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