Highly flexible metabolism of the marine euglenozoan protist Diplonema papillatum.

BACKGROUND: The 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. RESULTS: We 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, ß-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. CONCLUSIONS: The 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.

RNA-level unscrambling of fragmented genes in Diplonema mitochondria.

We previously reported a unique genome with systematically fragmented genes and gene pieces dispersed across numerous circular chromosomes, occurring in mitochondria of diplonemids. Genes are split into up to 12 short fragments (modules), which are separately transcribed and joined in a way that differs from known trans-splicing. Further, cox1 mRNA includes six non-encoded uridines indicating RNA editing. In the absence of recognizable cis-elements, we postulated that trans-splicing and RNA editing are directed by trans-acting molecules. Here, we provide insight into the post-transcriptional processes by investigating transcription, RNA processing, trans-splicing and RNA editing in cox1 and at a newly discovered site in cob. We show that module precursor transcripts are up to several thousand nt long and processed accurately at their 5' and 3' termini to yield the short coding-only regions. Processing at 5' and 3' ends occurs independently, and a processed terminus engages in trans-splicing even if the module's other terminus is yet unprocessed. Moreover, only cognate module transcripts join, though without directionality. In contrast, module transcripts requiring RNA editing only trans-splice when editing is completed. Finally, experimental and computational analyses suggest the existence of RNA trans-factors with the potential for guiding both trans-splicing and RNA editing.

Subunit composition of mitochondrial dehydrogenase complexes in diplonemid flagellates.

In eukaryotes, pyruvate, a key metabolite produced by glycolysis, is converted by a tripartite mitochondrial pyruvate dehydrogenase (PDH) complex to acetyl-coenzyme A, which is fed into the tricarboxylic acid cycle. Two additional enzyme complexes with analogous composition catalyze similar oxidative decarboxylation reactions albeit using different substrates, the branched-chain ketoacid dehydrogenase (BCKDH) complex and the 2-oxoglutarate dehydrogenase (OGDH) complex. Comparative transcriptome analyses of diplonemids, one of the most abundant and diverse groups of oceanic protists, indicate that the conventional E1, E2, and E3 subunits of the PDH complex are lacking. E1 was apparently replaced in the euglenozoan ancestor of diplonemids by an AceE protein of archaeal type, a substitution that we also document in dinoflagellates. Here, we demonstrate that the mitochondrion of the model diplonemid Paradiplonema papillatum displays pyruvate and 2-oxoglutarate dehydrogenase activities. Protein mass spectrometry of mitochondria reveal that the AceE protein is as abundant as the E1 subunit of BCKDH. This corroborates the view that the AceE subunit is a functional component of the PDH complex. We hypothesize that by acquiring AceE, the diplonemid ancestor not only lost the eukaryotic-type E1, but also the E2 and E3 subunits of the PDH complex, which are present in other euglenozoans. We posit that the PDH activity in diplonemids seems to be carried out by a complex, in which the AceE protein partners with the E2 and E3 subunits from BCKDH and/or OGDH.

Functional differentiation of Sec13 paralogues in the euglenozoan protists.

The ß-propeller protein Sec13 plays roles in at least three distinct processes by virtue of being a component of the COPII endoplasmic reticulum export vesicle coat, the nuclear pore complex (NPC) and the Seh1-associated (SEA)/GATOR nutrient-sensing complex. This suggests that regulatory mechanisms coordinating these cellular activities may operate via Sec13. The NPC, COPII and SEA/GATOR are all ancient features of eukaryotic cells, and in the vast majority of eukaryotes, a single Sec13 gene is present. Here we report that the Euglenozoa, a lineage encompassing the diplonemid, kinetoplastid and euglenid protists, possess two Sec13 paralogues. Furthermore, based on protein interactions and localization studies we show that in diplonemids Sec13 functions are divided between the Sec13a and Sec13b paralogues. Specifically, Sec13a interacts with COPII and the NPC, while Sec13b interacts with Sec16 and components of the SEA/GATOR complex. We infer that euglenozoan Sec13a is responsible for NPC functions and canonical anterograde transport activities while Sec13b acts within nutrient and autophagy-related pathways, indicating a fundamentally distinct organization of coatomer complexes in euglenozoan flagellates.

Phylogeny and Morphology of New Diplonemids from Japan.

Diplonemids were recently found to be the most species-rich group of marine planktonic protists. Based on phylogenetic analysis of 18S rRNA gene sequences and morphological observations, we report the description of new members of the genus Rhynchopus - R. humris sp. n. and R. serpens sp. n., and the establishment of two new genera - Lacrimia gen. n. and Sulcionema gen. n., represented by L. lanifica sp. n. and S. specki sp. n., respectively. In addition, we describe the organism formerly designated as Diplonema sp. 2 (ATCC 50224) as Flectonema neradi gen. n., sp. n. The newly described diplonemids share a common set of traits. Cells are sac-like but variable in shape and size, highly metabolic, and surrounded by a naked cell membrane, which is supported by a tightly packed corset of microtubules. They carry a single highly reticulated peripheral mitochondrion containing a large amount of mitochondrial DNA, with lamellar cristae. The cytopharyngeal complex and flagellar pocket are contiguous and have separate openings. Two parallel flagella are inserted sub-apically into a pronounced flagellar pocket. Rhynchopus species have their flagella concealed in trophic stages and fully developed in swimming stages, while they permanently protrude in all other known diplonemid species.