Project description:Anaerobic ammonium oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and mixotrophy beyond genome predictions. Here, we experimentally resolved the central carbon metabolism using metabolomics (LC-MS and GC-MS), metabolic flux analysis and proteomics (shot-gun proteomics).
Project description:The use of biofertilizers is becoming an economical and environmentally friendly alternative to promote sustainable agriculture. Biochar from microalgae can be applied to enhance the productivity of food crops through soil improvement, slow nutrient absorption and release, increased water uptake, and long-term mitigation of greenhouse gas sequestration. Therefore, the aim of this study was to evaluate the stimulatory effects of biochar produced from Spirulina platensis biomass on the development and seed production of rice plants. Biochar was produced by slow pyrolysis at 300°C, and characterization was performed through microscopy, chemical, and structural composition analyses. Molecular and physiological analyses were performed in rice plants submitted to different biochar concentrations (0.02, 0.1, and 0.5 mg mL-1) to assess growth and productivity parameters. Morphological and physicochemical characterization revealed a heterogeneous morphology and the presence of K and Mg minerals in the biochar composition. Chemical modification of compounds post-pyrolysis and a highly porous structure with micropores were observed. Rice plants submitted to 0.5 mg mL-1 of biochar presented a decrease in root length, followed by an increase in root dry weight. The same concentration influenced seed production, with an increase of 44% in the number of seeds per plant, 17% in the percentage of full seeds per plant, 12% in the weight of 1,000 full seeds, 53% in the seed weight per plant, and 12% in grain area. Differential proteomic analyses in shoots and roots of rice plants submitted to 0.5 mg mL-1 of biochar for 20 days revealed a fine-tuning of resource allocation towards seed production. These results suggest that biochar derived from Spirulina platensis biomass can stimulate rice seed production.
Project description:Anaerobic ammonium-oxidising (anammox) bacteria, members of the ‘Candidatus Brocadiaceae’ family, play an important role in the nitrogen cycle and are estimated to be responsible for about half of the oceanic nitrogen loss to the atmosphere. Anammox bacteria combine ammonium with nitrite and produce dinitrogen gas via the intermediates nitric oxide and hydrazine (anammox reaction) while nitrate is formed as a by-product. These reactions take place in a specialized, membrane-bound compartment called the anammoxosome. Therefore, the substrates ammonium, nitrite and product nitrate have to cross the outer-, cytoplasmic- and anammoxosome membranes to enter or exit the anammoxosome. The genomes of all anammox species harbour multiple copies of ammonium-, nitrite- and nitrate transporter genes. Here we investigated how the distinct genes for ammonium-, nitrite- and nitrate- transport were expressed during substrate limitation in membrane bioreactors. Transcriptome analysis of Kuenenia stuttgartiensis planktonic cells under ammonium-limitation showed that three of the seven ammonium transporter genes and one of the six nitrite transporter genes were significantly upregulated, while another ammonium and nitrite transporter gene were downregulated in nitrite limited growth conditions. The two nitrate transporters were expressed to similar levels in both conditions. In addition, genes encoding enzymes involved in the anammox reaction were differentially expressed, with those using nitrite as a substrate being upregulated under nitrite limited growth and those using ammonium as a substrate being upregulated during ammonium limitation. Taken together, these results give a first insight in the potential role of the multiple nutrient transporters in regulating transport of substrates and products in and out of the compartmentalized anammox cell.
Project description:The community composition (in terms of abundance, distribution and contribution of diverse clades) of bacteria involved in nitrogen transformations in the oxygen minimum zones may be related to the rates of fixed N loss in these systems. The abundance of both denirifying and anammox bacteria, and the assemblage composition of denitrifying bacteria were investigated in the Eastern Tropical South Pacific and the Arabian Sea using assays based on molecular markers for the two groups of bacteria. The abundance and distribution of bacteria associated with the fixed N removal processes denitrification and anammox were investigated using quantitative PCR for genes encoding nitrite reductase (nirK and nirS) in denitrifying bacteria and hydrazine oxidase(hzo) and 16S rRNA genesin anammox bacteria. All of these genes had depth distributions with maxima associated with the secondary nitrite maximum in low oxygen waters. NirS was mch more abundant than nirK, and much more abundant than the 16S rRNA gene from anammox bacteria. The ratio of hzo:16S rRNA for anammox was low and variable implying greater unexplored diversity in the the hzo gene. Assemblage composition of the abundant nirS-type denitrifiers was evaluated using a funcitonal gene microarray. Of the nirS archetypes represented on the microarray, very few occurred speficically in one region or depth interval, but the assemblages varied significantly. Community composition of denitrifiers based on microarray analysis of the nirS gene was most different between geographical regions. Within each region, the surface layer and OMZ assemblages clustered distinctly. Thus, in addition to spatial and temporal variation in denitrificaiton and anammox rates, both microbial abundance and community composition also vary between OMZ regions and depths.
Project description:We have used a combined transcriptome-proteome approach to describe how EPS affected the cargo of extracellular vesicles derived from myotubes from morbidly obese patients with T2D, and revealed several new factors, both miRs and proteins, that might act as exercise factors. During exercise, skeletal muscles release signaling factors that communicate with other organs and mediate beneficial effects of exercise. These factors include myokines, metabolites, and extracellular vesicles (EVs). In the present study, we have examined how electrical pulse stimulation (EPS) of myotubes, a model of exercise, affects the cargo of released EVs. Chronic low frequency EPS was applied for 24 h to human myotubes isolated and differentiated from biopsy samples from 6 morbidly obese females with T2D, and EVs, both exosomes and microvesicles (MV), were isolated from cell media 24 h thereafter.Protein content was assessed by high-resolution proteomic analysis (LC-MS/MS), non-coding RNA was quantified by Affymetrix GeneChip Multispecies miRNA-4_0 Array and Flash Tag TM Biotin RNA labelling, Thermo Fisher), and selected microRNAs (miRs) validated by real time RT-qPCR. We found that human myotubes secrete both exosomes and MV. EPS treatment of the myotubes, clearly changed the protein content of both exosomes and MV, whereas the miR content was changed only in exosomes. We suggest that skeletal muscle derived EVs can contribute to circulatory EVs and mediate beneficial effects of exercise in metabolically active organs.
Project description:In the search for LD-localized proteins that may mediate lipophagy, we purified LDs from HeLa cells treated with oleic acid (OA), which stimulates LD biogenesis and lipophagy, together with chloroquine (CQ), which inhibits lysosomal degradation. We incubated the LD lysates with purified GST-LC3B, and then pulled down GST-LC3B for mass spectrometry analysis