1. Bacterial single-cell approaches to the relationship between
diversity and function
in the Sea
C) Aproximacions a nivell de
cèl·lules individuals
B) Diversitat bacteriana
Funció biogeoquímica
dels bacteris
Relació

2. L’objectiu final del projecte
Roundicoccus
southamptii
Tinymonas
bremenensis
Dalibacter
banyuleus
(tots els noms són inventats)
Spirovibrio
kalmariensis
75% de la BCD
domina la
incorporació de
DMSP
consumit
preferentment
pels HNF
molt sensitiu a
l’atac viral

5. Ocean Projects in IGBP II
BASICS

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

7. Phyto
Zoo

8. Biogeochemical fluxes are a function of community structure

9. 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...

10. La clau: les tècniques d’anàlisi a
nivell individual

11. Horseradish-peroxidase-labeled FISH probes
and catalyzed reporter deposition (CARD)
(tyramide signal amplification, TSA)
HRP
Hybridization
HRP
protein
fluorescently labeled tyramide
Signal Amplification
Permeabilization
MPIMM

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

13. C C a a g g g a g C C a Protein Aminoacids HMW-DOM Cytophaga20 40 60 60 C C a a g
% active cells 40 g g a 20 g
Atlantic
Ocean C C a 20 40 60
% cells in sample
HMW-DOM
Cytophaga
g-Proteobacteria
LMW-DOM
a-Proteobacteria
Cottrell & Kirchman 2000

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

15. Standard station 41°40´ N, 2°48´ E 1 mile offshore, 20 m depth
One case-study, in the Bay of Blanes, July 2003
Half mile from harbour Depth of 20 m
Standard station 41°40´ N, 2°48´ E 1 mile offshore, 20 m depth

16. 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) • In summer, 1% attenuation depth of light 320 nm is at the bottom (20 m) • Relatively unaffected by human or freshwater influence • Episodic intrusions of oceanic waters caused by the Blanes canyon

17. The environment - Surface temperature10 12 14 16 18 20 22 24 26
180
360
540
720
900
1080
Temp
Temperature (°C)
Julian day
PICODIV
BASICS
20 Mar Nov 2002
13 Jan - 25 Nov 2003

18. The environment - Surface chlorophyll10 12 14 16 18 20 22 24 26
0.5 1
1.5 2
2.5
180
360
540
720
900
1080
Temp
Chlorophyll
Temperatura (°C)
Julian day
2003
Summer (June-September)’03
Total. Mean: 0.68 µg l Range: µg l-1
< 3µm. Mean: 0.27 µg l Range: µg l-1

19. The seasonal changes - Primary production
14 July 2003

20. The seasonal changes - Heterotrophic prokaryotes
14 July 2003
% CTC+ cells 5 10 15 20 25

21. The seasonal changes - Bacterial production & respiration
14 July 2003
BGE
2 %
63 %
20 %
3 %

22. The seasonal changes - Bacterial grazing losses
14 July 2003 10 12 14 16 18 20 22 24 26 30 40 50 90
180
270
360
Temperature (°C)
% bacteria day-1
% bacteria day -1
Julian day

23. The seasonal changes - Community metabolism
14 July 2003 10 12 14 16 18 20 22 24 26
Temperature (° C)

24. Bacterial diversity - Relative constancy...
1998

25. Biolog “diversity”
glycogen
Higher use of polymers in July

26. g g Bacterial diversity - Clon libraries SAR11 SAR11 SAR11 Roseobacter10 12 14 16 18 20 22 24 26
SAR11
Roseobacter
Other Alfas
SAR86
Other Gammas
CFB
Actinobacteria
Synechococcus
Chloroplasts
Other
5 10 5
1 10 6
1.5 10
Temperature (°C)
Bacteria 90
180
270
360
Julian day
SAR11
SAR11 g g

27. Bacterial diversity - CARD-FISH Bacteria & Euryarchaea20 40 60 80
100 90
180
270
360
Eub I-III
Eury808
% of DAPI cells
Julian day
14 July 2003
Bacteria: 70% (59-91%) Euryarchaea: 1.3% (0-3.6%)

28. Bacterial diversity - CARD-FISH main groups20 40 60 80 90
180
270
360
The three groups
Alf968
Gam42a
CF319a
% of DAPI cells
Julian day
14 July 2003

29. Bacterial diversity -  proteobacteria
14 July 2003
Alteromonas !

30. Conclusions in July
• Small bloom by flagellates, with no apparent nutrient increases
• Bacteria responded to this bloom with changes in activity and diversity.
• The whole community was affected (turned net autotrophic)
• Carbon was accumulated in the form of bacterial cells
The shift in bacterial community composition was towards g-proteobacteria (detectable as Alteromonas macleodii) which showed high metabolic versatility (used DMSP, had high exoenzyme levels, used polymers...), and relatively lower grazing pressure.
During this event, most C and S appeared to circulate through this very specific bacterial group, which is, therefore, associated to a particular pattern of biogeochemical C and S cycling

31. Experiments of Nutrient limition of bacterial production
-Monthly sampling
-250 ml unfiltered seawater
-Additions of:
20 µM C (glucose)
20 µM C (DMSP)
4 µM N (ammonium)
1 µM P (phosphate)
-24 h incubation
-Measure leucine incorp.

32. Shifts in nutrient limition of bacterial growth

33. Changes in dominant bacteria in seawater cultures - mimicking succession in situ
Jan
Jul
Mar
Apr
Jun
May
Aug
Sep
Oct
Jan
Nov
Dec
Feb
Mar
2003
2004

34. Identity of bacteria in the seawater cultures
Vibrio & Alteromonas
Roseobacter clade
Cryomorphaceae
Flavobacteria
Note the color code

35. Plate counts from Blanes Bay after stormy weather
10x
100x
Representatives of alpha- and gamma-proteobacteria and Bacteroidetes can all be found here

36. An all-together tree Sequenced by the Moore foundation
DGGE band sequences
Other Blanes isolates
Blanes clon libraries
An all-together tree

39. • Els aïllats seqüenciats semblen ser representatius
• Són abundants in situ ?
mirar-ho
• Com interaccionen amb l’entorn ? (nutrients, etc.)
cal mirar quan i com es desenvolupen
quines característiques enzimàtiques tenen
Per poder aprofitar la informació genòmica !
This is a joint venture by Jarone Pinhassi, Pep Gasol, Carles Pedrós-Alió & the Basics gang