The enigmatic RNase MRP of kinetoplastids.

The ribonucleoprotein RNase MRP is responsible for the processing of ribosomal RNA precursors. It is found in virtually all eukaryotes that have been examined. In the Euglenozoa, including the genera Euglena, Diplonema and kinetoplastids, MRP RNA and protein subunits have so far escaped detection using bioinformatic methods. However, we now demonstrate that the RNA component is widespread among the Euglenozoa and that these RNAs have secondary structures that conform to the structure of all other phylogenetic groups. In Euglena, we identified the same set of P/MRP protein subunits as in many other protists. However, we failed to identify any of these proteins in the kinetoplastids. This finding poses interesting questions regarding the structure and function of RNase MRP in these species.

Oxygenic Phototrophs Need ζ-Carotene Isomerase (Z-ISO) for Carotene Synthesis: Functional Analysis in Arthrospira and Euglena.

For carotenogenesis, two biosynthetic pathways from phytoene to lycopene are known. Most bacteria and fungi require only phytoene desaturase (PDS, CrtI), whereas land plants require four enzymes: PDS (CrtP), ζ-carotene desaturase (ZDS, CrtQ), ζ-carotene isomerase (Z-ISO) and cis-carotene isomerase (CrtISO, CrtH). The gene encoding Z-ISO has been functionally identified in only two species, Arabidopsis thaliana and Zea mays, and has been little studied in other organisms. In this study, we found that the deduced amino acid sequences of Arthrospira Z-ISO and Euglena Z-ISO have 58% and 62% identity, respectively, with functional Z-ISO from Arabidopsis. We studied the function of Z-ISO genes from the cyanobacterium Arthrospira platensis and eukaryotic microalga Euglena gracilis. The Z-ISO genes of Arthrospira and Euglena were transformed into Escherichia coli strains that produced mainly 9,15,9'-tri-cis-ζ-carotene in darkness. In the resulting E. coli transformants cultured under darkness, 9,9'-di-cis-ζ-carotene was accumulated predominantly as Z-ISO in Arabidopsis. This indicates that the Z-ISO genes were involved in the isomerization of 9,15,9'-tri-cis-ζ-carotene to 9,9'-di-cis-ζ-carotene in darkness. This is the first functional analysis of Z-ISO as a ζ-carotene isomerase in cyanobacteria and eukaryotic microalgae. Green sulfur bacteria and Chloracidobacterium also use CrtP, CrtQ and CrtH for lycopene synthesis as cyanobacteria, but their genomes did not comprise Z-ISO genes. Consequently, Z-ISO is needed in oxygenic phototrophs, whereas it is not found in anoxygenic species.

Molecular diversity of the ribulose-1,5-diphosphate carboxylase from photosynthetic microorganisms.

The ribulose-1,5-diphosphate carboxylases from green and blue-green algae and the purple sulfur photosynthetic bacterium Chromatium are proteins with high molecular weights and with sedimentation coefficients of 18 to 21 Svedberg units. The carboxylases of the Athiorhodaceae are smaller, that of Rhodospirillum rubrum being a 6.2S molecule, and those of the two species of Rhodopseudomonas are 12S and 14.5S.

Glyceraldehyde-3-phosphate dehydrogenase variants in phyletically diverse organisms.

Electrophoretically distinct forms of glyceraldehyde-3-phosphate dehydrogenase (TDH) have been detected in turtle, perch, trout, spinach, and yeast. Multiple forms were not detected in rat, rabbit, chicken, frog, honey bee, Euglena, or Escherichia coli. The combination of two different subunits into tetramers is a probable explanation for the five-membered sets usually detected in extracts exhibiting TDH multiplicity.

Recent horizontal intron transfer to a chloroplast genome.

Evidence is presented for the recent, horizontal transfer of a self-splicing, homing group II intron from a cyanobacteria to the chloroplast genome of Euglena myxocylindracea. The psbA gene of E.myxocylindracea was found to contain a single 2566 nt group II intron with a gene in domain 4 for a 575 amino acid maturase. The predicted secondary structure and tertiary interactions of the group II intron, as well as the derived maturase primary sequence, most closely resemble the homing intron of the cyanobacterium Calothrix and the rnl introns of Porphyra purpurea mitochondria, while being only distantly related to all other Euglena plastid introns and maturases. All main functional domains of the intron-encoded proteins of known homing introns are conserved, including reverse transcriptase domains 1-7, the zinc finger domain and domain X. The close relationship with cyanobacterial introns was confirmed by phylogenetic analysis. Both the full-length psbA intron and a Delta-maturase variant self-splice in vitro in two independent assays. The psbA intron is the first example of a self-splicing chloroplast group II intron from any organism. These results support the conclusion that the psbA intron is the result of a recent horizontal transfer into the E.myxocylindracea chloroplast genome from a cyanobacterial donor and should prompt a reconsideration of horizontal transfer mechanisms to account for the origin of other chloroplast genetic elements.

Identification and comparative analysis of the chloroplast alpha-subunit gene of DNA-dependent RNA polymerase from seven Euglena species.

When the sequence of the Euglena gracilis chloroplast genome was reported in 1993 the alpha-subunit gene (rpoA) of RNA polymerase appeared to be missing, based on a comparison of all putative reading frames to the then known rpoA loci. Since there has been a large increase in known rpoA sequences, the question of a Euglena chloroplast rpoA gene was re-examined. A previously described unknown reading frame of 161 codons was found to be part of an rpoA gene split by a single group III intron. This rpoA gene, which is highly variable from species to species, was then isolated and characterized in five other euglenoid species, Euglena anabaena, Euglena granulata, Euglena myxocylindracea, Euglena stellata and Euglena viridis, and in the Astasia longa plastid genome. All seven Euglena rpoA genes have either one or three group III introns. The rpoA gene products in Euglena spp. appear to be the most variable in this gene family when compared to the rpoA gene in other species of bacteria, algae and plants. Additionally, Euglena rpoA proteins lack a C-terminal domain required for interaction with some regulatory proteins, a feature shared only with some chlorophyte green algae. The E.gracilis rpoA gene is the distal cistron of a multigene cluster that includes genes for carbohydrate biosynthesis, photosynthetic electron transport, an antenna complex and ribosomal proteins. This study provides new insights into the transcription system of euglenoid plastids, the organization of the plastid genome, group III intron evolution and euglenoid phylogeny.

Gene Discovery for Synthetic Biology: Exploring the Novel Natural Product Biosynthetic Capacity of Eukaryotic Microalgae.

Eukaryotic microalgae are an incredibly diverse group of organisms whose sole unifying feature is their ability to photosynthesize. They are known for producing a range of potent toxins, which can build up during harmful algal blooms causing damage to ecosystems and fisheries. Genome sequencing is lagging behind in these organisms because of their genetic complexity, but transcriptome sequencing is beginning to make up for this deficit. As more sequence data becomes available, it is apparent that eukaryotic microalgae possess a range of complex natural product biosynthesis capabilities. Some of the genes concerned are responsible for the biosynthesis of known toxins, but there are many more for which we do not know the products. Bioinformatic and analytical techniques have been developed for natural product discovery in bacteria and these approaches can be used to extract information about the products synthesized by algae. Recent analyses suggest that eukaryotic microalgae produce many complex natural products that remain to be discovered.

Chemical cross-linking and its effect on fatty acid synthetase activity in intact chloroplasts from Euglena gracilis.

Intact chloroplasts were isolated from Euglena gracilis variety bacillaris, aliquots were exposed to several different chemical cross-linking reagents. The reagents penetrated the triple membrane of Euglena chloroplasts. This was shown by gradient acrylamide gel electrophoresis under denaturing conditions. The activity of the nonaggregated fatty acid synthetase of Euglena was located within the chloroplast stroma, and the effects of dimethylsuberimidate cross-linking on the activity of the enzyme system were examined. The acyl-carrier protein concentration in the chloroplast was measured at about 0.24 mM.

Acyl carrier protein interacts with melittin.

Acyl carrier protein (ACP) from Escherichia coli has been shown to form complexes with melittin, a cationic peptide from bee venom. ACP is a small (Mr 8847), acidic, Ca2(+)-binding protein, which possesses some characteristics resembling those of regulatory Ca2(+)-binding proteins including interaction with melittin. Complexing between melittin and ACP which occurred both in the presence and absence of Ca2+ was evident by chemical cross-linking the two peptides, fluorescence changes (including anisotropy measurements), and inhibition by melittin of the activity of a nonaggregated fatty acid synthetase from Euglena. Also, anti-Apis mellifera antibodies which contained antibodies against melittin specifically inhibited the same enzyme system activity relative to non-immune IgG.

Synthesis of diadenosine 5',5'''-P1,P4-tetraphosphate by organellar and cytoplasmic phenylalanyl-tRNA synthetases of Euglena gracilis.

Purified phenylalanyl-tRNA synthetases present in chloroplasts, mitochondria and cytoplasm of green and bleached Euglena gracilis strains, respectively, are able to synthesize diadenosine 5',5'''-P1,P4-tetraphosphate (Ap4A). Ap4A synthesis is strictly dependent on zinc ions. This is the first evidence that chloroplasts should be able to synthesize Ap4A. Synthesis of Ap4A by phenylalanyl-tRNA synthetases of the three compartments of a plant cell or by other enzymes such as Ap4A phosphorylase is discussed.

Role of cyclic GMP in the mediation of circadian rhythmicity of the adenylate cyclase-cyclic AMP-phosphodiesterase system in Euglena.

Cyclic AMP (cAMP) and cyclic GMP (cGMP) are two second messengers that have been proposed to act as a dualistic system in biological regulation. To determine if cGMP plays a role in the mediation of circadian rhythmicity of the adenylate cyclase (AC)-cAMP-phosphodiesterase (PDE) system in the achlorophyllous ZC mutant of the unicellular flagellate Euglena, the levels of cAMP and cGMP were monitored in synchronized cell populations, and the effects of the cGMP analog 8-bromo-cGMP (8-Br-cGMP) and the cGMP inhibitor 6-anilinoquinoline-5,8-quinone (LY 83583) on the activity of AC and PDE, as well as on the level of cAMP, were measured in vivo. A bimodal, 24-hr rhythm of cGMP content was found in both dividing and nondividing cultures in either a 12-hr:12-hr light-dark cycle or constant darkness. The peaks and troughs of the cGMP rhythm occurred 2 hr in advance of those of the cAMP rhythm that has been reported previously. The addition of 8-Br-cGMP at different circadian times increased the cAMP level in vivo by two to five times, whereas LY 83583 reduced the amplitude of the cAMP rhythm so that it disappeared. The effects of 8-Br-cGMP on the activity of AC and PDE were circadian phase-dependent and consistent with the changes in cAMP content. These findings suggest that cGMP may serve as an upstream effector that mediates the cAMP oscillation by regulation of the AC-cAMP-PDE system.

Ribulose bisphosphate-induced, slow conformational changes of spinach ribulose bisphosphate carboxylase cause the two types of inflections in the course of its carboxylase reaction.

The cause of the inflection in the course of the carboxylase reaction and the changes in the functioning form of spinach ribulose bisphosphate carboxylase (RuBisCO) during the reaction were elucidated by relating the activity to the protein conformation of RuBisCO using a fluorescence probe, 2-p-toluidinylnaphthalene sulfonate. The activity of RuBisCO in the linear phase was 50 to 60% of that in the initial burst at 0.5 to 1.0 mM ribulose bisphosphate (RuBP) and 65 to 80% at 2 to 5 mM RuBP. The amount and the progress of the decrease in the activity during the reaction had a close relationship to a change in the protein conformation of RuBisCO. The enzyme, the substrate binding sites of which were masked beforehand with carboxyarabinitol bisphosphate, still showed a change of its protein conformation upon addition of RuBP, suggesting that RuBisCO has two (substrate and regulatory) RuBP-binding sites per RuBisCO promoter. RuBisCO required over 2 mM RuBP for binding on the regulatory sites. Both sites also bound 6-phosphogluconate. When both sites were masked with 6-phosphogluconate beforehand, the course of the subsequent carboxylase reaction was linear with time. From these results, I propose that the inflection in the course of the reaction of spinach RuBisCO is a hysteretic response of the enzyme to RuBP bound to both substrate and regulatory sites.

Identification of a functional Gs protein in Euglena gracilis.

We found a Gs protein coupled to adenylyl cyclase in a free-living protist, Euglena gracilis. This Gs protein of approximately 42 kDa is substrate for cholera toxin and is recognized by an antibody against the C-terminal decapeptide of Gs. Furthermore, this protein is coupled to adenylyl cyclase, as shown by: (a) the activation of the enzyme by GTP-analogues and (b) the effect of cholera toxin on cAMP accumulation in intact cells and the continuous activation of adenylyl cyclase activity in membranes. These data indicate that the Gs-adenylyl cyclase-coupled system is already apparent in the protist kingdom.