The enigmatic nucleus of the marine dinoflagellate Prorocentrum cordatum.

The marine, bloom-forming dinoflagellate Prorocentrum cordatum CCMP 1329 (formerly P. minimum) has a genome atypical of eukaryotes, with a large size of ~4.15 Gbp, organized in plentiful, highly condensed chromosomes and packed in a dinoflagellate-specific nucleus (dinokaryon). Here, we apply microscopic and proteogenomic approaches to obtain new insights into this enigmatic nucleus of axenic P. cordatum. High-resolution focused ion beam/scanning electron microscopy analysis of the flattened nucleus revealed highest density of nuclear pores in the vicinity of the nucleolus, a total of 62 tightly packed chromosomes (~0.4-6.7 µm3), and interaction of several chromosomes with the nucleolus and other nuclear structures. A specific procedure for enriching intact nuclei was developed to enable proteomic analyses of soluble and membrane protein-enriched fractions. These were analyzed with geLC and shotgun approaches employing ion-trap and timsTOF (trapped-ion-mobility-spectrometry time-of-flight) mass spectrometers, respectively. This allowed identification of 4,052 proteins (39% of unknown function), out of which 418 were predicted to serve specific nuclear functions; additional 531 proteins of unknown function could be allocated to the nucleus. Compaction of DNA despite very low histone abundance could be accomplished by highly abundant major basic nuclear proteins (HCc2-like). Several nuclear processes including DNA replication/repair and RNA processing/splicing can be fairly well explained on the proteogenomic level. By contrast, transcription and composition of the nuclear pore complex remain largely elusive. One may speculate that the large group of potential nuclear proteins with currently unknown functions may serve yet to be explored functions in nuclear processes differing from those of typical eukaryotic cells. IMPORTANCE Dinoflagellates form a highly diverse group of unicellular microalgae. They provide keystone species for the marine ecosystem and stand out among others by their very large, unusually organized genomes embedded in the nuclei markedly different from other eukaryotic cells. Functional insights into nuclear and other cell biological structures and processes of dinoflagellates have long been hampered by the paucity of available genomic sequences. The here studied cosmopolitan P. cordatum belongs to the harmful algal bloom-forming, marine dinoflagellates and has a recently de novo assembled genome. We present a detailed 3D reconstruction of the P. cordatum nucleus together with comprehensive proteogenomic insights into the protein equipment mastering the broad spectrum of nuclear processes. This study significantly advances our understanding of mechanisms and evolution of the conspicuous dinoflagellate cell biology.

Light and prey influence the abundances of two rhodopsins in the dinoflagellate Oxyrrhis marina.

Antisera were raised against the C-terminal amino acid sequences of the two rhodopsins ADY17806 and AEA49880 of Oxyrrhis marina. The antisera and affinity-purified antibodies thereof were used in western immunoblotting experiments of total cell protein fractions from cultures grown either in darkness or in white, red, green, or blue light. Furthermore, the rhodopsin abundances were profiled in cultures fed with yeast or the prasinophyte Pyramimonas grossii. The immunosignals of ADY17806 and AEA49880 were similar when O. marina was grown in white, green, or blue light. Signal intensities were lower under conditions of red light and lowest in darkness. Higher amounts were registered for both rhodopsins when O. marina was fed with yeast compared to P. grossii. Furthermore, total cell protein of cultures of O. marina grown under all cultivation conditions was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by tryptic in-gel digestion and mass spectrometric analysis of the 25-kDa protein bands. The rhodopsin ADY17809 was detected in all samples of the light quality experiments and in 14 of the 16 samples of the prey quality experiments. The rhodopsin ABV22427 was not detected in one sample of the light quality experiments. It was detected in 15 of the 16 samples of the prey quality experiments. Peptide fragments of the other rhodopsins were detected less often, and no clear distribution pattern was evident with respect to the applied light quality or offered prey, indicating that none of them was exclusively formed under a distinct light regime or when feeding on yeast or the prasinophyte. Fluorescence light microscopy using the affinity-purified antibodies revealed significant labeling of the cell periphery and cell internal structures, which resembled vacuoles, tiny vesicles, and rather compact structures. Immunolabeling electron microscopy strengthened these results and showed that the cytoplasmic membrane, putative lysosome membranes, membranes encircling the food vacuole, and birefringent bodies became labeled.

Methanosaeta and "Candidatus Velamenicoccus archaeovorus".

The phylum "Candidatus Omnitrophica" (candidate division OP3) is ubiquitous in anaerobic habitats but is currently characterized only by draft genomes from metagenomes and single cells. We had visualized cells of the phylotype OP3 LiM in methanogenic cultures on limonene as small epibiotic cells. In this study, we enriched OP3 cells by double density gradient centrifugation and obtained the first closed genome of an apparently clonal OP3 cell population by applying metagenomics and PCR for gap closure. Filaments of acetoclastic Methanosaeta, the largest morphotype in the culture community, contained empty cells, cells devoid of rRNA or of both rRNA and DNA, and dead cells according to transmission electron microscopy (TEM), thin-section TEM, scanning electron microscopy (SEM), catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), and LIVE/DEAD imaging. OP3 LiM cells were ultramicrobacteria (200 to 300 nm in diameter) and showed two physiological stages in CARD-FISH fluorescence signals: strong signals of OP3 LiM cells attached to Bacteria and to Archaea indicated many rRNA molecules and an active metabolism, whereas free-living OP3 cells had weak signals. Metaproteomics revealed that OP3 LiM lives with highly expressed secreted proteins involved in depolymerization and uptake of macromolecules and an active glycolysis and energy conservation by the utilization of pyruvate via a pyruvate:ferredoxin oxidoreductase and an Rnf complex (ferredoxin:NAD oxidoreductase). Besides sugar fermentation, a nucleotidyl transferase may contribute to energy conservation by phosphorolysis, the phosphate-dependent depolymerization of nucleic acids. Thin-section TEM showed distinctive structures of predation. Our study demonstrated a predatory metabolism for OP3 LiM cells, and therefore, we propose the name "Candidatus Velamenicoccus archaeovorus" gen. nov., sp. nov., for OP3 LiM. IMPORTANCE Epibiotic bacteria are known to live on and off bacterial cells. Here, we describe the ultramicrobacterial anaerobic epibiont OP3 LiM living on Archaea and Bacteria. We detected sick and dead cells of the filamentous archaeon Methanosaeta in slowly growing methanogenic cultures. OP3 LiM lives as a sugar fermenter, likely on polysaccharides from outer membranes, and has the genomic potential to live as a syntroph. The predatory lifestyle of OP3 LiM was supported by its genome, the first closed genome for the phylum "Candidatus Omnitrophica," and by images of cell-to-cell contact with prey cells. We propose naming OP3 LiM "Candidatus Velamenicoccus archaeovorus." Its metabolic versatility explains the ubiquitous presence of "Candidatus Omnitrophica" 3 in anoxic habitats and gives ultramicrobacterial epibionts an important role in the recycling and remineralization of microbial biomass. The removal of polysaccharides from outer membranes by ultramicrobacteria may also influence biological interactions between pro- and eukaryotes.

Rhodopsins build up the birefringent bodies of the dinoflagellate Oxyrrhis marina.

The ultrastructure of the birefringent bodies of the dinoflagellate Oxyrrhis marina was investigated by transmission electron microscopy. Ultrathin sectioning revealed that the bodies consist of highly ordered and densely packed lamellae, which show a regular striation along their longitudinal axis. A lattice distance of 6.1 nm was measured for the densely packed lamellae by FFT (Fast Fourier Transformation) analysis. In addition, a rather faint and oblique running striation was registered. Lamellae sectioned rather oblique or almost close to the surface show a honeycombed structure with a periodicity of 7.2-7.8 nm. Freeze-fracture transmission electron microscopy revealed that the lamellae are composed of highly ordered, crystalline arrays of particles. Here, FFT analysis resulted in lattice distances of 7.0-7.6 nm. Freeze-fracture transmission electron microscopy further revealed that the bodies remained intact after cell rupture followed by ascending flotation of the membrane fractions on discontinuous sucrose gradients. The birefringent bodies most likely are formed by evaginations of membranes, which separate the cytoplasm from the food vacuoles. Distinct, slightly reddish-colored areas, which resembled the birefringent bodies with respect to size and morphology, were registered by bright field light microscopy within Oxyrrhis marina cells. An absorbance maximum at 540 nm was registered for these areas, indicating that they are composed of rhodopsins. This was finally proven by immuno-transmission electron microscopy, as antisera directed against the C-terminal amino acid sequences of the rhodopsins AEA49880 and ADY17806 intensely immunolabeled the birefringent bodies of Oxyrrhis marina.

The proteorhodopsins of the dinoflagellate Oxyrrhis marina: ultrastructure and localization by immunofluorescence light microscopy and immunoelectron microscopy.

At least 7 proteorhodopsin sequences of Oxyrrhis marina were recently proven in bands obtained by sucrose density gradient centrifugation, and MS analyses revealed that the bands consisted almost of pure, native proteorhodopsins (Rhiel et al. 2020). The proteorhodopsin fractions, i.e., bands B2, B3, and B4 were subjected to transmission electron microscopy. Negative staining revealed that band B2 consisted most likely of monomeric/oligomeric proteorhodopsins with particle dimensions of about 6 nm. Negative staining, freeze-fracture, and cryo-transmission electron microscopy revealed that bands B3 and B4 consisted of vesicular, sheet-like, and cup-shaped structures which all seemed to be composed of protein. Frequently, ring-like protein aggregates were registered at higher magnifications. They measured about 4 nm in diameter with a tiny hole of 1.5 nm in the middle. The bands B2, B3, and B4 were pooled and used to raise an antiserum. Immunoelectron microscopy resulted in intense labeling of the isolated structures. Immunofluorescence light microscopy of formaldehyde-fixed Oxyrrhis cells resulted in intense labeling of the cell periphery. Some cell internal structures became labeled, too. Immunoelectron microscopy of freeze-fractured cells revealed that most likely the membranes of the amphiesmal vesicles were labeled at the cell periphery, while the cell internal label seemed to originate from the food vacuoles.

A simple protocol for the isolation of proteorhodopsins of the dinoflagellate Oxyrrhis marina.

For the first time, native proteorhodopsins of the marine dinoflagellate Oxyrrhis marina were isolated. Total cell membrane fractions were minced in a bead beater and solubilized with the detergent Triton X-100. Subsequent sucrose density gradient centrifugation resulted in three or four red-colored bands. Nonsolubilized, but still red colored, membranes sedimented at the bottom. For each of these bands, absorbance maxima were registered at approximately 514-516 nm with shoulders toward shorter wavelengths (470-490 nm). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the uppermost band represented free retinal chromophore, as it contained no protein. The other bands were almost pure proteorhodopsin fractions as the banding patterns showed one major protein of 25 kDa. Tryptic, in-gel digestion of the 25 kDa proteins and of faint protein bands above and below 25 kDa was followed by mass spectrometry, confirming these protein bands to consist, nearly exclusively, proteorhodopsins. Only single peptides of few other proteins were detected. In total, at least seven predicted proteorhodopsin protein sequences were experimentally verified.

Biopearling of Interconnected Outer Membrane Vesicle Chains by a Marine Flavobacterium.

Large surface-to-volume ratios provide optimal nutrient uptake conditions for small microorganisms in oligotrophic habitats. The surface area can be increased with appendages. Here, we describe chains of interconnecting vesicles protruding from cells of strain Hel3_A1_48, affiliating with Formosa spp. within the Flavobacteriia and originating from coastal free-living bacterioplankton. The chains were up to 10 µm long and had vesicles emanating from the outer membrane with a single membrane and a size of 80 to 100 nm by 50 to 80 nm. Cells extruded membrane tubes in the exponential phase, whereas vesicle chains dominated on cells in the stationary growth phase. This formation is known as pearling, a physical morphogenic process in which membrane tubes protrude from liposomes and transform into chains of interconnected vesicles. Proteomes of whole-cell membranes and of detached vesicles were dominated by outer membrane proteins, including the type IX secretion system and surface-attached peptidases, glycoside hydrolases, and endonucleases. Fluorescein-labeled laminarin stained the cells and the vesicle chains. Thus, the appendages provide binding domains and degradative enzymes on their surfaces and probably storage volume in the vesicle lumen. Both may contribute to the high abundance of these Formosa-affiliated bacteria during laminarin utilization shortly after spring algal blooms.IMPORTANCE Microorganisms produce membrane vesicles. One synthesis pathway seems to be pearling that describes the physical formation of vesicle chains from phospholipid vesicles via extended tubes. Bacteria with vesicle chains had been observed as well as bacteria with tubes, but pearling was so far not observed. Here, we report the observation of, initially, tubes and then vesicle chains during the growth of a flavobacterium, suggesting biopearling of vesicle chains. The flavobacterium is abundant during spring bacterioplankton blooms developing after algal blooms and has a special set of enzymes for laminarin, the major storage polysaccharide of microalgae. We demonstrated with fluorescently labeled laminarin that the vesicle chains bind laminarin or contain laminarin-derived compounds. Proteomic analyses revealed surface-attached degradative enzymes on the outer membrane vesicles. We conclude that the large surface area and the lumen of vesicle chains may contribute to the ecological success of this marine bacterium.

On the morphology and predatory behavior of the dinoflagellate Oxyrrhis marina exposed to reduced salinity.

Changes in salinity are known to alter the morphology of protists, and we hypothesized that these changes subsequently alter also the predatory behavior of the dinoflagellate Oxyrrhis marina. Oxyrrhis was grown in media of 33, 25, 20, and 10% of the regular salinity of f/2 medium (31-32‰). In all cases, the cells discharged trichocysts and swelled. Cell surfaces and volumes increased with decreasing salinity, such that cell surface area at least doubled at 10% and the cell volume increased approximately fourfold. After 1 h, the cells started to regain their regular shape, which was almost completed after 24 h. Oxyrrhis immediately regained its regular shape when culture medium was added 5-10 min after the osmotic stress. When incubated with Pyramimonas grossii as prey, those short-term stressed cells showed no significant different prey uptake in comparison to non-stressed cells. In contrast, 24 h after the addition of prey, short-term stressed Oxyrrhis cells had, with weak statistical significance, more Pyramimonas cells engulfed than non-stressed cells. These results indicated that (1) trichocysts were most likely not involved in prey capture and (2) salinity-stressed Oxyrrhis either enhanced its capability to capture more prey, or its digestion apparatus was hampered.

Lectin-binding and dissociation/reconstitution studies on the trichocysts of the dinoflagellate Oxyrrhis marina.

Trichocyst-enriched fractions were isolated from the dinoflagellate Oxyrrhis marina and subjected to protein staining and lectin-binding studies, to dissociation experiments using heat, and to solubilization/reconstitution experiments using 6 M guanidine hydrochloride. The trichocysts could be stained by Alcian Blue and became labeled by the lectin Concanavalin A, but not by wheat germ agglutinin. The trichocysts did not dissociate when the fractions were heated for 5 min at 40 or 50 °C. Heating at 60 °C resulted in the dissociation of trichocysts into irregular filamentous structures. These filaments were still present when the fractions were incubated for 5 min at temperatures of 70 and 80 °C. Reassembly was not achieved by subsequent cooling steps. The disintegration of trichocysts was also achieved in 6 M guanidine hydrochloride, and reassembly into filamentous structures, similar to those obtained by heat, occurred after dialysis against distilled water. Electron microscopy revealed that the filaments created either by heat or using guanidine hydrochloride by far did not resemble native trichocysts. They were much thinner (5-7 nm in width), missed the characteristic striation of electron-dense and -transparent lines along the longitudinal axes, and showed much more bending. Furthermore, they tend to merge to thicker shapeless structures and blob-like aggregates. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that fractions enriched either in trichocysts or in reconstituted filamentous structures obtained in the guanidine hydrochloride solubilization experiments were dominated by proteins with relative molecular weights in the range of approximately 15 to 29 kDa. Minor amounts of larger proteins were also detected. Tryptic in gel digestion followed by mass spectrometry confirmed the presence of almost the same set of proteins within the both, the trichocyst-enriched fractions, and the fractions of filaments reconstituted thereafter. These proteins were previously proposed to represent the matrix polypeptides of the trichocysts of Oxyrrhis marina (Rhiel et al., Protoplasma 255: 217-230, 2018).

Candidates of trichocyst matrix proteins of the dinoflagellate Oxyrrhis marina.

Trichocysts are a common cell organelle of ciliates and dinoflagellates. They are composed of trichocyst matrix proteins and have been intensely investigated and characterized in ciliates. Here, for the first time, data have been obtained for trichocyst matrix proteins of a dinoflagellate. A DELTA-BLAST search using 14 available and complete amino acid sequences of mature trichocyst matrix proteins of the ciliate Paramecium tetraurelia resulted in 16 hits for the dinoflagellate Oxyrrhis marina when the E values and bit values to be scored were <10-4 and >40. They code for proteins with acidic pI values and exceeded the precursors of the trichocyst matrix proteins of the ciliate approximately twofold in length. The values calculated for coverage, identity, and positives ranged from 76 to 100, 21.5 to 28.3, and 44.9 to 53.9%, respectively. Protein conformation predictions indicate coiled-coil domains which are a common feature of mature ciliate trichocyst matrix proteins. As often several EST sequences of O. marina matched with a queried mature trichocyst matrix protein of P. tetraurelia, a multigene family can be assumed for trichocyst proteins in this dinophyte, too. Trichocyst-enriched fractions of O. marina were isolated and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. When samples were incubated with loading buffer without a reducing agent, the banding pattern was mainly composed of three regions in the range of >90, 75-60, and 50-35 kDa, with each region consisting of four to five bands. Tryptic in gel digestion of proteins excised from these three gel regions followed by mass spectrometry confirmed that up to 14 of the 16 predicted proteins were present within the trichocyst-enriched fractions. When the samples were reduced with either ß-mercaptoethanol or dithiothreitol, the proteins of the three regions disappeared almost completely and proteins in the range of 27 to 15 kDa became the dominating bands. Up to 12 of the predicted proteins were detected within these bands.

On the extrusomes of Oxyrrhis marina (Dinophyceae).

Oxyrrhis marina was subjected to conventional transmission electron microscopy, with emphasis being laid on its extrusomes. Mainly regular trichocysts were obvious in ultrathin sections. They were highly abundant, approximately 2 µm in length and 200 nm in width, and composed of the characteristic features, i.e., an anterior tip and the posterior crystalline body. The tip measures approximately 440 nm in length and is built by an outer less electron-dense concentric layer followed by an inner electron-dense core with a translucent center in the middle. The less electron-dense layer most likely ends up in a bundle of filaments which are concentrically placed around the electron-dense core in the transition zone between the tip and body. Trichocyst bodies which are sectioned along the longitudinal axis are approximately 1.5 µm in length and show a regular striation of electron-dense and electron-translucent lines with a spacing of 9 nm. Cross-sectioned bodies are square-shaped and show a crystalline lattice composed of particles which are 8-9 nm in size. Discharge of regular trichocysts results in long rigid rods. They are square-shaped, 54 nm broad, and with a regular striation of approximately 54 nm along their longitudinal axes. Besides regular trichocysts, an additional type of extrusome was registered. It is not as abundant as regular trichocysts, membrane-enclosed, 2 µm in length and 180 nm in width, and resembles two bullets adjacent to each other with the tips facing in opposite directions. The two parts are slightly of different lengths (anterior part, 740 nm; posterior part, 590 nm) and widths (anterior part, 126 nm; posterior part, 117 nm) and separated from each other by a gap of 30 nm. The anterior part is more electron-dense than the posterior one. A faint electron-dense sheet-like structure was registered between the envelope membrane and these two inner structures. In extrusomes which had been arrested in the process of discharge, the anterior part gives rise to an oozing, amorphous, fibrous blob, while the posterior part consists of twisted filaments which most likely function as the charge of a gun for the release of the anterior part.

On the biogenesis and degradation of ejectisomes in Pyramimonas grossii (Prasinophyceae).

The biogenesis, assembly, and degradation of ejectisomes of Pyramimonas grossii were investigated by conventional transmission electron microscopy. Premature ejectisomes were mainly found beneath the cell envelope, often in close proximity to the nucleus, and as vesicles with diameters of 100 to 400 nm. Ejectisomes in the early stages of development contained only a few (2-4) turns of the ejectisome tapes. In the course of the ejectisome development, the number of turns and the widths of the coiled tapes increased. It is likely that vesicles, which were up to 650 nm in diameter, with granule- and plate-like structures inside, delivered additional preassembled ejectisome polypeptides to these premature stages. Both types of vesicles, those containing early stages of ejectisomes and those delivering additional ejectisome material, are believed to originate directly from the endoplasmic reticulum. Mature ejectisomes were mainly registered at the apical periphery of the cells. Up to 11 ejectisomes were found within a single cell. Ejectisomes that were most likely being in the process of degradation were registered within the cytoplasm and within vesicles, often together with material which resembled body scales. Mature ejectisomes which were still furled or which were arrested in the process of discharge were also found outside the cells in the medium.

Permanent draft genome of 'Rhodopirellula islandica' strain K833.

The planctomycete strain K833 was isolated from cold waters at the coast of Island and is tentatively named 'Rhodopirellula islandica'. It has a lower temperature range for growth than other genome-sequenced Rhodopirellula strains affiliating to Rhodopirellula baltica and 'Rhodopirellula europaea'. The permanent draft genome of strain K833 was obtained as part of a larger study on the biogeography of Rhodopirellula species in European marine waters. The genome consists of 55 contigs with a genome size of 7,433,200 bp. With an average nucleotide identity of 81% to related genomes of R. baltica and 'R. europaea' and more than 4000 common genes, it will be a valuable source for the study of temperature adaptation of planctomycete genomes.

Dilution cultivation of marine heterotrophic bacteria abundant after a spring phytoplankton bloom in the North Sea.

The roles of individual bacterioplankton species in the re-mineralization of algal biomass are poorly understood. Evidence from molecular data had indicated that a spring diatom bloom in the German Bight of the North Sea in 2009 was followed by a rapid succession of uncultivated bacterioplankton species, including members of the genera Ulvibacter, Formosa, Polaribacter (class Flavobacteria) and Reinekea (class Gammaproteobacteria). We isolated strains from the same site during the diatom bloom in spring 2010 using dilution cultivation in an artificial seawater medium with micromolar substrate and nutrient concentrations. Flow cytometry demonstrated growth from single cells to densities of 10(4) -10(6) cells ml(-1) and a culturability of 35%. Novel Formosa, Polaribacter and Reinekea strains were isolated and had 16S rRNA gene sequence identities of > 99.8% with bacterioplankton in spring or summer 2009. Genomes of selected isolates were draft sequenced and used for read recruitment of metagenomes from bacterioplankton in 2009. Metagenome reads covered 93% of a Formosa clade B, 91% of a Reinekea and 74% of a Formosa clade A genome, applying a ≥ 94.5% nucleotide identity threshold. These novel strains represent abundant bacterioplankton species thriving on coastal phytoplankton blooms in the North Sea.

Isolation and characterisation of the trichocysts of the dinophyte Prorocentrum micans.

Trichocyst-enriched fractions were isolated from the marine dinophyte Prorocentrum micans. Transmission electron microscopy revealed that most of the trichocysts were discharged and had elongated to long filaments. Some trichocysts were still condensed. Fragments of discharged trichocysts measured up to 20 µm in length and 260 nm in width, those still condensed measured up to 1 µm in width and 16 µm in length. A distinct banding pattern with a transversal periodicity of approximately 16-18 nm and a periodic longitudinal striation of 3-4 nm could be measured along the trichocyst filaments. At higher magnifications, a fragile, alveolated, net-like organisation became obvious which resembled the one shown for the trichocysts of ciliates. When trichocyst-enriched fractions were treated with sodium dodecyl sulfate and centrifuged subsequently, no trichocysts were registered any longer in the sodium dodecyl sulfate-insoluble fraction by electron microscopy. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of trichocyst-enriched fractions and of the SDS-soluble fractions revealed a protein banding pattern which was dominated by polypeptides of 50-30, 12.5, and approximately 8.5 kDa. The polypeptide banding pattern deviated significantly from those registered for ejectisomes of cryptophytes and of the prasinophyte Pyramimonas grossii, for the Reb polypeptides which constitute the R-bodies of Caedibacter taeniospiralis, and also from the banding pattern of trichocysts of Paramecium. An antiserum directed against trichocysts of Paramecium did not cross-react with the polypeptides present in the trichocyst-enriched fraction of Prorocentrum micans.

Further investigations on the polypeptides and reconstitution of prasinophycean ejectisomes.

Ejectisome fragments were isolated from the prasinophyte Pyramimonas grossii and subjected to different treatments, i.e. Percoll density gradient centrifugation, incubation at pH 2.5 or at pH 10.8, or incubation in 6M guanidine hydrochloride. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that Percoll density gradient centrifugation did not improve the purity of the ejectisome fragment-enriched fractions. The ejectisome fragments withstood pH 2.5 and pH 10.8 treatment, and no loosely bound polypeptides became detached. The disintegration of ejectisome fragments was achieved in 6M guanidine hydrochloride, and reassembly into filamentous, ejectisome-like structures occurred after dialysis against distilled water. Fractions enriched either in ejectisome fragments or in reconstituted ejectisome-like structures were dominated by three polypeptides with relative molecular weights of approximately 12.5-19kDa and two additional polypeptides of 23 and 26kDa. A polyclonal antiserum directed against an ejectisome fragment-enriched fraction weakly cross-reacted with these polypeptides, and no significant immuno-labelling of ejectisome fragments was registered. A positive immuno-label was achieved using immunoglobulin (IgG) fractions which were gained by selectively incubating nitrocellulose stripes of these polypeptides with the antiserum.

Planktomarina temperata gen. nov., sp. nov., belonging to the globally distributed RCA cluster of the marine Roseobacter clade, isolated from the German Wadden Sea.

Four heterotrophic bacterial strains belonging to the globally distributed marine RCA (Roseobacter clade-affiliated) cluster (family Rhodobacteraceae, class Alphaproteobacteria) were obtained from coastal seawater samples. Strain RCA23(T) was isolated from a 10(-7) dilution culture inoculated with seawater from the German Wadden Sea (southern North Sea), reflecting the high abundance of RCA bacteria in this habitat. Strains IMCC1909, IMCC1923 and IMCC1933 were isolated from diluted seawater (10(-3)) of the Yellow Sea, South Korea. Based on 16S rRNA gene sequence comparison, Octadecabacter antarcticus 307(T) is the closest described relative of the RCA strains, with 95.4-95.5 % sequence similarity. Cells of RCA23(T), IMCC1909, IMCC1923 and IMCC1933 are small motile rods requiring sodium ions. Optimal growth of RCA23(T) occurs at 25 °C and within a very narrow pH range (pH 7-8, optimum pH 7.5). The DNA G+C base content of RCA23(T) is 53.67 mol%. The major respiratory lipoquinone is ubiquinone-10 (Q-10) and the dominant fatty acids (>1 %) are 12 : 1 3-OH, 16 : 1ω7c, 16 : 0, 18 : 1ω7c, 18 : 0 and 11-methyl 18 : 1ω7c. The polar lipid pattern indicated the presence of phosphatidylglycerol, two unidentified aminolipids and two unidentified phospholipids. On marine agar, RCA23(T) forms non-pigmented, transparent to light beige, small (<1 mm), circular, convex colonies. Strain RCA23(T) harbours all genes for the production of bacteriochlorophyll a (BChl a). Genes encoding the light-harvesting reaction centre of BChl a (pufM) were identified in all RCA strains. No visible pigmentation was observed for any of the strains under laboratory conditions, but spectrophotometric analysis revealed weak production of BChl a by RCA23(T). Morphological, physiological and genotypic features of strain RCA23(T) suggest that it represents a novel species of a new genus within the Rhodobacteraceae, for which we propose the name Planktomarina temperata gen. nov., sp. nov., described previously by Giebel et al. [ISME J 5 (2011), 8-19] as 'Candidatus Planktomarina temperata'. The type strain of Planktomarina temperata is RCA23(T) ( = DSM 22400(T) = JCM 18269(T)).

Isolation and characterization of the ejectisomes of the prasinophyte Pyramimonas grossii.

For the first time, ejectisome-enriched fractions were isolated from the marine prasinophyte Pyramimonas grossii. Transmission electron microscopy revealed that most of the ejectisomes were discharged and formed long, spirally twisted filaments. Some ejectisomes were still fully or partly furled. Discharged ejectisomes measured up to 26 µm in length and 200 nm in width; those still furled measured up to 900 nm in width and 1,000 nm in length. Particle periodicities of approximately 4.2 and 5.8 nm could be measured from freeze-fractured filaments. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a protein banding pattern, dominated by polypeptides of 16-20 kDa. These polypeptides were not glycosylated and did not cross-react with antisera directed against recombinant R-body polypeptides of Caedibacter taeniospiralis or directed against reconstituted cryptophycean ejectisomes.