the genome sequencing project of Alcanivorax borkumensis, which is a marine bacterium that uses exclusively petroleum oil hydrocarbons as sources of carbon and energy (therefore designated "hydrocarbonoclastic"). It is found in low numbers in all oceans of the world and in high numbers in oil-contaminated waters. Its ubiquity and unusual physiology suggest it is globally important in the removal of hydrocarbons from polluted marine systems. A functional genomics analysis of Alcanivorax borkumensis strain SK2 was recently initiated, and its genome sequence has just been completed. Annotation of the genome, metabolome modelling, and functional genomics, will soon reveal important insights into the genomic basis of the properties and physiology of this fascinating and globally important bacterium.

Alterations in the formation of lipopolysaccharide and membrane vesicles on the surface of Pseudomonas aeruginosa PAO1 under oxygen stress conditions.

It has been postulated that phenotypic variation in the relative expression of two chemically distinct types of lipopolysaccharide (LPS), a serotype-specific LPS (B-band) and a common antigen LPS (A-band) in Pseudomonas aeruginosa is an important mechanism enabling this opportunistic pathogen to alter its surface characteristics to mediate adhesion and to survive under extreme conditions. To further investigate this, the relative expression levels of the two distinct types of LPS in P. aeruginosa PAO1 were investigated with cells grown in a chemostat at different dissolved oxygen tensions (pO(2)). The A-band LPS was constitutively expressed as pO(2) was increased from nearly zero to 350 % of air saturation. In contrast, the B-band LPS showed a remarkable increase with increased pO(2). Almost no B-band LPS was found in cells grown at a pO(2) of less than 3 % of air saturation. Electron microscopic examination of cells revealed increased formation of membrane vesicles (MVs) on the surface of P. aeruginosa PAO1 under oxygen stress conditions. The toxicity of the supernatant of P. aeruginosa cultures to the growth of a hybridoma cell line significantly increased in samples taken from oxygen-stressed steady-state cultures. Furthermore, studies of adhesion in a continuous-flow biofilm culture revealed an increased adhesiveness for hydrophilic surfaces in P. aeruginosa PAO1 grown at a higher pO(2). The oxygen-dependent alterations of cell-surface components and properties observed in this work provide a possible explanation for the emergence of P. aeruginosa lacking the B-band LPS in chronically infected cystic fibrosis patients. The results are also useful for understanding the processes involved in the formation of MVs in P. aeruginosa.

Frozen motion of gliding bacteria outlines inherent features of the motility apparatus.

High-resolution data of actively gliding wild-type bacteria of four different species and of four different gliding mutants of Myxococcus xanthus were obtained from scanning electron micrographs. By shock freezing and freeze drying, motility-associated surface patterns could be fixed and consequently distinct intermediate states of motion could be observed for the first time. It is shown that these topographic patterns are immediately lost when gliding motility is stopped by blocking the respiratory chain with potassium cyanide or sodium azide. From the surface topography, the mode of action of the gliding apparatus of all four bacterial species examined can be described as a twisted circularly closed 'band'. During gliding, groups of nodes of the supertwisted apparatus show evidence of travelling like waves along the trichomes. However, the spacing between the nodes is not constant but varies within a certain range. This indicates that they are flexibly modulated as a consequence of the gliding state of the individual trichome.

Ultrastructure and composition of asteroid bodies.

PURPOSE: Asteroid hyalosis is a disease of the vitreous, characterized by brilliant reflecting particles, termed asteroid bodies, which are surrounded by a tightly adhering network of fibrils. The composition and mode of formation of asteroid bodies is not yet understood in detail. The purpose of this study was to investigate the ultrastructure of asteroid bodies and to identify the intrinsic inorganic and organic components that contribute to the nature and development of asteroid bodies. METHODS: Electron energy loss spectroscopy and energy-filtered transmission electron microscopy were used for the elemental analysis of asteroid bodies. The ultrastructural localization of glycosaminoglycans was investigated, using lectin and antibody conjugates in conjunction with transmission electron microscopy and epifluorescence microscopy. Anionic sites of glycosaminoglycans were detected with 15 nm cationic colloidal gold at low pH, applied as a postembedding technique. Ultrastructural details of asteroid bodies were documented using fast Fourier transform analysis of zero-loss filtered images. RESULTS: Element mapping of asteroid bodies by electron spectroscopic imaging revealed a homogeneous distribution of calcium, phosphorus, and oxygen. The electron energy loss spectra of these elements showed details similar to those found for hydroxyapatite. Additionally, high contrast and sensitivity against a calcium-specific chelator highlighted the crystalline, apatite-like nature of asteroid bodies. Immunofluorescence microscopy revealed the presence of chondroitin-6-sulfate at the periphery of asteroid bodies, which is in agreement with the ultrastructural colocalization of anionic sites. Fast Fourier transform analysis revealed that each 7-nm periodicity of asteroid lamellar stacks is divided by a fine, parallel-oriented line, separating each 7-nm layer into two halves of 3.5-nm thickness. Carbohydrates specific for hyaluronic acid were observed by lectin-gold labeling to be part of the inner matrix of asteroid bodies. CONCLUSIONS: The results of this study demonstrate the structural and elemental similarity of asteroid bodies with hydroxyapatite. Proteoglycans and their glycosaminoglycan side chains are implicated in playing a role in regulating the biomineralization process.

Determination of the systematic position of the genus Asticcacaulis Poindexter by a polyphasic analysis.

The genus Asticcacaulis, to date, comprises two species of unicellular, stalked bacteria, developing a stalk at a site which is not coincidental with the centre of the pole of the cell. Multiplication is similar to that demonstrated by the prosthecate species of the genera Caulobacter, Brevundimonas and Maricaulis. A polyphasic approach, comprising 16S rRNA gene sequencing, lipid analysis and NaCl tolerance characterizations, was used to clarify the taxonomy of the two Asticcacaulis species. From the analysis of the 16S rRNA gene sequences, a close phylogenetic relationship between the species that comprise the genera Asticcacaulis, Caulobacter and Brevundimonas could be deduced wherein the three genera form three distinct branches. The individual genera could also be discerned by different characteristic polar lipids. The species of Asticcacaulis differed from species of Caulobacter and Brevundimonas by the lack of 1,2-diacyl-3-O-[6'-phosphatidyl-alpha-D-glucopyranosyl]glycerol. They also did not contain 1,2-di-O-acyl-3-O-[D-glucopyranosyl-(1-->4)-alpha-D-glucuronopyranosyl]glycerol, which is found in most Brevundimonas species but not in strains of the genus Caulobacter. The morphological differences seen between the two species Asticcacaulis excentricus and Asticcacaulis biprosthecium are mirrored by the observed 16S rDNA sequence similarity value of 95.3%, which is relatively low compared to the interspecies similarity values observed within the genera Brevundimonas or Caulobacter.

Alcalilimnicola halodurans gen. nov., sp. nov., an alkaliphilic, moderately halophilic and extremely halotolerant bacterium, isolated from sediments of soda-depositing Lake Natron, East Africa Rift Valley.

An alkaliphilic, halotolerant, Gram-negative, heterotrophic, aerobic and rod-shaped organism was isolated from drying soda and at a water-covered site of Lake Natron, Tanzania, by means of the most-probable-number technique developed for anoxygenic, phototrophic sulfur bacteria. It had an absolute requirement for alkalinity, but not for salinity; growth occurred at salt concentrations of 0-28% (w/v), with optimal growth at 3-8% (w/v) NaCl. The bacterium preferentially metabolized volatile fatty acids and required vitamins for growth. The name Alcalilimnicola halodurans gen. nov., sp. nov. is proposed for the novel isolate, placed in the gamma-Proteobacteria within the family Ectothiorhodospiraceae on the basis of analysis of the 16S rDNA sequence, polar lipids, fatty acids and DNA base composition. Although Alcalilimnicola halodurans is closely related to the extreme anoxygenic, phototrophic sulfur bacteria of the genus Halorhodospira, it is not phototrophic.

Immunohistochemical and charge-specific localization of anionic constituents in pseudoexfoliation deposits on the central anterior lens capsule from individuals with pseudoexfoliation syndrome.

BACKGROUND: Pseudoexfoliation (PSX) syndrome is a degenerative systemic disorder that is characterized primarily by deposits of distinct fibrillar material on the surface lining the anterior and posterior chambers of the eye and is often associated with cataract and glaucoma. Although some components of the PSX material have been identified, the precise composition is obscure. METHODS: High-resolution scanning electron microscopy in conjunction with colloidal cationic gold labeling was used to localize anionic constituents at the surface of PSX aggregates. Transmission electron microscopy was applied for the immunocytochemical detection of glycosaminoglycans, and to monitor the charge-specific distribution of colloidal thorium dioxide and ferritin in PSX material. The specific binding of antibodies was confirmed by immunohistological staining of paraffin-embedded specimens. RESULTS: Paraffin-embedded tissue sections revealed immunoreactivity for keratan sulfate and dermatan sulfate proteoglycan within PSX material deposited on the surface of the anterior lens capsule. Post-embedding immunogold labeling of keratan sulfate demonstrated an intense label of PSX aggregates primarily associated with mature PSX fibrils, whereas dermatan sulfate proteoglycon appeared to be present in low quantities. Additionally, keratan sulfate was found at the humoral periphery of the lens capsules. To further investigate the distribution of anionic sites in PSX material, we used cationic colloidal tracers of different size, such as gold, thorium dioxide and ferritin. PSX aggregates exhibited a strong negative charge, resulting very likely from glycosaminoglycan chains of proteoglycans. The density of anionic sites was higher at the interfibrillar matrix. Lens capsules associated with PSX material revealed a diminished accumulation of cationic ferritin at the humoral surfaces. CONCLUSIONS: Increased amounts of different glycosaminoglycans identified in PSX material suggest an important role of proteoglycans for the pathogenic pathway in PSX.

Immunocytochemical localization of CDw60 antigens on human peripheral T cells.

About 25-35% of human T cells display the CDw60 ganglioside (9-O-acetyl-GD3) antigen at the cell surface [E.P. Rieber, in W. Knapp, B. Dörken, W.R. Gilks, E.P. Rieber, R.E. Schmidt, H. Stein, A.E.G.K. von dem Borne (Eds.), Leucocyte Typing IV, Oxford University, Oxford, 1989, p. 361.]. Other leucocytes do not express this antigen on the cell surface. This led us to investigate its presence by flow cytometry and immunoelectron microscopy (IEM). Flow cytometric analysis of isolated peripheral T cells showed 26% of the cell population to have the CDw60 antigen expressed on the cell surface whereas 74% did not. Similarly, IEM analysis of 262 random T cells by the preembedding immunogold labeling technique revealed CDw60 surface expression to be tetrapartite: (a) the majority of 63.7% of the T cells did not show any surface associated gold label; (b) 19.5% were of low CDw60 surface exposition, corresponding to a linear density of 0.05-2.0 gold markers per microm; (c) about 13.4% showed a medium surface exposition with a linear density of 2.1-4.5 gold markers per microm; and (d) a high exposition, ranging from 4.6 to 9.0 gold markers per microm, was seen at 3.4% of the T cells. From postembedding label experiments, which additionally make access to the antigen localized within the cytoplasm, it was found that nearly all T cells contained low levels of intracellular CDw60. Most of it was found to be associated with the cytoplasmic membrane or vesicles, derived from the Golgi. Immunogold conjugates associated with the cytoplasmic membrane showed a linear density up to 0.6 gold markers per microm. The asymmetric expression of the CDw60 antigen on human T cells and its occurrence in nearly all T cells suggests that its surface presentation is tightly regulated.

Propionispora vibrioides, nov. gen., nov. sp., a new gram-negative, spore-forming anaerobe that ferments sugar alcohols.

Anaerobic enrichment cultures, with erythritol as substrate, resulted in the isolation of a strain with properties not yet found in an existing genus in this combination. The strain, FKBS1, was strictly anaerobic, stained gram-negative and formed spores. Cells were small motile vibrios with flagella inserted at the concave side of the cell. Spores were located terminally and caused only slight swelling of the cells if compared to related spore-forming genera. FKBS1 fermented fructose, mannitol, sorbitol, xylitol and erythritol to propionic acid, acetic acid, CO2 and small amounts of H2 to balance the difference in the oxidation-reduction value between substrate and cell mass. The 16S rDNA sequence revealed relationship to the Sporomusa-Pectinatus-Selenomonas group. However, the phylogenetic distance to any of its members was too great to allow it to be placed in one of the existing genera. Morphologically the strain resembled Sporomusa, which, however, performs an acetogenic type of fermentation. The propionic-acid-forming genera of the group are either not spore-formers or, in the case of Dendrosporobacter quercicolus (syn. Clostridium quercicolum), morphologically different. It is therefore proposed to classify strain FKBS1 as a new genus and species, Propionispora vibrioides.

Structure and species composition of mercury-reducing biofilms.

Mercury-reducing biofilms from packed-bed bioreactors treating nonsterile industrial effluents were shown to consist of a monolayer of bacteria by scanning electron microscopy. Droplets of several micrometers in diameter which accumulated outside of the bacterial cells were identified as elemental mercury by electron-dispersive X-ray analysis. The monospecies biofilms of Pseudomonas putida Spi3 initially present were invaded by additional strains, which were identified to the species level by thermogradient gel electrophoresis (TGGE) and 16S rDNA sequencing. TGGE community fingerprints of the biofilms showed that they were composed of the effluent bacteria and did not contain uncultivable microorganisms. Of the 13 effluent bacterial strains, 2 were not mercury resistant, while all the others had resistance levels similar to or higher than the inoculant strain.

Effect of oxygen on formation and structure of Azotobacter vinelandii alginate and its role in protecting nitrogenase.

The activity of nitrogenase in the nitrogen-fixing bacterium Azotobacter vinelandii grown diazotrophically under aerobic conditions is generally considered to be protected against O(2) by a high respiration rate. In this work, we have shown that a high rate of respiration is not the prevailing mechanism for nitrogenase protection in A. vinelandii grown in phosphate-limited nitrogen-free chemostat culture. Instead, the formation of alginate appeared to play a decisive role in protecting the nitrogenase that is required for cell growth in this culture. Depending on the O(2) tension and cell growth rate, the formation rate and composition of alginate released into the culture broth varied significantly. Furthermore, transmission electron microscopic analysis of cell morphology and the cell surface revealed the existence of an alginate capsule on the surface of A. vinelandii. The composition, thickness, and compactness of this alginate capsule also varied significantly. In general, increasing O(2) tension led to the formation of alginate with a higher molecular weight and a greater L-guluronic acid content. The alginate capsule was accordingly thicker and more compact. In addition, the formation of the alginate capsule was found to be strongly affected by the shear rate in a bioreactor. Based on these experimental results, it is suggested that the production of alginate, especially the formation of an alginate capsule on the cell surface, forms an effective barrier for O(2) transfer into the cell. It is obviously the quality, not the quantity, of alginate that is decisive for the protection of nitrogenase.

Ferroplasma acidiphilum gen. nov., sp. nov., an acidophilic, autotrophic, ferrous-iron-oxidizing, cell-wall-lacking, mesophilic member of the Ferroplasmaceae fam. nov., comprising a distinct lineage of the Archaea.

An isolate of an acidophilic archaeon, strain YT, was obtained from a bioleaching pilot plant. The organism oxidizes ferrous iron as the sole energy source and fixes inorganic carbon as the sole carbon source. The optimal pH for growth is 1.7, although growth is observed in the range pH 1.3 to 2.2. The cells are pleomorphic and without a cell wall. 16S rRNA gene sequence analysis showed this strain to cluster phylogenetically within the order 'Thermoplasmales' sensu Woese, although with only 89.9 and 87.2% sequence identity, respectively, to its closest relatives, Picrophilus oshimae and Thermoplasma acidophilum. Other principal differences from described species of the 'Thermoplasmales' are autotrophy (strain YT is obligately autotrophic), the absence of lipid components typical of the ' Thermoplasmales' (no detectable tetraethers) and a lower temperature range for growth (growth of strain YT occurs between 15 and 45 degrees C). None of the sugars, amino acids, organic acids or other organic compounds tested was utilized as a carbon source. On the basis of the information described above, the name Ferroplasma acidiphilum gen. nov., sp. nov. is proposed for strain YT within a new family, the Ferroplasmaceae fam. nov. Strain YT is the type and only strain of F. acidiphilum. This is the first report of an autotrophic, ferrous-iron-oxidizing, cell-wall-lacking archaeon.

Reclassification of Clostridium quercicolum as Dendrosporobacter quercicolus gen. nov., comb. nov.

Morphological features, genomic DNA base composition and 16S rDNA sequence similarities, as well as a distinct phospholipid pattern, whole-cell fatty acid distribution and the occurrence of the lipoquinone 'lipid F', indicate that Clostridium quercicolum belongs to the Sporomusa-Pectinatus-Selenomonas phyletic group and possesses only a remote relationship to members of the genus Clostridium sensu stricto. On the basis of these results, the new genus and combination Dendrosporobacter quercicolus gen. nov., comb. nov. are proposed.