Project description:Biosynthesis of mitochondrial genome-encoded proteins is carried out by the mitoribosome, a specialized apparatus that has evolved and diverged dramatically since its bacterial origin. Recent studies across various eukaryotes have demonstrated widespread structural and compositional diversity of mitoribosomes. We used affinity pulldown of four mitoribosomal proteins to carry out a detailed analysis of mitoribosomes in Diplonema papillatum, the type species of diplonemids, a widespread group of single-celled marine flagellates. Using as baits mitoribosomal proteins integrating at distinct sites and phases during subunit maturation also allowed us to sample populations of mitoribosome assembly intermediates.
Project description:Biosynthesis of mitochondrial genome-encoded proteins is carried out by the mitoribosome, a specialized apparatus that has evolved and diverged dramatically since its bacterial origin. Recent studies across various eukaryotes have demonstrated widespread structural and compositional diversity of mitoribosomes. We used affinity pulldown of four mitoribosomal proteins to carry out a detailed analysis of mitoribosomes in Diplonema papillatum, the type species of diplonemids, a widespread group of single-celled marine flagellates. Using as baits mitoribosomal proteins integrating at distinct sites and phases during subunit maturation also allowed us to sample populations of mitoribosome assembly intermediates.
Project description:Biosynthesis of mitochondrial genome-encoded proteins is carried out by the mitoribosome, a specialized apparatus that has evolved and diverged dramatically since its bacterial origin. Recent studies across various eukaryotes have demonstrated widespread structural and compositional diversity of mitoribosomes. We used sucrose gradient centrifugation and Blue-Native PAGE to separate mitoribosomes of Diplonema papillatum, the type species of diplonemids, a widespread group of single-celled marine flagellates.
Project description:Diplonema papillatum represents a group of highly diverse and abundant marine protists with still unknown lifestyle and ecological functions. Based on alterations of the transcriptomic, proteomic and metabolomic profiles obtained from cells grown under different conditions we designed a metabolic map of its cellular bioenergetic pathways. Comparative analysis in the nutrient-rich and -poor media and in the absence and presence of oxygen revealed a capacity for major metabolic reprograming. D. papillatum is equipped with fundamental metabolic routes such as glycolysis, gluconeogenesis, TCA cycle, pentose phosphate pathway, respiratory complexes, β-oxidation and synthesis of fatty acid. While gluconeogenesis uniquely dominates over glycolysis, TCA cycle represents a combination of standard and unusual enzymes. The presence of typical anaerobic enzymes such as pyruvate:NADP+ oxidoreductase, fumarate reductase, opine dehydrogenase, enoyl-coenzyme A reductase, and lactate dehydrogenase reflects the ability to survive in low-oxygen environments. The metabolism quickly reacts to restricted carbon source, revealing unusual flexibility of diplonemids, also reflected in cell morphology and motility, which is in good correlation with their extreme ecological valence.
Project description:In this study the transcriptomes of Acinetobacter baumannii strains ATCC 17978 and 17978hm were compared. Strain 17978hm is a hns knockout derivative of strain ATCC 17978. Strain 17978hm displays a hyper-motile phenotype on semi-solid Mueller-Hinton (MH) media (0.25% agar). ATCC 17978 and 17978hm from an 37C overnight culture were transferred to the centre of the semi-solid MH plate and incubated at 37C for 8 hours. Only 17978hm cells displayed a motile phenotype and covered the complete surface of the plate. These motile 17978hm cells and the non-motile wild-type ATCC 17978 cells were harvested and RNA was isolated. The comparative transcriptome analysis was performed using the FairPlay labeling kit and a custom made Agilent MicroArray with probes designed to coding regions of the ATCC 17978 genome. The data was analyzed using Agilent GeneSpring GX9 and the significance analysis of microarray MS Excel add-on.
Project description:BackgroundThe phylum Euglenozoa is a group of flagellated protists comprising the diplonemids, euglenids, symbiontids, and kinetoplastids. The diplonemids are highly abundant and speciose, and recent tools have rendered the best studied representative, Diplonema papillatum, genetically tractable. However, despite the high diversity of diplonemids, their lifestyles, ecological functions, and even primary energy source are mostly unknown.ResultsWe designed a metabolic map of D. papillatum cellular bioenergetic pathways based on the alterations of transcriptomic, proteomic, and metabolomic profiles obtained from cells grown under different conditions. Comparative analysis in the nutrient-rich and nutrient-poor media, as well as the absence and presence of oxygen, revealed its capacity for extensive metabolic reprogramming that occurs predominantly on the proteomic rather than the transcriptomic level. D. papillatum is equipped with fundamental metabolic routes such as glycolysis, gluconeogenesis, TCA cycle, pentose phosphate pathway, respiratory complexes, β-oxidation, and synthesis of fatty acids. Gluconeogenesis is uniquely dominant over glycolysis under all surveyed conditions, while the TCA cycle represents an eclectic combination of standard and unusual enzymes.ConclusionsThe identification of conventional anaerobic enzymes reflects the ability of this protist to survive in low-oxygen environments. Furthermore, its metabolism quickly reacts to restricted carbon availability, suggesting a high metabolic flexibility of diplonemids, which is further reflected in cell morphology and motility, correlating well with their extreme ecological valence.