Effects of long-term radionuclide and heavy metal contamination on the activity of microbial communities, inhabiting uranium mining impacted soils.

Ore mining and processing have greatly altered ecosystems, often limiting their capacity to provide ecosystem services critical to our survival. The soil environments of two abandoned uranium mines were chosen to analyze the effects of long-term uranium and heavy metal contamination on soil microbial communities using dehydrogenase and phosphatase activities as indicators of metal stress. The levels of soil contamination were low, ranging from 'precaution' to 'moderate', calculated as Nemerow index. Multivariate analyses of enzyme activities revealed the following: (i) spatial pattern of microbial endpoints where the more contaminated soils had higher dehydrogenase and phosphatase activities, (ii) biological grouping of soils depended on both the level of soil contamination and management practice, (iii) significant correlations between both dehydrogenase and alkaline phosphatase activities and soil organic matter and metals (Cd, Co, Cr, and Zn, but not U), and (iv) multiple relationships between the alkaline than the acid phosphatase and the environmental factors. The results showed an evidence of microbial tolerance and adaptation to the soil contamination established during the long-term metal exposure and the key role of soil organic matter in maintaining high microbial enzyme activities and mitigating the metal toxicity. Additionally, the results suggested that the soil microbial communities are able to reduce the metal stress by intensive phosphatase synthesis, benefiting a passive environmental remediation and provision of vital ecosystem services.

Community level physiological profiles of bacterial communities inhabiting uranium mining impacted sites.

Bacterial activity and physiological diversity were characterized in mining and milling impacted soils collected from three abandoned uranium mine sites, Senokos, Buhovo and Sliven, using bacterial dehydrogenase activity and Biolog (EcoPlate) tests. The elemental composition of soils revealed high levels of uranium and heavy metals (sum of technogenic coefficients of contamination; TCC(sum) pollution as follows: Sliven (uranium - 374 mg/kg; TCC(sum) - 23.40) >Buhovo (uranium - 139.20mg/kg; TCC(sum) - 3.93) >Senokos (uranium - 23.01 mg/kg; TCC(sum) - 0.86). The physiological profiles of the bacterial community level were site specific, and indicated intensive utilization of polyols, carbohydrates and carboxylic acids in low and medium polluted environments, and i-erithrytol and 2-hydroxy-benzoic acid in the highly polluted environment of Sliven waste pile. Enzymes which take part in the biodegradation of recalcitrant substances were more resistant to pollution than these from the pathways of the easily degradable carbon sources. The Shannon index indicated that the physiological diversity of bacteria was site specific but not in line with the levels of pollution. A general tendency of increasing the importance of the number of utilizable substrates to bacterial physiological diversity was observed at less polluted sites, whereas in highly polluted sites the evenness of substrate utilization rate was more significant. Dehydrogenase activity was highest in Senokos upper soil layer and positively correlated (p<0.01) with the soil organic matter content. The bacterial activity (EcoPlate) and physiological diversity (Shannon index) correlated significantly and negatively with As, Cu, Zn, Pb and U, and Co, Cr, Ni and Mn, respectively. We concluded that the observed site specific shifts in bacterial communities were complex due to both the environmental peculiarities and the bacterial tolerance to the relevant level of pollution, rather than a strong indication of uranium and heavy metals toxicity.

Combined chemical and enzymatic stable isotope labeling for quantitative profiling of detergent-insoluble membrane proteins isolated using Triton X-100 and Brij-96.

Effective quantitative profiling of detergent-insoluble membrane proteins using high-throughput mass spectrometry (MS)-based proteomics would allow a better understanding of physiological and pathological processes that take place at the cell surface. To increase the coverage of proteins present in detergent-resistant membrane microdomains (DRMMs), a combination of 16O/18O and isotope coded affinity tags (ICAT) labeling was used in a comparative analysis of detergent-insoluble membrane proteins isolated from rat basophilic leukemia cells (RBL-2H3), with either Triton X-100 or Brij-96. The analysis resulted in the quantification of 738 unique proteins from Triton X-100 and Brij-96 isolated DRMMs, significantly exceeding the number of proteins quantified from either single labeling technique. Twenty-five noncysteine-containing proteins were quantified, as well as 32 cysteine-containing proteins that would have been missed if either 16O/18O or ICAT labeling had been used exclusively, which illustrate better proteome coverage and enhanced ability to quantitate. The comparative analysis revealed that proteins were more readily extracted using Triton X-100 than Brij-96; however, Triton X-100 also extracted larger quantities of non-DRMMs-associated proteins. This result confirms previous, targeted studies suggesting that DRMMs isolated using Triton X-100 and Brij-96 differ in their protein content.

Bacterial diversity in water samples from uranium wastes as demonstrated by 16S rDNA and ribosomal intergenic spacer amplification retrievals.

Bacterial diversity was assessed in water samples collected from several uranium mining wastes in Ger many and in the United States by using 16S rDNA and ribosomal intergenic spacer amplification retrievals. The results obtained using the 16S rDNA retrieval showed that the samples collected from the uranium mill tailings of Schlema/Alberoda, Germany, were predominated by Nitrospina-like bacteria, whereas those from the mill tailings of Shiprock, New Mexico, USA, were predominated by gamma-Pseudomonas and Frauteria spp. Additional smaller populations of the Cytophaga-Flavobacterium-Bacteroides group and alpha- and delta-Proteobacteria were identified in the Shiprock samples as well. Proteobacteria and Cytophaga-Flavobacterium-Bacteroides were also found in the third uranium mill tailings studied, Gittersee/Coschütz, Germany, but the groups of the predominant clones were rather small. Most of the clones of the Gittersee/Coschütz samples represented individual sequences, which indicates a high level of bacterial diversity. The samples from the fourth uranium waste studied, Steinsee Deponie B1, Germany, were predominantly occupied by Acinetobacter spp. The ribosomal intergenic spacer amplification retrieval provided results complementary to those obtained by the 16S rDNA analyses. For instance, in the Shiprock samples, an additional predominant bacterial group was identified and affiliated with Nitrosomonas sp., whereas in the Gittersee/Coschütz samples, anammox populations were identified that were not retrieved by the applied 16S rDNA approach.

Isolation and characterization of lipid rafts with different properties from RBL-2H3 (rat basophilic leukaemia) cells.

Lipid rafts are plasma-membrane microdomains that are enriched in certain lipids (sphingolipids, glycosphingolipids and cholesterol), as well as in lipid-modified proteins. Rafts appear to exist in the liquid-ordered phase, which contributes to their partitioning from the surrounding liquid-disordered glycerophospholipid environment. DRM (detergent-resistant membrane) fractions isolated from cells are believed to represent coalesced lipid rafts. We have employed extraction using two different non-ionic detergents, Brij-96 and Triton X-100, to isolate detergent-resistant lipid rafts from rat basophilic leukaemia cell line RBL-2H3, and compared their properties with each other and with plasma-membrane vesicles. DRM fractions were isolated as sealed unilamellar vesicles of similar size (135-170 nm diameter), using either sucrose-density-gradient sedimentation or gel-filtration chromatography. Lipid rafts isolated using Brij-96 and Triton X-100 differed in density, protein content and the distribution between high- and low-density fractions of the known raft constituents, Thy-1, and the non-receptor protein tyrosine kinases, Yes and Lyn. Lyn was found in the raft microdomains in predominantly phosphorylated form. The level of enrichment of the protein constituents of the isolated lipid rafts seemed to depend on the ratio of cell lipid/protein to detergent. As indicated by reactivity with anti-Thy-1 antibodies, lipid rafts prepared using Brij-96 appeared to consist of vesicles with primarily right-side-out orientation. Both Brij-96 and Triton X-100 appear to isolate detergent-insoluble raft microdomains from the rat basophilic leukaemia cell line RBL-2H3, but the observed differences suggest that either the detergents themselves play a role in determining the physicochemical characteristics of the resulting DRM fractions, or different subsets of rafts are isolated by the two detergents.