Bacterioplankton communities: single-cell characteristics and physiological structure

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Bacterioplankton communities single-cell
characteristics and physiological structure

• Paul del Giorgio
• Université du Québec à Montrèal

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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

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Ecosystem processes Carbon cycling Gas exchange
Trophic interactions Grazing (predation) Viral
mortality Competition
Bacterial community structure Bacterial
processes Production Respiration Nutrient
cycling
Resource supply the nature and amount of organic
matter and nutrients
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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

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From Cole et al. (1988)
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Bacterial response to changes in resources and
conditions
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Bacterial response to changes in resources and
conditions
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Bacterial response to changes in resources and
conditions
Changes in abundance
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Ducklow 1999
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Bacterial response to changes in resources and
conditions
Changes in composition of bacterial community
Changes in abundance
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Bacterial response to changes in resources and
conditions
Changes in composition of bacterial community
Changes in composition of bacterial community
Changes in abundance
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Bacterial response to changes in resources and
conditions
Changes in composition of bacterial community
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Changes in composition of bacterial community
Changes in abundance
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Bacterioplankton black box
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a
Phototrophic
or
heterotrophic
Bacterioplankton black box
Caja negra del picoplancton
Caja negra del picoplancton
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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

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Microbial bioenergetics maintenance versus growth
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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

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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
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The starvation sequence
Joux and Le Baron 2000
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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'.

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If I could do it all over again, I would be a
microbial ecologist. Ten billion bacteria live in
a gram of soil... They represent thousands of
species, almost none of each are known to
science Wilson, E.O. 1994. Naturalist. Island
Press
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Approaches to measuring single-cell properties
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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. 1996 Examples of cell and
capsule structure observed by TEM in
bacterioplankton samples
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Zweifel Hagström (1995)
Site BT (106) NuCC () MPN ()
Baltic Sea, NB1 2.5 - 3.2 4 - 6 0.1 - 0.3 Baltic
Sea, SR5 0.7 - 1.2 17 - 27 7 - 14 Baltic Sea,
US5b 0.6 - 2.7 12 - 27 6 - 15 North Sea,
Skagerrak-1 1.1 - 1.4 2 - 5 0.5 - 0.6 North Sea,
Skagerrak-2 0.2 - 0.8 4 - 32 0.2 -
0.8 Mediterranean, Point B 0.5 20 16
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Marie et al. 1997Marine picoplankton
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Cytometric enumeration of in situ aquatic
bacteria using green nucleic acid stains
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Cytometric detection of dead or injured bacteria
in situ using exclusion nucleic acid stains
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Cytometric detection of in situ bacteria with
depolarized membranes using the Oxonol DiBAC
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Cytometric detection of in situ actively
respiring bacteria using CTC
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In situ hybridation visualized with
epifluorescence microscopy
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RNA probing of bacterioplankton using
epifluorescence and cytometry
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Figs. 1 y 2 from Heissenberger et al. (1996)
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Autoradiography
From Hoppe (1976)
3H-AA
3H-thymidine
3H-aspartic
14C-glucose
CFU
Percentage of total cells
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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. 2001 River and coastal samples
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Longnecker et al. 2006
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del Giorgio and Gasol in press
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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
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The universe of DAPI-positive particles
Low activity Dormancy
High activity
Medium activity
Death
Lysis
No BT
TEM
PI (damage)
Dibac (depolarization)
CTC
Microautoradiography
DNA content
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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

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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?

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Bacterial composition
Fresh Salt
Fresh Salt
Relative abundance,
Distance downriver, Km
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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. 1990 Flagellate 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
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Gasol et al. (1995)
Total
Dapi
Percent
Active
González et al. 1990
Relative grazing efficiency
CTC
Chrzanowski Simek. 1990
Size (µm3)
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Selective grazing of live and active cells by
protists
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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
-0.43
Black box approach
1.09
0.87
0.08
-0.77
Using single-cell measurements
0.69
0.06
A
A
0.24
0.44
0.81
0.86
I
I
-0.19
-0.19
del Giorgio et al. 1996
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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)

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How do environmental and biological factors
interact to shape the physiological structure of
bacterioplankton?
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Experimental design
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Reducing protozoan grazers resulted in higher
proportions of CTC cells. The grazing effect may
be related to size-selective removal
No Grazers
Grazers
Grazers
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There was an interaction between grazing and
light (or UV) that affected the proportion of
CTC cells.
No grazers
No grazers
No grazers
Grazers
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The proportion of cells that took up the
exclusion strain TOPRO increased with UV exposure
PAR 80 UVA 70 UVB
PAR 30 UVA 0 UVB
PAR 0 UVB0 UBA
Maranger et al. 2001
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There is an inverse pattern of CTC and TOPRO
cells in relation to UV/light exposure
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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

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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

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Are there links between single-cell activity and
the phylogenetic affiliation of bacterial cells?
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Single-cell analyses that link composition with
activity and function
Hibridization (FISH) and in situ reverse
trabscription (ISRT) 16S rRNA mRNA Chen et al.
1997
In situ hibridization and microautoradiography
(MAR-FISH) 16S rRNA 3H-TdR Lee et al.
1999 Cottrell Kirchman 2000
Activity probes, cytometry cell sorting and
molecular analyses CTC, FACS, DGGE Bernard et
al. 2000 Zubkov et al. 2001


In situ hybridization and DNA synthesis
16S rRNA BrdU Pernthaler et al 2002
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Zubkov et al. 2001 Celtic Sea
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Does the active fraction (CTC) have the same
composition than the inactive fraction ?
Bernard et al. 2000
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Urbach et al. 1999 Used BromodeoxyUridine
(BrdU), an analog of thymidine, to detect growing
cells Cells incorporating BrdU can be detected
using immunofluorescence
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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
Hamasaki et al. 2004
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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
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del Giorgio and Gasol in press
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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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Microscale variability in coastal bacterial
community structure
Seymour et al. 2004
HDNA
Total BA
LDNA
D1 group
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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
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Variability in specific BP (BP / BA) and BR (BR /
BA) in marine waters
del Giorgio and Gasol in press
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Variability in abundance of microbial components
Landry and Kirchman 2002
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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.

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Microbial bioenergetics maintenance versus growth
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del Giorgio and Gasol in press
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del Giorgio and Gasol in press
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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?

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b-GAM
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Feedback at the population level
Protist biomass
Protist grazing
Bacterial Biomass/ production
?
Feedback at the cellular level
Protist single cell activity
Bacterioplankton structure
?
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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

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Zooplankton
300 l-1
Microphyto-
100 ml-1
Ciliates
2000 l-1
Flagellates
Nanophyto-
103 ml-1
1000 ml-1
107 ml-1
?
?
?
Picoplancton
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An important aspect of the functioning of
bacterial communities is social behavior
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Ducklow 2001
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Size does matter! Smaller organisms have higher
surface area (SA) to volume (V) ratios. Consider
a spherical microbe SA 4?r2 V 4/3 ?r3 So,
SAV 4?r2/4/3 ?r3 1/r That is, as organisms
get bigger, SAV gets smaller
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Approaches
Bacterial physiological parameters
intact membrane
Potentiel Membrane
Intégrité Membrane PI (Live/Dead Baclight)
damaged membrane
intact membrane DiOC6(3)
ADN
damaged membrane DiBAC4(3)
Content SYTO13
ADN
Turn over H3 Thymidine uptake
Turn over H3 Thymidine uptake
Protéines
Respiration
Protein
ETS
Turn over H3 Leucine uptake
CTC
Firefly Luciferase
Bioluminescence
enzyme
Enzymatic Activity
Luciferin
-Bulk metabolism -Single cell activity
Substrate Biolog