Horizontally Acquired Nitrate Reductase Realized Kleptoplastic Photoautotrophy of Rapaza viridis.

While photoautotrophic organisms utilize inorganic nitrogen as the nitrogen source, heterotrophic organisms utilize organic nitrogen and thus do not generally have an inorganic nitrogen assimilation pathway. Here, we focused on the nitrogen metabolism of Rapaza viridis, a unicellular eukaryote exhibiting kleptoplasty. Although belonging to the lineage of essentially heterotrophic flagellates, R. viridis exploits the photosynthetic products of the kleptoplasts and was therefore suspected to potentially utilize inorganic nitrogen. From the transcriptome data of R. viridis, we identified gene RvNaRL, which had sequence similarity to genes encoding nitrate reductases in plants. Phylogenetic analysis revealed that RvNaRL was acquired by a horizontal gene transfer event. To verify the function of the protein product RvNaRL, we established RNAi-mediated knock-down and CRISPR-Cas9-mediated knock-out experiments for the first time in R. viridis and applied them to this gene. The RvNaRL knock-down and knock-out cells exhibited significant growth only when ammonium was supplied. However, in contrast to the wild-type cells, no substantial growth was observed when nitrate was supplied. Such arrested growth in the absence of ammonium was attributed to impaired amino acid synthesis due to the deficiency of nitrogen supply from the nitrate assimilation pathway; this in turn resulted in the accumulation of excess photosynthetic products in the form of cytosolic polysaccharide grains, as observed. These results indicate that RvNaRL is certainly involved in nitrate assimilation by R. viridis. Thus, we inferred that R. viridis achieved its advanced kleptoplasty for photoautotrophy, owing to the acquisition of nitrate assimilation via horizontal gene transfer.

Predicted Structure of the BciC Enzyme Catalyzing the Removal of the C132-Methoxycarbonyl Group for Biosynthesis of Chlorosomal Chlorophylls: A Mechanism for Dual Catalytic Functions of Hydrolysis and Decarboxylation inside Its Active Site.

Green photosynthetic bacteria, one of the phototrophs, have the largest and most efficient light-harvesting antenna systems, called chlorosomes. The core part of chlorosomes consists of unique bacteriochlorophyll c/d/e molecules. In the biosynthetic pathway of these molecules, a BciC enzyme catalyzes the removal of the C132-methoxycarbonyl group of chlorophyllide a. Two sequential reactions have been proposed for the BciC enzymatic demethoxycarbonylation: the BciC enzyme would catalyze the hydrolysis of the C132-methoxycarbonyl group, and the resulting carboxylic acid would be rapidly decarboxylated to generate pyrochlorophyllide a. In this study, we computationally predicted the three-dimensional structure of the BciC protein. Its active site was proposed based on structural analysis using docking simulation. In vitro enzymatic reaction assays of mutated BciC supported the prediction. The BciC enzymatic hydrolysis would be an aspartic/glutamic acid hydrolase, which involves the amino residues E85 and D180. Furthermore, Y58 and H126 might depend on stabilization and/or recognition with the substrate. Most importantly, H137 would protonate 13-C═O or deprotonate C132-COOH in the hydrolyzed product to promote decarboxylation. In conclusion, the BciC enzyme has the dual functions of hydrolysis and decarboxylation.

Euglenozoan kleptoplasty illuminates the early evolution of photoendosymbiosis.

Kleptoplasts (kP) are distinct among photosynthetic organelles in eukaryotes (i.e., plastids) because they are routinely sequestered from prey algal cells and function only temporarily in the new host cell. Therefore, the hosts of kleptoplasts benefit from photosynthesis without constitutive photoendosymbiosis. Here, we report that the euglenozoan Rapaza viridis has only kleptoplasts derived from a specific strain of green alga, Tetraselmis sp., but no canonical plastids like those found in its sister group, the Euglenophyceae. R. viridis showed a dynamic change in the accumulation of cytosolic polysaccharides in response to light-dark cycles, and 13C isotopic labeling of ambient bicarbonate demonstrated that these polysaccharides originate in situ via photosynthesis; these data indicate that the kleptoplasts of R. viridis are functionally active. We also identified 276 sequences encoding putative plastid-targeting proteins and 35 sequences of presumed kleptoplast transporters in the transcriptome of R. viridis. These genes originated in a wide range of algae other than Tetraselmis sp., the source of the kleptoplasts, suggesting a long history of repeated horizontal gene transfer events from different algal prey cells. Many of the kleptoplast proteins, as well as the protein-targeting system, in R. viridis were shared with members of the Euglenophyceae, providing evidence that the early evolutionary stages in the green alga-derived secondary plastids of euglenophytes also involved kleptoplasty.

Genome evolution of a nonparasitic secondary heterotroph, the diatom Nitzschia putrida.

Secondary loss of photosynthesis is observed across almost all plastid-bearing branches of the eukaryotic tree of life. However, genome-based insights into the transition from a phototroph into a secondary heterotroph have so far only been revealed for parasitic species. Free-living organisms can yield unique insights into the evolutionary consequence of the loss of photosynthesis, as the parasitic lifestyle requires specific adaptations to host environments. Here, we report on the diploid genome of the free-living diatom Nitzschia putrida (35 Mbp), a nonphotosynthetic osmotroph whose photosynthetic relatives contribute ca. 40% of net oceanic primary production. Comparative analyses with photosynthetic diatoms and heterotrophic algae with parasitic lifestyle revealed that a combination of gene loss, the accumulation of genes involved in organic carbon degradation, a unique secretome, and the rapid divergence of conserved gene families involved in cell wall and extracellular metabolism appear to have facilitated the lifestyle of a free-living secondary heterotroph.

NADPH-to-NADH conversion by mitochondrial transhydrogenase is indispensable for sustaining anaerobic metabolism in Euglena gracilis.

Euglena gracilis produces ATP in the anaerobic mitochondria with concomitant wax ester formation, and NADH is essential for ATP formation and fatty acid synthesis in the mitochondria. This study demonstrated that mitochondrial cofactor conversion by nicotinamide nucleotide transhydrogenase (NNT), converting NADPH/NAD+ to NADP+ /NADH, is indispensable for sustaining anaerobic metabolism. Silencing of NNT genes significantly decreased wax ester production and cellular viability during anaerobiosis but had no such marked effects under aerobic conditions. An analogous phenotype was observed in the silencing of the gene encoding a mitochondrial NADP+ -dependent malic enzyme. These results suggest that the reducing equivalents produced in glycolysis are shuttled to the mitochondria as malate, where cytosolic NAD+ regeneration is coupled with mitochondrial NADPH generation.

Engineered chlorophyll catabolism conferring predator resistance for microalgal biomass production.

Production of valuable compounds including biofuels and pharmaceutical precursors derived from microalgae has garnered significant interest. Stable production of algal biomass is essential to make the microalgal industry commercially feasible. However, one of the largest issues is severe biological contamination by predators grazing the algal biomass, resulting in the crash of outdoor cultures. In the present study, we propose a novel engineering strategy for microalgae to cope with predators. The overexpression of plant chlorophyllase (CLH) in a microalga resulted in the enhancement of resistance to the predator. This result supported our hypothesis that CLH promotes chlorophyll breakdown in the chloroplasts of the microalgae when they are digested by the predator, generating the phototoxic catabolite chlorophyllide that damages the predator. To the best of our knowledge, this is the first study to establish predator-resistant microalgae by enhancing the CLH activity.

Bacterium Lacking a Known Gene for Retinal Biosynthesis Constructs Functional Rhodopsins.

Microbial rhodopsins, comprising a protein moiety (rhodopsin apoprotein) bound to the light-absorbing chromophore retinal, function as ion pumps, ion channels, or light sensors. However, recent genomic and metagenomic surveys showed that some rhodopsin-possessing prokaryotes lack the known genes for retinal biosynthesis. Since rhodopsin apoproteins cannot absorb light energy, rhodopsins produced by prokaryotic strains lacking genes for retinal biosynthesis are hypothesized to be non-functional in cells. In the present study, we investigated whether Aurantimicrobium minutum KNCT, which is widely distributed in terrestrial environments and lacks any previously identified retinal biosynthesis genes, possesses functional rhodopsin. We initially measured ion transport activity in cultured cells. A light-induced pH change in a cell suspension of rhodopsin-possessing bacteria was detected in the absence of exogenous retinal. Furthermore, spectroscopic analyses of the cell lysate and HPLC-MS/MS analyses revealed that this strain contained an endogenous retinal. These results confirmed that A. minutum KNCT possesses functional rhodopsin and, hence, produces retinal via an unknown biosynthetic pathway. These results suggest that rhodopsin-possessing prokaryotes lacking known retinal biosynthesis genes also have functional rhodopsins.

A non-photosynthetic green alga illuminates the reductive evolution of plastid electron transport systems.

BACKGROUND: Plastid electron transport systems are essential not only for photosynthesis but also for dissipating excess reducing power and sinking excess electrons generated by various redox reactions. Although numerous organisms with plastids have lost their photoautotrophic lifestyles, there is a spectrum of known functions of remnant plastids in non-photosynthetic algal/plant lineages; some of non-photosynthetic plastids still retain diverse metabolic pathways involving redox reactions while others, such as apicoplasts of apicomplexan parasites, possess highly reduced sets of functions. However, little is known about underlying mechanisms for redox homeostasis in functionally versatile non-photosynthetic plastids and thus about the reductive evolution of plastid electron transport systems. RESULTS: Here we demonstrated that the central component for plastid electron transport systems, plastoquinone/plastoquinol pool, is still retained in a novel strain of an obligate heterotrophic green alga lacking the photosynthesis-related thylakoid membrane complexes. Microscopic and genome analyses revealed that the Volvocales green alga, chlamydomonad sp. strain NrCl902, has non-photosynthetic plastids and a plastid DNA that carries no genes for the photosynthetic electron transport system. Transcriptome-based in silico prediction of the metabolic map followed by liquid chromatography analyses demonstrated carotenoid and plastoquinol synthesis, but no trace of chlorophyll pigments in the non-photosynthetic green alga. Transient RNA interference knockdown leads to suppression of plastoquinone/plastoquinol synthesis. The alga appears to possess genes for an electron sink system mediated by plastid terminal oxidase, plastoquinone/plastoquinol, and type II NADH dehydrogenase. Other non-photosynthetic algae/land plants also possess key genes for this system, suggesting a broad distribution of an electron sink system in non-photosynthetic plastids. CONCLUSION: The plastoquinone/plastoquinol pool and thus the involved electron transport systems reported herein might be retained for redox homeostasis and might represent an intermediate step towards a more reduced set of the electron transport system in many non-photosynthetic plastids. Our findings illuminate a broadly distributed but previously hidden step of reductive evolution of plastid electron transport systems after the loss of photosynthesis.

Inventory and Evolution of Mitochondrion-localized Family A DNA Polymerases in Euglenozoa.

The order Trypanosomatida has been well studied due to its pathogenicity and the unique biology of the mitochondrion. In Trypanosoma brucei, four DNA polymerases, namely PolIA, PolIB, PolIC, and PolID, related to bacterial DNA polymerase I (PolI), were shown to be localized in mitochondria experimentally. These mitochondrion-localized DNA polymerases are phylogenetically distinct from other family A DNA polymerases, such as bacterial PolI, DNA polymerase gamma (Polγ) in human and yeasts, "plant and protist organellar DNA polymerase (POP)" in diverse eukaryotes. However, the diversity of mitochondrion-localized DNA polymerases in Euglenozoa other than Trypanosomatida is poorly understood. In this study, we discovered putative mitochondrion-localized DNA polymerases in broad members of three major classes of Euglenozoa-Kinetoplastea, Diplonemea, and Euglenida-to explore the origin and evolution of trypanosomatid PolIA-D. We unveiled distinct inventories of mitochondrion-localized DNA polymerases in the three classes: (1) PolIA is ubiquitous across the three euglenozoan classes, (2) PolIB, C, and D are restricted in kinetoplastids, (3) new types of mitochondrion-localized DNA polymerases were identified in a prokinetoplastid and diplonemids, and (4) evolutionarily distinct types of POP were found in euglenids. We finally propose scenarios to explain the inventories of mitochondrion-localized DNA polymerases in Kinetoplastea, Diplonemea, and Euglenida.

Comparative Plastid Genomics of Cryptomonas Species Reveals Fine-Scale Genomic Responses to Loss of Photosynthesis.

Loss of photosynthesis is a recurring theme in eukaryotic evolution. In organisms that have lost the ability to photosynthesize, nonphotosynthetic plastids are retained because they play essential roles in processes other than photosynthesis. The unicellular algal genus Cryptomonas contains both photosynthetic and nonphotosynthetic members, the latter having lost the ability to photosynthesize on at least three separate occasions. To elucidate the evolutionary processes underlying the loss of photosynthesis, we sequenced the plastid genomes of two nonphotosynthetic strains, Cryptomonas sp. CCAC1634B and SAG977-2f, as well as the genome of the phototroph Cryptomonas curvata CCAP979/52. These three genome sequences were compared with the previously sequenced plastid genome of the nonphotosynthetic species Cryptomonas paramecium CCAP977/2a as well as photosynthetic members of the Cryptomonadales, including C. curvata FBCC300012D. Intraspecies comparison between the two C. curvata strains showed that although their genome structures are stable, the substitution rates of their genes are relatively high. Although most photosynthesis-related genes, such as the psa and psb gene families, were found to have disappeared from the nonphotosynthetic strains, at least ten pseudogenes are retained in SAG977-2f. Although gene order is roughly shared among the plastid genomes of photosynthetic Cryptomonadales, genome rearrangements are seen more frequently in the smaller genomes of the nonphotosynthetic strains. Intriguingly, the light-independent protochlorophyllide reductase comprising chlB, L, and N is retained in nonphotosynthetic SAG977-2f and CCAC1634B. On the other hand, whereas CCAP977/2a retains ribulose-1,5-bisphosphate carboxylase/oxygenase-related genes, including rbcL, rbcS, and cbbX, the plastid genomes of the other two nonphotosynthetic strains have lost the ribulose-1,5-bisphosphate carboxylase/oxygenase protein-coding genes.

Evaluation of δ13C and δ15N Uncertainties Associated with the Compound-Specific Isotope Analysis of Geoporphyrins.

Compound-specific isotope analyses of geoporphyrins, which are derivatives of chloropigments possessed by phototrophs, provide essential records of the biogeochemical cycle of past aquatic environments. Here, we evaluated uncertainties in carbon and nitrogen isotopic compositions (δ13C and δ15N) associated with high-performance liquid chromatography (HPLC) purification and isotopic measurements of geoporphyrins. Evaluation of total blank carbon and nitrogen for the HPLC and our sensitivity-improved elemental analyzer/isotope ratio mass spectrometer (nano-EA/IRMS) analysis confirmed that blank carbon can be corrected and that blank nitrogen is negligible compared to the mass of geoporphyrins required for the isotopic measurement. While geoporphyrins exhibited substantial in-peak carbon and nitrogen isotopic fractionations, no systematic changes in δ13C and δ15N values were observed during reversed- and normal-phase HPLC isolation of Ni- and VO-porphyrin standards, with the changes in δ13C and δ15N values being within ±0.6‰ and ±1.2‰ (2σ), respectively. These values are comparable to the instrumental precision of the nano-EA/IRMS system (±1.3‰ for 0.70 µgC and ±1.1‰ for 0.08 µgN, 2σ), confirming that no substantial artifact in the δ13C and δ15N values would be expected during the reversed- and normal-phase HPLC purification. The sensitivity and precision of our method enable us to determine δ13C and δ15N values of both major and minor geoporphyrins found in ancient sediments, which would provide detailed information on the photosynthetic primary producers and the carbon and nitrogen cycles in the past.

Taming chlorophylls by early eukaryotes underpinned algal interactions and the diversification of the eukaryotes on the oxygenated Earth.

Extant eukaryote ecology is primarily sustained by oxygenic photosynthesis, in which chlorophylls play essential roles. The exceptional photosensitivity of chlorophylls allows them to harvest solar energy for photosynthesis, but on the other hand, they also generate cytotoxic reactive oxygen species. A risk of such phototoxicity of the chlorophyll must become particularly prominent upon dynamic cellular interactions that potentially disrupt the mechanisms that are designed to quench photoexcited chlorophylls in the phototrophic cells. Extensive examination of a wide variety of phagotrophic, parasitic, and phototrophic microeukaryotes demonstrates that a catabolic process that converts chlorophylls into nonphotosensitive 132,173-cyclopheophorbide enols (CPEs) is phylogenetically ubiquitous among extant eukaryotes. The accumulation of CPEs is identified in phagotrophic algivores belonging to virtually all major eukaryotic assemblages with the exception of Archaeplastida, in which no algivorous species have been reported. In addition, accumulation of CPEs is revealed to be common among phototrophic microeukaryotes (i.e., microalgae) along with dismantling of their secondary chloroplasts. Thus, we infer that CPE-accumulating chlorophyll catabolism (CACC) primarily evolved among algivorous microeukaryotes to detoxify chlorophylls in an early stage of their evolution. Subsequently, it also underpinned photosynthetic endosymbiosis by securing close interactions with photosynthetic machinery containing abundant chlorophylls, which led to the acquisition of secondary chloroplasts. Our results strongly suggest that CACC, which allowed the consumption of oxygenic primary producers, ultimately permitted the successful radiation of the eukaryotes throughout and after the late Proterozoic global oxygenation.

Ultrafast excited state dynamics of nonfluorescent cyclopheophorbide-a enol, a catabolite of chlorophyll-a detoxified in algae-feeding aquatic microbes.

For photosynthetic organisms that nourish the earth's biosphere, chlorophylls (Chls) are the major pigments utilized for light harvesting and primary charge separation. Although Chl molecules are effective photosensitizers, they are inevitably phototoxic to living organisms due to the facile generation of highly oxidative singlet oxygen (1O2) through triplet energy transfer from their photoexcited states to oxygen molecules. Such phototoxicity of Chls is a major problem for translucent microbes that feed on photosynthetic algae. Recently, it has been reported that the metabolic conversion of Chls-a/b to 132,173-cyclopheophorbide-a/b enols (cPPB-a/bEs) is the detoxification mechanism for algivorous protists. cPPB-a/bEs are colored π-conjugated cyclic tetrapyrroles but are nonfluorescent due to efficient nonradiative decay. In this study, femtosecond time-resolved transient absorption spectroscopy was applied to cPPB-aE with the aim of understanding its quenching mechanism. As a result, we have captured the ultrafast generation of an intermediate state (∼140 fs) that leads to the rapid internal conversion to the ground state (∼450 fs).

In vivo and in vitro preparation of divinyl-132,173-cyclopheophorbide-a enol.

Divinyl-132,173-cyclopheophorbide-a enol was in vivo produced as a metabolite of divinyl-chlorophyll-a by protists and in vitro prepared by the intramolecular cyclization of methyl divinyl-pyropheophorbide-a, one of the divinyl-chlorophyll-a derivatives. The 1H NMR spectra in CDCl3 showed that the obtained product took exclusively its enol form in the solution. The intramolecular cyclization of chlorin π-system at the C132 and C173 positions affected the optical properties of such chlorophyll derivatives including the non-fluorescent emission of the enol.

Complete genome of a nonphotosynthetic cyanobacterium in a diatom reveals recent adaptations to an intracellular lifestyle.

The evolution of mitochondria and plastids from bacterial endosymbionts were key events in the origin and diversification of eukaryotic cells. Although the ancient nature of these organelles makes it difficult to understand the earliest events that led to their establishment, the study of eukaryotic cells with recently evolved obligate endosymbiotic bacteria has the potential to provide important insight into the transformation of endosymbionts into organelles. Diatoms belonging to the family Rhopalodiaceae and their endosymbionts of cyanobacterial origin (i.e., "spheroid bodies") are emerging as a useful model system in this regard. The spheroid bodies, which appear to enable rhopalodiacean diatoms to use gaseous nitrogen, became established after the divergence of extant diatom families. Here we report what is, to our knowledge, the first complete genome sequence of a spheroid body, that of the rhopalodiacean diatom Epithemia turgida. The E. turgida spheroid body (EtSB) genome was found to possess a gene set for nitrogen fixation, as anticipated, but is reduced in size and gene repertoire compared with the genomes of their closest known free-living relatives. The presence of numerous pseudogenes in the EtSB genome suggests that genome reduction is ongoing. Most strikingly, our genomic data convincingly show that the EtSB has lost photosynthetic ability and is metabolically dependent on its host cell, unprecedented characteristics among cyanobacteria, and cyanobacterial symbionts. The diatom-spheroid body endosymbiosis is thus a unique system for investigating the processes underlying the integration of a bacterial endosymbiont into eukaryotic cells.

13²,17³-Cyclopheophorbide b enol as a catabolite of chlorophyll b in phycophagy by protists.

Both 13(2),17(3)-cyclopheophorbide a and b enols were produced along with ingestion of green microalgae containing chlorophylls a and b by a centrohelid protist (phycophagy). The results suggest that chlorophyll b as well as chlorophyll a were directly degraded to colored yet non-phototoxic catabolites in the protistan phycophagic process. Such a simple process by the predators makes a contrast to the much sophisticated chlorophyll degradation process of land plants and some algae, where phototoxicity of chlorophylls was cancelled through the multiple enzymatic steps resulting in colorless and non-phototoxic catabolites.

Ubiquity and quantitative significance of detoxification catabolism of chlorophyll associated with protistan herbivory.

Chlorophylls are essential components of the photosynthetic apparati that sustain all of the life forms that ultimately depend on solar energy. However, a drawback of the extraordinary photosensitizing efficiency of certain chlorophyll species is their ability to generate harmful singlet oxygen. Recent studies have clarified the catabolic processes involved in the detoxification of chlorophylls in land plants, but little is understood about these strategies in aquatic ecosystem. Here, we report that a variety of heterotrophic protists accumulate the chlorophyll a catabolite 13(2),17(3)-cyclopheophorbide a enol (cPPB-aE) after their ingestion of algae. This chlorophyll derivative is nonfluorescent in solution, and its inability to generate singlet oxygen in vitro qualifies it as a detoxified catabolite of chlorophyll a. Using a modified analytical method, we show that cPPB-aE is ubiquitous in aquatic environments, and it is often the major chlorophyll a derivative. Our findings suggest that cPPB-aE metabolism is one of the most important, widely distributed processes in aquatic ecosystems. Therefore, the herbivorous protists that convert chlorophyll a to cPPB-aE are suggested to play more significant roles in the modern oceanic carbon flux than was previously recognized, critically linking microscopic primary producers to the macroscopic food web and carbon sequestration in the ocean.

Isolation and desalting with cation-exchange chromatography for compound-specific nitrogen isotope analysis of amino acids: application to biogeochemical samples.

We have established a procedure for removing interfering materials from extracts of geological and biological samples, in order to determine precise compound-specific nitrogen isotopic compositions of amino acids. We employed cation-exchange chromatography of protein and non-protein amino acids prior to derivatization for gas chromatographic separation. The average recovery of a standard amino acid solution was better than 94%, without nitrogen isotope fractionation during the cation-exchange chromatography. We applied the procedure to various environmental samples including 'hard' (calcareous, siliceous, rock and sediment samples) and 'soft' materials (aggregated microbial samples and biological soft tissue samples). We conclude that cation-exchange chromatography is a pre-treatment procedure which should be widely useful for the determination of compound-specific nitrogen isotopic compositions of amino acids.

An improved method for isolation and purification of sedimentary porphyrins by high-performance liquid chromatography for compound-specific isotopic analysis.

We describe an improved method for purification of sedimentary vanadyl and nickel porphyrins (i.e., naturally occurring metalloalkylporphyrins). For the purpose of compound-specific isotopic analyses, various sedimentary porphyrins were purified from the complex natural mixtures by the dual-step high-performance liquid chromatography (HPLC) method. The high-sample-capacity reversed-phase HPLCs by adding N,N-dimethylformamide to the mobile phase allow an efficient collection of fractions containing the target compounds even using analytical-scale columns. Furthermore, this method achieved improved chromatographic resolutions but significantly reduced the overall retention time down to 60% compared with the previous work. The target compounds were then isolated with the normal-phase HPLC with the baseline-resolution, which is necessary to avoid chromatographic isotopic fractionation. One of the advantages of this method is that it requires neither derivatization nor demetallation. The purity of these isolated compounds was demonstrated by various HPLC online detection methods utilizing a photodiode-array detector, a mass selective detector. The overall recoveries of Ni porphyrin, VO porphyrin, and porphyrin-free base, respectively, were estimated to be approximately 50-60%, 65%, and 85%.