Project description:Nitrogen fixation is an important metabolic process carried out by microorganisms, which converts molecular nitrogen into inorganic nitrogenous compounds such as ammonia (NH3). These nitrogenous compounds are crucial for biogeochemical cycles and for the synthesis of essential biomolecules, i.e. nucleic acids, amino acids and proteins. Azotobacter vinelandii is a bacterial non-photosynthetic model organism to study aerobic nitrogen fixation (diazotrophy) and hydrogen production. Moreover, the diazotroph can produce biopolymers like alginate and polyhydroxybutyrate (PHB) that have important industrial applications. However, many metabolic processes such as partitioning of carbon and nitrogen metabolism in A. vinelandii remain unknown to date.
Genome-scale metabolic models (M-models) represent reliable tools to unravel and optimize metabolic functions at genome-scale. M-models are mathematical representations that contain information about genes, reactions, metabolites and their associations. M-models can simulate optimal reaction fluxes under a wide variety of conditions using experimentally determined constraints. Here we report on the development of a M-model of the wild type bacterium A. vinelandii DJ (iDT1278) which consists of 2,003 metabolites, 2,469 reactions, and 1,278 genes. We validated the model using high-throughput phenotypic and physiological data, testing 180 carbon sources and 95 nitrogen sources. iDT1278 was able to achieve an accuracy of 89% and 91% for growth with carbon sources and nitrogen source, respectively. This comprehensive M-model will help to comprehend metabolic processes associated with nitrogen fixation, ammonium assimilation, and production of organic nitrogen in an environmentally important microorganism.
Project description:The aim of this study was to extend our analysis to the obligate human pathogen M. tuberculosis, which has to deal with a more restricted set of environmental variables in terms of nitrogen sources, and to delineate the GlnR regulon, by peforming global analysis of GlnR-DNA interactions by Chromatin Immunoprecipitation and high-throughput sequencing (ChIP-seq) over nitrogen run-out.
Project description:Small RNAs have been studied in detail in Bacteria and Eukarya domain, but in the case of Archaea domain the knowledge is scarce and the physiological function of the majority is still uncertain. To extend the knowledge of sRNAs in Archaea domain and its possible role in the regulation of the nitrogen assimilation metabolism in haloarchaea, Haloferax mediterranei has been used as a model microorganism. Bioinformatic approach has allowed to predict 295 putative sRNAs genes in the genome of H. mediterranei, 88 of which have been verified by means of RNA-seq. The secondary structure of putative sRNAs and its possible targets have been identified. Curiously, some of them present as possible targets genes related to the nitrogen assimilation, as glutamate dehydrogenase or regulatory nitrogen protein PII. Analysis of RNA-seq data has also revealed differences in the expression pattern of 16 sRNAs according to the nitrogen source. Consequently, RNomic and the bioinformatic approaches used in this work have allowed the identification of new sRNAs in Hfx. mediterranei, some of which show different expression pattern depending on the nitrogen source. It suggests that these sRNAs could be involved in the regulation of nitrogen assimilation, being able to constitute important gene regulatory network.
Project description:Gene regulatory networks play an important role in coordinating biochemical fluxes through diverse metabolic pathways. The modulation of enzyme levels enables efficient utilization of limited resources as organisms dynamically acclimate to nutritional fluctuations in their environment. Here we have identified and characterized a novel nutrient-responsive transcription factor from the halophilic archaea, AgmR. Like TrmB, its thermophilic archaeal homolog, AgmR regulates glycolytic and gluconeogenic pathways in response to sugar availability. However, using high throughput genome-scale experiments, we find that AgmR directly governs the transcription of nearly 100 additional genes encoding enzymes in diverse metabolic pathways. Genome-scale in vivo binding site location data reveals that >60% of these are direct targets. Integration of these systems-scale datasets with metabolic reconstruction models suggests that AgmR, a sequence-specific bacterial-like regulator, interacts with the general transcription factor machinery to coordinate nitrogen and carbon metabolism with the de novo synthesis of cognate cofactors and reducing equivalents, achieving system-wide redox and energy balance.
Project description:Considering the multi-targeting potential of furan-indole hybrids as anticancer agents, this study aimed to unveil the mechanism of new 3-(2-furoyl)-indole derivatives for their cytotoxicy on hepatocellular carcinoma cells. Initially, we conducted an in vitro cytotoxicity assay for thirteen 3-(2-furoyl)-indole derivatives on HepG2 and MCF-7 cells using MTT assay, correlating the results with in silico screening of the selected drug targets. Among them, compounds 4a, 4b, and 4c demonstrated better selectivity towards HepG2 cells than MCF-7 cells. This was in good agreement with docking studies, which showed the high binding affinity of compound 4a to the Chain A of IGF-1R at Ile1160, Glu 1080, Met 1082 and Asp-1086, similar to reference ligand. Molecular dynamic study inferred the stable and high binding affinity of compound 4a to IGF-1R. The RMSD and RMSF values were found to be acceptable, and accordingly, a pharmacophore model was constructed. Hence, we conducted label-free quantitative proteome profiling of HepG2 cells treated with active compounds using an in-solution trypsin digestion procedure followed by high-resolution mass spectrometric analysis. We noticed that compound 4a targets IGF-1R pathway by dysregulation of proteins such as SHC-transforming protein 1(P29353), Mitogen-activated protein kinase kinase 3 isoform B variant (Q53EZ9), dual specificity mitogen-activated protein kinase kinase 4 (P45985), stress-activated protein kinase JNK (A0A286YF97), and mitogen-activated protein kinase (E9PQW4). In addition, the apoptotic activities of 4a, 4b, and 4c were studied against HepG2 cells using FACS. These findings are in good agreement with our in silico results, proteome profiling, and also earlier reports on indole compounds. Furthermore, we predicted the in silico ADMET property of the compounds.