SufD - Bestandteil eines essentiellen plastidär lokalisierten Systems in Cyanobakterien, Cryptomonaden und höheren Pflanzen

Durch die vorliegende Arbeit konnte ein Beitrag zur funktionellen Charakterisierung des Nucleomorph-kodierten und plastidär lokalisierten Proteins ORF467 aus Guillardia theta geleistet werden. Dieses Protein konnte durch Sequenzhomologien als Bestandteil SufD des plastidären SUF-Systems identifiziert werden. Mit immunologischen Methoden konnte SufD in der Plastide von Guillardia theta nachgewiesen werden. Ein Gen für eine weitere Komponente des SUF-Systems, SufS, wurde durch die Auswertung von EST-Daten im Kerngenom lokalisiert. Die Cryptomonade Guillardia theta enthält damit ein über drei Genome, Kern, Plastide und Nucleomorph, verteiltes plastidäres SUF-System. Die heterogenomische Knock-out Mutation von SufD in Synechocystis führte zu einem Defekt der Zellteilung, der durch die Bildung von aus vier Zellen bestehenden Teilungsstadien gekennzeichnet war. In Importexperimenten mit radioaktivem Eisen wurde eine erhöhte Eisenaufnahme der SufD-Mutante gemessen. Die Analyse einer T-DNA Mutante des SufD-Homologen ATNAP6 in Arabidospis thaliana zeigte Defekte in der Embryonalentwicklung, eine veränderte Ultrastruktur der Chloroplasten, sowie ein reduziertes Wurzelwachstum und verringerten Chlorophyllgehalt der Pflanzen. Durch die in vivo Expression von GFP-Fusionsproteinen von ATNAP6 in Protoplasten konnte eine plastidäre Lokalisation von ATNAP6 nachgewiesen werden. Das Lokalisationsmuster zeigte eine Verteilung der GFP-Fluoreszenz in abgegrenzten subplastidären Bereichen, die für die subplastidäre Lokalisation notwendige beta-helikale Proteindomäne von ATNAP6 wurde durch die Generierung und in vivo Expression von modifizierten ATNAP6-GFP Fusionsproteinen identifiziert. In einer Expressionsanalyse mit einem Oligonukleotid-Genchip Microarray konnten signifikante Änderungen der Expression von regulativen Genen und von Genen in verschiedenen Stoffwechselwegen der ATNAP6 Knock-out Mutante gemessen werden. Die Expressionsdaten der ATNAP6 Knock-out Mutante und der komplexe Phänotyp von atnap6 konnten als Folge eines grundlegenden Defekts des plastidären SUF-Systems zur Synthese von Eisen-Schwefel Clustern interpretiert werden und ermöglichten es, ein Modell für die Funktion von ATNAP6 in Arabidopsis thaliana zu formulieren

Nitrogen fixation in eukaryotes – New models for symbiosis

BACKGROUND: Nitrogen, a component of many bio-molecules, is essential for growth and development of all organisms. Most nitrogen exists in the atmosphere, and utilisation of this source is important as a means of avoiding nitrogen starvation. However, the ability to fix atmospheric nitrogen via the nitrogenase enzyme complex is restricted to some bacteria. Eukaryotic organisms are only able to obtain fixed nitrogen through their symbiotic interactions with nitrogen-fixing prokaryotes. These symbioses involve a variety of host organisms, including animals, plants, fungi and protists. RESULTS: We have compared the morphological, physiological and molecular characteristics of nitrogen fixing symbiotic associations of bacteria and their diverse hosts. Special features of the interaction, e.g. vertical transmission of symbionts, grade of dependency of partners and physiological modifications have been considered in terms of extent of co-evolution and adaptation. Our findings are that, despite many adaptations enabling a beneficial partnership, most symbioses for molecular nitrogen fixation involve facultative interactions. However, some interactions, among them endosymbioses between cyanobacteria and diatoms, show characteristics that reveal a more obligate status of co-evolution. CONCLUSION: Our review emphasises that molecular nitrogen fixation, a driving force for interactions and co-evolution of different species, is a widespread phenomenon involving many different organisms and ecosystems. The diverse grades of symbioses, ranging from loose associations to highly specific intracellular interactions, might themselves reflect the range of potential evolutionary fates for symbiotic partnerships. These include the extreme evolutionary modifications and adaptations that have accompanied the formation of organelles in eukaryotic cells: plastids and mitochondria. However, age and extensive adaptation of plastids and mitochondria complicate the investigation of processes involved in the transition of symbionts to organelles. Extant lineages of symbiotic associations for nitrogen fixation show diverse grades of adaptation and co-evolution, thereby representing different stages of symbiont-host interaction. In particular cyanobacterial associations with protists, like the Rhopalodia gibba-spheroid body symbiosis, could serve as important model systems for the investigation of the complex mechanisms underlying organelle evolution

The cyanobacterial endosymbiont of the unicellular algae Rhopalodia gibba shows reductive genome evolution

<p>Abstract</p> <p>Background</p> <p>Bacteria occur in facultative association and intracellular symbiosis with a diversity of eukaryotic hosts. Recently, we have helped to characterise an intracellular nitrogen fixing bacterium, the so-called spheroid body, located within the diatom <it>Rhopalodia gibba</it>. Spheroid bodies are of cyanobacterial origin and exhibit features that suggest physiological adaptation to their intracellular life style. To investigate the genome modifications that have accompanied the process of endosymbiosis, here we compare gene structure, content and organisation in spheroid body and cyanobacterial genomes.</p> <p>Results</p> <p>Comparison of the spheroid body's genome sequence with corresponding regions of near free-living relatives indicates that multiple modifications have occurred in the endosymbiont's genome. These include localised changes that have led to elimination of some genes. This gene loss has been accompanied either by deletion of the respective DNA region or replacement with non-coding DNA that is AT rich in composition. In addition, genome modifications have led to the fusion and truncation of genes. We also report that in the spheroid body's genome there is an accumulation of deleterious mutations in genes for cell wall biosynthesis and processes controlled by transposases. Interestingly, the formation of pseudogenes in the spheroid body has occurred in the presence of intact, and presumably functional, <it>rec</it>A and <it>rec</it>F genes. This is in contrast to the situation in most investigated obligate intracellular bacterium-eukaryote symbioses, where at least either <it>rec</it>A or <it>rec</it>F has been eliminated.</p> <p>Conclusion</p> <p>Our analyses suggest highly specific targeting/loss of individual genes during the process of genome reduction and establishment of a cyanobacterial endosymbiont inside a eukaryotic cell. Our findings confirm, at the genome level, earlier speculation on the obligate intracellular status of the spheroid body in <it>Rhopalodia gibba</it>. This association is the first example of an obligate cyanobacterial symbiosis involving nitrogen fixation for which genomic data are available. It represents a new model system to study molecular adaptations of genome evolution that accompany a switch from free-living to intracellular existence.</p

Differential gene transfers and gene duplications in primary and secondary endosymbioses

BACKGROUND: Most genes introduced into phototrophic eukaryotes during the process of endosymbiosis are either lost or relocated into the host nuclear genome. In contrast, groEL homologues are found in different genome compartments among phototrophic eukaryotes. Comparative sequence analyses of recently available genome data, have allowed us to reconstruct the evolutionary history of these genes and propose a hypothesis that explains the unusual genome distribution of groEL homologues. RESULTS: Our analyses indicate that while two distinct groEL genes were introduced into eukaryotes by a progenitor of plastids, these particular homologues have not been maintained in all evolutionary lineages. This is of significant interest, because two chaperone proteins always co-occur in oxygenic photosynthetic organisms. We infer strikingly different lineage specific processes of evolution involving deletion, duplication and targeting of groEL proteins. CONCLUSION: The requirement of two groEL homologues for chaperon function in phototrophs has provided a constraint that has shaped convergent evolutionary scenarios in divergent evolutionary lineages. GroEL provides a general evolutionary model for studying gene transfers and convergent evolutionary processes among eukaryotic lineages

The Rhodomonas salina mitochondrial genome: bacteria-like operons, compact gene arrangement and complex repeat region

To gain insight into the mitochondrial genome structure and gene content of a putatively ancestral group of eukaryotes, the cryptophytes, we sequenced the complete mitochondrial DNA of Rhodomonas salina. The 48 063 bp circular-mapping molecule codes for 2 rRNAs, 27 tRNAs and 40 proteins including 23 components of oxidative phosphorylation, 15 ribosomal proteins and two subunits of tat translocase. One potential protein (ORF161) is without assigned function. Only two introns occur in the genome; both are present within cox1 belong to group II and contain RT open reading frames. Primitive genome features include bacteria-like rRNAs and tRNAs, ribosomal protein genes organized in large clusters resembling bacterial operons and the presence of the otherwise rare genes such as rps1 and tatA. The highly compact gene organization contrasts with the presence of a 4.7 kb long, repeat-containing intergenic region. Repeat motifs ∼40–700 bp long occur up to 31 times, forming a complex repeat structure. Tandem repeats are the major arrangement but the region also includes a large, ∼3 kb, inverted repeat and several potentially stable ∼40–80 bp long hairpin structures. We provide evidence that the large repeat region is involved in replication and transcription initiation, predict a promoter motif that occurs in three locations and discuss two likely scenarios of how this highly structured repeat region might have evolved

Complete DNA sequences of the plastid genomes of two parasitic flowering plant species, Cuscuta reflexa and Cuscuta gronovii

<p>Abstract</p> <p>Background</p> <p>The holoparasitic plant genus <it>Cuscuta </it>comprises species with photosynthetic capacity and functional chloroplasts as well as achlorophyllous and intermediate forms with restricted photosynthetic activity and degenerated chloroplasts. Previous data indicated significant differences with respect to the plastid genome coding capacity in different <it>Cuscuta </it>species that could correlate with their photosynthetic activity. In order to shed light on the molecular changes accompanying the parasitic lifestyle, we sequenced the plastid chromosomes of the two species <it>Cuscuta reflexa </it>and <it>Cuscuta gronovii</it>. Both species are capable of performing photosynthesis, albeit with varying efficiencies. Together with the plastid genome of <it>Epifagus virginiana</it>, an achlorophyllous parasitic plant whose plastid genome has been sequenced, these species represent a series of progression towards total dependency on the host plant, ranging from reduced levels of photosynthesis in <it>C. reflexa </it>to a restricted photosynthetic activity and degenerated chloroplasts in <it>C. gronovii </it>to an achlorophyllous state in <it>E. virginiana</it>.</p> <p>Results</p> <p>The newly sequenced plastid genomes of <it>C. reflexa </it>and <it>C. gronovii </it>reveal that the chromosome structures are generally very similar to that of non-parasitic plants, although a number of species-specific insertions, deletions (indels) and sequence inversions were identified. However, we observed a gradual adaptation of the plastid genome to the different degrees of parasitism. The changes are particularly evident in <it>C. gronovii </it>and include (a) the parallel losses of genes for the subunits of the plastid-encoded RNA polymerase and the corresponding promoters from the plastid genome, (b) the first documented loss of the gene for a putative splicing factor, MatK, from the plastid genome and (c) a significant reduction of RNA editing.</p> <p>Conclusion</p> <p>Overall, the comparative genomic analysis of plastid DNA from parasitic plants indicates a bias towards a simplification of the plastid gene expression machinery as a consequence of an increasing dependency on the host plant. A tentative assignment of the successive events in the adaptation of the plastid genomes to parasitism can be inferred from the current data set. This includes (1) a loss of non-coding regions in photosynthetic <it>Cuscuta </it>species that has resulted in a condensation of the plastid genome, (2) the simplification of plastid gene expression in species with largely impaired photosynthetic capacity and (3) the deletion of a significant part of the genetic information, including the information for the photosynthetic apparatus, in non-photosynthetic parasitic plants.</p

Microalgae as bioreactors for bioplastic production

<p>Abstract</p> <p>Background</p> <p>Poly-3-hydroxybutyrate (PHB) is a polyester with thermoplastic properties that is naturally occurring and produced by such bacteria as <it>Ralstonia eutropha </it>H16 and <it>Bacillus megaterium</it>. In contrast to currently utilized plastics and most synthetic polymers, PHB is biodegradable, and its production is not dependent on fossil resources making this bioplastic interesting for various industrial applications.</p> <p>Results</p> <p>In this study, we report on introducing the bacterial PHB pathway of <it>R. eutropha </it>H16 into the diatom <it>Phaeodactylum tricornutum</it>, thereby demonstrating for the first time that PHB production is feasible in a microalgal system. Expression of the bacterial enzymes was sufficient to result in PHB levels of up to 10.6% of algal dry weight. The bioplastic accumulated in granule-like structures in the cytosol of the cells, as shown by light and electron microscopy.</p> <p>Conclusions</p> <p>Our studies demonstrate the great potential of microalgae like the diatom <it>P. tricornutum </it>to serve as solar-powered expression factories and reveal great advantages compared to plant based production systems.</p

Enteroviruses in Respiratory Samples from Paediatric Patients of a Tertiary Care Hospital in Germany

Enteroviruses are associated with various diseases accompanied by rare but severe complications. In recent years, outbreaks of enterovirus D68 and enterovirus A71 associated with severe respiratory infections and neurological complications have been reported worldwide. Since information on molecular epidemiology in respiratory samples is still limited, the genetic diversity of enteroviruses was retrospectively analysed over a 4-year period (2013–2016) in respiratory samples from paediatric patients. Partial viral major capsid protein gene (VP1) sequences were determined for genotyping. Enteroviruses were detected in 255 (6.1%) of 4187 specimens. Phylogenetic analyses of 233 (91.4%) strains revealed 25 different genotypes distributed to Enterovirus A (39.1%), Enterovirus B (34.3%), and Enterovirus D (26.6%). The most frequently detected genotypes were enterovirus D68 (26.6%), coxsackievirus A6 (15.9%), and enterovirus A71 (7.3%). Enterovirus D68 detections were associated with lower respiratory tract infections and increased oxygen demand. Meningitis/encephalitis and other neurological symptoms were related to enterovirus A71, while coxsackievirus A6 was associated with upper respiratory diseases. Prematurity turned out as a potential risk factor for increased oxygen demand during enterovirus infections. The detailed analysis of epidemiological and clinical data contributes to the non-polio enterovirus surveillance in Europe and showed high and rapidly changing genetic diversity of circulating enteroviruses, including different enterovirus D68 variants

Complementation of a phycocyanin-bilin lyase from Synechocystis sp. PCC 6803 with a nucleomorph-encoded open reading frame from the cryptophyte Guillardia theta

<p>Abstract</p> <p>Background</p> <p>Cryptophytes are highly compartmentalized organisms, expressing a secondary minimized eukaryotic genome in the nucleomorph and its surrounding remnant cytoplasm, in addition to the cell nucleus, the mitochondrion and the plastid. Because the members of the nucleomorph-encoded proteome may contribute to essential cellular pathways, elucidating nucleomorph-encoded functions is of utmost interest. Unfortunately, cryptophytes are inaccessible for genetic transformations thus far. Therefore the functions of nucleomorph-encoded proteins must be elucidated indirectly by application of methods in genetically accessible organisms.</p> <p>Results</p> <p>Orf222, one of the uncharacterized nucleomorph-specific open reading frames of the cryptophyte <it>Guillardia theta</it>, shows homology to <it>slr</it>1649 of <it>Synechocystis </it>sp. PCC 6803. Recently a further homolog from <it>Synechococcus </it>sp. PCC 7002 was characterized to encode a phycocyanin-β155-bilin lyase. Here we show by insertion mutagenesis that the <it>Synechocystis </it>sp. PCC 6803 <it>slr</it>1649-encoded protein also acts as a bilin lyase, and additionally contributes to linker attachment and/or stability of phycobilisomes. Finally, our results indicate that the phycocyanin-β155-bilin lyase of <it>Synechocystis </it>sp. PCC 6803 can be complemented <it>in vivo </it>by the nucleomorph-encoded open reading frame <it>orf</it>222.</p> <p>Conclusion</p> <p>Our data show that the loss of phycocyanin-lyase function causes pleiotropic effects in <it>Synechocystis </it>sp. PCC 6803 and indicate that after separating from a common ancestor protein, the phycoerythrin lyase from <it>Guillardia theta </it>has retained its capacity to couple a bilin group to other phycobiliproteins. This is a further, unexpected example of the universality of phycobiliprotein lyases.</p