Volatiles of the Apicomplexan Alga Chromera velia and Associated Bacteria.

Volatiles released by the apicomplexan alga Chromera velia CCAP1602/1 and their associated bacteria have been investigated. A metagenome analysis allowed the identification of the most abundant heterotrophic bacteria of the phycosphere, but the isolation of additional strains showed that metagenomics underestimated the complexity of the algal microbiome, However, a culture-independent approach revealed the presence of a planctomycete that likely represents a novel bacterial family. We analysed algal and bacterial volatiles by open-system-stripping analysis (OSSA) on Tenax TA desorption tubes, followed by thermodesorption, cryofocusing and GC-MS-analysis. The analyses of the alga and the abundant bacterial strains Sphingopyxis litoris A01A-101, Algihabitans albus A01A-324, "Coraliitalea coralii" A01A-333 and Litoreibacter sp. A01A-347 revealed sulfur- and nitrogen-containing compounds, ketones, alcohols, aldehydes, aromatic compounds, amides and one lactone, as well as the typical algal products, apocarotenoids. The compounds were identified by gas chromatographic retention indices, comparison of mass spectra and syntheses of reference compounds. A major algal metabolite was 3,4,4-trimethylcyclopent-2-en-1-one, an apocarotenoid indicating the presence of carotenoids related to capsanthin, not reported from algae so far. A low overlap in volatiles bouquets between C. velia and the bacteria was found, and the xenic algal culture almost exclusively released algal components.

Filling the Gaps in the Cyanobacterial Tree of Life-Metagenome Analysis of Stigonema ocellatum DSM 106950, Chlorogloea purpurea SAG 13.99 and Gomphosphaeria aponina DSM 107014.

Cyanobacteria represent one of the most important and diverse lineages of prokaryotes with an unparalleled morphological diversity ranging from unicellular cocci and characteristic colony-formers to multicellular filamentous strains with different cell types. Sequencing of more than 1200 available reference genomes was mainly driven by their ecological relevance (Prochlorococcus, Synechococcus), toxicity (Microcystis) and the availability of axenic strains. In the current study three slowly growing non-axenic cyanobacteria with a distant phylogenetic positioning were selected for metagenome sequencing in order to (i) investigate their genomes and to (ii) uncover the diversity of associated heterotrophs. High-throughput Illumina sequencing, metagenomic assembly and binning allowed us to establish nearly complete high-quality draft genomes of all three cyanobacteria and to determine their phylogenetic position. The cyanosphere of the limnic isolates comprises up to 40 heterotrophic bacteria that likely coexisted for several decades, and it is dominated by Alphaproteobacteria and Bacteriodetes. The diagnostic marker protein RpoB ensured in combination with our novel taxonomic assessment via BLASTN-dependent text-mining a reliable classification of the metagenome assembled genomes (MAGs). The detection of one new family and more than a dozen genera of uncultivated heterotrophic bacteria illustrates that non-axenic cyanobacteria are treasure troves of hidden microbial diversity.

Optimized mRuby3 is a Suitable Fluorescent Protein for in vivo Co-localization Studies with GFP in the Diatom Phaeodactylum tricornutum.

Phaeodactylum tricornutum is an ecologically and evolutionarily relevant microalga that has developed into an important model for molecular biological studies on organisms with complex plastids. The diatom is particularly suitable for in vivo protein localization analyses via fluorescence microscopy in which the green fluorescent protein (GFP) and its derivatives are dominantly used. Whereas GFP fluorescence emission is usually measured between 500 and 520nm in confocal microscopy, the autofluorescence of the P. tricornutum plastid is detected above 625nm. Here we established the fluorescent protein mRuby3 as tag for efficient in vivo protein localization studies by expressing a codon-optimized gene in P. tricornutum. mRuby3 was directed to seven different subcellular localizations by means of full-length marker protein or N-/C-terminal targeting signal fusions; its emission was detected efficiently between 580 and 605nm, being unequivocally distinguishable from the plastid autofluorescence in vivo. Moreover, mRuby3 proved to be highly suitable for co-localization experiments using confocal laser scanning microscopy in which mRuby3 fusion proteins were expressed in parallel with GFP-tagged proteins. Our results show the potential of mRuby3 for its application in studying protein targeting and localization in P. tricornutum, particularly underlining its compatibility with GFP and the plastid autofluorescence in signal detection.

Identification and Localization of Peroxisomal Biogenesis Proteins Indicates the Presence of Peroxisomes in the Cryptophyte Guillardia theta and Other "Chromalveolates".

Peroxisomes are single-membrane-bound organelles with a huge metabolic versatility, including the degradation of fatty acids (ß-oxidation) and the detoxification of reactive oxygen species as most conserved functions. Although peroxisomes seem to be present in the majority of investigated eukaryotes, where they are responsible for many eclectic and important spatially separated metabolic reactions, knowledge about their existence in the plethora of protists (eukaryotic microorganisms) is scarce. Here, we investigated genomic data of organisms containing complex plastids with red algal ancestry (so-called "chromalveolates") for the presence of genes encoding peroxins-factors specific for the biogenesis, maintenance, and division of peroxisomes in eukaryotic cells. Our focus was on the cryptophyte Guillardia theta, a marine microalga, which possesses two phylogenetically different nuclei of host and endosymbiont origin, respectively, thus being of enormous evolutionary significance. Besides the identification of a complete set of peroxins in G. theta, we heterologously localized selected factors as GFP fusion proteins via confocal and electron microscopy in the model diatom Phaeodactylum tricornutum. Furthermore, we show that peroxins, and thus most likely peroxisomes, are present in haptophytes as well as eustigmatophytes, brown algae, and alveolates including dinoflagellates, chromerids, and noncoccidian apicomplexans. Our results indicate that diatoms are not the only "chromalveolate" group devoid of the PTS2 receptor Pex7, and thus a PTS2-dependent peroxisomal import pathway, which seems to be absent in haptophytes (Emiliania huxleyi) as well. Moreover, important aspects of peroxisomal biosynthesis and protein import in "chromalveolates"are highlighted.