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Bacterial single-cell approaches to the relationship between diversity and function in the Sea EVK3-CT2002-00078 November 2002 - October 2005 Marine Biodiversity Cluster Meeting - Brussels-July’03 Image: K. Jürgens 1 µm The project’s ultimate goal Roundicoccus southamptii Dalibacter banyuleus 75% of BCD preferentially grazed by HNF very sensitive to viral attack dominates DMSP uptake Spirovibrio kalmariensis Tinymonas bremenensis (all names are fiction) Bacterial single-cell approaches to the relationship between diversity and function in the Sea B) Bacterial diversity A) Bacterial biogeochemical function linkage C) Single-cell approaches By developing new methodologies, sampling different European seas and through laboratory and mesocosm experiments, we will address the main objectives of BASICS: The identification of the most important prokaryotic organisms associated with the biogeochemical functioning (in the carbon and sulfur cycling) of the sea, through the development of single-cell analysis techniques applied to pelagic microbes. BASICS will also study how resilient the link is between the diversity and the C and S biogeochemical cycling by bacterioplankton, in the face of the most important global environmental changes expected in European coastal waters. Objective 1: To describe bacterioplankton diversity in the coastal seas of Europe Objective 2: To describe the seasonal changes in the cycling of carbon and sulfur mediated by planktonic bacteria in surface waters of European coastal seas Objective 3: To design, test and fine-tune different methods and research strategies for the single-cell analysis of natural bacterioplankton organisms Objective 4: To link bacterial diversity and biogeochemical function (in the cycles of C and S) and identify the bacterial phylotypes responsible for the crucial steps in oceanic biogeochemical cycling, and to refine recently developed conceptual frameworks for the links between species richness (number of dominant coexisting species) and biogeochemical cycling Objective 5: To estimate the effect of environmental changes affecting the ocean’s bacterially-mediated biogeochemical function, global bacterial diversity and the link between bacterial diversity and C and S cycling -) Why bacteria ? A) Why bacterial diversity ? B) Why biogeochemical function ? • why the C cycle • why the S cycle C) Why study the linkage ? D) The key: Single-Cell methods E) Project organization F) Project partnership • The sampling sites • A few of the first results Bacteria are... • • • • • Most of Earth’s living biomass Most abundant living particles in the sea Only significant DOM transformers Responsible for most of ocean’s respiration Largest living surface in the ocean • The largest “unknown pool” of genomic and metabolic (i.e. functional) diversity • bacteria play far more important ecological roles in natural environments than their small sizes would suggest (Brock et al.’88) • “L’essentiel est invisible pour les yeux” (Antoine de Saint-Exupéry) • “small is beautiful !” Chisholm 2000 -) Why bacteria ? A) Why bacterial diversity ? B) Why biogeochemical function ? • why the C cycle • why the S cycle C) Why study the linkage ? D) The key: Single-Cell methods E) Project organization F) Project partnership • The sampling sites • A few of the first results Trying to make VISIBLE what is invisible Bacteria Archaea Stéphan Daigle National Geographic Hey, it’s me ! Pure culture Genes Proteins Activity Molecular biology Culturing native prokaryotes SYSTEM CULTURABILITY (%) Marine 0.001 - 0.1 Freshwater 0.25 Mesotrophic lake 0.1 - 1 Estuary 0.1 - 3 Activated sludge 1 - 15 Sediments 0.25 Soil 0.3 Are these few isolated bacteria relevant in plankton biogeochemistry ? Overview of techniques in molecular ecology Sea water microbes In situ hybridization Detection single cells Biomass collection Microbial biomass Nucleic acid extraction Comparison among communities DNA reassociation kinetics DNA cross-hybridization Fingerprinting: lmw RNA Bulk nucleic acid extract Quantitative rRNA hybridization Abundance some phylotypes PCR amplification Comparison among communities Fingerprinting DGGE ARDRA T-RFLP PCR product Cloning and sequencing Species composition BASICS partners will follow the seasonality of bacterial diversity in several sites in the North, Mediterranean and Baltic Seas and the English Channel We will use a variety of techniques: - fingerprinting techniques (DGGE, T-RFLP, SSCP...) - detection of single phylotypes/groups (FISH) - cloning and sequencing - culture isolation Objective 1: To describe bacterioplankton diversity in the coastal seas of Europe • Describe seasonality in “diversity” (what is there, who’s the most abundant) • Usage of different techniques (fingerprinting &clon libraries & isolation...) • Common framework • Characterization of isolates • Biotechnological exploitation of the isolates The BactLib (Bacterial Culture database) The ecologically-referenced phylotype database -) Why bacteria ? A) Why bacterial diversity ? B) Why biogeochemical function ? • why the C cycle • why the S cycle C) Why study the linkage ? D) The key: Single-Cell methods E) Project organization F) Project partnership • The sampling sites • A few of the first results To know the main routes of C circulation is a prerequisite... JGOFS ... for understanding the fluxes of C in the ocean It is the belief of BASICS that too much effort has been put in the past in describing bacterial diversity in the ocean... ... barely telling what the position and depth of the sample was... How can we understand the role that specific bacteria will play in nature then ??? Fig. 2.2. The feedback linking oceanic plankton and climate through the production of atmospheric sulfur. The original hypothesis postulated that production of dimethylsulfide (DMS) by phytoplankton, and its subsequent ventilation and oxidation in the atmosphere feeds cloud condensation nuclei in marine stratus, thereby increasing cloud albedo. If the consequent reduction in solar irradiance forced phytoplankton toproduce less DMS, then a negative feedback would operate, thus stabilizing climate. Recent advances suggest that it is not only phytoplankton but the whole food web (with bacteria playing a crucial role) that releases DMS. Kiene et al. 2000 - DMSP is a labile organic molecule which can represent 15% of BCD and close to 100% of S demand - DMS participates in climate feedback BASICS partners will follow the seasonality of microbial biogeochemical cycling in several sites in the North, Mediterranean and Baltic Seas and the English Channel We will measure a large amount of stocks and rates: - Bulk DOC and nutrients - Algal activity and biomass - Viral and protozoan stocks and activity - DMS and DMSP stocks and rates - etc. Objective 2: To describe the seasonal changes in the cycling of carbon and sulfur mediated by planktonic bacteria in surface waters of European coastal seas • Seasonal studies in C and S cycling • Key biogeochemical steps little studied Ocean Projects in IGBP II BASICS -) Why bacteria ? A) Why bacterial diversity ? B) Why biogeochemical function ? • why the C cycle • why the S cycle C) Why study the linkage ? D) The key: Single-Cell methods E) Project organization F) Project partnership • The sampling sites • A few of the first results P-Z-N Dynamics: Populations Change through Time T. Michaels Nitrate Zoop Phyto Zooplankton Phytoplankton Phyto T. Michaels Does it matter what biology is hidden within each box? Diatoms Prasinophytes Prymnesiophytes Prochlorococcus Synechococcus Phyto Zoo Phyto Dynamic Green Ocean Model Fe Si PO NO3 NH4 4 coccolith. N2 fixers diatoms CaCO3 DMS producers Nano phytoplankton DOM Buitenhuis et al. 2003 Biogeochemical fluxes are a function of community structure MicroZoo T. Michaels PicoPhyto NanoPhyto Bacteria Bacteria & & Nutrients Nutrients Stays Suspended MesoSALPS Zoo MicroPhyto Sinking Sinking Particles Particles Fecal Pellets salp D. Steinberg copepod euphausiid 1 mm Bacteria are abundant and important But We are unable of grouping them in biogeochemically relevant and distinct “boxes” Because we don’t know whether they all do the same, or not... Objective 4: To link bacterial diversity and biogeochemical function (in the cycles of C and S) and identify the bacterial phylotypes responsible for the crucial steps in oceanic biogeochemical cycling, and to refine recently developed conceptual frameworks for the links between species richness (number of dominant coexisting species) and biogeochemical cycling • Multistep strategy • Coocurrence analysis (Synthesis workshop !) • Design of oligonucleotide probes • Test of BGQ function in isolates • Test during an experimental algal bloom • Each approach is partial and has some risk of failure -) Why bacteria ? A) Why bacterial diversity ? B) Why biogeochemical function ? • why the C cycle • why the S cycle C) Why study the linkage ? D) The key: Single-Cell methods E) Project organization F) Project partnership • The sampling sites • A few of the first results Objective 3: To design, test and fine-tune different methods and research strategies for the single-cell analysis of natural bacterioplankton organisms • Flow cytometry sorting: standarization & controls • FISH improvements • Combination of techniques: MicroFISH, MicroACT, etc... • Capillary electrophoresis • X-Ray microanalysis • .... ??? The power is in the combination of methods Fluorescence in situ Hybridization: ("phylogenetic staining") Environmental sample Extracted nucleic acids DNA rRNA Hybridized Nucleic acid probe rDNA clones rDNA Sequences Comparative Analysis rDNA database Hybridization MPIMM Sequencing DAPI-stained MicroFISH 35S DMSP Roseobacter + AU ICM DAPI + AU Aminoacids Protein % active cells 60 C 40 g 20 0 60 C a g a 20 g 20 a 0 a g 40 40 60 0 Atlantic Ocean CC 0 20 40 60 % cells in sample HMW-DOM Cytophaga g-Proteobacteria LMW-DOM a-Proteobacteria Cottrell & Kirchman 2000 Cell sorting by FCM Prelabeling Radioactive substrates Nucleic acid probes Physiological probes Laser (488 nm) Further analyses of sorted fractions Trash FACSCalibur OOB FACSVantage High speed cell sorter * Activity (radioactivity) * Identification * Isolation * Chemical analyses (C; N; P,….) PML/SOC/MPIMM DMSP producing phytoplankton bloom in the North Sea Emiliania huxleyi y Prorocentrum minimum Flow Citometry Blue fluorescence (DNA) 103 FISH Cytophaga/Flavovacterium Roseobacter 102 SAR86 101 100 Threshold 101 102 103 Red fluorescence (protein) Abundance highly correlated with DMSP consumption Zubkov et al. 2001 PML/SOC/MPIMM Capillary electrophoresis Basic scheme Capillary Absorbance detector Data recording Sample + Power Supply Inlet buffer reservoir NIOZ Outlet buffer reservoir Protein separation + + + + + ++ + SDS - - + - - - - Detection window - + Sample - + Injection - + Separation - + Detection NIOZ UV absorbance Objective 5: To estimate the effect of environmental changes affecting the ocean’s bacterially-mediated biogeochemical function, global bacterial diversity and the link between bacterial diversity and C and S cycling • Functional redundancy, ecosystem stability... ... effects of env. change on - diversity - BGQ function - their linkage • Environmental perturbations in microcosms -) Why bacteria ? A) Why bacterial diversity ? B) Why biogeochemical function ? • why the C cycle • why the S cycle C) Why study the linkage ? D) The key: Single-Cell methods E) Project organization F) Project partnership • The sampling sites • A few of the first results WS0: Coordination WP1: Seasonal studies of bacterial diversity WP2: Explotaition of phylotypes and isolate information WP3: Seasonal studies of biogeochemical C & S cycling WP 4: SCA method development Phase 1 WS1: Synthesis WS on bacterial diversity in coastal euro pean seas and C & S cycling Phase 2 WP5: Linking bacterial diversity with biogeochemical function WP6: Experimental determination of the factors that regulate the link between bacterial diversity and function WS2: Mesocosm experiment WS3: Synthesis WS on SCA WP7: Functional stability of the link facing global change WS4: Microcosm experiments WS5: Summary Phase 3 WP1: Diversity WP2: Exploitation WP3: C/S-Cycling WP4: SCA dev WP5: Linking WP6: Factors regulate WP7: Funct. stabil. -) Why bacteria ? A) Why bacterial diversity ? B) Why biogeochemical function ? • why the C cycle • why the S cycle C) Why study the linkage ? D) The key: Single-Cell methods E) Project organization F) Project partnership • The sampling sites • A few of the first results Bergen Kalmar Plymouth Southampton Bremen Texel Toulouse Villefranche Banyuls Barcelona Barcelona Pep Gasol / Rafel Simó Banyuls Philippe Lebaron Texel Gerhard Herndl Kalmar Åke Hagström Toulouse Pascal Bordat Villefranche Markus Weinbauer Plymouth Steve Archer Bremen J. Pernthaler / B. Fuchs Bergen Frede Thingstad / Mikal Heldal Southampton Mike Zubkov / Peter Burkill -) Why bacteria ? A) Why bacterial diversity ? B) Why biogeochemical function ? • why the C cycle • why the S cycle C) Why study the linkage ? D) The key: Single-Cell methods E) Project organization F) Project partnership • The sampling sites • A few of the first results • Shelf = 20% of Ocean NPP; supports 90% of Marine Fisheries Production • Coastal population = 2.2 billion (40% of total) Before fishing • Nutrient inputs After fishing (Jackson et al. 2001) microbialization of food chains Sampling sites • Blanes Bay (ICM) • Banyuls - MOLA & MILA station (OOB) • Baltic proper landsort (UNIK) • North Sea Texel site (NIOZ) • Villefranche point “B” (LOV) • L4 English Channel station (PML & SOC) • Helgoland site G (MPIMM) General characteristics of Blanes Bay • Typical Mediterranean waters: warm, salty and nutrient-poor • Oligotrophic coastal system (annual average chlorophyll of 0.5 µg l-1) • Relatively unaffected by human or freshwater influence • Separated from oceanic waters by a southwest current associated with a front in the continental slope (10-20 miles offshore) • Episodic intrusions of oceanic waters caused by the Blanes canyon Half mile from harbour Depth of 20 m ICM Seasonality of phytoplankton Main peak of chlorophyll a during late winter, driven by high atmospheric pressures together with irradiances and temperatures higher than similar latitudes in the Atlantic ICM Seasonal succession of bacterioplankton 3 September 2July 29 July 9 October Summer Fall 4 November 1 Desember 26 March 29 April Spring 3 June 27 January 22 Desember 97 25 February 5 February 12 February 19 February ICM 3 March 1.0 18 March 11 March Winter Temporal dynamics of bacterial groups and populations ICM How representative is the Blanes site? CO Blanes-Jul Blanes-Nov Barcelona-Jul Masnou-Jul Masnou-Nov Harbour-Jan Barcelona-Jan Masnou-Jan Barcelona-Nov CC Harbour-Apr Barcelona-Apr Masnou-Apr Four sites 70 km appart Blanes-Apr One sample for season Blanes-Jan Comparison of bacterial composition by Harbour-Jul Harbour-Nov DGGE ICM 1.0 Summer Winter Spring PML/SOC seasonal sampling site Time series station since 1988 (Roger Harris and PML Zooplankton group) Measurements: (weekly throughout the year) •Environmental: Chl a, Temp, Salinity, Optics, POC, PON •Biological: Phytoplankton, Zooplankton, Bacteria, Viruses •Processes: Primary production (FRRF), Calanus egg production, L4 location: PML/SOC Western English Channel -) Why bacteria ? A) Why bacterial diversity ? B) Why biogeochemical function ? • why the C cycle • why the S cycle C) Why study the linkage ? D) The key: Single-Cell methods E) Project organization F) Project partnership • The sampling sites • A few of the first results MPIMM Horseradish-peroxidase-labeled FISH probes and catalyzed reporter deposition (CARD) (tyramide signal amplification, TSA) fluorescently labeled tyramide HRP HRP HRP HRP protein Permeabilization MPIMM Hybridization Signal Amplification conventional FISH FISH & signal amplification 10 seconds 1 second Sediment 10 seconds 1 second Surface water MPIMM Exposure time: Euryarchaeota in coastal North Sea surface waters DAPI-staining G2 Euryarchaea Coastal North Sea, % detection by FISH Probe detection rate [% of total cells] 50 G2-Euryarchaeota 40 30 20 10 0 MPIMM 03 05 07 Month 09 11 01 And still, other unplanned BASICS returns... • Development and optimization of molecular tools (useful with other microorganisms and in other ecosystems • Better knowledge of the spatial and temporal scales of bacterial biodiversity changes • Applicability of the “key species” concept to bacteria • Autoecology of bacteria • Indicator bacterial species of ecosystem perturbations • Biotechnological exploitation of effort made at the “pure science” (knowledge for its own sake) level • ... Bacterial single-cell approaches to the relationship between diversity and function in the Sea Bacterial diversity Bacterial biogeochemical function linkage Single-cell approaches www.icm.csic.es/bio/projects/basics