Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes numerous human pathologies. Biochemical evidence suggests that soluble misfolded proteins are retrotranslocated out of the ER, via the endoplasmic reticulum associated degradation (ERAD) pathway, for proteosome-mediated cytoplasmic degradation. Excess, misfolded- or insoluble proteins, are suggested to cause induction of “overflow” degradation pathways. For soluble proteins, overflow to vacuole-mediated destruction is suggested to occur via two Golgi-to-vacuole (Gvt) routes, the alkaline phosphatase (ALP), direct route, or, a carboxypeptidase Y- (CPY-), prevacuolar compartmentvacuole, indirect route, though only the CPY route is thought to degrade soluble proteins. Insoluble aggregate-containing structures are suggested to be degraded by engulfment by membranes of unknown origin and trafficking to the vacuole for destruction, via an autophagic pathway. To confirm biochemical evidence, wild-type (BY4742), autophagosome- (W303/ATG14), CPY- and autophagy pathway- (W303/VPS30), and proteosome (WCG/2) mutants of S. cerevisiae yeasts were transformed with a high expression pYES plasmid and mutant (Z) human alpha-1- proteinase inhibitor (A1PiZ), giving rise to the derivatives cells BY4742/Z, W303/ATG14/Z, W303/VPS30/Z and WCG/2/Z, respectively. Electron microscopy using gold labeling for A1PiZ, markers for the ER, the ERAD ER channel protein, Sec61, or the chaperone, binding protein (BiP), ALP for the ALP pathway, and CPY for the CPY pathway, was used. Overexpression of A1PiZ seems to result in targeting to the vacuole via a prevacuolar, CPY-like compartment (PVC, 200-500 nm), though CPY and A1PiZ appears not to colocalise, unconvincingly confirming collaborative biochemical data. Large amounts of A1PiZ localise in the cytosol, possibly indicating a largely proteasome-mediated degradation. ER-resident A1PiZ targeting to the vacuole seems also to occur by the budding of the ER and peripheral plasma membrane or ER membrane only. This occurs in all cells, but especially in ATG14 gene (ΔATG14) mutants, possibly indicating autophagosome-mediated degradation independence, in the latter mutants. The ATG14 mutation gave rise to crescent-shaped, initiating membranelike (IM-like) structures of approximately Cvt vesicle-diameter, possibly indicating that ΔATG14 blocks autophagosome- (500-1000 nm) and Cvt vesicle (100-200 nm) enclosure, after core IM formation. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Benzalkonium chlorides (BACs) are widely used, broad-spectrum disinfectants and frequently detected in the environment, even at toxic levels for life. Since such disinfectants can induce broad resistance capabilities, BACs may fuel the emergence of antibiotic resistance in the environment. A substantial body of literature has reported that exposure to BACs causes antibiotic resistance; yet, other studies suggest that the resistance linkage is rare, unsystematic, and/or clinically insignificant. Accordingly, whether or not disinfectant exposure mediates antibiotic resistance and, if so, what molecular mechanisms underlie the resistance link remains to be clearly elucidated. Further, understanding how microbial communities degrade BACs is important not only for alleviating the possible occurrence of antibiotic resistance but also reducing the potential risks to environmental and public health. An integrated strategy that combines metagenomics, metatranscriptomics, genetics, and traditional culture-dependent approaches was employed to provide novel insights into these issues. The integrative approach showed that a microbial community exposed to BACs can acquire antibiotic resistance through two mechanisms: i) horizontal transfer of previously uncharacterized efflux pump genes conferring resistance to BACs and antibiotics, which were encoded on a conjugative plasmid and co-selected together upon BACs and ii) selective enrichment of intrinsically multi-drug resistant organisms. Further, a microbial community adapts to BAC exposure via a variety of mechanisms, including selective enrichment of BAC-degrading species and amino acid substitutions and horizontal transfer of genes related to BAC resistance and degradation. The metatranscriptomic data suggests that the BAC-adapted microbial community metabolized BACs by cooperative interactions among its members. More specifically, Pseudomonas nitroreducens cleaved (i.e., dealkylated) BACs, metabolized the alkyl chain (the dealkylated product of BACs), and released benzyldimethylamine (the other product of BACs), which was further metabolized by other community members (e.g., Pseudomonas putida). Collectively, this study demonstrates the role of BACs in promoting antibiotic resistance and advances current understanding of a microbial community degrading BACs. The results of this work have important implications for (appropriate) usage of disinfectants and for assessing, predicting, and optimizing biological engineering processes treating BAC-bearing waste streams.

Northern peatlands are an important long-term sink for atmospheric carbon dioxide (CO2) and a contemporary source of methane (CH4). Under contemporary climate and environmental change, including enhanced nutrient deposition through industrialization and commercial peat harvesting, the microbial environment in peat is altered. Microorganisms are responsible for the net production of greenhouse gases in these sites, although controls on microbial activity and microbial communities are poorly understood, limiting our ability to predict greenhouse gas emissions. The objective of this thesis was to determine the microbial role in peat decomposition and greenhouse gas fluxes in northern peatlands. Nutrient, carbon (C) substrate, and microbial-ecological controls on microbial activity under natural climate variability, increased nutrient deposition, and commercial harvesting and restoration were explored in detail. Environmental change effects were evaluated in relation to processes and temporal variability in pristine sites. / The natural temporal variability of decomposition, microbial biomass, and nitrogen (N) was characterized in the Mer Bleue bog near Ottawa, ON over two years. In a warmer, drier year, lower water table position corresponded to increased N availability, which was in turn linked to enhanced microbial CO2 production, consistent with patterns in ecosystem respiration measured at the site level. It was shown that microbial activity can play an important role in inter-annual climate driven ecosystem respiration and net ecosystem CO2 exchange. / Through field and laboratory nutrient fertilization experiments, it was shown that increased nitrogen (N) deposition altered the heterotrophic microbial community at Met Bleue and led to decreased decomposition rates after one year, despite increased total microbial biomass. After the second year of fertilization, however, decomposition rates were elevated, presumably a result of a concomitant shift in moss species and supply of more bioavailable plant material. Comparison of fertilizations in the presence and absence of vegetation indicated that in oligotrophic sites, vegetation mediated elevated nutrient effects on decomposition and that N cycling occurred largely in the organic forms. / Aerobic and anaerobic microbial activity, peat organic and nutrient chemistry, microbial biomass, and methanogen, CH4-oxidizing bacteria, bacteria, and archaea were characterized in two sets of pristine, actively harvested, harvested and abandoned, and harvested and restored peatlands in Quebec and New Brunswick.

Protein disulfide isomerases (PDIs) catalyze the oxidation and isomerization of disulfide bonds in proteins passing through the endoplasmic reticulum (ER). Although as many as 20 enzymes are classified as PDI family members, their relative contributions to protein folding have remained an open question. Additionally, Ero1 has been characterized as the ER oxidase that transfers oxidizing equivalents from oxygen to PDI enzymes. However, knockout mice lacking the mammalian Ero1 isoforms, Ero1Lα and Ero1Lβ, are viable, and the role of other potential ER oxidases in maintaining an oxidative ER environment is now an important issue. By systematic depletion of ER PDI family members and potential ER oxidases and assessment of disulfide bond formation of secreted endogenous substrates, I have outlined the functional relationships among some of these enzymes. PDI family member depletion revealed that PDI, although not essential for complete disulfide bond formation in client proteins, is the most significant catalyst of oxidative folding. In comparison, ERp57 acts preferentially on glycosylated substrates, ERp72 functions in a more supplementary capacity, and P5 has no detectable role in formation of disulfide bonds for the substrates assayed. Initially, no impact of depletion of Ero1 was observed under steady state conditions, suggesting that other oxidase systems are working in parallel to support normal disulfide bond formation. Subsequent experiments incorporating a reductive challenge revealed that Ero1 depletion produces the strongest delay in re-oxidation of the ER and oxidation of substrate. Depletion of two other potential ER oxidases, peroxiredoxin 4 (PRDX4) and Vitamin K epoxide reductase (VKOR), showed more modest effects. Upon co-depletion of Ero1 and other oxidases, additive effects were observed, culminating in cell death following combined removal of Ero1, PRDX4, and VKOR activities. These studies affirm the predominant roles of Ero1 in ER oxidation processes and, for the first time, establish VKOR as a significant contributor to disulfide bond formation.

No description available.

Uranium contamination of soils and groundwater at Department of Energy facilities across the United States is a primary environmental concern and the development of effective remediation strategies is a major challenge. Bioremediation, or the use of microbial enzymatic activity to facilitate the remediation of a contaminant, offers a promising in situ approach that may be less invasive than traditional methods, such as pump and treat or excavation. This study demonstrates for the first time the successful biomineralization of uranium phosphate minerals as a result of microbial phosphatase activity at low pH in both aerobic and anaerobic conditions using pure cultures and soils from a contaminated waste site. Pure cultures of microorganisms isolated from soils of a low pH, high uranium- and nitrate-contaminated waste site, expressed constitutive phosphatase activity in response to an organophosphate addition in aerobic and anaerobic incubations. Sufficient phosphate was hydrolyzed to precipitate 73 to 95% total uranium as chernikovite identified by synchrotron X-ray absorption spectroscopy and X-ray diffraction. Highest rates of uranium precipitation and phosphatase activity were observed between pH 5.0 and 7.0. Indigenous microorganisms were also stimulated by organophosphate amendment in soils from a contaminated waste site using flow-through reactors. High phosphate concentrations (0.5 to 3 mmol L-1) in pore water effluents were observed within days of organophosphate addition. Highest rates of phosphatase activity occurred at pH 5.5 in naturally low pH soils in the presence of high uranium and nitrate concentrations. The precipitation of uranium phosphate was identified by a combination of pore water measurements, solid phase extractions, synchrotron-based X-ray spectroscopy, and a reactive transport model. The results of this study demonstrate that uranium is biomineralized to a highly insoluble uranyl phosphate mineral as a result of enzymatic hydrolysis of an organophosphate compound over a wide range of pH, in both aerobic and anaerobic conditions, and in the presence of high uranium and nitrate concentrations. The nonreductive biomineralization of U(VI) provides a promising new approach for in situ uranium bioremediation in low pH, high nitrate, and aerobic conditions that could be complementary to U(VI) bioreduction in high pH, low nitrate, and reducing environments.

<font face="Times New Roman"><font face="Times New Roman"> <p align="left">The popularization and health benefits associated with the &ldquo / Mediterranean diet&rdquo / saw a world wide increase in the production and consumption of processed olives and olive oil. During the brining of table olives large quantities of processed olive waste water is seasonally generated. This blackish-brown, malodours liquid is rich in organic and phenolic compounds, which cause environmental problems upon discarding. Currently, processed wastewater is discarded into large evaporation ponds where it poses serious environmental risks. The biodegradation of organic substrates present in the olive wastewater is inhibited by the high concentrations of phenolic compounds. <font face="Times New Roman">In order to identify organisms which could potentially be used in the bioremediation of olive wastewater, 36 microbial strains were isolated from evaporation ponds in the Boland region of South Africa. Twenty five isolates were capable of growth on 50% olive wastewater and their bioremediation potential as well as their ability to produce valuable intermediate compounds were subsequently characterized. Based on the RPHPLC results, which showed that a number of chemical intermediates were produced in fermentation of olive wastewater, isolate AS-35 was selected for further analysis. Strain AS-35, identified as a </font><font face="Times New Roman"><em>Bacillus megaterium,</em> </font><font face="Times New Roman">was significantly influenced by the exposure to olive waste. The total cellular protein profile, generation time and cellular morphology of this isolate were dramatically affected by the introduction of olive waste. <font face="Times New Roman">This study investigated the differential gene display of </font><font face="Times New Roman"><font face="Times New Roman"><em>Bacillus megaterium</em></font> </font><font face="Times New Roman">following exposure to olive wastewater. Proteomic and transcriptomic differences of the organism cultured in nutrient rich LB and olive wastewater were compared. These results indicated that AS-35 expressed genes involved in glycolysis, tryptophan and nucleotide synthesis as well as the chaperones GroEL and DnaK during its growth in LB. In contrast, genes induced following the abolishment of glucose dependent catabolite repression, genes involved in biotin synthesis and &szlig / -oxidation of fatty or organic acids as well as a gene whose expression is regulated by stress induced s</font><font face="Times New Roman" size="1">B</font><font face="Times New Roman">-dependent regulon were expressed during olive waste growth.</font></font></p> </font></font>

No description available.

Thesis (M.A.)--Texas State University-San Marcos, 2007. / Vita. Appendices: leaves 52-78. Includes bibliographical references (leaves 79-83).

Thesis (M.A.)--Texas State University-San Marcos, 2007. / Vita. Appendices: leaves 52-78. Includes bibliographical references (leaves 79-83).