Identification and characterization of uranium accumulation potential of a uranium mine isolated <Emphasis Type="Italic">Pseudomonas</Emphasis> strain

Uranium accumulation by a Pseudomonas strain was characterized in terms of kinetics, effect on cell viability and role of various regulatory factors (viz. pH, Co ions and metabolism). This strain was identified as Pseudomonas aeruginosa based on 16S rRNA gene and Fatty Acid Methyl Ester (FAME) analyses. The bacterium exhibited rapid, concentration and pH dependent, uranium accumulation with maximum loading of 275 mg uranium g⁻¹ dry wt. at pH 4.0. Survival studies during the period of uranium exposure indicated a viable but non growing state of the cells. Uranium accumulation remains largely insensitive to metabolic activity and presence of other co-ions except Fe³⁺. Transmission electron microscopy (TEM) confirmed the deposition of bioaccumulated uranium by live cells within the cell envelope region. Our findings suggest that the P. aeruginosa J007 has intrinsic uranium-removal and -resistance properties. Accumulated uranium is deposited within the cell envelope region providing a possible survival strategy to the bacterium. Our study contributed significantly to gain insight into bacterial interaction with uranium and will be useful in understanding the role of native microorganisms inhabiting in uranium contaminated sites in biogeochemical cycling of uranium and in bioremediation applications.     

Effects of selected physical and chemical parameters on uranium uptake by the bacterium Chryseomonas MGF-48

The effect of selected physical and chemical parameters, including media composition, uranium concentration, and metabolic inhibitors, on uranium uptake from aqueous solution by Chryseomonas sp. MGF-48, a bacterium isolated from electroplating effluent, was analysed. It was found that 198 mg/g (dry weight) of uranium was taken up from a 150 ppm (mg/l) solution of uranium when cells were maintained under starvation conditions. Uranium was released from the cells by addition of sodium carbonate. Uptake of uranium was reduced when cells were subjected to pretreatment with ultraviolet (u.v.) light (263.7 nm, 10 min) or heat (100 °C, 15 min), i.e., 99.8 and 57.5 mg/g, respectively. Addition of 2, 4-dinitrophenol (10 mM) or sodium azide (0.1%) resulted in decreased uptake (129 and 123 mg/g, respectively), whereas addition of glycerol-2-phosphate (G-2-P) resulted in deposition of uranium. Maximum uptake of uranium occurred at pH 6.5. Uranium uptake in the presence of selected carbohydrates decreased as follows: xylose > arabinose > mannose > maltose > glucose. When Chryseomonas sp. MGF-48 was exposed to 5, 50, or 100 mg/l uranium prior to uptake experiments, a decrease in uranium uptake was noted. Furthermore, immobilization of the bacterial cells in a calcium-alginate medium reduced the efficiency of uptake, yielding only 60% uranium uptake. It is concluded that uranium uptake and accumulation by this bacterium involves both metabolism-independent absorption and metabolism-dependent absorption, the former being considered to be more efficient.     

Natural uranium impairs the differentiation and the resorbing function of osteoclasts

BackgroundUranium is a naturally occurring radionuclide ubiquitously present in the environment. The skeleton is the main site of uranium long-term accumulation. While it has been shown that natural uranium is able to perturb bone metabolism through its chemical toxicity, its impact on bone resorption by osteoclasts has been poorly explored. Here, we examined for the first time in vitro effects of natural uranium on osteoclasts.MethodsThe effects of uranium on the RAW 264.7 monocyte/macrophage mouse cell line and primary murine osteoclastic cells were characterized by biochemical, molecular and functional analyses.ResultsWe observed a cytotoxicity effect of uranium on osteoclast precursors. Uranium concentrations in the μM range are able to inhibit osteoclast formation, mature osteoclast survival and mineral resorption but don't affect the expression of the osteoclast gene markers Nfatc1, Dc-stamp, Ctsk, Acp5, Atp6v0a3 or Atp6v0d2 in RAW 274.7 cells. Instead, we observed that uranium induces a dose-dependent accumulation of SQSTM1/p62 during osteoclastogenesis.ConclusionsWe show here that uranium impairs osteoclast formation and function in vitro. The decrease in available precursor cells, as well as the reduced viability of mature osteoclasts appears to account for these effects of uranium. The SQSTM1/p62 level increase observed in response to uranium exposure is of particular interest since this protein is a known regulator of osteoclast formation. A tempting hypothesis discussed herein is that SQSTM1/p62 dysregulation contributes to uranium effects on osteoclastogenesis.General significanceWe describe cellular and molecular effects of uranium that potentially affect bone homeostasis.     

Biosorption of heavy metals by Saccharomyces cerevisiae

Abundant and common yeast biomass has been examined for its capacity to sequester heavy metals from dilute aqueous solutions. Live and non-living biomass of Saccharomyces cerevisiae differs in the uptake of uranium, zinc and copper at the optimum pH 4–5. Culture growth conditions can influence the biosorbent metal uptake capacity which normally was: living and non-living brewer's yeast: U > Zn > Cd > Cu; non-living baker's yeast: Zn > (Cd) > U > Cu; living baker's yeast: Zn > Cu (Cd) > U. Non-living brewer's yeast biomass accumulated 0.58 mmol U/g. The best biosorbent of zinc was non-living baker's yeast ( 0.56 mmol Zn/g). Dead cells of S. cerevisiae removed approximately 40% more uranium or zinc than the corresponding live cultures. Biosorption of uranium by S. cerevisiae was a rapid process reaching 60% of the final uptake value within the first 15 min of contact. Its deposition differing from that of other heavy metals more associated with the cell wall, uranium was deposited as fine-needle-like crystals both on the inside and outside of the S. cerevisiae cells.     

Uranium uptake by immobilized cells of Pseudomonas strain EPS 5028

Polyacrylamide-gel-immobilized cells of Pseudomonas strain EPS 5028 were effective in the removal of uranium (U) from synthetic effluents. Metal accumulation was performed in an open system in columns filled with immobilized cells that were challenged with continuous flows containing U. Possible variables of the system were studied. Uranium uptake by the immobilized cells of this microorganism was affected by pH but not by temperature or flow rate. In addition, U binding could be interpreted in terms of the Freundlich adsorption isotherm indicating single-layer adsorption. The feasibility of reusing the immobilized cells was suggested after the recovery of U with a solution of 0.1 m sodium carbonate. Correspondence to: M. C. Fusté     

Direct Microbial Reduction and Subsequent Preservation of Uranium in Natural Near-Surface Sediment

The fate of uranium in natural systems is of great environmental importance. X-ray absorption near-edge spectroscopy (XANES) revealed that U(VI) was reduced to U(IV) in shallow freshwater sediment at an open pit in an inactive uranium mine. Geochemical characterization of the sediment showed that nitrate, Fe(III), and sulfate had also been reduced in the sediment. Observations of the sediment particles and microbial cells by scanning and transmission electron microscopy, coupled with elemental analysis by energy dispersive spectroscopy, revealed that uranium was concentrated at microbial cell surfaces. U(IV) was not associated with framboidal pyrite or nanometer-scale iron sulfides, which are presumed to be of microbial origin. Uranium concentrations were not detected in association with algal cells. Phylogenetic analyses of microbial populations in the sediment by the use of 16S rRNA and dissimilatory sulfite reductase gene sequences detected organisms belonging to the families Geobacteraceae and Desulfovibrionaceae. Cultivated members of these lineages reduce U(VI) and precipitate iron sulfides. The association of uranium with cells, but not with sulfide surfaces, suggests that U(VI) is reduced by the enzymatic activities of microorganisms. Uranium was highly enriched (760 ppm) in a subsurface black layer in unsaturated sediment sampled from a pit which was exposed to seasonal fluctuations in the pond level. XANES analysis showed that the majority of uranium in this layer was U(IV), indicating that uranium is preserved in its reduced form after burial.     

Indentation with atomic force microscope,Saccharomyces cerevisiae cell gains elasticity under ethanol stress

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane is the first target to be attacked by the accumulated ethanol. In such a prominent position, S. cerevisiae cell membrane could reversely provide protection through changing fluidity or elasticity secondary to remodeled membrane components or structure during the fermentation process. However, there is yet to be a direct observation of the real effect of the membrane compositional change. In this study, atomic force microscope-based strategy was performed to determine Young's modulus of S. cerevisiae to directly clarify ethanol stress-associated changes and roles of S. cerevisiae cell membrane fluidity and elasticity. Cell survival rate decreased while the cell swelling rate and membrane permeability increased as ethanol concentration increased from 0% to 20% v/v. Young's modulus decreased continuously from 3.76 MPa to 1.53 MPa while ethanol stress increased from 0% to 20% v/v, indicating that ethanol stress induced the S. cerevisiae membrane fluidity and elasticity changes. Combined with the fact that membrane composition varies under ethanol stress, to some extent, this could be considered as a forced defensive act to the ethanol stress by S. cerevisiae cells. On the other hand, the ethanol stress induced loosening of cell membrane also caused S. cerevisiae cell to proactively remodel membrane to make cell membrane more agreeable to the increase of environmental threat. Increased ethanol stress made S. cerevisiae cell membrane more fluidized and elastic, and eventually further facilitated yeast cell’s survival.     

Jen1p: A High Affinity Selenite Transporter in Yeast

Selenium is a micronutrient in most eukaryotes, including humans, which is well known for having an extremely thin border between beneficial and toxic concentrations. Soluble tetravalent selenite is the predominant environmental form and also the form that is applied in the treatment of human diseases. To acquire this nutrient from low environmental concentrations as well as to avoid toxicity, a well-controlled transport system is required. Here we report that Jen1p, a proton-coupled monocarboxylate transporter in S. cerevisiae, catalyzes high-affinity uptake of selenite. Disruption of JEN1 resulted in selenite resistance, and overexpression resulted in selenite hypersensitivity. Transport assay showed that overexpression of Jen1p enables selenite accumulation in yeast compared with a JEN1 knock out strain, indicating the Jen1p transporter facilitates selenite accumulation inside cells. Selenite uptake by Jen1p had a K_m of 0.91 mM, which is comparable to the K_m for lactate. Jen1p transported selenite in a proton-dependent manner which resembles the transport mechanism for lactate. In addition, selenite and lactate can inhibit the transport of each other competitively. Therefore, we postulate selenite is a molecular mimic of monocarboxylates which allows selenite to be transported by Jen1p.     

Effects of single and combined cell treatments based on low pH and high concentrations of ethanol on the growth and fermentation of Dekkera bruxellensis and Saccharomyces cerevisiae

The alcoholic fermentation in Brazil displays some peculiarities because the yeast used is recycled in a non-aseptic process. After centrifugation, the cells are treated with acid to control the bacterial growth. However, it is difficult to manage the indigenous yeasts without affecting the main culture of Saccharomyces cerevisiae. This work evaluated how the cell treatment could be modified to combat contaminant yeasts based on the differential sensitivities to low pH and high concentrations of ethanol displayed by an industrial strain of S. cerevisiae and three strains of Dekkera bruxellensis, which are common contaminant yeasts in Brazilian fermentation processes. The tests were initially performed in rich medium with a low pH or a high concentration of ethanol to analyse the yeast growth profile. Then, the single and combined effects of low pH and ethanol concentration on the yeast cell viability were evaluated under non-proliferative conditions. The effects on the fermentation parameters were also verified. S. cerevisiae grew best when not subjected to the stresses, but this yeast and D. bruxellensis had similar growth kinetics when exposed to a low pH or increased ethanol concentrations. However, the combined treatments of low pH (2.0) and ethanol (11 or 13 %) resulted in a decrease of D. bruxellensis cell viability almost three times higher than of S. cerevisiae, which was only slightly affected by all cell treatments. The initial viability of the treated cells was restored within 8 h of growth in sugar cane juice, with the exception of the combined treatment for D. bruxellensis. The ethanol-based cell treatment, in despite of slowing the fermentation, could decrease and maintain D. bruxellensis population under control while S. cerevisiae was taking over the fermentation along six fermentative cycles. These results indicate that it may be possible to control the growth of D. bruxellensis without major effects on S. cerevisiae. The cells could be treated between the fermentation cycles by the parcelled addition of 13 % ethanol to the tanks in which the yeast cream is treated with sulphuric acid at pH 2.0.     

Lectin-stimulated cellular iron uptake and toxin generation in the freshwater cyanobacterium Microcystis aeruginosa

The lectin family is composed of mono- and oligosaccharide binding proteins that could activate specific cellular activities, such as cell-cell attachment and toxin production. In the present study, the effect of the external addition of lectins to culture media containing the freshwater cyanobacterium Microcystis aeruginosa on its metabolic activities, such as iron uptake and toxin production was investigated. Among the three lectins examined in this study (concanavalin A [Con A], wheat germ agglutinin [WGA] and peanut agglutinin [PNA]), PNA substantially increased the accumulated intracellular and extracellular iron content. The binding of PNA and Con A to M. aeruginosa cells was visualized via fluorescence microscopy using a lectin adjunct with fluorescein isothiocyanate, and resulted in carbohydrate and protein accumulation in the cellular capsule. Given that the highest carbohydrate accumulation was seen in the Con A system (where iron accumulation was relatively lower), carbohydrate quality is likely important factor that influences cellular iron accumulation. Since PNA specifically binds to sugars such as galactose and N-acetylgalactosamine, these saccharide species could be important candidates for intracellular and extracellular iron accumulation and transport. Microcystin biosynthesis was stimulated in the presence of PNA and WGA, whereas cellular iron uptake increased only in the presence of PNA. Thus, the iron uptake was not necessarily congruent with the upregulation of microcystin synthesis, which suggested that the positive effect of lectin on iron uptake is probably attributable to the PNA-assisted iron accumulation around the cell surface. Overall, the present study provides insights into the interactions of lectin that influence cellular metabolic activities such as iron uptake, extracellular polymeric substance accumulation, and toxin production.     

Onsite bio-detoxification of steam-exploded corn stover for cellulosic ethanol production

In the process of ethanol production from steam-exploded corn stover (SECS), a cellulose-degradation strain of Aspergillus nidulans (FLZ10) was investigated whether it could remove the inhibitors released from steam exploded pretreatment , and thereby be used for biological detoxification on Saccharomycescerevisiae. The results showed that FLZ10 removed 75.2% formic acid, 53.6% acetic acid, and 100% hydroxymethyl furfural (5-HMF) and furfural from the hydrolysate washed from SECS after 72 h cultivation. A cellulase activity of 0.49 IU/ml was simultaneously produced while the biological detoxification occurred. An ethanol yield of 0.45 g/g on glucose was obtained in the hydrolysate biodetoxified by FLZ10. The glucose consumption rate of FLZ10 was much lower than that of S. cerevisiae, thereby it had little competition with S. cerevisiae on glucose consumption. Based on SECS to ethanol mass balance analysis, with the onsite bio-detoxification, fermentation using S. cerevisiae effectively converted monomeric glucose with 94.4% ethanol yield.     

Regulation of Lactobacillus plantarum contamination on the carbohydrate and energy related metabolisms of Saccharomyces cerevisiae during bioethanol fermentation

During the industrial bioethanol fermentation, Saccharomyces cerevisiae cells are often stressed by bacterial contaminants, especially lactic acid bacteria. Generally, lactic acid bacteria contamination can inhibit S. cerevisiae cell growth through secreting lactic acid and competing with yeast cells for micronutrients and living space. However, whether are there still any other influences of lactic acid bacteria on yeast or not? In this study, Lactobacillus plantarum ATCC 8014 was co-cultivated with S. cerevisiae S288c to mimic the L. plantarum contamination in industrial bioethanol fermentation. The contaminative L. plantarum-associated expression changes of genes involved in carbohydrate and energy related metabolisms in S. cerevisiae cells were determined by quantitative real-time polymerase chain reaction to evaluate the influence of L. plantarum on carbon source utilization and energy related metabolism in yeast cells during bioethanol fermentation. Contaminative L. plantarum influenced the expression of most of genes which are responsible for encoding key enzymes involved in glucose related metabolisms in S. cerevisiae. Specific for, contaminated L. plantarum inhibited EMP pathway but promoted TCA cycle, glyoxylate cycle, HMP, glycerol synthesis pathway, and redox pathway in S. cerevisiae cells. In the presence of L. plantarum, the carbon flux in S. cerevisiae cells was redistributed from fermentation to respiratory and more reducing power was produced to deal with the excess NADH. Moreover, L. plantarum contamination might confer higher ethanol tolerance to yeast cells through promoting accumulation of glycerol. These results also highlighted our knowledge about relationship between contaminative lactic acid bacteria and S. cerevisiae during bioethanol fermentation.     

Cysteine-125 is the catalytic residue of Saccharomyces cerevisiae free methionine-R-sulfoxide reductase

Free methionine-R-sulfoxide reductase (fRMsr) is a new type of methionine sulfoxide reductase that catalyzes the reduction of free methionine-R-sulfoxide to methionine. This enzyme cannot reduce oxidized methionine residues in proteins. While three Cys residues, Cys-91, Cys-101 and Cys-125, have been demonstrated to be involved in the catalysis by Saccharomyces cerevisiae fRMsr, their specific functions have not been fully established. In this work, we performed in vivo growth complementation experiments using S. cerevisiae cells lacking all three known methionine sulfoxide reductases. Cells containing a C125S construct, in which Cys-125 in fRMsr was replaced with Ser, did not grow in methionine sulfoxide medium, whereas cells containing C91S, C101S, or C91/101S constructs could grow in this medium. In addition, when assayed with thioredoxin and glutaredoxin reduction systems, the C125S form was inactive, whereas C91S and C101S had 1-2% and 9-10%, respectively, of the activity of the wild-type fRMsr. These data show that Cys-125 is the catalytic residue in fRMsr.     

Genome-Scale NAD(H/+) Availability Patterns as a Differentiating Feature between Saccharomyces cerevisiae and Scheffersomyces stipitis in Relation to Fermentative Metabolism

Scheffersomyces stipitis is a yeast able to ferment pentoses to ethanol, unlike Saccharomyces cerevisiae, it does not present the so-called overflow phenomenon. Metabolic features characterizing the presence or not of this phenomenon have not been fully elucidated. This work proposes that genome-scale metabolic response to variations in NAD(H/⁺) availability characterizes fermentative behavior in both yeasts. Thus, differentiating features in S. stipitis and S. cerevisiae were determined analyzing growth sensitivity response to changes in available reducing capacity in relation to ethanol production capacity and overall metabolic flux span. Using genome-scale constraint-based metabolic models, phenotypic phase planes and shadow price analyses, an excess of available reducing capacity for growth was found in S. cerevisiae at every metabolic phenotype where growth is limited by oxygen uptake, while in S. stipitis this was observed only for a subset of those phenotypes. Moreover, by using flux variability analysis, an increased metabolic flux span was found in S. cerevisiae at growth limited by oxygen uptake, while in S. stipitis flux span was invariant. Therefore, each yeast can be characterized by a significantly different metabolic response and flux span when growth is limited by oxygen uptake, both features suggesting a higher metabolic flexibility in S. cerevisiae. By applying an optimization-based approach on the genome-scale models, three single reaction deletions were found to generate in S. stipitis the reducing capacity availability pattern found in S. cerevisiae, two of them correspond to reactions involved in the overflow phenomenon. These results show a close relationship between the growth sensitivity response given by the metabolic network and fermentative behavior.     

Extreme chemical mutagen sensitivity of respiratory adaptation in yeast

The respiratory adaptation process (i.e essentially mitochondrial biogenesis) in the cells of both wild-type Saccharomyces cerevisiae and strains sensitive to ultraviolet radiation (UV) undergoing transition from the anaerobic to the aerobic state (1–2 h aeration) could be arrested by a prior incubation for 15–30 min with several chemical mutagens and other DNA-acting chemicals at very low concentrations (10^?7 to 10^?6 M added to cells suspended at the density of 10⁷ cells/ml). At the same concentrations, these chemicals also inhibited DNA and RNA biosynthesis in maturing mitochondria during respiratory adaptation. This provides suggestive evidence for the view that the inhibitory effect of the chemical mutagens on respiratory adaptation could be due to lesions introduced into the DNA of promitochondria in the anaerobic cells. The system of respiratory adaptation in S. cerevisiae cells could serve as a rapid test for ascertaining the potentiality of a chemical to affect DNA and probably, in turn, its potentiality to be mutagenic.