RESUMEN

The genomes of peridinin-containing dinoflagellate chloroplasts have a very unusual organisation. These genomes are highly fragmented and greatly reduced, with most of the usual complement of chloroplast genes relocated to the nucleus. Dinoflagellate chloroplasts highlight evolutionary changes that are found to varying extents in a number of other organelle genomes. These include the chloroplast genome of the green alga Boodlea and other Cladophorales, and the mitochondrial genomes of blood-sucking and chewing lice, the parasitic plant Rhopalocnemis phalloides, the red alga Rhodosorus marinus and other members of the Stylonematophyceae, diplonemid flagellates, and some Cnidaria. Consideration of the coding content of the remnant chloroplast genomes indicates that organelles may preferentially retain genes for proteins important in initiating assembly of complexes, and the same is largely true for mitochondria. We propose a new principle, of CO-location for COntrol of Assembly (COCOA), indicating the importance of retaining these genes in the organelle. This adds to, but does not invalidate, the existing hypotheses of the multisubunit completion principle, CO-location for Redox Regulation (CORR) and Control by Epistasy of Synthesis (CES).

Asunto(s)

Cloroplastos , Dinoflagelados , Dinoflagelados/genética , Cloroplastos/genética , Cloroplastos/metabolismo , Genoma del Cloroplasto

RESUMEN

Many chloroplast-bearing plants and algae lost their photosynthetic activity during evolution but retained their chloroplasts for other functions. A group of dinoflagellate algae apparently lost one half of their photosynthetic machinery but retained the other, providing a novel mechanism for light perception.

Asunto(s)

Dinoflagelados , Plastidios , Plastidios/genética , Plastidios/metabolismo , Cloroplastos/metabolismo , Plantas , Fotosíntesis , Evolución Molecular

RESUMEN

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.

Asunto(s)

ADN/administración & dosificación , Eucariontes/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Biología Marina , Modelos Biológicos , Transformación Genética , Biodiversidad , Ecosistema , Ambiente , Eucariontes/clasificación , Especificidad de la Especie

RESUMEN

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

RESUMEN

The plastid genomes of peridinin-containing dinoflagellates are highly unusual, possessing very few genes, which are located on small chromosomal elements termed "minicircles". These minicircles may contain genes, or no recognisable coding information. Transcripts produced from minicircles may undergo unusual processing events, such as the addition of a 3' poly(U) tail. To date, little is known about the genetic or transcriptional diversity of non-coding sequences in peridinin dinoflagellate plastids. These sequences include empty minicircles, and regions of non-coding DNA in coding minicircles. Here, we present an integrated plastid genome and transcriptome for the model peridinin dinoflagellate Amphidinium carterae, identifying a previously undescribed minicircle. We also profile transcripts covering non-coding regions of the psbA and petB/atpA minicircles. We present evidence that antisense transcripts are produced within the A. carterae plastid, but show that these transcripts undergo different end cleavage events from sense transcripts, and do not receive 3' poly(U) tails. The difference in processing events between sense and antisense transcripts may enable the removal of non-coding transcripts from peridinin dinoflagellate plastid transcript pools.

Asunto(s)

Dinoflagelados/clasificación , Dinoflagelados/genética , Genoma de Protozoos/genética , Plastidios/genética , Transcriptoma/genética , Perfilación de la Expresión Génica , Genómica

RESUMEN

Coral reefs are some of the most important and ecologically diverse marine environments. At the base of the reef ecosystem are dinoflagellate algae, which live symbiotically within coral cells. Efforts to understand the relationship between alga and coral have been greatly hampered by the lack of an appropriate dinoflagellate genetic transformation technology. By making use of the plasmid-like fragmented chloroplast genome, we have introduced novel genetic material into the dinoflagellate chloroplast genome. We have shown that the introduced genes are expressed and confer the expected phenotypes. Genetically modified cultures have been grown for 1 year with subculturing, maintaining the introduced genes and phenotypes. This indicates that cells continue to divide after transformation and that the transformation is stable. This is the first report of stable chloroplast transformation in dinoflagellate algae.

Asunto(s)

Cloroplastos/genética , Dinoflagelados/genética , Genética Microbiana/métodos , Transformación Genética , Dinoflagelados/crecimiento & desarrollo , Expresión Génica , Inestabilidad Genómica , Fenotipo

RESUMEN

Modern transformation and genome editing techniques have shown great success across a broad variety of organisms. However, no study of successfully applied genome editing has been reported in a dinoflagellate despite the first genetic transformation of Symbiodinium being published about 20 years ago. Using an array of different available transformation techniques, we attempted to transform Symbiodinium microadriaticum (CCMP2467), a dinoflagellate symbiont of reef-building corals, with the view to performing subsequent CRISPR-Cas9 mediated genome editing. Plasmid vectors designed for nuclear transformation containing the chloramphenicol resistance gene under the control of the CaMV p35S promoter as well as several putative endogenous promoters were used to test a variety of transformation techniques including biolistics, electroporation and agitation with silicon carbide whiskers. Chloroplast-targeted transformation was attempted using an engineered Symbiodinium chloroplast minicircle encoding a modified PsbA protein expected to confer atrazine resistance. We report that we have been unable to confer chloramphenicol or atrazine resistance on Symbiodinium microadriaticum strain CCMP2467.

Asunto(s)

Núcleo Celular/genética , Cloroplastos/genética , Dinoflagelados/genética , Transformación Genética , Animales , Antozoos/microbiología , Atrazina/farmacología , Núcleo Celular/metabolismo , Cloranfenicol/farmacología , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Dinoflagelados/metabolismo , Resistencia a Medicamentos/genética

RESUMEN

The persistence of coral reef ecosystems relies on the symbiotic relationship between scleractinian corals and intracellular, photosynthetic dinoflagellates in the genus Symbiodinium. Genetic evidence indicates that these symbionts are biologically diverse and exhibit discrete patterns of environmental and host distribution. This makes the assessment of Symbiodinium diversity critical to understanding the symbiosis ecology of corals. Here, we applied pyrosequencing to the elucidation of Symbiodinium diversity via analysis of the internal transcribed spacer 2 (ITS2) region, a multicopy genetic marker commonly used to analyse Symbiodinium diversity. Replicated data generated from isoclonal Symbiodinium cultures showed that all genomes contained numerous, yet mostly rare, ITS2 sequence variants. Pyrosequencing data were consistent with more traditional denaturing gradient gel electrophoresis (DGGE) approaches to the screening of ITS2 PCR amplifications, where the most common sequences appeared as the most intense bands. Further, we developed an operational taxonomic unit (OTU)-based pipeline for Symbiodinium ITS2 diversity typing to provisionally resolve ecologically discrete entities from intragenomic variation. A genetic distance cut-off of 0.03 collapsed intragenomic ITS2 variants of isoclonal cultures into single OTUs. When applied to the analysis of field-collected coral samples, our analyses confirm that much of the commonly observed Symbiodinium ITS2 diversity can be attributed to intragenomic variation. We conclude that by analysing Symbiodinium populations in an OTU-based framework, we can improve objectivity, comparability and simplicity when assessing ITS2 diversity in field-based studies.

Asunto(s)

Antozoos , Dinoflagelados/genética , Variación Genética , Simbiosis , Animales , ADN Espaciador Ribosómico/genética , Dinoflagelados/clasificación , Ecosistema , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADN

RESUMEN

Dinoflagellate algae of the genus Symbiodinium form important symbioses within corals and other benthic marine animals. Dinoflagellates possess an extremely reduced plastid genome relative to those examined in plants and other algae. In dinoflagellates the plastid genes are located on small plasmids, commonly referred to as 'minicircles'. However, the chloroplast genomes of dinoflagellates have only been extensively characterised from a handful of species. There is also evidence of considerable variation in the chloroplast genome organisation across those species that have been examined. We therefore characterised the chloroplast genome from an environmental coral isolate, in this case containing a symbiont belonging to the Symbiodinium sp. clade C3. The gene content of the genome is well conserved with respect to previously characterised genomes. However, unlike previously characterised dinoflagellate chloroplast genomes we did not identify any 'empty' minicircles. The sequences of this chloroplast genome show a high rate of evolution relative to other algal species. Particularly notable was a surprisingly high level of sequence divergence within the core polypeptides of photosystem I, the reasons for which are currently unknown. This chloroplast genome also possesses distinctive codon usage and GC content. These features suggest that chloroplast genomes in Symbiodinium are highly plastic.

Asunto(s)

Alveolados/genética , Variación Genética , Genoma del Cloroplasto , Alveolados/clasificación , Composición de Base , Análisis por Conglomerados , Codón , ADN de Cloroplastos/química , ADN de Cloroplastos/genética , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN

RESUMEN

Although transcription and transcript processing in the chloroplasts of plants have been extensively characterised, the RNA metabolism of other chloroplast lineages across the eukaryotes remains poorly understood. In this paper, we use RT-PCR to study transcription and transcript processing in the chloroplasts of Amphidinium carterae, a model peridinin-containing dinoflagellate. These organisms have a highly unusual chloroplast genome, with genes located on multiple small 'minicircle' elements, and a number of idiosyncratic features of RNA metabolism including transcription via a rolling circle mechanism, and 3' terminal polyuridylylation of transcripts. We demonstrate that transcription occurs in A. carterae via a rolling circle mechanism, as previously shown in the dinoflagellate Heterocapsa, and present evidence for the production of both polycistronic and monocistronic transcripts from A. carterae minicircles, including several regions containing ORFs previously not known to be expressed. We demonstrate the presence of both polyuridylylated and non-polyuridylylated transcripts in A. carterae, and show that polycistronic transcripts can be terminally polyuridylylated. We present a model for RNA metabolism in dinoflagellate chloroplasts where long polycistronic precursors are processed to form mature transcripts. Terminal polyuridylylation may mark transcripts with the correct 3' end.

Asunto(s)

Dinoflagelados/genética , Dinoflagelados/metabolismo , Genes del Cloroplasto , Genes Protozoarios , Cloroplastos/genética , ADN Circular/genética , ADN Circular/metabolismo , ADN Protozoario/genética , ADN Protozoario/metabolismo , Modelos Biológicos , Poli U/metabolismo , Procesamiento Postranscripcional del ARN , ARN Protozoario/genética , ARN Protozoario/metabolismo

RESUMEN

Protist mitochondrial genomes show a very wide range of gene content, ranging from three genes for respiratory chain components in Apicomplexa and dinoflagellates to nearly 100 genes in Reclinomonas americana. In many organisms the rRNA genes are fragmented, although still functional. Some protist mitochondria encode a full set of tRNAs, while others rely on imported molecules. There is similarly a wide variation in mitochondrial genome organization, even among closely related groups. Mitochondrial gene expression and control are generally poorly characterized. Transcription probably relies on a 'viral-type' RNA polymerase, although a 'bacterial-type' enzyme may be involved in some cases. Transcripts are heavily edited in many lineages. The chloroplast genome generally shows less variation in gene content and organization, although greatly reduced genomes are found in dinoflagellate algae and non-photosynthetic organisms. Genes in the former are located on small plasmids in contrast to the larger molecules found elsewhere. Control of gene expression in chloroplasts involves transcriptional and post-transcriptional regulation. Redox poise and the ATP/ADP ratio are likely to be important determinants. Some protists have an additional extranuclear genome, the nucleomorph, which is a remnant nucleus. Nucleomorphs of two separate lineages have a number of features in common.

Asunto(s)

Evolución Biológica , Genoma , Orgánulos/genética , Expresión Génica , Genoma del Cloroplasto , Genoma Mitocondrial , ARN/genética , Simbiosis/genética

RESUMEN

Dinoflagellate algae are notorious for their highly unusual organization of nuclear and chloroplast genomes. Early studies on the dinoflagellate mitochondrial genome indicated that it encodes the same three protein-coding genes found in Plasmodium spp., but with a complex organization and transcript editing. Recent work has extended this view, showing that the dinoflagellate mitochondrial genome contains a wide array of gene fragments and genes interspersed with noncoding inverted repeats. The genome seems to require noncanonical start and stop codons, as well as high levels of editing, trans-splicing and the addition of oligonucleotide caps at the 5' and 3' ends of transcripts. Despite its small coding content, the dinoflagellate mitochondrial genome is one of the most complex known.

Asunto(s)

Dinoflagelados/genética , Genoma Mitocondrial/genética , Genoma de Protozoos/genética , Animales , ADN Mitocondrial/genética , Sistemas de Lectura Abierta/genética , ARN/genética

RESUMEN

SUMMARY: The sensitive to freezing2 (SFR2) gene has an important role in freezing tolerance in Arabidopsis thaliana. We show that homologous genes are present, and expressed, in a wide range of terrestrial plants, including species not able to tolerate freezing. Expression constructs derived from the cDNAs of a number of different plant species, including examples not tolerant to freezing, are able to complement the freezing sensitivity of the Arabidopsis sfr2 mutant. In Arabidopsis the SFR2 protein is localized to the chloroplast outer envelope membrane, as revealed by the analysis of transgenic plants expressing SFR2 fusions to GFP, by confocal microscopy, and by the immunological analysis of isolated chloroplasts treated with thermolysin protease. Moreover, the chloroplasts of the sfr2 mutant show clear evidence of rapid damage after a freezing episode, suggesting a role for SFR2 in the protection of the chloroplast.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cloroplastos/fisiología , beta-Glucosidasa/genética , Secuencia de Aminoácidos , Arabidopsis/fisiología , Proteínas de Arabidopsis/fisiología , Cloroplastos/genética , Congelación , Genes de Plantas , Genes Reporteros , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/fisiología , Membranas Intracelulares , Microscopía Confocal , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Filogenia , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/fisiología , ARN de Planta/genética , Proteínas Recombinantes/genética , Alineación de Secuencia , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , beta-Glucosidasa/fisiología

RESUMEN

Dinoflagellates are an economically and ecologically important eukaryotic algal group. The organization of their chloroplast genome appears to be radically different from that in plants and other algae. The gene content has been dramatically reduced in dinoflagellates, with the large-scale transfer of genes to the nucleus. Most of the remaining genes encode subunits of Photosystems I and II, the cytochrome b(6)f complex, and ATP synthase, as well as rRNAs and a few tRNAs. Whereas conventional chloroplast genomes have all their genes physically linked on one molecule, dinoflagellate chloroplast genes are located on small plasmids, termed 'minicircles'. Each minicircle has at most a few genes, and a distinguishable 'core' region. Genes are always in the same orientation with respect to the core region. There are also non-coding minicircles, including aberrant forms of minicircles apparently derived from other minicircles by rearrangement. The evidence that the minicircles are located in the chloroplast and that there is no conventional chloroplast genome in addition to the minicircles is discussed. Transcription of minicircles is probably initiated close to the core, generating transcripts corresponding to an almost entire minicircle. The transcripts are then cleaved to molecules corresponding to individual genes. Post-transcriptional modifications include editing and addition of a polyU tail. It is discussed why these particular genes have been retained in the dinoflagellate chloroplast, together with the possibility that the chloroplast supplies fMet-tRNA to the mitochondrion.

Asunto(s)

Dinoflagelados/genética , Genoma del Cloroplasto , Genoma de Protozoos , Animales , ADN de Cloroplastos/genética , ADN Protozoario/genética , Evolución Molecular , Datos de Secuencia Molecular , Plásmidos , Transcripción Genética

RESUMEN

The dinoflagellate chloroplast genome is fragmented into a number of plasmid-like minicircles, mostly containing one or more genes, and with a conserved core. We show here that, in addition to the transcripts of similar sizes to individual genes that have been reported previously, there are larger transcripts beginning and ending close to the core region. These may give rise to the smaller transcripts by processing. We also show that previously reported ORFs (open reading frames) are represented by transcripts that are significantly more abundant than those for non-coding regions, indicating that the ORFs are indeed functional. We show that 'empty' minicircles are also transcribed. We propose a model for linkage of DNA replication and transcription in dinoflagellate chloroplasts.

Asunto(s)

ADN de Cloroplastos/genética , Dinoflagelados/genética , Plastidios/genética , Transcripción Genética , Animales , Northern Blotting , Modelos Genéticos , Plásmidos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

We have characterized the mitochondrial genome of the dinoflagellate Amphidinium carterae. It contains just 3 identifiable protein-coding genes: cox1, cox3, and cob. No evidence for rRNA or tRNA genes was found. Expressed sequence tags (EST) sequences for the 3 genes suggest that RNA editing occurs in 2 cases removing an in-frame stop codon. Two of the transcripts (cob and cox1) lack a stop codon at the end of the gene. The genome contains a large amount of noncoding DNA including many fragmented copies of all the 3 genes and large numbers of inverted repeats. The genome, which contains about 70% AT, has undergone extensive recombination, possibly due to the inverted repeats. The highly reduced mitochondrial gene content supports the relationship of the dinoflagellates and apicomplexa as sister groups.

Asunto(s)

Dinoflagelados/genética , Genes Mitocondriales , Animales , Secuencia de Bases , Evolución Molecular , Genómica , Datos de Secuencia Molecular , Edición de ARN , Análisis de Secuencia de ADN

RESUMEN

BACKGROUND: Peridinin-containing dinoflagellates have a highly reduced chloroplast genome, which is unlike that found in other chloroplast containing organisms. Genome reduction appears to be the result of extensive transfer of genes to the nuclear genome. Unusually the genes believed to be remaining in the chloroplast genome are found on small DNA 'minicircles'. In this study we present a comparison of sets of minicircle sequences from three dinoflagellate species. RESULTS: PCR was used to amplify several minicircles from Amphidinium carterae so that a homologous set of gene-containing minicircles was available for Amphidinium carterae and Amphidinium operculatum, two apparently closely related peridinin-containing dinoflagellates. We compared the sequences of these minicircles to determine the content and characteristics of their chloroplast genomes. We also made comparisons with minicircles which had been obtained from Heterocapsa triquetra, another peridinin-containing dinoflagellate. These in silico comparisons have revealed several genetic features which were not apparent in single species analyses. The features include further protein coding genes, unusual rRNA genes, which we show are transcribed, and the first examples of tRNA genes from peridinin-containing dinoflagellate chloroplast genomes. CONCLUSION: Comparative analysis of minicircle sequences has allowed us to identify previously unrecognised features of dinoflagellate chloroplast genomes, including additional protein and RNA genes. The chloroplast rRNA gene sequences are radically different from those in other organisms, and in many ways resemble the rRNA genes found in some highly reduced mitochondrial genomes. The retention of certain tRNA genes in the dinoflagellate chloroplast genome has important implications for models of chloroplast-mitochondrion interaction.

Asunto(s)

Cloroplastos/genética , Dinoflagelados/genética , Genoma de Protozoos , ARN Protozoario/análisis , ARN Ribosómico/genética , ARN de Transferencia/genética , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Alineación de Secuencia

RESUMEN

Dinoflagellate algae of the genus Symbiodinium are important symbionts within corals and other benthic marine animals. The molecular diversity of Symbiodinium has been described mainly by use of ribosomal DNA sequence data. We tested whether minicircle sequences, which appear to form the chloroplast genome in many dinoflagellates, could be used as a marker for molecular diversity among symbionts found in corals and sea anemones. Partial and full-length sequences for psbA were obtained from environmental samples of coral and sea anemones of wide-ranging geographical distribution. Phylogenetic trees constructed with partial psbA sequences were consistent with the known phylotypes of the isolates. Further sequencing suggested that the psbA gene is present on a minicircle in all Symbiodinium phylotypes. The length and DNA sequence of the non-coding portion of the minicircles varied considerably among Symbiodinium phylotypes. In two Symbiodinium isolates from different phylotypes an elaborate pattern of repeat sequences of unknown function was found in the non-coding region. Phylogenetic analysis of the non-coding region of the psbA minicircle indicates that minicircle sequences could be a useful chloroplast-derived marker for differentiating both closely related and distantly related Symbiodinium isolates.

Asunto(s)

Cnidarios/parasitología , ADN de Cinetoplasto/genética , Dinoflagelados/genética , Complejo de Proteína del Fotosistema II/genética , Agua de Mar/microbiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Protozoario/genética , ADN Ribosómico/genética , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/genética , Alineación de Secuencia , Simbiosis

RESUMEN

The evolution of the plastid from a photosynthetic bacterial endosymbiont involved a dramatic reduction in the complexity of the plastid genome, with many genes either discarded or transferred to the nucleus of the eukaryotic host. However, this evolutionary process has not gone to completion and a subset of genes remains in all plastids examined to date. The various hypotheses put forward to explain the retention of the plastid genome have tended to focus on the need for photosynthetic organisms to retain a genetic system in the chloroplast, and they fail to explain why heterotrophic plants and algae, and the apicomplexan parasites all retain a genome in their non-photosynthetic plastids. Here we consider two additional explanations: the 'essential tRNAs' hypothesis and the 'transfer-window' hypothesis.

Asunto(s)

Genoma , Fotosíntesis , Plastidios/genética , Animales , Apicomplexa/genética , Apicomplexa/metabolismo , Genes de Plantas , Genes de ARNr , Hemo/biosíntesis , Mitocondrias/metabolismo , Fotosíntesis/genética , Biosíntesis de Proteínas , ARN de Transferencia/fisiología , ARN de Transferencia de Ácido Glutámico/fisiología , ARN de Transferencia de Metionina/fisiología

RESUMEN

Recent studies have demonstrated that multiple co-occurring global changes can alter the abundance, diversity, and productivity of plant communities. Below ground processes, often mediated by soil microorganisms, are central to the response of these communities to global change. Very little is known, however, about the effects of multiple global changes on microbial communities. We examined the response of ammonia-oxidizing bacteria (AOB), microorganisms that mediate the transformation of ammonium into nitrite, to simultaneous increases in atmospheric CO2, precipitation, temperature, and nitrogen deposition, manipulated on the ecosystem level in a California grassland. Both the community structure and abundance of AOB responded to these simulated global changes. Increased nitrogen deposition significantly altered the structure of the ammonia-oxidizing community, consistently shifting the community toward dominance by bacteria most closely related to Nitrosospira sp. 2. This shift was most pronounced when temperature and precipitation were not increased. Total abundance of AOB significantly decreased in response to increased atmospheric CO2. This decrease was most pronounced when precipitation was also increased. Shifts in community composition were associated with increases in nitrification, but changes in abundance were not. These results demonstrate that microbial communities can be consistently altered by global changes and that these changes can have implications for ecosystem function.

Asunto(s)

Amoníaco/metabolismo , Betaproteobacteria/metabolismo , Efecto Invernadero , Betaproteobacteria/clasificación , Betaproteobacteria/genética , Betaproteobacteria/aislamiento & purificación , California , Dióxido de Carbono/metabolismo , Ecosistema , Genes Bacterianos , Datos de Secuencia Molecular , Oxidación-Reducción , Oxidorreductasas/genética , Filogenia , Poaceae/crecimiento & desarrollo , Poaceae/metabolismo , Microbiología del Suelo , Temperatura