Genetic Basis of Ammonium Toxicity Resistance in a Sake Strain of Yeast: A Mendelian Case.

High concentrations of ammonium at physiological concentrations of potassium are toxic for the standard laboratory strain of Saccharomyces cerevisiae In the original description of this metabolic phenotype, we focused on the standard laboratory strains of Saccharomyces In this study, we screened a large collection of S. cerevisiae natural isolates and identified one strain that is resistant to high concentrations of ammonium. This strain, K12, was isolated in sake breweries. When the K12 strain was crossed to the standard laboratory strain (FY4), the resulting tetrads displayed 2:2 segregation of the resistance phenotype, suggesting a single gene trait. Using a bulk segregant analysis strategy, we mapped this trait to a 150-kb region on chromosome X containing the TRK1 gene. This gene encodes a transporter required for high-affinity potassium transport in S. cerevisiae Data from reciprocal hemizygosity experiments with TRK1 deletion strains in K12 and BY backgrounds, as well as analysis of the deletion of this gene in the K12 strain, demonstrate that the K12 allele of TRK1 is responsible for ammonium toxicity resistance. Furthermore, we determined the minimal amount of potassium required for both the K12 and laboratory strain needed for growth. These results demonstrate that the gene encoded by the K12 allele of TRK1 has a greater affinity for potassium than the standard allele of TRK1 found in Saccharomyces strains. We hypothesize that this greater-affinity allele of the potassium transporter reduces the flux of ammonium into the yeast cells under conditions of ammonium toxicity. These findings further refine our understanding of ammonium toxicity in yeast and provide an example of using natural variation to understand cellular processes

Ammonium Toxicity and Potassium Limitation in Yeast

DNA microarray analysis of gene expression in steady-state chemostat cultures limited for potassium revealed a surprising connection between potassium and ammonium: potassium limits growth only when ammonium is the nitrogen source. Under potassium limitation, ammonium appears to be toxic for Saccharomyces cerevisiae. This ammonium toxicity, which appears to occur by leakage of ammonium through potassium channels, is recapitulated under high-potassium conditions by over-expression of ammonium transporters. Although ammonium toxicity is well established in metazoans, it has never been reported for yeast. To characterize the response to ammonium toxicity, we examined the filtrates of these cultures for compounds whose excretion might serve to detoxify the ammonium (such as urea in mammals). Using liquid chromatography–tandem mass spectrometry to assay for a wide array of metabolites, we detected excreted amino acids. The amounts of amino acids excreted increased in relation to the severity of growth impairment by ammonium, suggesting that amino acid excretion is used by yeast for ammonium detoxification

Response and variability of Arctic soils exposed to nitrogenous compounds

Increased development in Canada’s northern environments has increased the need for accurate methods to detect adverse impacts on tundra ecosystems. Ammonium nitrate is a common water pollutant associated with many industrial and municipal activities, including diamond mining, and is of special concern due to the toxicity of ammonia in aquatic systems. One solution to reduce exposure of sensitive aquatic systems to nitrogenous compounds is to atomize (atmospherically disperse in fine particles) contaminated water over the arctic tundra which will reduce N loading to surface water. However, the toxicity of ammonium nitrate to arctic soils is poorly understood. In this study I investigate the potential toxicity of ammonium nitrate solutions to arctic soil functions such as carbon mineralization, nitrification and plant growth, to determine concentrations that can be applied without causing significant inhibition to these processes. Arctic ecosystems are based on a soil type termed a cryosol that has an underlying permafrost layer. Often these soils are subject to cryoturbation, a process which heaves and mixes the soil, bringing the mineral horizons to the surface. I hypothesized that phytotoxicity test results in arctic soils would be highly variable compared to other terrestrial ecosystems due to the cryoturbation process and subsequent range of soil characteristics. The variability associated with phytotoxicity tests was evaluated using Environment Canada’s standardized plant toxicity test in three cryoturbated soils from Canada’s arctic exposed to a reference toxicant, boric acid. The phytotoxicity of boric acid to northern wheatgrass (Elymus lanceolatus ) in cryosols was much greater than commonly reported in other soils, with less than 150 ug boric acid g-1 soil needed to inhibit root and shoot growth by 20%. There was also large variability in the phytotoxicity test results, with coefficients of variation for 10 samples ranging from 160 to 79%. Due to this variability in cryoturbated arctic soils, more than 30 samples should be collected from each control and potentially impacted area to accurately assess contaminant effects, and ensure that false negatives of toxicant impacts in arctic soils are minimized. To characterize the toxicity of ammonium nitrate I exposed a variety of arctic soils and a temperate soil to different concentrations of ammonium nitrate solution over a 90 day time period. Dose responses of carbon mineralization, nitrification and phytotoxicity test parameters were estimated for ammonium nitrate applications. In addition to direct toxicity, the effect of ammonium nitrate on ecosystem resistance was investigated by dosing nitrogen impacted soils with boric acid. Ammonium nitrate solutions had no effect on carbon mineralization activity, and affected nitrification rates in only one soil, a polar desert soil from Cornwallis Island. In contrast, ammonium nitrate applications (43 mmol N L-1 soil water) significantly impaired seedling emergence, root length and shoot length of northern wheatgrass. Concentrations of ammonium nitrate in soil water that inhibited plant parameters by 20% varied between 43 to 280 mmol N L-1 soil water, which corresponds with 2,100 to 15,801 mg L-1 in the application water. Arctic soils were more resistant to ammonium nitrate toxicity than the temperate soil under these study conditions. However, it is not clear if this represents a general trend for all polar soils, and because nitrogen is an essential macro-nutrient, nitrogenous toxicity should likely be considered a special case for soil toxicity. As soil concentrations could be maintained under inhibitory levels with continual application of low concentrations of ammonium nitrate over the growing season, atomization of wastewater contaminated with ammonium nitrate is a promising technology for mitigation of nitrogen pollution in polar environments. Increased development in Canada’s northern environments has increased the need for accurate methods to detect adverse impacts on tundra ecosystems. Ammonium nitrate is a common water pollutant associated with many industrial and municipal activities, including diamond mining, and is of special concern due to the toxicity of ammonia in aquatic systems. One solution to reduce exposure of sensitive aquatic systems to nitrogenous compounds is to atomize (atmospherically disperse in fine particles) contaminated water over the arctic tundra which will reduce N loading to surface water. However, the toxicity of ammonium nitrate to arctic soils is poorly understood. In this study I investigate the potential toxicity of ammonium nitrate solutions to arctic soil functions such as carbon mineralization, nitrification and plant growth, to determine concentrations that can be applied without causing significant inhibition to these processes. Arctic ecosystems are based on a soil type termed a cryosol that has an underlying permafrost layer. Often these soils are subject to cryoturbation, a process which heaves and mixes the soil, bringing the mineral horizons to the surface. I hypothesized that phytotoxicity test results in arctic soils would be highly variable compared to other terrestrial ecosystems due to the cryoturbation process and subsequent range of soil characteristics. The variability associated with phytotoxicity tests was evaluated using Environment Canada’s standardized plant toxicity test in three cryoturbated soils from Canada’s arctic exposed to a reference toxicant, boric acid. The phytotoxicity of boric acid to northern wheatgrass (Elymus lanceolatus ) in cryosols was much greater than commonly reported in other soils, with less than 150 ug boric acid g-1 soil needed to inhibit root and shoot growth by 20%. There was also large variability in the phytotoxicity test results, with coefficients of variation for 10 samples ranging from 160 to 79%. Due to this variability in cryoturbated arctic soils, more than 30 samples should be collected from each control and potentially impacted area to accurately assess contaminant effects, and ensure that false negatives of toxicant impacts in arctic soils are minimized. To characterize the toxicity of ammonium nitrate I exposed a variety of arctic soils and a temperate soil to different concentrations of ammonium nitrate solution over a 90 day time period. Dose responses of carbon mineralization, nitrification and phytotoxicity test parameters were estimated for ammonium nitrate applications. In addition to direct toxicity, the effect of ammonium nitrate on ecosystem resistance was investigated by dosing nitrogen impacted soils with boric acid. Ammonium nitrate solutions had no effect on carbon mineralization activity, and affected nitrification rates in only one soil, a polar desert soil from Cornwallis Island. In contrast, ammonium nitrate applications (43 mmol N L-1 soil water) significantly impaired seedling emergence, root length and shoot length of northern wheatgrass. Concentrations of ammonium nitrate in soil water that inhibited plant parameters by 20% varied between 43 to 280 mmol N L-1 soil water, which corresponds with 2,100 to 15,801 mg L-1 in the application water. Arctic soils were more resistant to ammonium nitrate toxicity than the temperate soil under these study conditions. However, it is not clear if this represents a general trend for all polar soils, and because nitrogen is an essential macro-nutrient, nitrogenous toxicity should likely be considered a special case for soil toxicity. As soil concentrations could be maintained under inhibitory levels with continual application of low concentrations of ammonium nitrate over the growing season, atomization of wastewater contaminated with ammonium nitrate is a promising technology for mitigation of nitrogen pollution in polar environments

A Potentiometric Flow Biosensor Based on Ammonia-Oxidizing Bacteria for the Detection of Toxicity in Water

A flow biosensor for the detection of toxicity in water using the ammonia-oxidizing bacterium (AOB) Nitrosomonas europaea as a bioreceptor and a polymeric membrane ammonium-selective electrode as a transducer is described. The system is based on the inhibition effects of toxicants on the activity of AOB, which can be evaluated by measuring the ammonium consumption rates with the ammonium-selective membrane electrode. The AOB cells are immobilized on polyethersulfone membranes packed in a holder, while the membrane electrode is placed downstream in the flow cell. Two specific inhibitors of the ammonia oxidation. allylthiourea and thioacetamide. have been tested. The IC50 values defined as the concentration of an inhibitor causing a 50% reduction in the ammonia oxidation activity have been measured as 0.17 mu M and 0.46 mu M for allylthiourea and thioacetamide, respectively. The proposed sensor offers advantages of simplicity, speed and high sensitivity for measuring toxicity in water.A flow biosensor for the detection of toxicity in water using the ammonia-oxidizing bacterium (AOB) Nitrosomonas europaea as a bioreceptor and a polymeric membrane ammonium-selective electrode as a transducer is described. The system is based on the inhibition effects of toxicants on the activity of AOB, which can be evaluated by measuring the ammonium consumption rates with the ammonium-selective membrane electrode. The AOB cells are immobilized on polyethersulfone membranes packed in a holder, while the membrane electrode is placed downstream in the flow cell. Two specific inhibitors of the ammonia oxidation. allylthiourea and thioacetamide. have been tested. The IC50 values defined as the concentration of an inhibitor causing a 50% reduction in the ammonia oxidation activity have been measured as 0.17 mu M and 0.46 mu M for allylthiourea and thioacetamide, respectively. The proposed sensor offers advantages of simplicity, speed and high sensitivity for measuring toxicity in water

A Marriage of Old and New: Chemostats and Microarrays Identify a New Model System for Ammonium Toxicity

Toxicity is related to an organism's ability to rid itself of the offending molecules. This primer provides insights into how this can be monitored by highlighting the case of ammonium toxicity

Electrochemical technology enables nutrient recovery and ammonia toxicity control in anaerobic digestion

The aim of this study was to investigate the impact of an electrochemical system (ES) on the performance of an anaerobic digester during both low and high ammonium (NH4+) loading rates. For this, a Test (with ES) and Control (without ES) setup was used. Ammonia (NH3), in equilibrium with NH4+, is a toxic compound to the methanogenic community, limits the substrate loading rate and endangers process stability. We hypothesized that, through coupling of an ES to a digester, NH3 toxicity can be controlled with simultaneous recovery of this nutrient. The ES always had a temporary negative effect when switched on. However, during periods of high ammonium loading rates the CH4 production of the Test reactor was at maximum a factor 4.5 higher compared to the Control reactor, which could be explained through a combination of NH4+ extraction and electrochemical pH control. A nitrogen flux of 47 g N m-2 membrane d-1 could be obtained in the Test reactor, resulting in a current and removal efficiency of 38±5% and 28±2%, respectively. For this, an electrochemical power input 17±2 kWh kg-1 N was necessary. In addition, anodic oxidation of sulphide resulted in a significantly lower H2S emission

A mathematical model of bacteria capable of complete oxidation of ammonium predicts improved nitrogen removal and reduced production of nitrous oxide

The removal of excess nutrients from water ecosystems requires oxidation of toxic ammonium by two types of bacteria; one oxidizes ammonium to nitrite and the other oxidizes nitrite to nitrate. The oxidation of ammonium is often incomplete and nitrite accumulates. Nitrite is also toxic, and is converted by the ammoniumoxidizing bacteria to nitrous oxide, a powerful greenhouse gas. Here we use mathematical modeling to analyze a potential solution to the problems related to incomplete oxidation of ammonium. We propose that a single engineered nitrifying bacterium should be capable of complete oxidation of high concentrations of ammonium to nitrate. Our model is based on available data on ammonium- and nitrite-oxidizing bacteria. The model predicts that insertion of highly expressed genes of a nitrite oxidation system into the genome of an ammonia-oxidation bacterium should result in complete oxidation of ammonium to nitrate in nutrient-overloaded conditions. Due to its increased capacity to fully oxidize ammonium to nitrate, the proposed bacterium would display dramatically reduced production of nitrous oxide, and therefore might have great potential to reduce the greenhouse effect of nutrient-overloaded water system

Ammonia toxicity: from head to toe?

Ammonia is diffused and transported across all plasma membranes. This entails that hyperammonemia leads to an increase in ammonia in all organs and tissues. It is known that the toxic ramifications of ammonia primarily touch the brain and cause neurological impairment. However, the deleterious effects of ammonia are not specific to the brain, as the direct effect of increased ammonia (change in pH, membrane potential, metabolism) can occur in any type of cell. Therefore, in the setting of chronic liver disease where multi-organ dysfunction is common, the role of ammonia, only as neurotoxin, is challenged. This review provides insights and evidence that increased ammonia can disturb many organ and cell types and hence lead to dysfunction

Dibucaine in Ionic-Gradient Liposomes: Biophysical, Toxicological, and Activity Characterization

Administration of local anesthetics is one of the most effective pain control techniques for postoperative analgesia. However, anesthetic agents easily diffuse into the injection site, limiting the time of anesthesia. One approach to prolong analgesia is to entrap local anesthetic agents in nanostructured carriers (e.g., liposomes). Here, we report that using an ammonium sulphate gradient was the best strategy to improve the encapsulation (62.6%) of dibucaine (DBC) into liposomes. Light scattering and nanotracking analyses were used to characterize vesicle properties, such as, size, polydispersity, zeta potentials, and number. In vitro kinetic experiments revealed the sustained release of DBC (50% in 7 h) from the liposomes. In addition, in vitro (3T3 cells in culture) and in vivo (zebrafish) toxicity assays revealed that ionic-gradient liposomes were able to reduce DBC cyto/cardiotoxicity and morphological changes in zebrafish larvae. Moreover, the anesthesia time attained after infiltrative administration in mice was longer with encapsulated DBC (27 h) than that with free DBC (11 h), at 320 μM (0.012%), confirming it as a promising long-acting liposome formulation for parenteral drug administration of dibucaine.Fil: Couto, Verônica M.. Universidade Estadual de Campinas; BrasilFil: Prieto, Maria Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB | Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB | Universidad Nacional de la Plata. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB; ArgentinaFil: Igartúa, Daniela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB | Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB | Universidad Nacional de la Plata. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB; ArgentinaFil: Feas, Daniela Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB | Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB | Universidad Nacional de la Plata. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB; ArgentinaFil: Ribeiro, Lígia N.M.. Universidade Estadual de Campinas; BrasilFil: Silva, Camila M.G.. Universidade Estadual de Campinas; BrasilFil: Castro, Simone R.. Universidade Estadual de Campinas; BrasilFil: Guilherme, Viviane A.. Universidade Estadual de Campinas; BrasilFil: Dantzger, Darlene D.. Universidade Estadual de Campinas; BrasilFil: Machado, Daisy. Universidade Estadual de Campinas; BrasilFil: Alonso, Silvia del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB | Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB | Universidad Nacional de la Plata. Instituto Multidisciplinario de Biología Celular. Grupo Vinculado al IMBICE - Grupo de Biología Estructural y Biotecnología-Universidad Nacional de Quilmes - GBEyB; ArgentinaFil: de Paula, Eneida. Universidade Estadual de Campinas; Brasi