The Cryptic Plastid of Euglena longa Defines a New Type of Nonphotosynthetic Plastid Organelle.

Most secondary nonphotosynthetic eukaryotes have retained residual plastids whose physiological role is often still unknown. One such example is Euglena longa, a close nonphotosynthetic relative of Euglena gracilis harboring a plastid organelle of enigmatic function. By mining transcriptome data from E. longa, we finally provide an overview of metabolic processes localized to its elusive plastid. The organelle plays no role in the biosynthesis of isoprenoid precursors and fatty acids and has a very limited repertoire of pathways concerning nitrogen-containing metabolites. In contrast, the synthesis of phospholipids and glycolipids has been preserved, curiously with the last step of sulfoquinovosyldiacylglycerol synthesis being catalyzed by the SqdX form of an enzyme so far known only from bacteria. Notably, we show that the E. longa plastid synthesizes tocopherols and a phylloquinone derivative, the first such report for nonphotosynthetic plastids studied so far. The most striking attribute of the organelle could be the presence of a linearized Calvin-Benson (CB) pathway, including RuBisCO yet lacking the gluconeogenetic part of the standard cycle, together with ferredoxin-NADP+ reductase (FNR) and the ferredoxin/thioredoxin system. We hypothesize that the ferredoxin/thioredoxin system activates the linear CB pathway in response to the redox status of the E. longa cell and speculate on the role of the pathway in keeping the redox balance of the cell. Altogether, the E. longa plastid defines a new class of relic plastids that is drastically different from the best-studied organelle of this category, the apicoplast.IMPORTANCE Colorless plastids incapable of photosynthesis evolved in many plant and algal groups, but what functions they perform is still unknown in many cases. Here, we study the elusive plastid of Euglena longa, a nonphotosynthetic cousin of the familiar green flagellate Euglena gracilis We document an unprecedented combination of metabolic functions that the E. longa plastid exhibits in comparison with previously characterized nonphotosynthetic plastids. For example, and truly surprisingly, it has retained the synthesis of tocopherols (vitamin E) and a phylloquinone (vitamin K) derivative. In addition, we offer a possible solution of the long-standing conundrum of the presence of the CO2-fixing enzyme RuBisCO in E. longa Our work provides a detailed account on a unique variant of relic plastids, the first among nonphotosynthetic plastids that evolved by secondary endosymbiosis from a green algal ancestor, and suggests that it has persisted for reasons not previously considered in relation to nonphotosynthetic plastids.

Orientation of FtsH protease homologs in Trypanosoma brucei inner mitochondrial membrane and its evolutionary implications.

Trypanosoma brucei is an important human pathogen. In this study, we have focused on the characterization of FtsH protease, ATP-dependent membrane-bound mitochondrial enzyme important for regulation of protein abundance. We have determined localization and orientation of all six putative T.brucei FtsH homologs in the inner mitochondrial membrane by in silico analyses, by immunofluorescence, and with protease assay. The evolutionary origin of these homologs has been tested by comparative phylogenetic analysis. Surprisingly, some kinetoplastid FtsH proteins display inverted orientation in the mitochondrial membrane compared to related proteins of other examined eukaryotes. Moreover, our data strongly suggest that during evolution the orientation of FtsH protease in T. brucei varied due to both loss and acquisition of the transmembrane domain.

Peculiar features of the plastids of the colourless alga Euglena longa and photosynthetic euglenophytes unveiled by transcriptome analyses.

Euglenophytes are a familiar algal group with green alga-derived secondary plastids, but the knowledge of euglenophyte plastid function and evolution is still highly incomplete. With this in mind we sequenced and analysed the transcriptome of the non-photosynthetic species Euglena longa. The transcriptomic data confirmed the absence of genes for the photosynthetic machinery, but provided candidate plastid-localised proteins bearing N-terminal bipartite topogenic signals (BTSs) of the characteristic euglenophyte type. Further comparative analyses including transcriptome assemblies available for photosynthetic euglenophytes enabled us to unveil salient aspects of the basic euglenophyte plastid infrastructure, such as plastidial targeting of several proteins as C-terminal translational fusions with other BTS-bearing proteins or replacement of the conventional eubacteria-derived plastidial ribosomal protein L24 by homologs of archaeo-eukaryotic origin. Strikingly, no homologs of any key component of the TOC/TIC system and the plastid division apparatus are discernible in euglenophytes, and the machinery for intraplastidial protein targeting has been simplified by the loss of the cpSRP/cpFtsY system and the SEC2 translocon. Lastly, euglenophytes proved to encode a plastid-targeted homolog of the termination factor Rho horizontally acquired from a Lambdaproteobacteria-related donor. Our study thus further documents a substantial remodelling of the euglenophyte plastid compared to its green algal progenitor.

An intact plastid genome is essential for the survival of colorless Euglena longa but not Euglena gracilis.

Euglena gracilis growth with antibacterial agents leads to bleaching, permanent plastid gene loss. Colorless Euglena (Astasia) longa resembles a bleached E. gracilis. To evaluate the role of bleaching in E. longa evolution, the effect of streptomycin, a plastid protein synthesis inhibitor, and ofloxacin, a plastid DNA gyrase inhibitor, on E. gracilis and E. longa growth and plastid DNA content were compared. E. gracilis growth was unaffected by streptomycin and ofloxacin. Quantitative PCR analyses revealed a time dependent loss of plastid genes in E. gracilis demonstrating that bleaching agents produce plastid gene deletions without affecting cell growth. Streptomycin and ofloxacin inhibited E. longa growth indicating that it requires plastid genes to survive. This suggests that evolutionary divergence of E. longa from E. gracilis was triggered by the loss of a cytoplasmic metabolic activity also occurring in the plastid. Plastid metabolism has become obligatory for E. longa cell growth. A process termed "intermittent bleaching", short term exposure to subsaturating concentrations of reversible bleaching agents followed by growth in the absence of a bleaching agent, is proposed as the molecular mechanism for E. longa plastid genome reduction. Various non-photosynthetic lineages could have independently arisen from their photosynthetic ancestors via a similar process.

RuBisCO in Non-Photosynthetic Alga Euglena longa: Divergent Features, Transcriptomic Analysis and Regulation of Complex Formation.

Euglena longa, a close relative of the photosynthetic model alga Euglena gracilis, possesses an enigmatic non-photosynthetic plastid. Its genome has retained a gene for the large subunit of the enzyme RuBisCO (rbcL). Here we provide new data illuminating the putative role of RuBisCO in E. longa. We demonstrated that the E. longa RBCL protein sequence is extremely divergent compared to its homologs from the photosynthetic relatives, suggesting a possible functional shift upon the loss of photosynthesis. Similarly to E. gracilis, E. longa harbors a nuclear gene encoding the small subunit of RuBisCO (RBCS) as a precursor polyprotein comprising multiple RBCS repeats, but one of them is highly divergent. Both RBCL and the RBCS proteins are synthesized in E. longa, but their abundance is very low compared to E. gracilis. No RBCS monomers could be detected in E. longa, suggesting that processing of the precursor polyprotein is inefficient in this species. The abundance of RBCS is regulated post-transcriptionally. Indeed, blocking the cytoplasmic translation by cycloheximide has no immediate effect on the RBCS stability in photosynthetically grown E. gracilis, but in E. longa, the protein is rapidly degraded. Altogether, our results revealed signatures of evolutionary degradation (becoming defunct) of RuBisCO in E. longa and suggest that its biological role in this species may be rather unorthodox, if any.

Impact of a high magnetic field on the orientation of gravitactic unicellular organisms--a critical consideration about the application of magnetic fields to mimic functional weightlessness.

The gravity-dependent behavior of Paramecium biaurelia and Euglena gracilis have previously been studied on ground and in real microgravity. To validate whether high magnetic field exposure indeed provides a ground-based facility to mimic functional weightlessness, as has been suggested earlier, both cell types were observed during exposure in a strong homogeneous magnetic field (up to 30 T) and a strong magnetic field gradient. While swimming, Paramecium cells were aligned along the magnetic field lines; orientation of Euglena was perpendicular, demonstrating that the magnetic field determines the orientation and thus prevents the organisms from the random swimming known to occur in real microgravity. Exposing Astasia longa, a flagellate that is closely related to Euglena but lacks chloroplasts and the photoreceptor, as well as the chloroplast-free mutant E. gracilis 1F, to a high magnetic field revealed no reorientation to the perpendicular direction as in the case of wild-type E. gracilis, indicating the existence of an anisotropic structure (chloroplasts) that determines the direction of passive orientation. Immobilized Euglena and Paramecium cells could not be levitated even in the highest available magnetic field gradient as sedimentation persisted with little impact of the field on the sedimentation velocities. We conclude that magnetic fields are not suited as a microgravity simulation for gravitactic unicellular organisms due to the strong effect of the magnetic field itself, which masks the effects known from experiments in real microgravity.

[Endocellular aminopeptidase from Astasia longa].

A new aminopeptidase was isolated from the biomass of the flagellate Astasia longa by precipitation with ammonium sulfate, gel filtration, and affinity chromatography on Arginine-Silochrome in 41% yield and with purification degree 490. The enzyme is irreversible inhibited by mercury chloride, EDTA, o-phenanthroline and, partially, bestatin and zinc chloride. It has an optimum pH 8.5 toward the hydrolysis of a synthetic chromogenic substrate Ala-pNA. The enzyme molecular mass is 45 kDa, isoelectric point 5.5, and temperature optimum 45 degrees C. The enzyme most effectively hydrolyzes p-nitroanilides of alanine, arginine, and leucine; it is classified as metalloaminopeptidase.

Gravitactic signal transduction elements in Astasia longa investigated during parabolic flights.

Euglena gracilis and its close relative Astasia longa show a pronounced negative gravitactic behavior. Many experiments revealed that gravitaxis is most likely mediated by an active physiological mechanism. The goal of the present study was to examine elements in the sensory transduction by means of inhibitors of gravitaxis and the intracellular calcium concentration during short microgravity periods. During the course of six parabolic flights (ESA 31th parabolic flight campaign and DLR 6th parabolic flight campaign) the effects of trifluoperazine (calmodulin inhibitor), caffeine (phosphodiesterase inhibitor) and gadolinium (blocks mechano-sensitive ion channels) was investigated. Due to the extreme parabolic flight maneuvers of the aircraft alternating phases of 1.8 x g(n) (about 20 s) and microgravity (about 22 s) were achieved (g(n): acceleration of Earth's gravity field). The duration of the microgravity periods was sufficient to detect a loss of cell orientation in the samples. In the presence of gadolinium impaired gravitaxis was found during acceleration, while caffeine-treated cells showed, compared to the controls, a very precise gravitaxis and faster reorientation in the 1.8 x g(n) period following microgravity. A transient increase of the intracellular calcium upon increased acceleration was detected also in inhibitor-treated samples. Additionally, it was found that the cells showed a higher calcium signal when they deviated from the vertical swimming direction. In the presence of trifluoperazine a slightly higher general calcium signal was detected compared to untreated controls, while gadolinium was found to decrease the intracellular calcium concentration. In the presence of caffeine no clear changes of intracellular calcium were detected compared to the control.