Klaus-Ulrich Valentin

Picoeukaryotic Plankton Diversity at the Helgoland Time Series Site as Assessed by Three Molecular Methods

We analyzed picoeukaryote assemblages in the German Bight at the Helgoland time series site by sequencing cloned eukaryotic 18S rRNA genes in six genetic libraries plus one library from the Orkney Islands from a cruise of opportunity. The libraries were constructed from environmental samples collected at different periods of the year. The same samples were also analyzed using a fingerprinting technique, single-strand conformational polymorphism (SSCP), and DNA microarrays with class-level oligonucleotide probes. One hundred unique clones were analyzed from each library, thus insuring over 85% coverage of the library. The V4 region of the 18S rRNA gene was sequenced from each of these clones, thus providing the most discrimination among the clones. The nonphotosynthetic picoeukaryotic component dominated over the photosynthetic one and was represented by the ciliates at 45% and group II alveolates at 42%. Prasinophytes dominated the photosynthetic group at 40%, but other picoplankton groups, such as bolidomonads and chrysophytes, were also present. Totally novel groups were found in the cryptomonads and in the dinoflagellates. A new algal group sister to the cryptophyte nuclear gene and the glaucocystophytes was also found. These three groups have been found in other picoeukaryotic planktonic clone libraries. SSCP analyses at closer time intervals suggest that clone libraries should be made at weekly intervals if succession in the picoeukaryotic plankton community is to be monitored accurately. A comparison of annual samples suggests thatthere appears to be an annual cycle with regard to species composition. Microarray analysis supported the clone library data and offered a faster means of community analysis, which can be performed with similar accuracy and with higher throughput for a more in-depth analysis.

Antifreeze proteins in polar sea ice diatoms: diversity and gene expression in the genus Fragilariopsis

Fragilariopsis is a dominating psychrophilic diatom genus in polar sea ice. The two species Fragilariopsis cylindrus and Fragilariopsis curta are able to grow and divide below freezing temperature of sea water and above average sea water salinity. Here we show that antifreeze proteins (AFPs), involved in cold adaptation in several psychrophilic organisms, are widespread in the two polar species. The presence of AFP genes (afps) as a multigene family indicated the importance of this group of genes for the genus Fragilariopsis, possibly contributing to its success in sea ice. Protein phylogeny showed the potential mobility of afps, which appear to have crossed kingdom and domain borders, occurring in Bacteria, diatoms, crustaceans and fungi. Our results revealed a broad distribution of AFPs not only in polar organisms but also in taxa apparently not related to cold environments, suggesting that these proteins may be multifunctional. The relevance of AFPs to Fragilariopsis was also shown by gene expression analysis. Under stress conditions typical for sea ice, with subzero temperatures and high salinities, F. cylindrus and F. curta strongly expressed selected afps. An E/G point mutation in the Fragilariopsis AFPs may play a role in gene expression activity and protein function.

PHOTOSYNTHESIS AND COLD ACCLIMATION: MOLECULAR EVIDENCE FROM A POLAR DIATOM

The psychrophilic diatom Fragilariopsis cylindrus (Grunow) Krieger in Helmcke & Krieger was used to investigate photosynthesis and growth under freezing temperatures. Gene expression during a temperature shift from +5° C to −1.8° C was studied under 3 and 35 μmol photons·m−2·s−1 by using a macroarray. These measurements were paralleled by determination of fluorescence induction at PSII and pigment analysis. The shift to −1.8° C at 35 μmol photons·m−2·s−1 caused a marginal decrease of photosynthetic quantum yield (Fv/Fm) from 0.61 to 0.52 with fast recovery after 1 day. The ratio of chl c to chl a increased from 3.1 to 5.5, and the ratio of diatoxanthin to diadinoxanthin increased from 0.7 to 5.0. Genes encoding proteins of PSII (psbA, psbC) and for carbon fixation (rbcL) were down‐regulated, whereas genes encoding chaperons (hsp70) and genes for plastid protein synthesis and turnover (elongation factor EfTs, ribosomal protein rpS4, ftsH protease) were up‐regulated. In contrast, cold exposure at 3 μmol photons·m−2·s−1 induced a marginal increase in Fv/Fm from 0.61 to 0.63 and a strong increase in fucoxanthin concentrations from 0.04 up to 0.12 pg·cell−1. This was paralleled by up‐regulation of fcp genes. The ratio of chl c to chl a also increased from 3.1 to 4.2, as did the ratio of diatoxanthin to diadinoxanthin from 0.7 to 2.2. Down‐regulation of psbA, psbC, and rbcL could also be measured but not up‐regulation of hsp70, EfTs, rpS4, and the ftsH protease. The latter genes are probably necessary to avoid cold shock photoinhibition only at higher light intensities.

Transcriptomic Analysis of Acclimation to Temperature and Light Stress in Saccharina latissima (Phaeophyceae)

Kelps, brown algae of the order Laminariales, dominate rocky shores and form huge kelp beds which provide habitat and nurseries for various marine organisms. Whereas the basic physiological and ecophysiological characteristics of kelps are well studied, the molecular processes underlying acclimation to different light and temperature conditions are still poorly understood. Therefore we investigated the molecular mechanisms underlying the physiological acclimation to light and temperature stress. Sporophytes of S. latissima were exposed to combinations of light intensities and temperatures, and microarray hybridizations were performed to determine changes in gene expression patterns. This first large-scale transcriptomic study of a kelp species shows that S. latissima responds to temperature and light stress with a multitude of transcriptional changes: up to 32% of genes showed an altered expression after the exposure experiments. High temperature had stronger effects on gene expression in S. latissima than low temperature, reflected by the higher number of temperature-responsive genes. We gained insights into underlying molecular processes of acclimation, which includes adjustment of the primary metabolism as well as induction of several ROS scavengers and a sophisticated regulation of Hsps. We show that S. latissima, as a cold adapted species, must make stronger efforts for acclimating to high than to low temperatures. The strongest response was caused by the combination of high temperatures with high light intensities, which proved most harmful for the alga.

A new class of ice-binding proteins discovered in a salt-stress-induced cDNA library of the psychrophilic diatom Fragilariopsis cylindrus (Bacillariophyceae)

The psychrophilic pennate diatom Fragilariopsis cylindrus is a dominant polar diatom adapted to grow at extremely low temperatures and high salinities prevailing in the brine channels of sea ice. As a basis for deeper investigations of this physiological trait, we used an EST approach to find candidate genes involved in acclimation to salt stress in this diatom. From 2880 cDNA clones sequenced from the 5′ end, 1691 high-quality tentative unique sequences were assembled and analysed. Only 62% of these sequences have homologues in the genomes of two mesophilic diatoms, Thalassiosira pseudonana (centric) and Phaeodactylum tricornutum (pennate), which share 73–78% of their genes. Of the 1691 sequences, 44.2% could be functionally characterized by comparison with the Swiss-Prot and RefSeq databases. These include sequences encoding different ionic transporters and antiporters, reflecting the requirement to re-establish the ion homeostasis disturbed by exogenous salt stress. Furthermore, numerous genes encoding heat shock proteins (hsps), genes related to oxidative stress, and three key genes involved in the proline synthesis pathway, the most important organic osmolyte synthesized in F. cylindrus were identified. A major outcome of this analysis is the finding of four full-length ORFs showing significant similarities to ice-binding proteins (IBPs), which have been shown in a parallel study to be specific to sea ice diatoms, giving evidence of their ability to shape their habitat.

Genome Variations Associated with Viral Susceptibility and Calcification in Emiliania huxleyi

Emiliania huxleyi, a key player in the global carbon cycle is one of the best studied coccolithophores with respect to biogeochemical cycles, climatology, and host-virus interactions. Strains of E. huxleyi show phenotypic plasticity regarding growth behaviour, light-response, calcification, acidification, and virus susceptibility. This phenomenon is likely a consequence of genomic differences, or transcriptomic responses, to environmental conditions or threats such as viral infections. We used an E. huxleyi genome microarray based on the sequenced strain CCMP1516 (reference strain) to perform comparative genomic hybridizations (CGH) of 16 E. huxleyi strains of different geographic origin. We investigated the genomic diversity and plasticity and focused on the identification of genes related to virus susceptibility and coccolith production (calcification). Among the tested 31940 gene models a core genome of 14628 genes was identified by hybridization among 16 E. huxleyi strains. 224 probes were characterized as specific for the reference strain CCMP1516. Compared to the sequenced E. huxleyi strain CCMP1516 variation in gene content of up to 30 percent among strains was observed. Comparison of core and non-core transcripts sets in terms of annotated functions reveals a broad, almost equal functional coverage over all KOG-categories of both transcript sets within the whole annotated genome. Within the variable (non-core) genome we identified genes associated with virus susceptibility and calcification. Genes associated with virus susceptibility include a Bax inhibitor-1 protein, three LRR receptor-like protein kinases, and mitogen-activated protein kinase. Our list of transcripts associated with coccolith production will stimulate further research, e.g. by genetic manipulation. In particular, the V-type proton ATPase 16 kDa proteolipid subunit is proposed to be a plausible target gene for further calcification studies.

rRNA and rDNA based assessment of sea ice protist biodiversity from the central Arctic Ocean

Abstract Sea ice is a large and diverse ecosystem contributing significantly to primary production in ice-covered regions. In the Arctic Ocean, sea ice consists of mixed multi-year ice (MYI), often several metres thick, and thinner first-year ice (FYI). Current global warming is most severe in Arctic regions; as a consequence, summer sea ice cover is decreasing and MYI is disappearing at an alarming rate. Despite its apparent hostility, sea ice is inhabited by a diverse microbial community of bacteria and protists, many of which are photosynthetic. Here we present an assessment of eukaryotic biodiversity in MYI and FYI from the central Arctic Ocean using high-throughput 454 sequencing of 18S rRNA and rDNA amplicons. We compared the rDNA-based ‘total’ biodiversity with the ‘active’ biodiversity from rRNA amplicons and found differences between them including an over-representation of Ciliophora, Bicosoecida and Bacillariophyceae operational taxonomic units (OTUs) in the active part of the community. Differences between the two libraries are more pronounced at the lower taxonomic level: certain genera, such as Melosira, are more abundant in the rRNA library, indicating activity of these genera. Furthermore, we found that one FYI station showed a higher activity of potential grazers which was probably due to the advanced stage of melt evident by higher ice temperatures and highly porous ice compared with the other stations.

Nucleic acid isolation from environmental aqueous samples

The application of molecular techniques has revolutionized freshwater and marine ecology, especially for plankton research. Methods, such as denatured gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), and single-strand conformation polymorphism (SSCP), together with environmental clone libraries, have unraveled an unexpected biodiversity of organisms in the water column. Molecular probes are just entering the field of commercialization for monitoring toxic algal blooms. Genomics and metagenomics were recently introduced into marine biology. At the basis of all molecular approaches is the isolation of nucleic acids from cultures, tissue, or environmental samples. Here, we summarize methods, quality controls, and hints for sample treatment to reliably isolate nucleic acids, both DNA and RNA, from environmental aqueous samples. This chapter not only is directed to researchers inexperienced with such methods but also is an aid to those already working in the field. It may be used as a step-by-step guide for nucleic acid isolation from field samples, and we make suggestions for subsequent use of the DNA/RNA.

Heterologous expression, refolding and functional characterization of two antifreeze proteins from Fragilariopsis cylindrus (Bacillariophyceae)

Antifreeze proteins (AFPs) provide protection for organisms subjected to the presence of ice crystals. The psychrophilic diatom Fragilariopsis cylindrus which is frequently found in polar sea ice carries a multitude of AFP isoforms. In this study we report the heterologous expression of two antifreeze protein isoforms from F. cylindrus in Escherichia coli. Refolding from inclusion bodies produced proteins functionally active with respect to crystal deformation, recrystallization inhibition and thermal hysteresis. We observed a reduction of activity in the presence of the pelB leader peptide in comparison with the GS-linked SUMO-tag. Activity was positively correlated to protein concentration and buffer salinity. Thermal hysteresis and crystal deformation habit suggest the affiliation of the proteins to the hyperactive group of AFPs. One isoform, carrying a signal peptide for secretion, produced a thermal hysteresis up to 1.53°C±0.53°C and ice crystals of hexagonal bipyramidal shape. The second isoform, which has a long preceding N-terminal sequence of unknown function, produced thermal hysteresis of up to 2.34°C±0.25°C. Ice crystals grew in form of a hexagonal column in presence of this protein. The different sequences preceding the ice binding domain point to distinct localizations of the proteins inside or outside the cell. We thus propose that AFPs have different functions in vivo, also reflected in their specific TH capability.

In situ expression of eukaryotic ice-binding proteins in microbial communities of Arctic and Antarctic sea ice

Ice-binding proteins (IBPs) have been isolated from various sea-ice organisms. Their characterisation points to a crucial role in protecting the organisms in sub-zero environments. However, their in situ abundance and diversity in natural sea-ice microbial communities is largely unknown. In this study, we analysed the expression and phylogenetic diversity of eukaryotic IBP transcripts from microbial communities of Arctic and Antarctic sea ice. IBP transcripts were found in abundances similar to those of proteins involved in core cellular processes such as photosynthesis. Eighty-nine percent of the IBP transcripts grouped with known IBP sequences from diatoms, haptophytes and crustaceans, but the majority represented novel sequences not previously characterized in cultured organisms. The observed high eukaryotic IBP expression in natural eukaryotic sea ice communities underlines the essential role of IBPs for survival of many microorganisms in communities living under the extreme conditions of polar sea ice.