2-Homoectoine: An Additional Member of the Ectoine Family from Phyto- and Bacterioplankton Involved in Osmoadaptation.

Ectoine is a central osmolyte in marine plankton due to its excellent cytoprotective traits and its multifunctional roles supporting the survival of microalgae and bacteria under unfavorable environmental conditions. The protective effect of ectoine toward several kinds of stresses stirred interest in biotechnology, pharmacy, and other fields including cosmetics. Also, its hydroxylated derivative, 5-hydroxyectoine, exhibits functions similar to ectoine. Here we introduce a molecular networking-based approach to expand the family of ectoine derivatives from phyto- and bacterioplankton. A ZIC-HILIC separation protocol coupled with HRMS/MS-based molecular networking allowed us to identify the new ectoine derivative 1,4,5,6-tetrahydro-2-ethyl-4-pyrimidinecarboxylic acid, or 2-homoectoine (1). 1 is found in many algae including dinoflagellates, chlorophytes, and haptophytes. In axenic strains, the content of 1 is substantially lower. In accordance, we found that marine bacteria are prolific producers of the compound as well. This suggests that the microalgae with their associated microbiome have to be considered as sources of the compound. Increasing concentrations of the compound under high salinity suggest a role as a protectant against osmotic stress.

The metabolite dimethylsulfoxonium propionate extends the marine organosulfur cycle.

Algae produce massive amounts of dimethylsulfoniopropionate (DMSP), which fuel the organosulfur cycle1,2. On a global scale, several petagrams of this sulfur species are produced annually, thereby driving fundamental processes and the marine food web1. An important DMSP transformation product is dimethylsulfide, which can be either emitted to the atmosphere3,4 or oxidized to dimethylsulfoxide (DMSO) and other products5. Here we report the discovery of a structurally unusual metabolite, dimethylsulfoxonium propionate (DMSOP), that is synthesized by several DMSP-producing microalgae and marine bacteria. As with DMSP, DMSOP is a low-molecular-weight zwitterionic metabolite that carries both a positively and a negatively charged functional group. Isotope labelling studies demonstrate that DMSOP is produced from DMSP, and is readily metabolized to DMSO by marine bacteria. DMSOP was found in near nanomolar amounts in field samples and in algal culture media, and thus represents-to our knowledge-a previously undescribed biogenic source for DMSO in the marine environment. The estimated annual oceanic production of oxidized sulfur from this pathway is in the teragram range, similar to the calculated dimethylsulfide flux to the atmosphere3. This sulfoxonium metabolite is therefore a key metabolite of a previously undescribed pathway in the marine sulfur cycle. These findings highlight the importance of DMSOP in the marine organosulfur cycle.

Cell surface composition, released polysaccharides, and ionic strength mediate fast sedimentation in the cyanobacterium Synechococcus elongatus PCC 7942.

Cyanobacteria are photosynthetic prokaryotes of high ecological and biotechnological relevance that have been cultivated in laboratories around the world for more than 70 years. Prolonged laboratory culturing has led to multiple microevolutionary events and the appearance of a large number of 'domesticated' substrains among model cyanobacteria. Despite its widespread occurrence, strain domestication is still largely ignored. In this work we describe Synechococcus elongatus PCC 7942-KU, a novel domesticated substrain of the model cyanobacterium S. elongatus PCC 7942, which presents a fast-sedimenting phenotype. Under higher ionic strengths the sedimentation rate increased leading to complete sedimentation in just 12 h. Through whole genome sequencing and gene deletion, we demonstrated that the Group 3 alternative sigma factor F plays a key role in cell sedimentation. Further analysis showed that significant changes in cell surface structures and a three-fold increase in released polysaccharides lead to the appearance of a fast-sedimenting phenotype. This work sheds light on the determinants of the planktonic to benthic transitions and provides genetic targets to generate fast-sedimenting strains that could unlock cost-effective cyanobacterial harvesting at scale.

Structure Elucidation of the First Sex-Inducing Pheromone of a Diatom.

Diatoms are abundant unicellular microalgae, responsible for ≈20 % of global photosynthetic CO2 fixation. Nevertheless, we know little about fundamental aspects of their biology, such as their sexual reproduction. Pheromone-mediated chemical communication is crucial for successful mating. An attraction pheromone was identified in the diatom Seminavis robusta, but metabolites priming cells for sex and synchronizing search and mating behavior remained elusive. These sex-inducing pheromones (SIP) induce cell cycle arrest and trigger the production of the attraction pheromone. Here we describe the challenging structure elucidation of an S. robusta SIP. Guided by metabolomics, a candidate metabolite was identified and elucidated by labeling experiments, NMR, ESI MSn analyses, and chemical transformations. The use of negative ion mode MS was essential to decipher the unprecedented hydroxyproline and ß-sulfated aspartate-containing cyclic heptapeptide that acts in femtomolar concentrations.

Development of a Highly Sensitive Luciferase-Based Reporter System To Study Two-Step Protein Secretion in Cyanobacteria.

Cyanobacteria, ubiquitous oxygenic photosynthetic bacteria, interact with the environment and their surrounding microbiome through the secretion of a variety of small molecules and proteins. The release of these compounds is mediated by sophisticated multiprotein complexes, also known as secretion systems. Genomic analyses indicate that protein and metabolite secretion systems are widely found in cyanobacteria; however, little is known regarding their function, regulation, and secreted effectors. One such system, the type IVa pilus system (T4aPS), is responsible for the assembly of dynamic cell surface appendages, type IVa pili (T4aP), that mediate ecologically relevant processes such as phototactic motility, natural competence, and adhesion. Several studies have suggested that the T4aPS can also act as a two-step protein secretion system in cyanobacteria akin to the homologous type II secretion system in heterotrophic bacteria. To determine whether the T4aP are involved in two-step secretion of nonpilin proteins, we developed a NanoLuc (NLuc)-based quantitative secretion reporter for the model cyanobacterium Synechocystis sp. strain PCC 6803. The NLuc reporter presented a wide dynamic range with at least 1 order of magnitude more sensitivity than traditional immunoblotting. Application of the reporter to a collection of Synechocystis T4aPS mutants demonstrated that the two-step secretion of NLuc is independent of T4aP. In addition, our data suggest that secretion differences typically observed in T4aPS mutants are likely due to a disruption of cell envelope homeostasis. This study opens the door to exploring protein secretion in cyanobacteria further. IMPORTANCE Protein secretion allows bacteria to interact and communicate with the external environment. Secretion is also biotechnologically relevant, where it is often beneficial to target proteins to the extracellular space. Due to a shortage of quantitative assays, many aspects of protein secretion are not understood. Here, we introduce an NLuc-based secretion reporter in cyanobacteria. NLuc is highly sensitive and can be assayed rapidly and in small volumes. The NLuc reporter allowed us to clarify the role of type IVa pili in protein secretion and identify mutations that increase secretion yield. This study expands our knowledge of cyanobacterial secretion and offers a valuable tool for future studies of protein secretion systems in cyanobacteria.

Dynamic Diatom-Bacteria Consortia in Synthetic Plankton Communities.

Microalgae that form phytoplankton live and die in a complex microbial consortium in which they co-exist with bacteria and other microorganisms. The dynamics of species succession in the plankton depends on the interplay of these partners. Bacteria utilize substrates produced by the phototrophic algae, while algal growth can be supported by bacterial exudates. Bacteria might also use chemical mediators with algicidal properties to attack algae. To elucidate whether specific bacteria play universal or context-specific roles in the interaction with phytoplankton, we investigated the effect of cocultured bacteria on the growth of 8 microalgae. An interaction matrix revealed that the function of a given bacterium is highly dependent on the cocultured partner. We observed no universally algicidal or universally growth-promoting bacteria. The activity of bacteria can even change during the aging of an algal culture from inhibitory to stimulatory or vice versa. We further established a synthetic phytoplankton/bacteria community with the centric diatom, Coscinodiscus radiatus, and 4 phylogenetically distinctive bacterial isolates, Mameliella sp., Roseovarius sp., Croceibacter sp., and Marinobacter sp. Supported by a Lotka-Volterra model, we show that interactions within the consortium are specific and that the sum of the pairwise interactions can explain algal and bacterial growth in the community. No synergistic effects between bacteria in the presence of the diatom was observed. Our survey documents highly species-specific interactions that are dependent on algal fitness, bacterial metabolism, and community composition. This species specificity may underly the high complexity of the multi-species plankton communities observed in nature. IMPORTANCE The marine food web is fueled by phototrophic phytoplankton. These algae are central primary producers responsible for the fixation of ca. 40% of the global CO2. Phytoplankton always co-occur with a diverse bacterial community in nature. This diversity suggests the existence of ecological niches for the associated bacteria. We show that the interaction between algae and bacteria is highly species-specific. Furthermore, both, the fitness stage of the algae and the community composition are relevant in determining the effect of bacteria on algal growth. We conclude that bacteria should not be sorted into algicidal or growth supporting categories; instead, a context-specific function of the bacteria in the plankton must be considered. This functional diversity of single players within a consortium may underly the observed diversity in the plankton.

Orchestrated Response of Intracellular Zwitterionic Metabolites in Stress Adaptation of the Halophilic Heterotrophic Bacterium Pelagibaca bermudensis.

Osmolytes are naturally occurring organic compounds that protect cells against various forms of stress. Highly polar, zwitterionic osmolytes are often used by marine algae and bacteria to counteract salinity or temperature stress. We investigated the effect of several stress conditions including different salinities, temperatures, and exposure to organic metabolites released by the alga Tetraselmis striata on the halophilic heterotrophic bacterium Pelagibaca bermudensis. Using ultra-high-performance liquid chromatography (UHPLC) on a ZIC-HILIC column and high-resolution electrospray ionization mass spectrometry, we simultaneously detected and quantified the eleven highly polar compounds dimethylsulfoxonium propionate (DMSOP), dimethylsulfoniopropionate (DMSP), gonyol, cysteinolic acid, ectoine, glycine betaine (GBT), carnitine, sarcosine, choline, proline, and 4-hydroxyproline. All compounds are newly described in P. bermudensis and potentially involved in physiological functions essential for bacterial survival under variable environmental conditions. We report that adaptation to various forms of stress is accomplished by adjusting the pattern and amount of the zwitterionic metabolites.

Propentdyopents as Heme Degradation Intermediates Constrict Mouse Cerebral Arterioles and Are Present in the Cerebrospinal Fluid of Patients With Subarachnoid Hemorrhage.

RATIONALE: Delayed ischemic neurological deficit is the most common cause of neurological impairment and unfavorable prognosis in patients with subarachnoid hemorrhage (SAH). Despite the existence of neuroimaging modalities that depict the onset of the accompanying cerebral vasospasm, preventive and therapeutic options are limited and fail to improve outcome owing to an insufficient pathomechanistic understanding of the delayed perfusion deficit. Previous studies have suggested that BOXes (bilirubin oxidation end products), originating from released heme surrounding ruptured blood vessels, are involved in arterial vasoconstriction. Recently, isolated intermediates of oxidative bilirubin degradation, known as PDPs (propentdyopents), have been considered as potential additional effectors in the development of arterial vasoconstriction. OBJECTIVE: To investigate whether PDPs and BOXes are present in hemorrhagic cerebrospinal fluid and involved in the vasoconstriction of cerebral arterioles. METHODS AND RESULTS: Via liquid chromatography/mass spectrometry, we measured increased PDP and BOX concentrations in cerebrospinal fluid of SAH patients compared with control subjects. Using differential interference contrast microscopy, we analyzed the vasoactivity of PDP isomers in vitro by monitoring the arteriolar diameter in mouse acute brain slices. We found an arteriolar constriction on application of PDPs in the concentration range that occurs in the cerebrospinal fluid of patients with SAH. By imaging arteriolar diameter changes using 2-photon microscopy in vivo, we demonstrated a short-onset vasoconstriction after intrathecal injection of either PDPs or BOXes. Using magnetic resonance imaging, we observed a long-term PDP-induced delay in cerebral perfusion. For all conditions, the arteriolar narrowing was dependent on functional big conductance potassium channels and was absent in big conductance potassium channels knockout mice. CONCLUSIONS: For the first time, we have quantified significantly higher concentrations of PDP and BOX isomers in the cerebrospinal fluid of patients with SAH compared to controls. The vasoconstrictive effect caused by PDPs in vitro and in vivo suggests a hitherto unrecognized pathway contributing to the pathogenesis of delayed ischemic deficit in patients with SAH.

12-Oxo-10-glutathionyl-5,8,14-eicosatrienoic acid (TOG10), a novel glutathione-containing eicosanoid generated via the 12-lipoxygenase pathway in human platelets.

Biologically active glutathione (GSH) conjugates of oxygenated fatty acids comprise a group of pro- and anti-inflammatory lipid mediators. While arachidonic acid (AA)-derived conjugates, as the cysteinyl leukotrienes (cys-LTs) and eoxins (EXs) have pro-inflammatory properties, conjugates in tissue regeneration (CTRs) biosynthesized from docosahexaenoic acid (DHA) exhibit pro-resolving activity. Human platelets express abundant amounts of platelet-type 12-lipoxygenase (pt12-LOX) and leukotriene C4 synthase (LTC4S). However, the only two described GSH conjugates formed by platelets are the AA-derived cys-LTs and the recently reported maresin CTRs (MCTRs). While cys-LTs are biosynthesized in a transcellular mechanism via the action of 5-LOX and LTC4S, MCTR1 is formed by 12-LOX and a yet unidentified GSH S-transferase (GST). Here, we present a novel GSH conjugate formed from AA via the 12-LOX pathway in human platelets. The 12-oxo-glutathione adduct, 12-oxo-10-glutathionyl-5,8,14-eicosatrienoic acid (TOG10), was identified by mass spectrometry using positive electrospray ionization. The structural proposal is supported by fragmentation data of the labeled metabolite obtained after incubation of deuterated AA (AA-d8). In platelets as well as in HEK293 cells stably expressing pt12-LOX, TOG10 biosynthesis was inhibited by the 12-LOX inhibitor ML-355 (5 µM), which confirms the involvement of pt12-LOX. Interestingly, TOG10 was formed independently of LTC4S in platelets. This is in accordance with the observation that the conjugate was also generated by AA-stimulated HEK_12-LOX cells in absence of LTC4S. Nevertheless, TOG10 can also be formed by LTC4S as the biosynthesis in HEK_12-LOX_LTC4S cells was reduced by the specific LTC4S inhibitor TK04a. In summary, TOG10 was identified as a new AA-derived GSH conjugate generated in human platelets via the action of pt12-LOX in combination with a GST.

Pheromone Mediated Sexual Reproduction of Pennate Diatom Cylindrotheca closterium.

Benthic diatoms dominate primary production in marine subtidal and intertidal environments. Their extraordinary species diversity and ecological success is thought to be linked with their predominantly heterothallic sexual reproduction. Little is known about pheromone involvement during mating of pennate diatoms. Here we describe pheromone guided mating in the coastal raphid diatom Cylindrotheca closterium. We show that the two mating types (mt+ and mt-) have distinct functions. Similar to other benthic diatoms, mt+ cells are searching for the mt- cells to pair. To enhance mating efficiency mt- exudes an attraction pheromone which we proved by establishing a novel capillary assay. Further, two more pheromones produced by mt- promote the sexual events. One arrests the cell cycle progression of mt+ while the other induces gametogenesis of mt+. We suggest that C. closterium shares a functionally similar pheromone system with other pennate diatoms like Seminavis robusta and Pseudostaurosira trainorii which synchronize sexual events and mate attraction. Remarkably, we found no evidence of mt+ producing pheromones, which differentiates C. closterium from other pennates and suggests a less complex pheromone system in C. closterium.

Cysteinolic Acid Is a Widely Distributed Compatible Solute of Marine Microalgae.

Phytoplankton rely on bioactive zwitterionic and highly polar small metabolites with osmoregulatory properties to compensate changes in the salinity of the surrounding seawater. Dimethylsulfoniopropionate (DMSP) is a main representative of this class of metabolites. Salinity-dependent DMSP biosynthesis and turnover contribute significantly to the global sulfur cycle. Using advanced chromatographic and mass spectrometric techniques that enable the detection of highly polar metabolites, we identified cysteinolic acid as an additional widely distributed polar metabolite in phytoplankton. Cysteinolic acid belongs to the class of marine sulfonates, metabolites that are commonly produced by algae and consumed by bacteria. It was detected in all dinoflagellates, haptophytes, diatoms and prymnesiophytes that were surveyed. We quantified the metabolite in different phytoplankton taxa and revealed that the cellular content can reach even higher concentrations than the ubiquitous DMSP. The cysteinolic acid concentration in the cells of the diatom Thalassiosira weissflogii increases significantly when grown in a medium with elevated salinity. In contrast to the compatible solute ectoine, cysteinolic acid is also found in high concentrations in axenic algae, indicating biosynthesis by the algae and not the associated bacteria. Therefore, we add this metabolite to the family of highly polar metabolites with osmoregulatory characteristics produced by phytoplankton.

Untargeted Metabolomics Unveil Changes in Autotrophic and Mixotrophic Galdieria sulphuraria Exposed to High-Light Intensity.

The thermoacidophilic red alga Galdieria sulphuraria has been optimizing a photosynthetic system for low-light conditions over billions of years, thriving in hot and acidic endolithic habitats. The growth of G. sulphuraria in the laboratory is very much dependent on light and substrate supply. Here, higher cell densities in G. sulphuraria under high-light conditions were obtained, although reductions in photosynthetic pigments were observed, which indicated this alga might be able to relieve the effects caused by photoinhibition. We further describe an extensive untargeted metabolomics study to reveal metabolic changes in autotrophic and mixotrophic G. sulphuraria grown under high and low light intensities. The up-modulation of bilayer lipids, that help generate better-ordered lipid domains (e.g., ergosterol) and keep optimal membrane thickness and fluidity, were observed under high-light exposure. Moreover, high-light conditions induced changes in amino acids, amines, and amide metabolism. Compared with the autotrophic algae, higher accumulations of osmoprotectant sugars and sugar alcohols were recorded in the mixotrophic G. sulphuraria. This response can be interpreted as a measure to cope with stress due to the high concentration of organic carbon sources. Our results indicate how G. sulphuraria can modulate its metabolome to maintain energetic balance and minimize harmful effects under changing environments.

A new glance at the chemosphere of macroalgal-bacterial interactions: In situ profiling of metabolites in symbiosis by mass spectrometry.

Symbiosis is a dominant form of life that has been observed numerous times in marine ecosystems. For example, macroalgae coexist with bacteria that produce factors that promote algal growth and morphogenesis. The green macroalga Ulva mutabilis (Chlorophyta) develops into a callus-like phenotype in the absence of its essential bacterial symbionts Roseovarius sp. MS2 and Maribacter sp. MS6. Spatially resolved studies are required to understand symbiont interactions at the microscale level. Therefore, we used mass spectrometry profiling and imaging techniques with high spatial resolution and sensitivity to gain a new perspective on the mutualistic interactions between bacteria and macroalgae. Using atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionisation high-resolution mass spectrometry (AP-SMALDI-HRMS), low-molecular-weight polar compounds were identified by comparative metabolomics in the chemosphere of Ulva. Choline (2-hydroxy-N,N,N-trimethylethan-1-aminium) was only determined in the alga grown under axenic conditions, whereas ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) was found in bacterial presence. Ectoine was used as a metabolic marker for localisation studies of Roseovarius sp. within the tripartite community because it was produced exclusively by these bacteria. By combining confocal laser scanning microscopy (cLSM) and AP-SMALDI-HRMS, we proved that Roseovarius sp. MS2 settled mainly in the rhizoidal zone (holdfast) of U. mutabilis. Our findings provide the fundament to decipher bacterial symbioses with multicellular hosts in aquatic ecosystems in an ecologically relevant context. As a versatile tool for microbiome research, the combined AP-SMALDI and cLSM imaging analysis with a resolution to level of a single bacterial cell can be easily applied to other microbial consortia and their hosts. The novelty of this contribution is the use of an in situ setup designed to avoid all types of external contamination and interferences while resolving spatial distributions of metabolites and identifying specific symbiotic bacteria.

High CO2 concentration and iron availability determine the metabolic inventory in an Emiliania huxleyi-dominated phytoplankton community.

Ocean acidification (OA), a consequence of anthropogenic carbon dioxide (CO2 ) emissions, strongly impacts marine ecosystems. OA also influences iron (Fe) solubility, affecting biogeochemical and ecological processes. We investigated the interactive effects of CO2 and Fe availability on the metabolome response of a natural phytoplankton community. Using mesocosms we exposed phytoplankton to ambient (390 µatm) or future CO2 levels predicted for the year 2100 (900 µatm), combined with ambient (4.5 nM) or high (12 nM) dissolved iron (dFe). By integrating over the whole phytoplankton community, we assigned functional changes based on altered metabolite concentrations. Our study revealed the complexity of phytoplankton metabolism. Metabolic profiles showed three stages in response to treatments and phytoplankton dynamics. Metabolome changes were related to the plankton group contributing respective metabolites, explaining bloom decline and community succession. CO2 and Fe affected metabolic profiles. Most saccharides, fatty acids, amino acids and many sterols significantly correlated with the high dFe treatment at ambient pCO2 . High CO2 lowered the abundance of many metabolites irrespective of Fe. However, sugar alcohols accumulated, indicating potential stress. We demonstrate that not only altered species composition but also changes in the metabolic landscape affecting the plankton community may change as a consequence of future high-CO2 oceans.

Mammalian-Like Inflammatory and Pro-Resolving Oxylipins in Marine Algae.

Oxylipins constitute a family of oxidized fatty acids, that are well known as tissue hormones in mammals. They contribute to inflammation and its resolution. The major classes of these lipid mediators are inflammatory prostaglandins (PGs) and leukotrienes (LTs) as well as pro-resolving resolvins (Rvs). Understanding their biosynthetic pathways and modes of action is important for anti-inflammatory interventions. Besides mammals, marine algae also biosynthesize mammalian-like oxylipins and thus offer new opportunities for oxylipin research. They provide prolific sources for these compounds and offer unique opportunities to study alternative biosynthetic pathways to the well-known lipid mediators. Herein, we discuss recent findings on the biosynthesis of oxylipins in mammals and algae including an alternative pathway to prostaglandin E2 , a novel pathway to a precursor of leukotriene B4 , and the production of resolvins in algae. We evaluate the pharmacological potential of the algal metabolites with implications in health and disease.

Identification to species level of live single microalgal cells from plankton samples with matrix-free laser/desorption ionization mass spectrometry.

INTRODUCTION: Marine planktonic communities are complex microbial consortia often dominated by microscopic algae. The taxonomic identification of individual phytoplankton cells usually relies on their morphology and demands expert knowledge. Recently, a live single-cell mass spectrometry (LSC-MS) pipeline was developed to generate metabolic profiles of microalgae. OBJECTIVE: Taxonomic identification of diverse microalgal single cells from collection strains and plankton samples based on the metabolic fingerprints analyzed with matrix-free laser desorption/ionization high-resolution mass spectrometry. METHODS: Matrix-free atmospheric pressure laser-desorption ionization mass spectrometry was performed to acquire single-cell mass spectra from collection strains and prior identified environmental isolates. The computational identification of microalgal species was performed by spectral pattern matching (SPM). Three similarity scores and a bootstrap-derived confidence score were evaluated in terms of their classification performance. The effects of high and low-mass resolutions on the classification success were evaluated. RESULTS: Several hundred single-cell mass spectra from nine genera and nine species of marine microalgae were obtained. SPM enabled the identification of single cells at the genus and species level with high accuracies. The receiver operating characteristic (ROC) curves indicated a good performance of the similarity measures but were outperformed by the bootstrap-derived confidence scores. CONCLUSION: This is the first study to solve taxonomic identification of microalgae based on the metabolic fingerprints of the individual cell using an SPM approach.

Pyrrolic and Dipyrrolic Chlorophyll Degradation Products in Plants and Herbivores.

The degradation of chlorophyll, the omnipresent green pigment, has been investigated intensively over the last 30 years resulting in many elucidated tetrapyrrolic degradation products. With a comparison to the degradation of the structurally similar heme, we hereby propose a novel additional chlorophyll degradation mechanism to mono- and dipyrrolic products. This is the first proof of the occurrence of a family of mono- and dipyrrols in leaves that are previously only known as heme degradation products. This product family is also found in spit and feces of herbivores with specific metabolomic patterns reflecting the origin of the samples. Based on chromatographic and mass spectrometric evidence as well as on mechanistic considerations we also suggest several tentative new degradation products. One of them, dihydro BOX A, was fully confirmed as a novel natural product by synthesis and comparison of its spectroscopic data.

Ectoine from Bacterial and Algal Origin Is a Compatible Solute in Microalgae.

Osmoregulation in phytoplankton is attributed to several highly polar low-molecular-weight metabolites. A widely accepted model considers dimethylsulfoniopropionate (DMSP) as the most important and abundant osmotically active metabolite. Using an optimized procedure for the extraction and detection of highly polar metabolites, we expand the group of phytoplankton osmolytes by identifying ectoine in several microalgae. Ectoine is known as a bacterial compatible solute, but, to the best of our knowledge, was never considered as a phytoplankton-derived product. Given the ability of microalgae to take up zwitterions, such as DMSP, we tested the hypothesis that the algal ectoine is derived from associated bacteria. We therefore analyzed methanol extracts of xenic and axenic cultures of two different species of microalgae and could detect elevated concentrations of ectoine in those that harbor associated bacteria. However, also microalgae without an associated microbiome contain ectoine in smaller amounts, pointing towards a dual origin of this metabolite in the algae from their own biosynthesis as well as from uptake. We also tested the role of ectoine in the osmoadaptation of microalgae. In the model diatoms Thalassiosira weissflogii and Phaeodactylum tricornutum, elevated amounts of ectoine were found when cultivated in seawater with salinities of 50 PSU compared to the standard culture conditions of 35 PSU. Therefore, we add ectoine to the family of osmoadaptive metabolites in phytoplankton and prove a new, potentially synergistic metabolic interplay of bacteria and algae.

Sulfonium Acids Loaded onto an Unusual Thiotemplate Assembly Line Construct the Cyclopropanol Warhead of a Burkholderia Virulence Factor.

Pathogenic bacteria of the Burkholderia pseudomallei group cause severe infectious diseases such as glanders and melioidosis. Malleicyprols were identified as important bacterial virulence factors, yet the biosynthetic origin of their cyclopropanol warhead has remained enigmatic. By a combination of mutational analysis and metabolomics we found that sulfonium acids, dimethylsulfoniumpropionate (DMSP) and gonyol, known as osmolytes and as crucial components in the global organosulfur cycle, are key intermediates en route to the cyclopropanol unit. Functional genetics and in vitro analyses uncover a specialized pathway to DMSP involving a rare prokaryotic SET-domain methyltransferase for a cryptic methylation, and show that DMSP is loaded onto the NRPS-PKS hybrid assembly line by an adenylation domain dedicated to zwitterionic starter units. Then, the megasynthase transforms DMSP into gonyol, as demonstrated by heterologous pathway reconstitution in E. coli.

The Algicidal Bacterium Kordia algicida Shapes a Natural Plankton Community.

Plankton communities consist of complex microbial consortia that change over time. These fluctuations can be only partially explained by limiting resources. Biotic factors such as herbivores and pathogens also contribute to the control of algal blooms. Here we address the effects of algicidal bacteria on a natural plankton community in an indoor enclosure experiment. The algicidal bacteria, introduced into plankton taken directly from the North Sea during a diatom bloom, caused the rapid decline of the bloom-forming Chaetoceros socialis within only 1 day. The haptophyte Phaeocystis, in contrast, is resistant to the lytic bacteria and could benefit from the removal of the competitor, as indicated by an onset of a bloom in the treated enclosures. This cascading effect caused by the bacterial pathogen accelerated the succession of Phaeocystis, which bloomed with a delay of only several weeks in the in situ waters at Helgoland Roads in the North Sea. The algicidal bacteria can thus modulate the community within the limits of the abiotic and biotic conditions of the local environment. Implications of our findings for plankton ecosystem functioning are discussed.IMPORTANCE Plankton communities change on a seasonal basis in temperate systems, with distinct succession patterns; this is mainly due to algal species that have their optimal timing relative to environmental conditions. We know that bacterial populations are also instrumental in the decay and termination of phytoplankton blooms. Here, we describe algicidal bacteria as modulators of this important species succession. Upon treatment of a natural plankton consortium with an algicidal bacterium, we observed a strong shift in the phytoplankton community structure, compared to controls, resulting in formation of a succeeding Phaeocystis bloom. Blooms of this alga have a substantial impact on global biogeochemical and ecological cycles, as they are responsible for a substantial proportion of primary production during spring in the North Sea. We propose that one of the key factors influencing such community shifts may be algicidal bacteria.