A novel cost effective and high-throughput isolation and identification method for marine microalgae

Background: Marine microalgae are of major ecologic and emerging economic importance. Biotechnological screening schemes of microalgae for specific traits and laboratory experiments to advance our knowledge on algal biology and evolution strongly benefit from culture collections reflecting a maximum of the natural inter- and intraspecific diversity. However, standard procedures for strain isolation and identification, namely DNA extraction, purification, amplification, sequencing and taxonomic identification still include considerable constraints increasing the time required to establish new cultures. Results: In this study, we report a cost effective and high-throughput isolation and identification method for marine microalgae. The throughput was increased by applying strain isolation on plates and taxonomic identification by direct PCR (dPCR) of phylogenetic marker genes in combination with a novel sequencing electropherogram based screening method to assess the taxonomic diversity and identity of the isolated cultures. For validation of the effectiveness of this approach, we isolated and identified a range of unialgal cultures from natural phytoplankton communities sampled in the Arctic Ocean. These cultures include the isolate of a novel marine Chlorophyceae strain among several different diatoms. Conclusions: We provide an efficient and effective approach leading from natural phytoplankton communities to isolated and taxonomically identified algal strains in only a few weeks. Validated with sensitive Arctic phytoplankton, this approach overcomes the constraints of standard molecular characterisation and establishment of unialgal cultures

Physiology, syntrophy and viral interplay in the marine sponge holobiont

Holobionts result from intimate associations of eukaryotic hosts and microbes and are now widely accepted as ubiquitous and important elements of nature. Marine sponge holobionts combine simple morphology and complex microbiology whilst diverging early in the animal kingdom. As filter feeders, sponges feed on planktonic bacteria, but also harbour stable species-specific microbial consortia. This interaction with bacteria renders sponges to exciting systems to study basal determinants of animal-microbe symbioses. While inventories of symbiont taxa and gene functions continue to grow, we still know little about the symbiont physiology, cellular interactions and metabolic currencies within sponges. This limits our mechanistic understanding of holobiont stability and function. Therefore, this PhD thesis set out to study the questions of what individual symbionts actually do and how they interact. The first part of this thesis focuses on the cell physiology of cosmopolitan sponge symbionts. For the first time, I characterised the ultrastructure of dominant sponge symbiont clades within sponge tissue by establishing fluorescence in situ hybridization-correlative light and electron microscopy (FISH-CLEM). In combination with genome-centred metatranscriptomics, this approach revealed structural adaptations of symbionts to process complex holobiont-derived nutrients (i.e., bacterial microcompartments and bipolar storage polymers). Next, we unravelled complementary symbiont physiologies and cell co-localisation indicating vivid symbiont-symbiont metabolic interactions within the holobiont. This suggests strategies of nutritional resource partitioning and syntrophy to dominate over spatial segregation to avoid competitive exclusion- a mechanistic framework to sustain high microbial diversity. By combining stable isotope pulse-chase experiments with metabolic imaging, we demonstrated that symbionts can account for up to 60 % of the heterotrophic carbon and nitrogen assimilation in sponges. Thus, sponge symbiont action determines sponge-driven biochemical cycles in marine ecosystems. Finally, I explored the role of phages in the sponge holobiont focussing on tripartie phage-microbe-host interplay. Sponges appeared as rich reservoirs of novel viral diversity with 491 previously unidentified genus-level viral clades. Further, sponges harboured highly individual, yet species-specific viral communities. Importantly, I discovered that phages, termed “Ankyphages”, abundantly encode ankyrin proteins. Such “Ankyphages” I found to be widespread in host-associated environments, including humans. Using macrophage infection assays I showed that phage ankyrins aid bacteria in eukaryote immune evasion by downregulating eukaryotic antibacterial immunity. Thus, I identified a potentially widespread mechanism of tripartite phage-prokaryote-host interplay where phages foster animal-microbe symbioses. Altogether, I draw three main conclusions: The sponge holobiont is a metabolically intertwined ecosystem, with symbiont action impacting the environment, and tripartite phage-prokaryote-eukaryote interplay fostering symbiosis

Confinement at Weak Coupling

The free energy of U(N) and SU(N) gauge theory was recently found to be of order N^0 to all orders of a perturbative expansion about a center-symmetric orbit of vanishing curvature. Here I consider extended models for which this expansion is perturbatively stable. The extreme case of an SU(2) gauge theory whose configuration space is restricted to center-symmetric orbits has recently been investigated on the lattice hep-lat/0509156. In extension of my talk, a discussion and possible interpretation of the observed finite temperature phase transition is given. The transfer matrix of constrained SU(N) lattice gauge theory is constructed for any finite temperature.Comment: 8 pages, no figures, updated talk given at LC2005 in Cairns, Australi

Heterotrophy in the earliest gut: a single-cell view of heterotrophic carbon and nitrogen assimilation in sponge-microbe symbioses

Sponges are the oldest known extant animal-microbe symbiosis. These ubiquitous benthic animals play an important role in marine ecosystems in the cycling of dissolved organic matter (DOM), the largest source of organic matter on Earth. The conventional view on DOM cycling through microbial processing has been challenged by the interaction between this efficient filter-feeding host and its diverse and abundant microbiome. Here we quantify, for the first time, the role of host cells and microbial symbionts in sponge heterotrophy. We combined stable isotope probing and nanoscale secondary ion mass spectrometry to compare the processing of different sources of DOM (glucose, amino acids, algal-produced) and particulate organic matter (POM) by a high-microbial abundance (HMA) and low-microbial abundance (LMA) sponge with single-cell resolution. Contrary to common notion, we found that both microbial symbionts and host choanocyte (i.e. filter) cells and were active in DOM uptake. Although all DOM sources were assimilated by both sponges, higher microbial biomass in the HMA sponge corresponded to an increased capacity to process a greater variety of dissolved compounds. Nevertheless, in situ feeding data demonstrated that DOM was the primary carbon source for both the LMA and HMA sponge, accounting for ~90% of their heterotrophic diets. Microbes accounted for the majority (65–87%) of DOM assimilated by the HMA sponge (and ~60% of its total heterotrophic diet) but <5% in the LMA sponge. We propose that the evolutionary success of sponges is due to their different strategies to exploit the vast reservoir of DOM in the ocean

Marine Sponges as Chloroflexi Hot Spots: Genomic Insights and High-Resolution Visualization of an Abundant and Diverse Symbiotic Clade

Members of the widespread bacterial phylum Chloroflexi can dominate high-microbial-abundance (HMA) sponge microbiomes. In the Sponge Microbiome Project, Chloroflexi sequences amounted to 20 to 30% of the total microbiome of certain HMA sponge genera with the classes/clades SAR202, Caldilineae, and Anaerolineae being the most prominent. We performed metagenomic and single-cell genomic analyses to elucidate the functional gene repertoire of Chloroflexi symbionts of Aplysina aerophoba. Eighteen draft genomes were reconstructed and placed into phylogenetic context of which six were investigated in detail. Common genomic features of Chloroflexi sponge symbionts were related to central energy and carbon converting pathways, amino acid and fatty acid metabolism, and respiration. Clade-specific metabolic features included a massively expanded genomic repertoire for carbohydrate degradation in Anaerolineae and Caldilineae genomes, but only amino acid utilization by SAR202. While Anaerolineae and Caldilineae import cofactors and vitamins, SAR202 genomes harbor genes encoding components involved in cofactor biosynthesis. A number of features relevant to symbiosis were further identified, including CRISPR-Cas systems, eukaryote-like repeat proteins, and secondary metabolite gene clusters. Chloroflexi symbionts were visualized in the sponge extracellular matrix at ultrastructural resolution by the fluorescence in situ hybridization-correlative light and electron microscopy (FISH-CLEM) method. Carbohydrate degradation potential was reported previously for “Candidatus Poribacteria” and SAUL, typical symbionts of HMA sponges, and we propose here that HMA sponge symbionts collectively engage in degradation of dissolved organic matter, both labile and recalcitrant. Thus, sponge microbes may not only provide nutrients to the sponge host, but they may also contribute to dissolved organic matter (DOM) recycling and primary productivity in reef ecosystems via a pathway termed the sponge loop. IMPORTANCE Chloroflexi represent a widespread, yet enigmatic bacterial phylum with few cultivated members. We used metagenomic and single-cell genomic approaches to characterize the functional gene repertoire of Chloroflexi symbionts in marine sponges. The results of this study suggest clade-specific metabolic specialization and that Chloroflexi symbionts have the genomic potential for dissolved organic matter (DOM) degradation from seawater. Considering the abundance and dominance of sponges in many benthic environments, we predict that the role of sponge symbionts in biogeochemical cycles is larger than previously thought

A Phage Protein Aids Bacterial Symbionts in Eukaryote Immune Evasion

Highlights: • Sponges, evolutionary basal animals, represent a reservoir of novel viral diversity • Viromes of neighboring sponges are individually unique and species specific • Phages encode ankyrins to aid bacteria in evading the eukaryotic immune system • Such “Ankyphages” are widespread in host-associated environments, including humans Summary: Phages are increasingly recognized as important members of host-associated microbiomes, with a vast genomic diversity. The new frontier is to understand how phages may affect higher order processes, such as in the context of host-microbe interactions. Here, we use marine sponges as a model to investigate the interplay between phages, bacterial symbionts, and eukaryotic hosts. Using viral metagenomics, we find that sponges, although massively filtering seawater, harbor species-specific and even individually unique viral signatures that are taxonomically distinct from other environments. We further discover a symbiont phage-encoded ankyrin-domain-containing protein, which is widely spread in phages of many host-associated contexts including human. We confirm in macrophage infection assays that the ankyrin protein (ANKp) modulates the eukaryotic host immune response against bacteria. We predict that the role of ANKp in nature is to facilitate coexistence in the tripartite interplay between phages, symbionts, and sponges and possibly many other host-microbe associations

An Enrichment of CRISPR and Other Defense-Related Features in Marine Sponge-Associated Microbial Metagenomes

Many marine sponges are populated by dense and taxonomically diverse microbial consortia. We employed a metagenomics approach to unravel the differences in the functional gene repertoire among three Mediterranean sponge species, Petrosia ficiformis, Sarcotragus foetidus, Aplysina aerophoba and seawater. Different signatures were observed between sponge and seawater metagenomes with regard to microbial community composition, GC content, and estimated bacterial genome size. Our analysis showed further a pronounced repertoire for defense systems in sponge metagenomes. Specifically, clustered regularly interspaced short palindromic repeats, restriction modification, DNA phosphorothioation and phage growth limitation systems were enriched in sponge metagenomes. These data suggest that defense is an important functional trait for an existence within sponges that requires mechanisms to defend against foreign DNA from microorganisms and viruses. This study contributes to an understanding of the evolutionary arms race between viruses/phages and bacterial genomes and it sheds light on the bacterial defenses that have evolved in the context of the sponge holobiont

Proteomic Characterization of Cellular and Molecular Processes that Enable the Nanoarchaeum equitans-Ignicoccus hospitalis Relationship

Nanoarchaeum equitans, the only cultured representative of the Nanoarchaeota, is dependent on direct physical contact with its host, the hyperthermophile Ignicoccus hospitalis. The molecular mechanisms that enable this relationship are unknown. Using whole-cell proteomics, differences in the relative abundance of >75% of predicted protein-coding genes from both Archaea were measured to identify the specific response of I. hospitalis to the presence of N. equitans on its surface. A purified N. equitans sample was also analyzed for evidence of interspecies protein transfer. The depth of cellular proteome coverage achieved here is amongst the highest reported for any organism. Based on changes in the proteome under the specific conditions of this study, I. hospitalis reacts to N. equitans by curtailing genetic information processing (replication, transcription) in lieu of intensifying its energetic, protein processing and cellular membrane functions. We found no evidence of significant Ignicoccus biosynthetic enzymes being transported to N. equitans. These results suggest that, under laboratory conditions, N. equitans diverts some of its host's metabolism and cell cycle control to compensate for its own metabolic shortcomings, thus appearing to be entirely dependent on small, transferable metabolites and energetic precursors from I. hospitalis

Observation of a J^PC = 1-+ exotic resonance in diffractive dissociation of 190 GeV/c pi- into pi- pi- pi+

The COMPASS experiment at the CERN SPS has studied the diffractive dissociation of negative pions into the pi- pi- pi+ final state using a 190 GeV/c pion beam hitting a lead target. A partial wave analysis has been performed on a sample of 420000 events taken at values of the squared 4-momentum transfer t' between 0.1 and 1 GeV^2/c^2. The well-known resonances a1(1260), a2(1320), and pi2(1670) are clearly observed. In addition, the data show a significant natural parity exchange production of a resonance with spin-exotic quantum numbers J^PC = 1-+ at 1.66 GeV/c^2 decaying to rho pi. The resonant nature of this wave is evident from the mass-dependent phase differences to the J^PC = 2-+ and 1++ waves. From a mass-dependent fit a resonance mass of 1660 +- 10+0-64 MeV/c^2 and a width of 269+-21+42-64 MeV/c^2 is deduced.Comment: 7 page, 3 figures; version 2 gives some more details, data unchanged; version 3 updated authors, text shortened, data unchange