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.

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.

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.

Laminariales Host Does Impact Lipid Temperature Trajectories of the Fungal Endophyte Paradendryphiella salina (Sutherland.).

Kelps are colonized by a wide range of microbial symbionts. Among them, endophytic fungi remain poorly studied, but recent studies evidenced yet their high diversity and their central role in algal defense against various pathogens. Thus, studying the metabolic expressions of kelp endophytes under different conditions is important to have a better understanding of their impacts on host performance. In this context, fatty acid composition is essential to a given algae fitness and of interest to food web studies either to measure its nutritional quality or to infer about its contribution to consumers diets. In the present study, Paradendryphiella salina, a fungal endophyte was isolated from Saccharina latissima (L.) and Laminaria digitata (Hudson.) and its fatty acid composition was assessed at increasing salinity and temperature conditions. Results showed that fungal composition in terms of fatty acids displayed algal-dependent trajectories in response to temperature increase. This highlights that C18 unsaturated fatty acids are key components in the host-dependant acclimation of P. salina to salinity and temperature changes.

Secondary Metabolites from the Culture of the Marine-derived Fungus Paradendryphiella salina PC 362H and Evaluation of the Anticancer Activity of Its Metabolite Hyalodendrin.

High-throughput screening assays have been designed to identify compounds capable of inhibiting phenotypes involved in cancer aggressiveness. However, most studies used commercially available chemical libraries. This prompted us to explore natural products isolated from marine-derived fungi as a new source of molecules. In this study, we established a chemical library from 99 strains corresponding to 45 molecular operational taxonomic units and evaluated their anticancer activity against the MCF7 epithelial cancer cell line and its invasive stem cell-like MCF7-Sh-WISP2 counterpart. We identified the marine fungal Paradendryphiella salina PC 362H strain, isolated from the brown alga Pelvetia caniculata (PC), as one of the most promising fungi which produce active compounds. Further chemical and biological characterizations of the culture of the Paradendryphiella salina PC 362H strain identified (-)-hyalodendrin as the active secondary metabolite responsible for the cytotoxic activity of the crude extract. The antitumor activity of (-)-hyalodendrin was not only limited to the MCF7 cell lines, but also prominent on cancer cells with invasive phenotypes including colorectal cancer cells resistant to chemotherapy. Further investigations showed that treatment of MCF7-Sh-WISP2 cells with (-)-hyalodendrin induced changes in the phosphorylation status of p53 and altered expression of HSP60, HSP70 and PRAS40 proteins. Altogether, our study reveals that this uninvestigated marine fungal crude extract possesses a strong therapeutic potential against tumor cells with aggressive phenotypes and confirms that members of the epidithiodioxopiperazines are interesting fungal toxins with anticancer activities.

An Integrative Approach to Decipher the Chemical Antagonism between the Competing Endophytes Paraconiothyrium variabile and Bacillus subtilis.

An integrative approach combining traditional natural products chemistry, molecular networking, and mass spectrometry imaging has been undertaken to decipher the molecular dialogue between the fungus Paraconiothyrium variabile and the bacterium Bacillus subtilis, which were isolated as endophytes from the conifer Cephalotaxus harringtonia and are characterized by a strong and mutual antibiosis. From this study, we highlight that bacterial surfactins and a fungal tetronic acid are involved in such competition and that the fungus is able to hydrolyze surfactins to fight against the bacterial partner.

Molecular Analysis of the embCAB Locus and embR Gene Involved in Ethambutol Resistance in Clinical Isolates of Mycobacterium tuberculosis in France.

Modification of codon 306 in embB is regarded as the main mechanism leading to ethambutol (ETB) resistance in clinical isolates of Mycobacterium tuberculosis. However, numerous mutations elsewhere in the embCAB locus and in embR, a putative transcriptional activator of this locus, have been reported to be involved in ETB resistance. Here, we investigated the diversity of nucleotide variations observed in embCAB and embR in M. tuberculosis complex isolates from France. These regions were sequenced in 71 ETB-resistant (ETB-R) and 60 ETB-susceptible (ETB-S) clinical isolates of known phylogenetic lineages. The 131 isolates had 12 mutations corresponding to phylogenetic markers. Among the 60 ETB-S isolates, only 3 (5%) had nonsynonymous mutations that were not phylogenetic markers. Among the 71 ETB-R isolates, 98% had mutations in embCAB that likely contribute to ETB resistance: 70% had mutations located in embB codon 306, 406, or 497; 13% had mutations located outside these three positions between codons 296 and 426; and 15% had mutations corresponding to mutations in the embC-embA intergenic region. We found a strong association between resistance to ETB and the presence of mutations in embB and the embC-embA intergenic region (P < 0.001). In contrast, the mutations detected in embC and embA were not involved in ETB resistance, and no mutation was detected in embR. These results strongly suggest that the sensitivity of diagnostic assays for detecting ETB resistance based on testing of embB codon 306 can be increased by testing of the embB region between codons 296 and 497 and by including the embC-embA intergenic region between positions -8 and -21.

Bacteria modulate microalgal aging physiology through the induction of extracellular vesicle production to remove harmful metabolites.

The bloom and bust patterns of microalgae in aquatic systems contribute massively to global biogeochemical cycles. The decline of algal blooms is mainly caused by nutrient limitation resulting in cell death, the arrest of cell division and the aging of surviving cells. Nutrient intake can re-initiate proliferation, but the processes involved are poorly understood. Here we characterize how the bloom-forming diatom Coscinodiscus radiatus recovers from starvation after nutrient influx. Rejuvenation is mediated by extracellular vesicles that shuttle reactive oxygen species, oxylipins and other harmful metabolites out of the old cells, thereby re-enabling their proliferation. By administering nutrient pulses to aged cells and metabolomic monitoring of the response, we show that regulated pathways are centred around the methionine cycle in C. radiatus. Co-incubation experiments show that bacteria mediate aging processes and trigger vesicle production using chemical signalling. This work opens new perspectives on cellular aging and rejuvenation in complex microbial communities.

Temporal and Spatial Signaling Mediating the Balance of the Plankton Microbiome.

The annual patterns of plankton succession in the ocean determine ecological and biogeochemical cycles. The temporally fluctuating interplay between photosynthetic eukaryotes and the associated microbiota balances the composition of aquatic planktonic ecosystems. In addition to nutrients and abiotic factors, chemical signaling determines the outcome of interactions between phytoplankton and their associated microbiomes. Chemical mediators control essential processes, such as the development of key morphological, physiological, behavioral, and life-history traits during algal growth. These molecules thus impact species succession and community composition across time and space in processes that are highlighted in this review. We focus on spatial, seasonal, and physiological dynamics that occur during the early association of algae with bacteria, the exponential growth of a bloom, and its decline and recycling. We also discuss how patterns from field data and global surveys might be linked to the actions of metabolic markers in natural phytoplankton assemblages.

Author Correction: The oomycete Lagenisma coscinodisci hijacks host alkaloid synthesis during infection of a marine diatom.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Live Single-Cell Metabolomics With Matrix-Free Laser/Desorption Ionization Mass Spectrometry to Address Microalgal Physiology.

Unicellular phototrophic algae can form massive blooms with up to millions of individual cells per milliliter in freshwater and marine ecosystems. Despite the temporal dominance of bloom formers many algal species can co-exist and compete for nutrients and space, creating a complex and diverse community. While microscopy and single cell genomics can address the taxonomic inventory, the cellular metabolome has yet to be thoroughly explored to determine the physiological status of microalgae. This might, however, provide a key to understand the observed species diversity in the homogeneous environment. Here, we introduce an effective, rapid and versatile method to analyze living single cells from aqueous substrata with laser-desorption/ionization mass spectrometry (LDI-MS) using a simple and inexpensive matrix-free support. The cells deposited on a cultivation-medium wetted support are analyzed with minimal disturbance as they remain in their natural viable state until their disruption during LDI-MS. Metabolites desorbed from single cells are analyzed on High-Resolution Mass Spectrometry (HR-MS) using the Orbitrap FT-MS technology to fingerprint cellular chemistry. This live single-cell mass spectrometry (LSC-MS) allows assessing the physiological status and strain-specifics of different microalgae, including marine diatoms and freshwater chlorophytes, at the single-cell level. We further report a reliable and robust data treatment pipeline to perform multivariate statistics on the replicated LSC-MS data. Comparing single cell MS spectra from natural phytoplankton samples and from laboratory strains allows the identification and discrimination of inter and intra-specific metabolic variability and thereby has promising applications in addressing highly complex phytoplankton communities. Notably, the herein described matrix-free live-single-cell LDI-HR-MS approach enables monitoring dynamics of the plankton and might explain why key-players survive, thrive, avoid selective feeding or pathogenic virus and bacteria, while others are overcome and die.

Biocontrol Potential of a Novel Endophytic Bacterium From Mulberry (Morus) Tree.

Mulberry (Morus) is an economically important woody tree that is suitable for use in sericulture as forage and in medicine. However, this broad-leaved tree is facing multiple threats ranging from phytopathogens to insect pests. Here, a Gram-positive, endospore-forming bacterium (ZJU1) was frequently isolated from healthy mulberry plants by screening for foliar endophytes showing antagonism against pathogens and pests. Whole-genome sequencing and annotation resulted in a genome size of 4.06 Mb and classified the bacterium as a novel strain of Bacillus amyloliquefaciens that has rarely been identified from tree leaves. An integrative approach combining traditional natural product chemistry, activity bioassays, and high-resolution mass spectrometry confirmed that strain ZJU1 uses a blend of antimicrobials including peptides and volatile organic compounds to oppose Botrytis cinerea, a major phytopathogenic fungus causing mulberry gray mold disease. We showed that the inoculation of endophyte-free plants with ZJU1 significantly decreased both leaf necrosis and mortality under field conditions. In addition to the direct interactions of endophytes with foliar pathogens, in planta studies suggested that the inoculation of endophytes also induced plant systemic defense, including high expression levels of mulberry disease resistance genes. Moreover, when applied to the generalist herbivore Spodoptera litura, ZJU1 was sufficient to reduce the pest survival rate below 50%. A previously undiscovered crystal toxin (Cry10Aa) could contribute to this insecticidal effect against notorious lepidopteran pests. These unique traits clearly demonstrate that B. amyloliquefaciens ZJU1 is promising for the development of successful strategies for biocontrol applications. The search for new plant-beneficial microbes and engineering microbiomes is therefore of great significance for sustainably improving plant performance.

Bacterial-Fungal Interactions in the Kelp Endomicrobiota Drive Autoinducer-2 Quorum Sensing.

Brown macroalgae are an essential component of temperate coastal ecosystems and a growing economic sector. They harbor diverse microbial communities that regulate algal development and health. This algal holobiont is dynamic and achieves equilibrium via a complex network of microbial and host interactions. We now report that bacterial and fungal endophytes associated with four brown algae (Ascophyllum nodosum, Pelvetia canaliculata, Laminaria digitata, and Saccharina latissima) produce metabolites that interfere with bacterial autoinducer-2 quorum sensing, a signaling system implicated in virulence and host colonization. Additionally, we performed co-culture experiments combined to a metabolomic approach and demonstrated that microbial interactions influence production of metabolites, including metabolites involved in quorum sensing. Collectively, the data highlight autoinducer-2 quorum sensing as a key metabolite in the complex network of interactions within the algal holobiont.

The oomycete Lagenisma coscinodisci hijacks host alkaloid synthesis during infection of a marine diatom.

Flagellated oomycetes frequently infect unicellular algae, thus limiting their proliferation. Here we show that the marine oomycete Lagenisma coscinodisci rewires the metabolome of the bloom-forming diatom Coscinodiscus granii, thereby promoting infection success. The algal alkaloids ß-carboline and 4-carboxy-2,3,4,9-tetrahydro-1H-ß-carboline are induced during infection. Single-cell profiling with AP-MALDI-MS and confocal laser scanning microscopy reveals that algal carbolines accumulate in the reproductive form of the parasite. The compounds arrest the algal cell division, increase the infection rate and induce plasmolysis in the host. Our results indicate that the oomycete manipulates the host metabolome to support its own multiplication.

Chemically-Mediated Interactions Between Macroalgae, Their Fungal Endophytes, and Protistan Pathogens.

Filamentous fungi asymptomatically colonize the inner tissues of macroalgae, yet their ecological roles remain largely underexplored. Here, we tested if metabolites produced by fungal endophytes might protect their host against a phylogenetically broad spectrum of protistan pathogens. Accordingly, the cultivable fungal endophytes of four brown algal species were isolated and identified based on LSU and SSU sequencing. The fungal metabolomes were tested for their ability to reduce the infection by protistan pathogens in the algal model Ectocarpus siliculosus. The most active metabolomes effective against the oomycetes Eurychasma dicksonii and Anisolpidium ectocarpii, and the phytomixid Maullinia ectocarpii were further characterized chemically. Several pyrenocines isolated from Phaeosphaeria sp. AN596H efficiently inhibited the infection by all abovementioned pathogens. Strikingly, these compounds also inhibited the infection of nori (Pyropia yezoensis) against its two most devastating oomycete pathogens, Olpidiopsis pyropiae, and Pythium porphyrae. We thus demonstrate that fungal endophytes associated with brown algae produce bioactive metabolites which might confer protection against pathogen infection. These results highlight the potential of metabolites to finely-tune the outcome of molecular interactions between algae, their endophytes, and protistan pathogens. This also provide proof-of-concept toward the applicability of such metabolites in marine aquaculture to control otherwise untreatable diseases.

A Reactive Eremophilane and Its Antibacterial 2(1H)-Naphthalenone Rearrangement Product, Witnesses of a Microbial Chemical Warfare.

Two sesquiterpenes, 4-epi-microsphaeropsisin (1) and a dihydrofurano-2(1H)-naphthalenone (variabilone, 2) which represents a new skeleton, were isolated from endophytic fungus Paraconiothyrium variabile. Reactivity studies showed that eremophilane 1 is a precursor of 2 through acid-promoted methyl 1,2-migration and aromatization. An electrophilic intermediate of this transformation was intercepted by N-acetylcysteamine, a biomimetic nucleophile. Only compound 2 was antibacterial against endophytic bacterium Bacillus subtilis (coisolated with P. variabile), suggesting a role in the microbial competition in plants.