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Ba cterial si ngle- c ell approaches to the relationship between diversity and function

Ba cterial si ngle- c ell approaches to the relationship between diversity and function in the S ea. EVK3-CT2002-00078 November 2002 - October 2005. Marine Biodiversity Cluster Meeting - Brussels-July’03. Image: K. Jürgens. 1 µm. Roundicoccus southamptii. Dalibacter

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Ba cterial si ngle- c ell approaches to the relationship between diversity and function

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  1. 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

  2. Image: K. Jürgens 1 µm

  3. 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) The project’s ultimate goal

  4. A) Bacterial biogeochemical function B) Bacterial diversity C) Single-cell approaches Bacterial single-cell approaches to the relationship between diversity and function in the Sea linkage

  5. 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.

  6. 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

  7. -) 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

  8. 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 !”

  9. Chisholm 2000

  10. -) 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

  11. Bacteria Archaea Trying to make VISIBLE what is invisible

  12. Stéphan Daigle National Geographic

  13. Hey, it’s me ! Pure culture Molecular biology Genes Proteins Activity

  14. 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 ?

  15. Overview of techniques in molecular ecology

  16. 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

  17. 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

  18. The BactLib (Bacterial Culture database)

  19. The ecologically-referenced phylotype database

  20. -) 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

  21. To know the main routes of C circulation is a prerequisite...

  22. ... for understanding the fluxes of C in the ocean JGOFS

  23. 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 ???

  24. 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.

  25. - DMSP is a labile organic molecule which can represent 15% of BCD and close to 100% of S demand - DMS participates in climate feedback Kiene et al. 2000

  26. 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.

  27. 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

  28. BASICS Ocean Projects in IGBP II

  29. -) 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

  30. P-Z-N Dynamics: Populations Change through Time Nitrate Zoop T. Michaels Phyto Zooplankton Phytoplankton

  31. Diatoms Prasinophytes Prymnesiophytes Prochlorococcus Synechococcus Phyto Does it matter what biology is hidden within each box? T. Michaels Phyto

  32. Zoo Phyto

  33. Dynamic Green Ocean Model Si PO4 NO3 NH4 Fe coccolith. Nano phytoplankton DMS producers N2 fixers diatoms CaCO3 DOM Buitenhuis et al. 2003

  34. Biogeochemical fluxes are a function of community structure

  35. Meso-Zoo Micro-Zoo SALPS Micro-Phyto Pico-Phyto Nano-Phyto Bacteria & Nutrients Sinking Particles Sinking Particles Bacteria & Nutrients Stays Suspended T. Michaels

  36. Fecal Pellets salp copepod euphausiid 1 mm D. Steinberg

  37. 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...

  38. 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

  39. -) 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

  40. 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

  41. Environmental sample Extracted nucleic acids DNA rRNA rDNA clones Nucleic acid probe rDNA Sequences Comparative Analysis rDNA database Hybridization Sequencing Fluorescence in situ Hybridization: ("phylogenetic staining") Hybridized DAPI-stained MPIMM

  42. DAPI + AU MicroFISH 35S DMSP Roseobacter + AU ICM

  43. 60 40 20 0 0 20 40 60 Aminoacids Protein 60 C C a a g % active cells 40 g g g 20 a Atlantic Ocean C C a 0 0 20 40 60 % cells in sample HMW-DOM Cytophaga g-Proteobacteria a-Proteobacteria LMW-DOM Cottrell & Kirchman 2000

  44. Cell sorting by FCM Laser (488 nm) Trash Radioactive substrates Nucleic acid probes Physiological probes Prelabeling Further analyses of sorted fractions * Activity (radioactivity) * Identification * Isolation * Chemical analyses (C; N; P,….) FACSVantage High speed cell sorter FACSCalibur OOB

  45. PML/SOC/MPIMM

  46. Flow Citometry 103 Cytophaga/Flavovacterium FISH 102 Blue fluorescence (DNA) Roseobacter Threshold 101 100 101 102 103 Red fluorescence (protein) Abundance highly correlated with DMSP consumption SAR86 DMSP producing phytoplankton bloom in the North Sea Emiliania huxleyi y Prorocentrum minimum Zubkov et al. 2001 PML/SOC/MPIMM

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