c) aproximacions a nivell de cèl·lules individuals cèl·lules individuals relació b) diversitat...
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C) Aproximacions a nivell deC) Aproximacions a nivell de cèl·lules individualscèl·lules individuals
RelacióRelacióB) Diversitat bacterianaB) Diversitat bacteriana
A)A) Funció biogeoquímica Funció biogeoquímica dels bacterisdels bacteris
BaBacterial cterial sisingle-ngle-ccell ell approaches to the approaches to the relationship between relationship between diversity and function diversity and function in the in the SSeaea
L’objectiu final del projecteL’objectiu final del projecte
Roundicoccus Roundicoccus southamptiisouthamptii
TinymonasTinymonas bremenensisbremenensis
Dalibacter Dalibacter banyuleusbanyuleus
(tots els noms són inventats)(tots els noms són inventats)
SpirovibrioSpirovibrio kalmariensiskalmariensis
75% de la BCD75% de la BCD
domina la domina la incorporació de incorporació de DMSPDMSP
consumit consumit preferentment preferentment
pels HNFpels HNF
molt sensitiu a molt sensitiu a l’atac virall’atac viral
Ocean Projects in IGBP II
BASICSBASICS
Does it matter what biology is hidden within each box?Does it matter what biology is hidden within each box?
Phyto
DiatomsPrasinophytesPrymnesiophytes
ProchlorococcusSynechococcus
Phyto
T. M
ichaels
PhytoPhyto
ZooZoo
Biogeochemical fluxes are a function of community structureBiogeochemical fluxes are a function of community structure
Bacteria are abundant and importantBacteria are abundant and important
ButBut
We are unable of grouping them in We are unable of grouping them in biogeochemically relevant and biogeochemically relevant and distinctdistinct““boxes”boxes”
we don’t know whether they all do the same, or we don’t know whether they all do the same, or not...not...
BecauseBecause
La clau: les tècniques d’anàlisi aLa clau: les tècniques d’anàlisi anivell individualnivell individual
Horseradish-peroxidase-labeled FISH probes and catalyzed reporter deposition (CARD)
(tyramide signal amplification, TSA)
HRP
protein
fluorescently labeled tyramide
Signal AmplificationPermeabilization
HRP
HRP HRP
Hybridization
MPIMMMPIMM
DAPI + AU
Roseobacter + AU
MicroFISH
35S DMSP
ICMICM
Cottrell & Kirchman 2000Cottrell & Kirchman 2000
HMW-DOMHMW-DOM
LMW-DOMLMW-DOM
CytophagaCytophaga
-Proteobacteria-Proteobacteria
-Proteobacteria-Proteobacteria
% a
ctiv
e c
ells
% a
ctiv
e c
ells
% cells in sample
0
20
40
60
0 20 40 60
Protein
C C
0
20
40
60
0 20 40 60
Aminoacids
C CAtlanticAtlanticOceanOcean
Blu
e fl
uore
scence
(D
NA
)
103
102
Threshold101
100 101 102 103
Red fluorescence (protein)
Flow Citometry
DMSP producing phytoplankton bloom in the North SeaEmiliania huxleyi y Prorocentrum minimum
FISH
Roseobacter
Cytophaga/Flavovacterium
SAR86
Zubkov et al. 2001Zubkov et al. 2001
PML/SOC/MPIMMPML/SOC/MPIMM
Abundance highly correlated with
DMSP consumption
Standard stationStandard station
41°40´ N, 2°48´ E
1 mile offshore, 20 m
depth
One case-study, in the Bay of Blanes, July 2003One case-study, in the Bay of Blanes, July 2003
Half mile from harbour
Depth of 20 m
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
The environmentThe environment - Surface temperature - Surface temperature
10
12
14
16
18
20
22
24
26
0 180 360 540 720 900 1080
TempT
em
pe
ratu
re (
°C)
Julian day
PICODIVPICODIV BASICSBASICS20 Mar 2001 - 27 Nov 2002 13 Jan - 25 Nov 2003
10
12
14
16
18
20
22
24
26
0
0.5
1
1.5
2
2.5
0 180 360 540 720 900 1080
Temp ChlorophyllT
em
pe
ratu
ra (
°C)
Ch
loro
ph
yll
Julian day
Total. Total. Mean: 0.68 µg lMean: 0.68 µg l-1-1 Range: 0.21 - 2.09 µg l Range: 0.21 - 2.09 µg l--
11
Temp ChlorophyllChlorophyll <3µm
The environmentThe environment - Surface chlorophyll - Surface chlorophyll
< 3µm. < 3µm. Mean: 0.27 µg lMean: 0.27 µg l-1-1 Range: 0.10 - 0.63 µg l Range: 0.10 - 0.63 µg l-1-1
Summer (June-September)’03Summer (June-September)’03
20032003
0
0.5
1
1.5
2
2.5
0
1
2
3
4
5
0 90 180 270 360
Chl a (µg l-1) PP (mgC m-3 h-1)
Chl a (µg l
-1)
PP (mgC m
-3 h
-1)
Julian day
The seasonal changesThe seasonal changes - Primary production - Primary production
14 July 2003
JUNE JULYµgC/L µgC/L
Diatoms 0.5 5.7Flagellates 7.1 69.7Picoplankton 11.5 23.6
10
12
14
16
18
20
22
24
26
5 105
1 106
1.5 106
0 90 180 270 360
Temperature (°C) Bacteria
Temperature (°C)
Bacteria
Julian day
The seasonal changesThe seasonal changes - Heterotrophic prokaryotes - Heterotrophic prokaryotes
14 July 2003 % CTC+ cells
0
5
10
15
20
25
% C
TC
+ ce
lls
0
0.5
1
1.5
2
2.5
0
20
40
60
80
0 90 180 270 360
Chl a (µg l-1) Bacterial RespirationBacterial Production
Temperature (°C)
Bacterial Production/Respiration
(µgC l
-1 d
-1)
Julian day
The seasonal changesThe seasonal changes - Bacterial production & respiration - Bacterial production & respiration
14 July 2003
BGE 2 % 63 % 20 % 3 %
10
12
14
16
18
20
22
24
26
0
10
20
30
40
50
0 90 180 270 360
Temperature (°C) % bacteria day-1
Te
mp
era
ture
(°C
) % b
acte
ria d
ay
-1
Julian day
The seasonal changesThe seasonal changes - Bacterial grazing - Bacterial grazing
losseslosses
14 July 2003
0
0.5
1
1.5
2
2.5
0
0.5
1
1.5
2
2.5
3
3.5
0 90 180 270 360
Chl a (µg l-1) P/R
Chlorophyll
a (µg l
-1)
Net Production / Total Respiration
Julian day
The seasonal changesThe seasonal changes - Community metabolism - Community metabolism
10
12
14
16
18
20
22
24
26
Te
mp
era
ture
(°
C)
14 July 2003
19981998
Bacterial diversityBacterial diversity - Relative constancy... - Relative constancy...
Higher use of polymers in JulyHigher use of polymers in July
Biolog Biolog “diversity”“diversity”
glycogenglycogen
13 May 2003 4 August 2003
Bacterial diversityBacterial diversity - Clon libraries - Clon libraries
SAR11RoseobacterOther AlfasSAR86
Other GammasCFBActinobacteriaSynechococcusChloroplastsOther
10
12
14
16
18
20
22
24
26
5 105
1 106
1.5 106
0 90 180 270 360
Te
mp
era
ture
(°C
)
Bacte
ria
Julian day
SAR11SAR11
SAR11SAR11
Bacterial diversityBacterial diversity - CARD-FISH Bacteria & Euryarchaea - CARD-FISH Bacteria & Euryarchaea
14 July 2003
0
20
40
60
80
100
0 90 180 270 360
Eub I-IIIEury808
% o
f D
AP
I ce
lls
Julian day
Bacteria: Bacteria: 70% (59-91%) 70% (59-91%)
Euryarchaea:Euryarchaea: 1.3% (0-3.6%)1.3% (0-3.6%)
14 July 2003
Bacterial diversityBacterial diversity - CARD-FISH main groups - CARD-FISH main groups
0
20
40
60
80
0 90 180 270 360
The three groupsAlf968Gam42aCF319a
% o
f D
AP
I ce
lls
Julian day
Bacterial diversityBacterial diversity - - proteobacteriaproteobacteria
14 July 2003
0
10
20
30
40
50
0 90 180 270 360
Gam42aSAR86Alt1416NOR5
Julian day
Alteromonas !Alteromonas !
ConclusionsConclusions in July 2003...in July 2003...
• • Small bloom by flagellates, with no apparent nutrient increasesSmall bloom by flagellates, with no apparent nutrient increases
• • Bacteria responded to this bloom with changes in activity and Bacteria responded to this bloom with changes in activity and diversity. diversity.
• • The whole community was affected (turned net autotrophic)The whole community was affected (turned net autotrophic)
• • Carbon was accumulated in the form of bacterial cellsCarbon was accumulated in the form of bacterial cells
The shift in bacterial community composition was towards The shift in bacterial community composition was towards --proteobacteria (detectable as proteobacteria (detectable as Alteromonas macleodiiAlteromonas macleodii) which showed ) which showed
high metabolic versatility (used DMSP, had high exoenzyme levels, high metabolic versatility (used DMSP, had high exoenzyme levels, used polymers...), and relatively lower grazing pressure.used polymers...), and relatively lower grazing pressure.
During this event, most C and S appeared to circulate through this very During this event, most C and S appeared to circulate through this very specific bacterial group, which is, therefore, associated to a particular specific bacterial group, which is, therefore, associated to a particular pattern of biogeochemical C and S cyclingpattern of biogeochemical C and S cycling
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.
0
1
2
3
4
Treatment
24 May 2004
Shifts in nutrient limition of bacterial growth
0
2
4
6Ctrl
C
P
2003 2004
0
3
6
9
12C+P
DMSP+P
Jan
Mar
Apr
May Jun
Jul
Aug
Mar
Sep
FebOct
Jan
Nov
Dec
20042003
Changes in dominant bacteria in seawater cultures - mimicking succession in situ
Identity of bacteria in the seawater cultures
Flavobacteria
Cryomorphaceae
Vibrio & Alteromonas
Roseobacter clade
Note the color codeNote the color code
100x10x
Plate counts from Blanes Bay after stormy weather
Representatives of alpha- and gamma-proteobacteria and Bacteroidetes can all be found here
An all-together treeAn all-together tree
Sequenced by the Sequenced by the Moore foundationMoore foundation
DGGE band sequencesDGGE band sequences
Other Blanes isolatesOther Blanes isolates
Blanes clon librariesBlanes clon libraries
• • Els aïllats seqüenciats semblen ser representatiusEls aïllats seqüenciats semblen ser representatius• • Són abundants in situ ?Són abundants in situ ?
mirar-homirar-ho• • Com interaccionen amb l’entorn ? (nutrients, etc.) Com interaccionen amb l’entorn ? (nutrients, etc.)
cal mirar quan i com es desenvolupencal mirar quan i com es desenvolupenquines característiques enzimàtiques tenenquines característiques enzimàtiques tenen
Per poder aprofitar la informació genòmica !Per poder aprofitar la informació genòmica !
This is a joint venture by Jarone Pinhassi, Pep Gasol, Carles Pedrós-Alió & the Basics gangThis is a joint venture by Jarone Pinhassi, Pep Gasol, Carles Pedrós-Alió & the Basics gang