bacterioplankton communities: single-cell characteristics and physiological structure paul del...

Bacterioplankton communities: single-cell characteristics and

physiological structure

Paul del Giorgio

Université du Québec à Montrèal

Why study aquatic bacteria?• They are responsible for much of organic matter and

nutrient transformation and mineralization• Bacteria are responsible for much of the aerobic respiration

and all of anaerobic respiration in aquatic systems• Aquatic bacteria are one of the largest living reservoirs of

carbon, P, N, Fe and other materials• Aquatic bacteria represent the largest surface in oceans and

lakes• Bacterial biomass may be a significant food resource in

aquatic food webs• Some bacteria pose sanitary or environmental problems

Resource supply: the nature and amountof organic matter and nutrients

Bacterial community structureBacterial processes:

ProductionRespiration

Nutrient cycling

Ecosystem processesCarbon cyclingGas exchange

Trophic interactions:Grazing (predation)

Viral mortalityCompetition

What is community structure at the microbial level?

• Bacterial biomass• Bacterial cell size and morphology• Attached versus free-living cells• The distribution of cells with different functions • Taxonomic (phylogenetic) composition• The distribution of cells with different growth and

metabolic rates

From Cole et al. (1988)From Cole et al. (1988)

1

10

100

10 100 1000

NPP mgC m-3 d -1

BP

mgC

m-3 d

-1

Bacterial response to changes in resources and conditions

Δ EnvironmentΔ bacterial community metabolism

?



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Changes in abundance

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

Ducklow 1999



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Changes in composition of bacterial community




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Bacterioplankton black boxBacterioplankton black box

Caja negra del picoplanctonCaja negra del picoplancton

Aliveor

dead

Activeor

dormant

z

zzz

Smallor

large

Large virusesor

small bacteria

Attachedor

free-living

Phototrophicor

heterotrophic

hO2

O2

Aerobicor

quimioautotrophsor

fermentersCH4

O2

With or w/oexternalstructure

Bacterioplankton black boxBacterioplankton black box

Starvation, dormancy, slow growth

• Dormancy, starvation-survival, slow growth, and inactivity are often used interchangeably to denote low levels of cellular activity in marine bacteria, but these terms are not synonyms and refer to different states

Microbial bioenergetics: maintenance versus growth

Growth rate µ (h -1)

Spec substrate consumption

} me (µ = 0.0 h-1)

m (µ)

Dormancy

e (µ)

}Death

}

Starvation survival

• Under conditions of extreme substrate and energy deprivation, marine bacteria may undergo a “starvation” response

• The starvation response is regulated by specific genes and involves cell miniaturization, and profound changes in macromolecular composition, with the synthesis of specialized protective proteins

• Prolonged starvation may lead to cell “dormancy”, which is a state of complete metabolic arrest that allows long-term survival under unfavorable conditions. Cells in a dormant state are still more resistant to other environmental stresses

• There are costs and benefits associated to entering dormancy as opposed to maintaining a slow level of metabolic activity and growth as a response to low substrate availability

• Resource patchiness and temporal variability play a major role in shaping the survival strategies of marine bacteria, whether it is slow growth, starvation response or dormancy

The distribution of cells into different physiological categories is termed the

“physiological structure” of bacterioplankton• Within a bacterial community there is a continuum of activity, from

dead to highly active cells• The categories used to describe the physiological structure are

operational and depend on the methods used• The physiological structure is related, albeit in complex ways, to the

size structure of the community, as well as to the phylogenetic structure, i.e. the distribution of cells into operational taxonomical units

• The physiological structure is dynamic, i.e. the proportions of cells in various physiological states may vary at short time scales and small spatial scales



Nyström et al. 1992

The starvation sequence





Joux and Le Baron 2000

The reality of our disciplne:

• Thomas Brock's classic microbial ecology text (Brock 1966) is prefaced by a quote attributed as a graduate student motto. The motto simply states, 'microbial ecology is microbial physiology under the worst possible conditions'.

““If I could do it all over again, I would be a microbial If I could do it all over again, I would be a microbial ecologist. Ten billion bacteria live in a gram of soil... They ecologist. Ten billion bacteria live in a gram of soil... They

represent thousands of species, almost none of each are represent thousands of species, almost none of each are known to science”known to science”

Wilson, E.O. 1994. Naturalist. Island PressWilson, E.O. 1994. Naturalist. Island Press

Approaches to measuring single-cell properties

Phylogenetic composition:Fluorescence In Situ Hybridization (FISH)

Ribosomes

MetabolismC respiration (O2 consumption)C production (3H-thym incorporation)Bacterial Growth Efficiency (BGE)

O2 CO

2

3H3H

Abundance:Nucleic acid staining (SYTO13)

DNA

Physiological state:Altered membrane (BackLight)

Physiological state:Depolarized cells (DiBac)

ETS

Physiological stateHighly active cells (CTC, HDNA)

Some approaches used to assess bacterial characteristics in situ that are culture

independent

• Microautoradiography to assess uptake of radiolabeled organic compounds

• RNA (and other macromolecular) contents• Vital stains as indices of cell metabolism

(Fluorescein, Calcein, INT, CTC)• Stains that reflect membrane polarization and

integrity (PI, Oxonol, SYTOX, TOPRO)• Structural integrity under TEM



Heissenberger et al. 1996Examples of cell and capsule structure observed by TEM in bacterioplankton samples

Zweifel & Hagström (1995)Zweifel & Hagström (1995)

Baltic Sea, NB1Baltic Sea, NB1 2.5 - 3.22.5 - 3.2 4 - 64 - 6 0.1 - 0.30.1 - 0.3Baltic Sea, SR5Baltic Sea, SR5 0.7 - 1.20.7 - 1.2 17 - 2717 - 27 7 - 147 - 14Baltic Sea, US5bBaltic Sea, US5b 0.6 - 2.70.6 - 2.7 12 - 2712 - 27 6 - 156 - 15North Sea, Skagerrak-1North Sea, Skagerrak-1 1.1 - 1.41.1 - 1.4 2 - 52 - 5 0.5 - 0.60.5 - 0.6North Sea, Skagerrak-2North Sea, Skagerrak-2 0.2 - 0.80.2 - 0.8 4 - 324 - 32 0.2 - 0.80.2 - 0.8Mediterranean, Point BMediterranean, Point B 0.50.5 2020 1616

SiteSite BT (10BT (1066)) NuCC (%)NuCC (%) MPN (%)MPN (%)

Marie et al. 1997Marine picoplankton



Cytometric enumeration of in situ aquatic bacteria using green nucleic acid

stains

Cytometric detection of dead or injured bacteria in situ using exclusion nucleic

acid stains

Cytometric detection of in situ bacteria with depolarized membranes using the

Oxonol DiBAC

Cytometric detection of in situ actively respiring bacteria using CTC

In situ hybridation visualized with epifluorescence microscopy

RNA probing of bacterioplankton using epifluorescence and cytometry

Figs. 1 y 2 from Heissenberger et al. (1996)Figs. 1 y 2 from Heissenberger et al. (1996)

0

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60

1 2 3 4 5 6

Station in a gradientStation in a gradient

% o

f ba

cte

rial

co

mm

uni

ty%

of b

act

eri

al c

om

mu

nity

Intact cellsIntact cellsDamaged cellsDamaged cells

Empty cellsEmpty cells

Autoradiography

From Hoppe (1976)From Hoppe (1976)

0 20 40 60 80 100

CFU

14C-glucose

3H-aspartic

3H-thymidine

3H-AA

Percentage of total cells

Smith and del Giorgio 2003



Bouvier et al. 2007



Bouver et al. 2007







Lebaron et al. 2001River and coastal samples



Longnecker et al. 2006





aaa

Memb+

CTC+

DVCNucC

103104105106107

105 106 107

100%10%1%

All bacteria (cells ml-1)

103104105106107Est+

MAR+

HNA 105 106 107All bacteria (cells ml-1)

100%10%1%

100%10%1%

100%10%1%103104105106107

103104105106107

100%10%1%

100%10%1%

100%10%1%

MethodNlog-log sloper2NucC2121.35 ± 0.060.73DVC2041.27 ± 0.070.63Est+1100.63 ± 0.050.61CTC+4981.21 ± 0.050.58MAR+3151.11 ± 0.040.70Memb+4471.02 ± 0.030.72HNA8041.02 ± 0.010.86

del Giorgio and Gasol in press

a

All bacteriaMembrane+FISH-EUB+MAR+INT+

Direct viable countEsterase activityCTC+% (average, SE, 25 and 75% quartiles and range)020406080100

MotileHNANucCCapsule





Søndergaard and Danielsen 2001

The highly active “CTC” fraction is seasonally much more dynamic than the total bacterial abundance in lakes

aaaa

+++++------Membrane polarization

rRNA probesMembranepermeabilityDNARibosomesETSchainRespirationprobesNA probesDNA DuplicationMetabolism:organic substrate incorporationCO2, O2 exchange...

Enzymeprobesesterases********

CTC

Microautoradiography

DNA content

Dibac (depolarization)

PI (damage)

TEM

High activity

Medium activity

Low activityDormancy

Death Lysis

The universe of DAPI-positive particlesThe universe of DAPI-positive particles

No BT

The regulation of the physiological structure of bacterioplankton communities has three main components

• Environmental factors that influence the individual level of metabolic activity and cell integrity and damage, such as substrate and nutrient availability, UV and temperature

• Physical and biological factors that influence the persistence and loss of the various physiological fractions, such as selective grazing and viral infection, and selective degradation

• Intrinsic phylogenetic characteristics that modulate the response of different bacterial strains to the above factors

Example: Bacterial succession along the transition between fresh and salt waters

• Does bacterial composition change abruptly along a salinity gradient in an estuary?

• Is the compositional succession accompanied by changes in the physiological structure of the community along this salinity gradient?

Bacterial composition

Rel

ativ

e ab

unda

nce,

%

0

25

50March May

Upper Middle Lower

0 20 40 60 80 1000

25

50July

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Sept

BETA

ALPHA

Upper Middle Lower Fresh Salt Fresh Salt

Distance downriver, Km

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Salinity BP

River sites----------Estuarine sites

TurbidityMaximum 8 Km

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05101520

Salinity %CTC



-2

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05101520

Salinity %Dibac +


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Salinity %Dead


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05101520

Salinity %EUB +



Environmental stress influences the physiological structure of bacterioplankton

• What about biological interactions, such as grazing and viral infection



Viles and Sieracki 1992



Fukuda et al. 1998



Gonzalez et al. 1990Flagellate and ciliate grazing is strongly size-dependent.

This had strong implications on the influence of bacterial structure on food web interactions within the microbial loop

Hahn and Höfle 1999Grazing influences the size distribution within individual bacterial taxa.

Great morphological plasticity in bacteria QuickTime™ and a

TIFF (LZW) decompressorare needed to see this picture.

Gasol et al. (1995)Gasol et al. (1995)

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0.01 0.1 1

Rel

ativ

e gr

azi

ng

effic

ienc

y

Size (µm3)

ActiveActive

TotalTotal

González et al. 1990González et al. 1990

Chrzanowski & Simek. 1990Chrzanowski & Simek. 1990

Dapi +Dapi +

CTC +CTC +

aa

2 1054 1056 1058 1051 1061.2 1061.4 1061.6 1061.8 106

020004000600080001 104

02468

Time (d)4550556065707580

10203040506070

02468

2 1054 1056 1058 1051 1061.2 1061.4 106

01 1052 1053 1054 1055 1056 1057 105

02468

0500100015002000250030003500

00.511.522.53

02468

A

B

C

D

Time (d)

Selective grazing of live and active cells by protists

Black box approachBlack box approach

AA

II

0.870.87 1.091.090.080.08

-0.43-0.43

0.810.81 0.860.86

0.690.69

-0.19-0.19

0.00.066

0.20.2440.440.44

-0.77-0.77

-0.19-0.19

AA

II

In situ dyalysis bag experiments in the Mediterranean Sea to follow the dynamics of active and inactive cells in the presence and absence of protistan grazing showed selective grazing and significant cell inactivation

Using single-cellUsing single-cellmeasurementsmeasurements

del Giorgio et al. 1996del Giorgio et al. 1996

Lake Microcosm Experiments:(with David Bird, Rox Maranger and Yves Prairie, UQÀM)

• Water samples were filtered through 0.8 µm (to remove grazers), or unfiltered

• Water samples were incubated in dialysis bags in situ in Lac Cromwell (Québec)

• Three UV/light treatments

• We followed thee abundance of highly active cells (CTC+) and injured/dead cells (TOPRO+)

How do environmental and biological factors interact to

shape the physiological structure of bacterioplankton?

Experimental design

Surface Plexiglass

Deep1.5 m depth

5 cm depth

Lake surface

Unfiltered water

Filtered water (0.8 µm)

Reducing protozoan grazers resulted in higher proportions of CTC+ cells. The grazing effect may

be related to size-selective removal

0

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Deep Filt Deep Unfilt Lake

No Grazers

Grazers

Grazers

There was an interaction between grazing and light (or UV) that affected the proportion of CTC+ cells.

0

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Surface Filt Plexi Filt Deep Filt Lake

No grazers

No grazers

No grazers

Grazers

The proportion of cells that took up the exclusion strain TOPRO increased with UV exposure

0

5

10

15

Surface Filt Plexi Filt Deep Filt Lake

W

PAR80% UVA70% UVB

PAR30% UVA0% UVB PAR

0% UVB0% UBA

Maranger et al. 2001

There is an inverse pattern of CTC+ and TOPRO+ cells in relation to UV/light

exposure

0

5

10

15

20

25

30CTC%TOP%

TreatmentSURF S+P DEEP LAKE

Some conclusions regarding the link between grazing and

bacterioplankton activity (I)

• Grazing and UV radiation both affect the physiological structure of bacterioplankton

• Grazing is highly selective and preferentially removes active cells

• Active cells appear to be on average larger than less active or dormant cells

• Grazing selectivity may be based on size

Some general ecological patterns in microbial (II)

• In aquatic microbial communities, small size and low activity represent a refuge against predation and perhaps viral infection

• Large cells must find alternative refuges: attachment, parasitism, chemical defenses

• In other types of communities it is often the the small and the weak that are selectively removed

• General allometric rules, i.e. size versus specific activity, do not necessarily apply to aquatic microbial communities

Are there links between single-cell activity and the phylogenetic

affiliation of bacterial cells?

Single-cell analyses that link composition withSingle-cell analyses that link composition with activity and functionactivity and function

• • In situ hibridization and microautoradiography (MAR-FISH)In situ hibridization and microautoradiography (MAR-FISH)16S rRNA & 16S rRNA & 33H-TdRH-TdR Lee et al. 1999Lee et al. 1999

Cottrell & Kirchman 2000Cottrell & Kirchman 2000

• • Hibridization (FISH) and in situ reverse trabscription (ISRT)Hibridization (FISH) and in situ reverse trabscription (ISRT)16S rRNA & mRNA16S rRNA & mRNA Chen et al. 1997Chen et al. 1997

• • Activity probes, cytometry cell sorting and molecular analysesActivity probes, cytometry cell sorting and molecular analysesCTC, FACS, DGGECTC, FACS, DGGE Bernard et al. 2000Bernard et al. 2000

Zubkov et al. 2001Zubkov et al. 2001

• • In situ hybridization and DNA synthesisIn situ hybridization and DNA synthesis 16S rRNA & BrdU16S rRNA & BrdU Pernthaler et al 2002Pernthaler et al 2002





Zubkov et al. 2001Celtic Sea

Bernard et al. 2000Bernard et al. 2000

Does the active fraction (CTC+) have the same compositionDoes the active fraction (CTC+) have the same compositionthan the inactive fraction ?than the inactive fraction ?





Urbach et al. 1999

Used BromodeoxyUridine (BrdU), an analog of thymidine, to detect growing cellsCells incorporating BrdU can be detected using immunofluorescence



Hamasaki et al. 2004

Linking growth to phylogeny: BrdU-incorporation

• Found that the BrdU-incorporating (growing) communities were

substantially different from the total communities

• This suggests that the numerically dominant groups are not necessarily those that are the most active

Hamasaki et al. 2007





Cottrel and Kirchman 2003

Showed that the contribution of the major groups to Tdr and Leu assimilation varied greatly along a salinity gradient

Also showed that some groups contribute disproportionately to total bacterial activity

aaa

020406080

LeucineMixed aminoacidsGlucoseProteinThymidine

Bacteroidetesgammaproteobacteria

% incorporating the substrate

Bacterial subgroupalfaproteobacteriaSAR11Roseobacter


What is the link between single-cell activity and phylogenetic affiliation?

• MAR-FISH analysis analyses show that in most cases there is a mixture of cells that are active and inactive in substrate uptake within any given bacterial group, suggesting that the level of single-cell activity is not intrinsic but rather that members of the same group may express very different levels of activity depending on their microenvironment and of their immediate history

• This scenario would further suggest that resource microheterogeneity may play a key role in determining the distribution of activity within bacterial assemblages

• Alternatively, the heterogeneity of single-cell activity detected within broad phylogenetic groups may indicate that within these groups there is a wide range of genetic diversity, that is expressed as a wide range in metabolic responses of different cells to the same set of environmental conditions

• This establishes two extreme scenarios, i.e. the physiological structure entirely due to environmental heterogeneity, microscale patchiness and temporal variability, versus physiological heterogeneity due entirely to genetic/phenotypic diversity. Where along this gradient lie natural bacterioplankton assemblages is still a matter of study



Azam 1998



% HDNA

Seymour et al. 2004

Microscale variability in coastal bacterial community structure



Total BA

LDNA D1 group

Some general ecological conclusions from these examples:• There are intense bacterioplankton phylogenetic

successions along environmental gradients, associated to physiological stress and possibly cell mortality

• Predation is a major structuring factor in microbial communities, but predator-prey interactions may be distinct in microbial systems

• Some general ecological notions, such as allometric relationships, refuge and succession theory, may not effectively describe the microbial world



Ducklow 2001

aaa

05101520253035

Cell specific BR and BP (fgC cell-1 h-1), log scale05101520253035

0.00030.0010.0030.010.030.10.3131030100BP BR

793 individual measurements of BP, 324 of BR


Variability in specific BP (BP / BA) and BR (BR / BA) in marine waters

Region Parameter HBACT PRO SYN PEUK

HNLC Equator Mean 716,000 145,000 9,800 6,300S.D. 126,000 38,000 3,400 1,800C.V. 18% 26% 35% 28%

Western Equator Mean 172,000 2,300 870S.D. 72,000 2,600 450C.V. 42% 113% 51%

HOT Mean 444,000 183,000 1,700 720S.D. 119,000 45,000 1,100 360C.V. 27% 25% 65% 50%

Variability in abundance of microbial components

Landry and Kirchman 2002

We know that there is an upper limit to bacterial growth rate, but how slowly can a bacterial cell grow?

• There are thermodynamic constraints that determine both the upper and lower limits of cell growth

• Slow growth still requires the operation of tranport systems, the maintenance of cell membranes, and the turnover of proteins and nucleic acids.

Microbial bioenergetics: maintenance versus growth

Growth rate µ (h -1)

Spec substrate consumption

} me (µ = 0.0 h-1)

m (µ)

Dormancy

e (µ)

}Death

}

aaa

AA I IA


aaa

B

P P PLow growth,Low yieldcellsHigh growth,High yieldcellsAll cells have equal growthrates and growth yield

RR RBulk BGEBulk BGE

HomogeneousHeterogeneous

aa

0.10.20.30.40.5

05101520% CTC+ cells0.10.20.30.40.5

11.522.533.544.55Fluorescence of the CTC+ cells(relative to beads)0.10.20.30.40.5

01020304050607080% CTC+ x single-cell fluorescence

A B C

0.10.20.30.40.5


What about other components of the microbial food web: The coupling between

protist predators and their bacterial prey

• There is evidence that protist grazing may profoundly affect the physiological (and taxonomic) structure of bacterioplankton

• But does the distribution of single cell activity affect protist activity?

0

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1 104

1.5 104

2 104

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1.2 107

A

Heterotrofic flagellatesBacteriaHigh-DNALow-DNA

0 20 40 60 80 100 120

HNF abundance (cells ml

-1)

Bacterial abundance (cells ml

-1)

10

100

10 100 1000 104 105

%High-DNA%CTC+

Heterotrophic flagellates (ml-1)

0.001

0.01

0.1

1

10

10 100 1000 104 105

Total enzyme activity (nmol l

-1 h -1)

Heterotrofic flagellates (cells ml -1)

TEA = 0.005 x HNF 0.52

r2 = 0.18

Beta-glucosaminidase activity-GAM

10-6

10-5

0.0001

0.001

1 10 100

Specific enzyme (nmol HNF

-1 h

-1)

%CTC+ bacteria

SE = 5.84 x CTC 1.68

r2 = 0.94

Protist biomass Protist grazingBacterialBiomass/

production

Protist single cell activity

Bacterioplanktonstructure?

?

Feedback at the population level

Feedback at thecellular level

Some patterns concerning protist-bacteria interactions:

• Microbial predators can respond to prey fluctuations at the population level, like predators in other types of systems

• But microbial predators can also respond at the level of cellular metabolism

• This response is much faster and allows microbial predator-prey systems to be more tightly coupled than any other system

• This tight coupling provides overall stability to the ecosystem

ZooplanktonZooplankton

Microphyto-Microphyto-

Nanophyto-Nanophyto-

100 ml100 ml-1-1

300 l300 l-1-1

1000 ml1000 ml-1-1

2000 l2000 l-1-1 CiliatesCiliates

101033 ml ml-1-1

FlagellatesFlagellates

101077 ml ml-1-1

PicoplanctonPicoplancton

?? ????

An important aspect of the functioning of bacterial communities is social behavior





Ducklow 2001

Size does matter!

Smaller organisms have higher surface area (SA) to volume (V) ratios. Consider a spherical microbe:

SA= 4r2

V= 4/3 r3

So, SA:V = 4r2/4/3 r3 ~ 1/r

That is, as organisms get bigger, SA:V gets smaller

14

Approaches

Bacterial physiological parameters

Turn over: H3 Thymidine uptake

ATPETS

CTC

Potentiel Membrane

damaged membraneDiBAC4(3)

intact membraneDiOC6(3)

Respiration

ADN

Intégrité MembranePI (Live/Dead Baclight)

damaged membrane

intact membrane

Luciferin

Firefly Luciferase

Bioluminescence

Content: SYTO13

ADN

ProtéinesProtein

-Bulk metabolism-Single cell activity

Turn over: H3 Leucine uptake

Enzymatic Activity Substrate

Biolog

enzyme

Turn over: H3 Thymidine uptake

bacterioplankton communities: single-cell characteristics and physiological structure paul del...

Documents

bacterial response

abundance slide

growth slide

abundance changes

lakes bacterial biomass

qubec montral slide

starvation response

small bacteria