High growth potential of long-term starved deep ocean opportunistic heterotrophic bacteria

Experiments with bacteria in culture have shown that they often display “feast and famine” strategies that allow them to respond with fast growth upon pulses in resource availability, and enter a growth-arrest state when resources are limiting. Although feast responses have been observed in natural communities upon enrichment, it is unknown whether this blooming ability is maintained after long periods of starvation, particularly in systems that are energy limited like the bathypelagic ocean. Here we combined bulk and single-cell activity measurements with 16S rRNA gene amplicon sequencing to explore the response of a bathypelagic community, that had been starved for 1.6 years, to a sudden organic carbon supply. We observed a dramatic change in activity within 30 h, with leucine incorporation rates increasing over two orders of magnitude and the number of translationally active cells (mostly Gammaproteobacteria) increasing 4-fold. The feast response was driven by a single operational taxonomic unit (OTU) affiliated with the Marinobacter genus, which had remained rare during 7 months of starvation. Our work suggests that bathypelagic communities harbor a seed bank of highly persistent and resourceful “feast and famine” strategists that might disproportionally contribute to carbon fluxes through fast responses to occasional pulses of organic matter

Regularidades estacionales en la producción primaria y los parámetros fotosintéticos en una estación costera del NO Mediterráneo

We carried out monthly photosynthesis-irradiance (P-E) experiments with the 14C-method for 12 years (2003–2014) to determine the photosynthetic parameters and primary production of surface phytoplankton in the Blanes Bay Microbial Observatory, a coastal sampling station in the NW Mediterranean Sea. Our goal was to obtain seasonal trends and to establish the basis for detecting future changes of primary production in this oligotrophic area. The maximal photosynthetic rate PBmax ranged 30-fold (0.5-15 mg C mg Chl a–1 h–1), averaged 3.7 mg C mg Chl a–1 h–1 (±0.25 SE) and was highest in August and lowest in April and December. We only observed photoinhibition twice. The initial or light-limited slope of the P-E relationship, αB, was low, averaging 0.007 mg C mg Chl a–1 h–1 (μmol photons m–2 s–1)–1 (±0.001 SE, range 0.001-0.045) and showed the lowest values in spring (April-June). The light saturation parameter or saturation irradiance, EK, averaged 711 μmol photons m–2 s–1 (± 58.4 SE) and tended to be higher in spring and lower in winter. Phytoplankton assemblages were typically dominated by picoeukaryotes in early winter, diatoms in late autumn and late winter, dinoflagellates in spring and cyanobacteria in summer. Total particulate primary production averaged 1.45 mg C m-3 h–1 (±0.13 SE) with highest values in winter (up to 8.50 mg C m-3 h–1) and lowest values in summer (summer average, 0.30 mg C m-3 h–1), while chlorophyll-specific primary production averaged 2.49 mg C mg Chl a–1 h–1 (±0.19, SE) and peaked in summer (up to 12.0 mg C mg Chl a–1 h–1 in August). 14C-determined phytoplankton growth rates varied between ca. 0.3 d–1 in winter and 0.5 d–1 in summer and were within 60-80% of the maximal rates of growth, based on PBmax. Chlorophyll a was a good predictor of primary production only in the winter and autumn. Seasonality appeared to explain most of the variability in the studied variables, while phytoplankton composition played a minor role. Daily integrated primary production was fairly constant throughout the year: similar to previous oxygen-based estimates in winter but considerably lower than these in summer. The difference between 14C- and oxygen-based estimates of primary production could be explained by community respiration. Annually integrated primary production amounted to a rather modest 48 g C m–2 yr–1 (equivalent to 130 mg C m–2 d–1). Although no interannual patterns were detected, our work soundly establishes the seasonal trends for the coastal NW Mediterranean, therefore setting the basis for future detection of change.Hemos llevado a cabo mediciones mensuales de la relación entre fotosíntesis e irradiancia (curvas P-E) con el método del 14C durante 12 años (2003-2014), para obtener los parámetros fotosintéticos y la producción primaria del fitoplancton superficial en el Observatorio Microbiano de la Bahía de Blanes, una estación de muestreo costera en el noroeste del Mediterráneo. Nuestro objetivo era determinar las tendencias estacionales y establecer la línea de base para detectar cambios futuros de la producción primaria en esta área oligotrófica. La tasa fotosintética máxima PBmaxosciló 30 veces (0.5 a 15 mg C mg Chl a–1 h–1), con un promedio de 3.7 mg C mg Chl a–1 h–1 (±0.25, error estándar), y fue máxima en agosto y mínima en abril y diciembre. Sólo se observó fotoinhibicion dos veces. La pendiente inicial de la curva P-E, αB, fue baja, con un promedio de 0.007 mg C mg Chl a–1 h–1 (µmol fotones m–2 s–1)–1 (error estándar ±0.001, rango de 0.001-0.045) y presentó los valores más bajos en primavera (abril-junio). El parámetro de saturación de irradiancia, EK, presentó un promedio de 711 µmol fotones m–2 s–1 (±58.4, error estándar) y tendió a ser mayor en primavera y menor en invierno. El fitoplancton estuvo típicamente dominado por picoeucariotas a principios de invierno, diatomeas a finales de otoño y en el invierno avanzado, dinoflagelados en primavera, y cianobacterias en verano. La producción primaria particulada total promedió 1.45 m–3 h–1 (±0.13 error estándar) con máximos en invierno (hasta 8.50 mg C m–3 h–1) y mínimos en verano (media en verano, 0.30 mg C m–3 h–1), mientras que la producción primaria por unidad de clorofila promedió 2.49 mg C mg Chl a–1 h–1 (±0.19, error estándar) y alcanzó su punto máximo en verano (hasta 12.0 mg C mg Chl a–1 h–1 en agosto). Las tasas de crecimiento del fitoplancton determinadas por 14C oscilaron entre ca. 0.3 d–1 en invierno y 0.5 d–1 en verano, siendo 60-80% de las tasas máximas de crecimiento basadas en los valores de PBmax. La clorofila a resultó ser un buen predictor de la producción primaria sólo en el invierno y el otoño. La estacionalidad explica la mayor parte de la variabilidad en las variables estudiadas, mientras que la composición del fitoplancton juega un papel menor. La producción primaria integrada diaria fue bastante constante durante todo el año, similar a estimaciones anteriores con mediciones de oxígeno en invierno, pero considerablemente más bajas en verano. La diferencia entre las estimaciones de la producción primaria por 14C y las estimaciones en base al oxígeno podrían explicarse por la respiración de la comunidad. Anualmente la producción primaria integrada ascendió a valores más bien modestos de 48 g C m–2 año–1 (equivalentes a 130 mg C m–2 d–1). Aunque no se detectaron patrones interanuales, nuestro trabajo establece las tendencias estacionales para la producción primaria en el litoral Mediterráneo noroccidental, y supone la línea de base para la detección de cambios futuros

High Growth Potential of Long-Term Starved Deep Ocean Opportunistic Heterotrophic Bacteria

Experiments with bacteria in culture have shown that they often display “feast and famine” strategies that allow them to respond with fast growth upon pulses in resource availability, and enter a growth-arrest state when resources are limiting. Although feast responses have been observed in natural communities upon enrichment, it is unknown whether this blooming ability is maintained after long periods of starvation, particularly in systems that are energy limited like the bathypelagic ocean. Here we combined bulk and single-cell activity measurements with 16S rRNA gene amplicon sequencing to explore the response of a bathypelagic community, that had been starved for 1.6 years, to a sudden organic carbon supply. We observed a dramatic change in activity within 30 h, with leucine incorporation rates increasing over two orders of magnitude and the number of translationally active cells (mostly Gammaproteobacteria) increasing 4-fold. The feast response was driven by a single operational taxonomic unit (OTU) affiliated with the Marinobacter genus, which had remained rare during 7 months of starvation. Our work suggests that bathypelagic communities harbor a seed bank of highly persistent and resourceful “feast and famine” strategists that might disproportionally contribute to carbon fluxes through fast responses to occasional pulses of organic matter

High amino acid osmotrophic incorporation by marine eukaryotic phytoplankton revealed by click-chemistry

pages, 7 figures.-- Data Availability Statement: Raw sequences are publicly available at the European Nucleotide Archive (https://www.ebi.ac.uk/ena) under the accession number PRJEB63614. The datasets generated for this study are available on request to the corresponding authors.The osmotrophic uptake of dissolved organic compounds in the ocean is considered to be dominated by heterotrophic prokaryotes, whereas the role of planktonic eukaryotes is still unclear. We explored the capacity of natural eukaryotic plankton communities to incorporate the synthetic amino acid L-homopropargylglycine (HPG, analogue of methionine) using biorthogonal noncanonical amino acid tagging (BONCAT), and compared it with prokaryotic HPG use throughout a 9-day survey in the NW Mediterranean. BONCAT allows to fluorescently identify translationally active cells, but it has never been applied to natural eukaryotic communities. We found a large diversity of photosynthetic and heterotrophic eukaryotes incorporating HPG into proteins, with dinoflagellates and diatoms showing the highest percentages of BONCAT-labelled cells (49 ± 25% and 52 ± 15%, respectively). Among them, pennate diatoms exhibited higher HPG incorporation in the afternoon than in the morning, whereas small (≤5 μm) photosynthetic eukaryotes and heterotrophic nanoeukaryotes showed the opposite pattern. Centric diatoms (e.g., Chaetoceros, Thalassiosira, Lauderia spp.) dominated the eukaryotic HPG incorporation due to their high abundances and large sizes, accounting for up to 86% of the eukaryotic BONCAT signal, and strongly correlating with bulk 3H-leucine uptake rates. When including prokaryotes, eukaryotes were estimated to account for 19–31% of the bulk BONCAT signal. Our results evidence a large complexity in the osmotrophic uptake of HPG, which varies over time within and across eukaryotic groups, and highlights the potential of BONCAT to quantify osmotrophy and protein synthesis in complex eukaryotic communitiesThis work was funded by the Spanish Ministry of Science, Innovation and Universities (MICINN) through the MIAU (RTI2018-101025-30-B-I00) and MICOLOR (PID2021-125469NB-C31) projects and the Ramon y Cajal contract to C.R.G. (RYC2019-026758-I), with funding from the Spanish Government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S). C.M. was supported by the Juan de la Cierva-formación fellowship (FJC2021-047745-34 I), funded by the Spanish Ministry of Science and Innovation (MCIN/AEI) and the EU (“NextGenerationEU”/PRTR)Peer reviewe

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing

International audienceAlthough protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date

Detection of cannabinoid receptor type 2 in native cells and zebrafish with a highly potent, cell-permeable fluorescent probe.

Despite its essential role in the (patho)physiology of several diseases, CB2R tissue expression profiles and signaling mechanisms are not yet fully understood. We report the development of a highly potent, fluorescent CB2R agonist probe employing structure-based reverse design. It commences with a highly potent, preclinically validated ligand, which is conjugated to a silicon-rhodamine fluorophore, enabling cell permeability. The probe is the first to preserve interspecies affinity and selectivity for both mouse and human CB2R. Extensive cross-validation (FACS, TR-FRET and confocal microscopy) set the stage for CB2R detection in endogenously expressing living cells along with zebrafish larvae. Together, these findings will benefit clinical translatability of CB2R based drugs

Maquetes 3D d'organismes del plàncton

5 pages, figuresEn el marc del projecte "PlànctON: motor de vida al planeta oceà" hem recopilat diferents models 3D d'organismes microscòpics (microalgues, bacteris i virus) per representar la diversitat de formes d'alguns dels organismes més petits del plàncton i fomentar l'enfoc STEM (Science, Technology, Engineering and Mathematics). Aquestes maquetes son de gran utilitat per acompanyar les explicacions a l'aula o els tallers d'observació de mostres de plàncton amb microscopis. La col·lecció que tenim a dia d'avui s'ha fet recopilant models de diferents fonts amb 'objectiu de tenir la major diversitat possible de la fracció més petita del plàncton. Quan els models no existien prèviament, els hem dissenyat en el marc del projecte PlànctON i els hem posat a disposició de la comunitat penjant-los a Thingiverse. Aquest és el cas de l'alga Pyramimonas i del bacteri en forma de coc, però ens agradaria poder ampliar la col·lecció en futurs projectes. Les 3 fonts d'on provenen els models són: - Planktomania, un projecte educatiu que preten crear recursos per descubrir la infinitat del plancton a través de la tecnologia (realitat augmentada i virtual, objectes 3D) (http://planktomania.org/es/). - US National Institutes of Health (https://3dprint.nih.gov/discover/) un web interactiu per cercar, navegar, descarregar i compartir models biomèdics d’impressió en 3D, tutorials de modelatge i material educatiu. - MakerBot’s Thingiverse (thingiverse.com) és una comunitat de dissenyadors per descobrir, fer i compartir tot tipus de coses imprimibles en 3D"PlànctON: motor de vida al planeta oceà" (FCT-20-15876). Un projecte de l'Institut de Ciències del Mar (ICM-CSIC) i l'Escola del Mar (Ajuntament de Badalona), amb la col·laboració de la Fundación Española para la Ciencia y la Tecnología (FECYT) - Ministerio de Ciencia e Innovación, i amb el suport institucional de l'acreditació AEI "Severo Ochoa Centre of Excellence" (CEX2019-000928-S)Peer reviewe

Growth of uncultured heterotrophic flagellates in unamended seawater incubations

10 pages, 5 figures, 2 tablesUnamended dark incubations of 3 µm-filtered seawater were prepared with surface samples from the Norwegian Sea, the NW Mediterranean and the Indian Ocean. Except for the very oligotrophic Indian Ocean samples, this simple treatment promoted the growth of heterotrophic flagellates, with rates (0.3 to 1.2 d–1) typical of natural assemblages. Marine stramenopile (MAST)-1 and MAST-4 cell counts, obtained by fluorescence in situ hybridization (FISH), increased in most incubations. These yet uncultured protists were first detected in molecular surveys of marine picoplankton and have been recently shown to be globally distributed heterotrophic flagellates. Three incubations from the Norwegian Sea were studied in more detail by denaturing gradient gel electrophoresis (DGGE), 18S rRNA gene clone libraries and FISH counts for 5 MAST groups. Protist diversity changed gradually during the incubation, but the DGGE bands selected were already present at the beginning of the incubation. Clone libraries from the peaks in abundance of heterotrophic flagellates were dominated by MAST sequences. FISH counts revealed MAST-1B to be a very successful organism in the 3 incubations, accounting for 15 to 30% of heterotrophic flagellates after 6 to 8 d; MAST-1A and -1C cells were also abundant. MAST-4 cells peaked before the other groups and MAST-2 was the least represented. We concluded that unamended seawater incubations can select for heterotrophic flagellates abundant in situ but not yet isolated in pure culture. Therefore, they allow investigation of the growth requirements and dynamics of these uncultured protists, and provide promising preliminary stages for their isolationThis study was supported by projects ESTRAMAR (CTM2004-12631/MAR, MEC) and TRANSINDICO (REN2002-10951-E/MAR, MCyT) granted to R.M. and ARCTIC-2000 (REN2001-4909-E/ANT, MCyT) and Marine Genomics (FP6- 505403, EU) to C.P.A. Chief-scientists R. Ingvaldsen and D. Blackman and the crews of RV ‘Johan Hjort’ (Norwegian Marine Research Institute) and RV ‘Melville’ (Scripps Institution of Oceanography) provided an excellent sampling environmentPeer reviewe

Accessing the ecology of uncultured picoeukaryotes through a high-throughput automatic cell enumeration approach

VII European Congress of Protistology (ECP) in partnership with The International Society of Protistologists (ISOP), 5-10 September 2015, Sevilla, SpainSmall planktonic protists (i.e. picoeukaryotes, cells of 0.8-3 µm) have fundamental roles in the functioning of marine ecosystems, both as primary producers and as microbial grazers, and are likely the most abundant eukaryotes on Earth. Over the last fifteen years, technological progresses in molecular ecology and environmental sequencing have substantially boosted our understanding of these marine microbes, unveiling an unexpected diversity and notably the existence of new uncultured clades. These methodological improvements have notably offered the possibility to analyse simultaneously a substantial number of samples, allowing a more accurate description of their diversity in time and space. Faced with this growing amount of data available on microbial diversity, it is now challenging to design parallel molecular tools combined with microscopy to acccess their quantititive importance in environmental systems. However, this quantitative approach is relatively expensive, time-consuming and observant-dependant. Furthermore, their tiny size, their lack of specific morphological traits and the changing number of 18S rDNA copies between taxa among the picosize protistan community do not allow the use of classical microscopy and SSU-based molecular approaches (qPCR) to access their abundance in a large numbers of samples. Within this study we tested a newly developed automatic image acquisition and subsequently cell enumeration system of picoeukaryotic organisms. Two uncultured groups of picoeukaryotes differing by their cell size, MAST-4 (2 µm) and MAST-1C (5 µm), have been considered here to optimize this high-throughput quantitative approach for picoeukarytic cells. After targeting these groups by TSA-FISH using specific oligonucleotide probes, microscopic images were acquired fully automatically and cells enumerated using the program ACMEtool 2.0. In general, direct microscopic cell counts were in agreement with our automatic cell counting approach. The automated method can process a larger number of fields of view (FOVs) and consequently analyzes more cells, so it provides an estimate that is closer to true cell abundance. We were further able to test this automatic microscope and cell enumeration system on a spatial survey with a wide geographical coverage, about 100 stations from a circumglobal expedition, by depicting the spatial distribution of the two above-mentioned MAST groupsPeer Reviewe

Using click-chemistry for visualizing in situ changes of translational activity in planktonic marine bacteria

11 pages, 8 figures, supplementary material https://doi.org/10.3389/fmicb.2017.02360A major challenge in microbial ecology is linking diversity and function to determine which microbes are actively contributing to processes occurring in situ. Bioorthogonal non-canonical amino acid tagging (BONCAT) is a promising technique for detecting and quantifying translationally active bacteria in the environment. This technique consists of incubating a bacterial sample with an analog of methionine and using click-chemistry to identify the cells that have incorporated the substrate. Here, we established an optimized protocol for the visualization of protein-synthesizing cells in oligotrophic waters that can be coupled with taxonomic identification using Catalyzed Reporter Deposition Fluorescent in Situ Hybridization. We also evaluated the use of this technique to track shifts in translational activity by comparing it with leucine incorporation, and used it to monitor temporal changes in both cultures and natural samples. Finally, we determined the optimal concentration and incubation time for substrate incorporation during BONCAT incubations at an oligotrophic site. Our results demonstrate that BONCAT is a fast and powerful semi-quantitative approach to explore the physiological status of marine bacteriaThis work was supported by grants EcoRARE (CTM2014-60467-JIN), funded by the Spanish Government and the European Regional Development Fund (ERDF), and REMEI (CTM2015-70340-R) funded by the Spanish GovernmentPeer Reviewe