bacterioplankton communities: single-cell characteristics and physiological structure paul del...
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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
?
Bacterial response to changes in resources and conditions
Δ EnvironmentΔ bacterial community metabolism
?
Bacterial response to changes in resources and conditions
Δ EnvironmentΔ bacterial community metabolism
?
Changes in abundance
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Ducklow 1999
Bacterial response to changes in resources and conditions
Δ EnvironmentΔ bacterial community metabolism
?
Changes in composition of bacterial community
Changes in abundance
Bacterial response to changes in resources and conditions
Δ EnvironmentΔ bacterial community metabolism
?
Changes in composition of bacterial community
Changes in abundance
Changes in composition of bacterial community
Bacterial response to changes in resources and conditions
Δ EnvironmentΔ bacterial community metabolism
?
Changes in composition of bacterial community
Changes in abundance
Changes in composition of bacterial community
?
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
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Nyström et al. 1992
The starvation sequence
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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
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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
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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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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
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Bouvier et al. 2007
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Bouvier et al. 2007
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Bouver et al. 2007
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Lebaron et al. 2001River and coastal samples
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Longnecker et al. 2006
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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
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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
0 20 40 60 80 100
Sept
BETA
ALPHA
Upper Middle Lower Fresh Salt Fresh Salt
Distance downriver, Km
-2
0
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0
0.5
1
1.5
2
2.5
05101520
Salinity BP
River sites----------Estuarine sites
TurbidityMaximum 8 Km
-2
0
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05101520
Salinity %CTC
TurbidityMaximum 8 Km
River sites----------Estuarine sites
-2
0
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05101520
Salinity %Dibac +
River sites----------Estuarine sites
-2
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05101520
Salinity %Dead
River sites----------Estuarine sites
-2
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05101520
Salinity %EUB +
River sites----------Estuarine sites
TurbidityMaximum 8 Km
Environmental stress influences the physiological structure of bacterioplankton
• What about biological interactions, such as grazing and viral infection
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Viles and Sieracki 1992
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Fukuda et al. 1998
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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
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Hahn and Höfle 1999Grazing influences the size distribution within individual bacterial taxa.
Great morphological plasticity in bacteria QuickTime™ and a
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Gasol et al. (1995)Gasol et al. (1995)
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50P
erc
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0
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0.01 0.1 1
Rel
ativ
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azi
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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
5
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35
40
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
5
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35
40
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
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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 ?
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Urbach et al. 1999
Used BromodeoxyUridine (BrdU), an analog of thymidine, to detect growing cellsCells incorporating BrdU can be detected using immunofluorescence
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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
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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
del Giorgio and Gasol in press
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
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Azam 1998
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% HDNA
Seymour et al. 2004
Microscale variability in coastal bacterial community structure
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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
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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
del Giorgio and Gasol in press
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
del Giorgio and Gasol in press
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
del Giorgio and Gasol in press
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
5000
1 104
1.5 104
2 104
0
2 106
4 106
6 106
8 106
1 107
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
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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
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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