Physiological plasticity of marine phytoplankton revealed by untargeted metabolomics

The majority of the earth's surface is covered by oceans. Within their photic zones, microscopically small algae are fixing carbon dioxide in form of organic molecules, thus building the base of the marine food web. The metabolic plasticity of these algae allows them to respond to ecological changes such as grazing, thereby increasing their fitness and survival. However, most of the metabolites produced by microalgae are not yet known. State-of-the-art metabolomics techniques allow the untargeted and sensitive analysis of intra- and extracellular metabolites both in laboratory cultures and the ocean. The aim of this thesis was to describe physiological plasticity of marine microalgae with regard to grazing interactions. Thus, I conducted a comprehensive review on metabolomics in the field of chemical ecology, and introduced a systematically optimized and standardized protocol for the metabolic analysis of marine algae. Furthermore, I developed a UHPLC-APCI-HRMS analysis for the simultaneous profiling of volatile and non-volatile oxylipins - two compound classes that are released by microalgae as grazing defence, and that had to be analysed separately in the past. To better understand the influence of algal physiology on trophic interactions with marine grazers, I described the physiological plasticity of the abundant cold-water species Phaeocystis pouchetii in laboratory cultures, identified by an untargeted metabolomics approach endo- and exometabolites that are potential physiological markers for different growth phases, and used these markers to characterize natural P. pouchetii blooms in the Northeast Atlantic. In addition, I showed that during filtration of P. pouchetii metabolites can be released which influence the specific growth of marine bacteria. In summary, this thesis provides new insights into the role of physiological and thus functional parameters in the ecology of marine algal communities.Der Großteil der Erde wird von Meerwasser bedeckt, in welchem mikroskopisch kleine Algen Kohlenstoffdioxid in Form organischer Moleküle fixieren, und somit die Basis der marinen Nahrungskette bilden. Ihr plastischer Zellstoffwechsel ermöglicht es Algen, auf ökologische Veränderungen wie z.B. Fraß zu reagieren, und erhöht somit deren Fitness und Überleben. Moderne Metabolomics-Methoden erlauben die nichtselektive, sensitive Messung intra- und extrazellulärer Metabolite sowohl in Laborkulturen als auch im Meer. Das Ziel dieser Arbeit war es, die physiologische Plastizität mariner Mikroalgen in besonderem Hinblick auf Räuber-Beute-Beziehungen zu beschreiben. So verfasste ich einen Übersichtsartikel zur Anwendung von Metabolomics im Bereich der chemischen Ökologie, und stellte ein systematisch optimiertes und standardisiertes Protokoll zur metabolomischen Analyse mariner Algen in Form eines Buchkapitels zusammen. Weiterhin entwickelte ich eine UHPLC-APCI-HRMS Methode zur simultanen Messung flüchtiger und nichtflüchtiger Oxylipine - zwei Substanzklassen, die von Kieselalgen zum Fraßschutz freigesetzt werden, und die in der Vergangenheit nur getrennt analysiert werden konnten. Um den Einfluss der Zellphysiologie auf trophische Interaktionen mit marinem Zooplankton besser zu verstehen, beschrieb ich die physiologische Plastizität der abundanten Kaltwasserart Phaeocystis pouchetii in Laborkulturen, identifizierte mithilfe einer nichtselektiven Metabolomanalytik intra- und extrazelluläre Metabolite, die sich als physiologische Marker für verschiedene Wachstumsphasen eignen, und nutzte diese Marker zur Charakterisierung natürlicher P. pouchetii-Blüten im Nordostatlantik. Weiterhin zeigte ich, dass bei Filtration von P. pouchetii Stoffwechselmetabolite freigesetzt werden, welche das spezifische Wachstum von Bakterien beeinflussen. Diese Arbeit gewährt neue Einblicke in die Bedeutung physiologischer und somit funktioneller Parameter für die Ökologie mariner Algengemeinschaften

Grazing on Marine Viruses and Its Biogeochemical Implications

Viruses are the most abundant biological entities in the ocean and show great diversity in terms of size, host specificity, and infection cycle. Lytic viruses induce host cell lysis to release their progeny and thereby redirect nutrients from higher to lower trophic levels. Studies continue to show that marine viruses can be ingested by nonhost organisms. However, not much is known about the role of viral particles as a nutrient source and whether they possess a nutritional value to the grazing organisms. This review seeks to assess the elemental composition and biogeochemical relevance of marine viruses, including roseophages, which are a highly abundant group of bacteriophages in the marine environment. We place a particular emphasis on the phylum Nucleocytoviricota (NCV) (formerly known as nucleocytoplasmic large DNA viruses [NCLDVs]), which comprises some of the largest viral particles in the marine plankton that are well in the size range of prey for marine grazers. Many NCVs contain lipid membranes in their capsid that are rich carbon and energy sources, which further increases their nutritional value. Marine viruses may thus be an important nutritional component of the marine plankton, which can be reintegrated into the classical food web by nonhost organism grazing, a process that we coin the “viral sweep.” Possibilities for future research to resolve this process are highlighted and discussed in light of current technological advancements.publishedVersio

Bacterial lifestyle switch in response to algal metabolites

Unicellular algae, termed phytoplankton, greatly impact the marine environment by serving as the basis of marine food webs and by playing central roles in the biogeochemical cycling of elements. The interactions between phytoplankton and heterotrophic bacteria affect the fitness of both partners. It is becoming increasingly recognized that metabolic exchange determines the nature of such interactions, but the underlying molecular mechanisms remain underexplored. Here, we investigated the molecular and metabolic basis for the bacterial lifestyle switch, from coexistence to pathogenicity, in Sulfitobacter D7 during its interaction with Emiliania huxleyi, a cosmopolitan bloom-forming phytoplankter. To unravel the bacterial lifestyle switch, we analyzed bacterial transcriptomes in response to exudates derived from algae in exponential growth and stationary phase, which supported the Sulfitobacter D7 coexistence and pathogenicity lifestyles, respectively. In pathogenic mode, Sulfitobacter D7 upregulated flagellar motility and diverse transport systems, presumably to maximize assimilation of E. huxleyi-derived metabolites released by algal cells upon cell death. Algal dimethylsulfoniopropionate (DMSP) was a pivotal signaling molecule that mediated the transition between the lifestyles, supporting our previous findings. However, the coexisting and pathogenic lifestyles were evident only in the presence of additional algal metabolites. Specifically, we discovered that algae-produced benzoate promoted the growth of Sulfitobacter D7 and hindered the DMSP-induced lifestyle switch to pathogenicity, demonstrating that benzoate is important for maintaining the coexistence of algae and bacteria. We propose that bacteria can sense the physiological state of the algal host through changes in the metabolic composition, which will determine the bacterial lifestyle during interaction.ISSN:2050-084

A simple adjustment to test reliability of bacterivory rates derived from the dilution method

Quantification of grazing losses of marine heterotrophic bacteria is critical for understanding nutrient and carbon pathways in aquatic systems. The dilution method is a commonly used experimental approach for quantifying bacterivory. However, valid estimates of grazing rates obtained using this method depend on several methodological assumptions including that the method does not influence specific growth rates of bacteria. Here, we hypothesize that filtration during the set-up of a dilution experiment has the potential to release allelochemicals from phytoplankton cells and thereby stimulate or inhibit bacterial growth with the consequence of biased grazing estimates. We tested this hypothesis during a natural Phaeocystis pouchetii bloom at two different locations within an Arctic fjord. Results from the dilution experiments suggest higher gross growth rate and grazing impact for bacteria in the outer fjord compared with the inner fjord. However, specific growth rates estimated by bacterial production cell−1 were significantly elevated in dilutions of water from the outer fjord but not the inner fjord. The analysis of dissolved metabolites in the seawater from both experiments prior and after filtration revealed altered metabolic profiles after filtration at both stations. As unaffected specific growth of prey on dilution is one of three fundamental assumptions of the dilution method, we conclude that it is important that empirically estimated bacterial specific growth rates be routinely included when using the dilution method to quantify bacterivory

Microsatellite Stable Colorectal Cancers in Clinically Suspected Hereditary Nonpolyposis Colorectal Cancer Patients without Vertical Transmission of Disease Are Unlikely to Be Caused by Biallelic Germline Mutations in MYH

Microsatellite analysis and immunohistochemistry are commonly used initial screening tests for hereditary nonpolyposis colorectal cancer. However, tumors in roughly one-half of the patients fulfilling the Bethesda guidelines are microsatellite stable. In addition, normal mismatch repair protein expression in these tumors suggests that a defect in the mismatch repair system is unlikely. Because biallelic MYH mutations occur in patients with both high and low numbers of adenomas, we hypothesized that MYH is involved in the tumorigenesis of microsatellite stable colorectal cancers in patients without vertical transmission of disease and who fulfill the Bethesda guidelines. MYH was analyzed in 50 cancer patients and 116 healthy controls by complete genomic DNA sequencing. No biallelic germline mutations were identified. One patient was a heterozygous carrier for the p.G382D missense mutation, and another patient was a heterozygous carrier for the novel missense mutation p.Q484H. We identified six common variants, three in the coding region (p.V22M, p.Q324H, and p.S501F) and three in adjacent intronic regions (c.157+30A>G, c.462+35G>A, and c.1435–40G>C). In summary, biallelic germline mutations of MYH are unlikely to cause colorectal cancer in patients sharing clinical features with hereditary nonpolyposis colorectal cancer families without mismatch repair defect and therefore cannot fill the molecular diagnostic gap in this subgroup of Bethesda-positive patients

Viral infection switches the balance between bacterial and eukaryotic recyclers of organic matter during coccolithophore blooms

Algal blooms are hotspots of marine primary production and play central roles in microbial ecology and global elemental cycling. Upon demise of the bloom, organic carbon is partly respired and partly transferred to either higher trophic levels, bacterial biomass production or sinking. Viral infection can lead to bloom termination, but its impact on the fate of carbon remains largely unquantified. Here, we characterize the interplay between viral infection and the composition of a bloom-associated microbiome and consequently the evolving biogeochemical landscape, by conducting a large-scale mesocosm experiment where we monitor seven induced coccolithophore blooms. The blooms show different degrees of viral infection and reveal that only high levels of viral infection are followed by significant shifts in the composition of free-living bacterial and eukaryotic assemblages. Intriguingly, upon viral infection the biomass of eukaryotic heterotrophs (thraustochytrids) rivals that of bacteria as potential recyclers of organic matter. By combining modeling and quantification of active viral infection at a single-cell resolution, we estimate that viral infection causes a 2–4 fold increase in per-cell rates of extracellular carbon release in the form of acidic polysaccharides and particulate inorganic carbon, two major contributors to carbon sinking into the deep ocean. These results reveal the impact of viral infection on the fate of carbon through microbial recyclers of organic matter in large-scale coccolithophore blooms

Viral infection switches the balance between bacterial and eukaryotic recyclers of organic matter during coccolithophore blooms

Algal blooms are hotspots of marine primary production and play central roles in microbial ecology and global elemental cycling. Upon demise of the bloom, organic carbon is partly respired and partly transferred to either higher trophic levels, bacterial biomass production or sinking. Viral infection can lead to bloom termination, but its impact on the fate of carbon remains largely unquantified. Here, we characterize the interplay between viral infection and the composition of a bloom-associated microbiome and consequently the evolving biogeochemical landscape, by conducting a large-scale mesocosm experiment where we monitor seven induced coccolithophore blooms. The blooms show different degrees of viral infection and reveal that only high levels of viral infection are followed by significant shifts in the composition of free-living bacterial and eukaryotic assemblages. Intriguingly, upon viral infection the biomass of eukaryotic heterotrophs (thraustochytrids) rivals that of bacteria as potential recyclers of organic matter. By combining modeling and quantification of active viral infection at a single-cell resolution, we estimate that viral infection causes a 2–4 fold increase in per-cell rates of extracellular carbon release in the form of acidic polysaccharides and particulate inorganic carbon, two major contributors to carbon sinking into the deep ocean. These results reveal the impact of viral infection on the fate of carbon through microbial recyclers of organic matter in large-scale coccolithophore blooms