Project description:Geobacter sulfurreducens is a widely explored microorganism recognized by its metabolic versatility able to reduce a number of external electron acceptors. In the present study the capacity of this strain to reduce nitrate was evaluated along with its transcriptomic profile under nitrate-reducing conditions and the catalytic role of Pd nanoparticles on the reductive pathway. Results demonstrated that G. sulfurreducens was able to reduce nitrate and important kinetic differences related to the time response were found among the electron donors used (acetate and hydrogen). When using acetate, a delay response on nitrate reduction of 4 days and reduction of 94% of nitrate was achieved, while nitrite was not detected, and all the nitrogen was recovered as ammonium (79.6 ± 5.7 %). The use of hydrogen as electron donor increased 2-fold the maximum rate of nitrate reduction, leading to 93% reduction of nitrate during the first 20 h with recovery of 45% as ammonium, while nitrite was not detected. In addition, transcriptome profiling analysis of G. sulfurreducens under nitrate-reducing conditions using hydrogen or acetate as an electron donor at 2 and 6 days reveals that a core of 146 genes (69 upregulated and 77 downregulated) are differentially expressed in all conditions. Genes related to nitrogen metabolism, such as nrfA and nrfH, gdhA, and amtB, were upregulated in the incubations and RT-qPCR data confirmed upregulations of these genes. Experiments performed with biologically synthesized Pd (Bio-Pd) + G. sulfurreducens cells demonstrated synergistic input of Bio-Pd and the metabolic capacity of G. sulfurreducens. These results expand the metabolic versatility of G. sulfurreducens, which may have important implications in nitrogen cycling in natural environments and engineered systems.
Project description:Acididesulfobacillus acetoxydans is an acidophilic sulfate reducer that can dissimilatory reduce nitrate to ammonia (DNRA). However, no known nitrite reductase is encoded. This study was performed to investigate how A. acetoxydans reduces nitrate to nitrite and elucidated a novel DNRA mechanism and potential nitrosative stress resistance mechanisms in acidophiles.
Project description:Nitrogen metabolism in Aspergillus nidulans is subject to regulation by the GATA transcription factor AreA which is required for the utilization of a wide range of nitrogen sources other than glutamine or ammonium. The level of AreA activity is regulated by intracellular glutamine levels that vary in response to nitrogen supplementation. For nitrate assimilation, which involves two transporters (CrnA, CrnB), nitrate reductase (NiaD) and nitrite reductase (NiiA), the respective genes are subject to regulation at the level of transcription, including nitrogen metabolite repression mediated by AreA and induction mediated by nitrite or nitrate, mediated by a second transcription factor, NirA. Both transcription factors act synergistically to regulate the expression of all four structural genes when nitrogen is limiting or either nitrate or nitrite is available. In this study we dissect the nitrogen limitation effect mediated by AreA form the nitrate/nitrite specific effect mediated by NirA on the transcriptome level. Keywords: Nitrate/nitrogen limitation response
Project description:Staphylococcus xylosus is one of the major starter cultures used for meat fermentation because of its crucial role in the reduction of nitrate to nitrite, which contributes to color and flavor development. Despite the long use of these additives, their impact on the physiology of S. xylosus has not yet been explored. We present the first in situ global gene expression profile of S. xylosus in meat supplemented with nitrate and nitrite. More than 600 genes of S. xylosus were differentially expressed at 24 or 72 hours of incubation. They represent more than 20% of the total genes and led us to suppose that addition of nitrate and nitrite to meat leads to a global change in gene expression. This profile revealed that S. xylosus is subject to nitrosative stress caused by reactive nitrogen species generated from nitrate and nitrite. To overcome this stress, S. xylosus has developed several oxidative stress resistance mechanisms, such as modulation of the expression of several genes involved in iron homeostasis and in antioxidant defense. Most of these genes belong to the Fur and PerR regulons respectively. S. xylosus has also counteracted this stress by developing DNA and protein repair. Furthermore, it has adapted its metabolic response—carbon and nitrogen metabolism, energy production and cell wall biogenesis—to the alterations produced by nitrosative stress.
Project description:Staphylococcus xylosus is one of the major starter cultures used for meat fermentation because of its crucial role in the reduction of nitrate to nitrite, which contributes to color and flavor development. Despite the long use of these additives, their impact on the physiology of S. xylosus has not yet been explored. We present the first in situ global gene expression profile of S. xylosus in meat supplemented with nitrate and nitrite. More than 600 genes of S. xylosus were differentially expressed at 24 or 72 hours of incubation. They represent more than 20% of the total genes and led us to suppose that addition of nitrate and nitrite to meat leads to a global change in gene expression. This profile revealed that S. xylosus is subject to nitrosative stress caused by reactive nitrogen species generated from nitrate and nitrite. To overcome this stress, S. xylosus has developed several oxidative stress resistance mechanisms, such as modulation of the expression of several genes involved in iron homeostasis and in antioxidant defense. Most of these genes belong to the Fur and PerR regulons respectively. S. xylosus has also counteracted this stress by developing DNA and protein repair. Furthermore, it has adapted its metabolic responseM-bM-^@M-^Tcarbon and nitrogen metabolism, energy production and cell wall biogenesisM-bM-^@M-^Tto the alterations produced by nitrosative stress. Microarray was used to evaluate modification in the transcriptome of S. xylosus C2a strain in the presence (N) or absence (V) of nitroso compounds. Three biological replicates collected on separate days for each meat matrix and labelled following a dye-switch design; for each condition one labeling in Cy3 and one in Cy5.
Project description:The effect of nitrate reduction (anaerobic cultivation in the presence of heme, vitamin K2 and nitrate) was compared with anaerobic cultivation supplemented with citrate (Lactobacillus plantarum). The medium was chemically defined medium with mannitol as main carbon source Two-condition experiment, nitrate vs citrate reducing cells. Biological replicates: 4 nitrate reducing cultures, 4 citrate reducing cultures, independently grown and harvested. Two slides were used, each slide contained 8 Arrays. Citrate reducing cultures are called reactor 1-4, Nitrate reducing cultures are called reactor A-D
Project description:Here we report a metatranscriptomic analysis of gene expression and regulation of “Candidatus Accumulibacter”-enriched lab-scale sludge during enhanced biological phosphorus removal (EBPR). Medium density oligonucleotide microarrays were generated with probes targeting most predicted genes hypothesized to be important for the EBPR phenotype. The objectives were to investigate which genes were expressed during EBPR and which genes were differentially expressed between the early stage of anaerobic and aerobic phases (defined as 15 min after acetate addition and 15 min after switching to aeration respectively).
Project description:The genomic response to low levels of nitrate was studied in Arabidopsis using the Affymetrix ATH1 chip containing more than 22,500 probe sets. Arabidopsis plants were grown hydroponically in sterile liquid culture on ammonium as the sole source of nitrogen for 10 d, then treated with 250 um nitrate for 20 min. The response to nitrate was much stronger in roots (1,176 genes showing increased or decreased mRNA levels) than in shoots (183 responding genes). In addition to known nitrate-responsive genes (e.g. those encoding nitrate transporters, nitrate reductase, nitrite reductase, ferredoxin reductase, and enzymes in the pentose phosphate pathway), genes encoding novel metabolic and potential regulatory proteins were found. These genes encode enzymes in glycolysis (glucose-6-phosphate isomerase and phosphoglycerate mutase), in trehalose-6-P metabolism (trehalose-6-P synthase and trehalose-6-P phosphatase), in iron transport/metabolism (nicotianamine synthase), and in sulfate uptake/reduction. In many cases, only a few select genes out of several in small gene families were induced by nitrate. These results show that the effect of nitrate on gene expression is substantial (affecting almost 10% of the genes with detectable mRNA levels) yet selective and affects many genes involved in carbon and nutrient metabolism. Keywords: Expression profilling by array
Project description:The genomic response to low levels of nitrate was studied in Arabidopsis using the Affymetrix ATH1 chip containing more than 22,500 probe sets. Arabidopsis plants were grown hydroponically in sterile liquid culture on ammonium as the sole source of nitrogen for 10 d, then treated with 250 um nitrate for 20 min. The response to nitrate was much stronger in roots (1,176 genes showing increased or decreased mRNA levels) than in shoots (183 responding genes). In addition to known nitrate-responsive genes (e.g. those encoding nitrate transporters, nitrate reductase, nitrite reductase, ferredoxin reductase, and enzymes in the pentose phosphate pathway), genes encoding novel metabolic and potential regulatory proteins were found. These genes encode enzymes in glycolysis (glucose-6-phosphate isomerase and phosphoglycerate mutase), in trehalose-6-P metabolism (trehalose-6-P synthase and trehalose-6-P phosphatase), in iron transport/metabolism (nicotianamine synthase), and in sulfate uptake/reduction. In many cases, only a few select genes out of several in small gene families were induced by nitrate. These results show that the effect of nitrate on gene expression is substantial (affecting almost 10% of the genes with detectable mRNA levels) yet selective and affects many genes involved in carbon and nutrient metabolism. Keywords: Expression profilling by array 8 samples were used in this experiment
Project description:Alicycliphilus denitrificans is a versatile, ubiquitous, facultative anaerobic bacterium. A. denitrificans strain BC can use chlorate, nitrate and oxygen as electron acceptor for growth. Cells display a prolonged lag-phase when transferred from nitrate to chlorate and vice versa. Furthermore, cells adapted to aerobic growth do not easily use nitrate or chlorate as electron acceptor. We further investigated these responses of strain BC by differential proteomics, transcript analysis and enzyme activity assays. In nitrate-adapted cells transferred to chlorate and vice versa, appropriate electron acceptor reduction pathways need to be activated. In oxygen-adapted cells, adaptation to the use of chlorate or nitrate is likely difficult due to the poorly active nitrate reduction pathway and low active chlorate reduction pathway. We deduce that the Nar-type nitrate reductase of strain BC also reduces chlorate, which may result in toxic levels of chlorite if cells are transferred to chlorate. Furthermore, the activities of nitrate reductase and nitrite reductase appear to be not balanced when oxygen-adapted cells a shifted to nitrate as electron acceptor, leading to the production of a toxic amount of nitrite. These data suggest that strain BC encounters metabolic challenges in environments with fluctuations in the availability of electron acceptors. Proteomic samples were prepared from Alicycliphilus denitrificans grown in the presence of different electron acceptors: chlorate, nitrate and oxygen. Proteins were separated by a short SDS gel and for each sample 4 in-gel digest slices were prepared. Peptide samples were measured by nLC LTQ-Orbitrap and the data were analysed with MaxQuant using default settings (1% FDR on peptide and protein level) and filtered extra to keep only proteins identified with at least 2 peptides and 1 unique and 1 unmodified peptide.