AARHUS UNIVERSITY
DEPARTMENT OF ENVIRONMENTAL SCIENCE
DEPARTMENT OF ENVIRONMENTAL SCIENCE
July 8th 2014
Interaction between
bacteria and protozoa
in soil
Anne Winding, senior scientist
Dept. of Environmental Science, Aarhus University, Denmark
UNI
VERSITET
AARHUS UNIVERSITY
DEPARTMENT OF ENVIRONMENTAL SCIENCE
Outline of presentation: bacteria and protozoa
Protozoa
Effect of protozoa on bacteria
Effect of bacteria on protozoa
Interactions in vitro
Interactions in soil
Diversity of soil protozoa
Bacterial pathogens and soil protozoa
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Protozoa
› Protista: unicellular eukaryotic organisms: protozoa, unicellular
algae, and slime molds.
› Proto = first, zoa = animals.
› Aquatic – motile – water films
› 5-500 µm
› Non-monophylogenetic origin
› Ciliates, flagellates, heliozoans (aquatic, with exopodia), and
amoebas.
› Trophozoites: physiological active stage
› Cysts: inactive resting stage, recalcitrant. Water stress; Cyst
bank
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Distinct morphological groups
Several phylum
• Flagellates,
1-few flagella, asexual reproduction
Ekelund, 2002
Tikhonenkov, 2010
• Amoebae,
No flagella, asexual reproduction
(sexual reproduction rare)
Smirnov and Brown, 2004
Foissner and Al-Rasheid, 2007
• Ciliates,
Many tiny flagella, sexual and asexual
reproduction
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Trophic interactions btw bacteria and predators
Bacterial-feeding
nematodes
Predatory
nematodes
Protozoa
Omnivorous
nematodes
Bacteria
Enchytraeids
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Protozoa: feeding behaviour
› Suspended bacteria:
– Direct interception: flagellates, individual bacteria in
suspension
– Filter feeding: in soil few ciliates
– Diffusion feeding: stationary protozoa, rare in soil
› Attached bacteria
– Raptorial feeding: food searching mobile protozoa (up to
69%)
– Grasping: protozoa feeding on attached bacteria (biofilm),
common in soil (up to 30%).
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Predator – prey interaction
Jousset 2012
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Food uptake
› Phagocytosis
› Food vacuoles
› Enzymatic digestion
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Protozoa affect abundance
of bacteria
Old news
(Sinclair and Alexander 1989)
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Avoidance of predation
(Pernthaler 2005)
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Neocercomonas jutlandica
In vitro studies:
controlled model systems
Cercomonas longicauda
Bodo designis
Bodo caudatus
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Microbial Pest Control Agents:
effects on fungi and insects
• antagonistic
• effects on predatory protozoa?
Pseudomonas spp. against root pathogenic fungi
Means of microbial pest control:
- Secondary metabolites
- Competition of ressources
- Degradation of pathogenicity factors
- Production of enzymes
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Growth of amoebae and bacteria in vitro
Amoebae
Bacteria
(Andersen and Winding 2004)
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DEPARTMENT OF ENVIRONMENTAL SCIENCE
N.N.jutlandica
jutlantica
C. longicauda
109
B. caudatus
pseudomonads
108
E. aerogenes
107
CFU ml-1
106
protozoa
105
104
103
Control
P. chlororaphis ATCC43928
P. fluorescens DR54
P. fluorescens CHA0
102
101
0
1
2
3
Days
4
5
6
0
2
4
6
8
10
Days
0
2
4
6
8
10
Days
Flagellates: different sensitivity
Figure
1: 2010)
Pedersen
(Pedersen
et al.
et al.
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109
DSM50090T
107
105
103
Spent bacterial growth media
show effects on C. longicauda
growth - depending on bacteria
109
-1
Flagellate or bacteria (cells ml )
CHA0
C. longicauda
P. fluorescens
E. aerogenes
107
105
Washed
103
Not washed
0
2
4
6
Time (days)
8
10
Time (days)
(Pedersen et al. 2010)
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Effects of secondary metabolites
Growth of soil
protozoa
inhibited by
DR54 cell
extract
(Andersen and Winding 2004)
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Cercomonas longicauda swimming
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DEPARTMENT OF ENVIRONMENTAL SCIENCE
jutlandica
N.N.jutlantica
106
-1
Flagellates (cells ml )
107
9 protozoa grown on 7 bacteria:
- 4 secondary metabolite producing
- 3 non-producing
105
104
103
102
101
-1
Flagellates (cells ml )
B. designis 23
106
105
104
103
102
101
-1
Flagellates (cells ml )
Spumella sp.
106
105
104
no bacteria added
E. aerogenes
P. chlororaphis ATCC 43928
P. fluorescens DSM 50090
P. fluorescens DR54
P. fluorescens CHA0
Pseudomonas sp. DSS73
P. chlororaphis MA342
103
102
101
(Pedersen et al. 2011)
0
2
4
6
Time (days)
8
10
18
(Pedersen et al. 2011)
en
s
50
09
0
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43
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c
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average growth rate (day-1)
Food quality
P.
P.
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DEPARTMENT OF ENVIRONMENTAL SCIENCE
2.5
a
2.0
1.5
1.0
a
Resulting average growth rate
when fed to nine different protozoa
b
c
d
e
0.5
f
g
Food bacterium
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DEPARTMENT OF ENVIRONMENTAL SCIENCE
1.0
Dependence on type
of protozoa
a
Ratio between growth rate on 4 metabolite
producing and 3 non producing bacteria
Ratio
0.8
0.6
b
b
bc
bc
0.4
cd
d
de
0.2
om
on
N
as
eo
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rc
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23
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C
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c
_____ ___
Rhizaria
(Cercomonadidae)
(Pedersen et al. 2011)
____
__
Chromalveolata
Excavata
(Bodonidae)
Amoebozoa
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Flow cytometry for counting
(Pedersen et al 2009)
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Difference in food
selectivity by protozoa
and nematode:
protozoa select,
nematodes don’t
(Pedersen et al 2009)
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Interactions in vitro
› Difference in food quality of bacteria
– Correlating with secondary metabolite production
– Importance of membrane bound vs unbound metabolites
– Unknown compounds?
› Feeding behaviour
– Difference between protozoa in growth on the same bacteria
– Difference in selectivity between protozoa and nematode
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DEPARTMENT OF ENVIRONMENTAL SCIENCE
Location in soil
Ciliates
Flagellates
Bacteria
Interaction between bacteria and protozoa in soil
Anne Winding.
July 8th 2014
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DEPARTMENT OF ENVIRONMENTAL SCIENCE
Location in soil
1
2
3
soil pore
1. Plenty of water: trophozoites and viable bacteria
2. Less water: cysts and viable bacteria
3. Little water: cysts and spores
Interaction between bacteria and protozoa in soil
Anne Winding.
July 8th 2014
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DEPARTMENT OF ENVIRONMENTAL SCIENCE
Biochar as protection against bacterial predators?
Tetrahymena
Valentina Imparato
Pseudomonas
Interaction between bacteria and protozoa in soil
Anne Winding.
July 8th 2014
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DEPARTMENT OF ENVIRONMENTAL SCIENCE
Nutrients and
hormones
Microflora
Bacteria
Fungi
Protozoa
Nematodes
Root
exudates
Organic
matter
Earthworms
M. Bonkowski et al. / Eur. J. Soil Biol. 36 (2000) 135–147
Soil food web
Up to 100,000 individuals/gram soil
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Interactions in rhizosphere
Prashar et al. 2014
Interaction between bacteria and protozoa in soil
Anne Winding.
July 8th 2014
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fenpropimorph
Interaction btw prey
and predator
Thirup et al 2000
Interaction between bacteria and protozoa in soil
Anne Winding.
July 8th 2014
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DEPARTMENT OF ENVIRONMENTAL SCIENCE
Soil and rhizosphere inoculated with P. fluorescens DR54:
Positive effect on fast-responding protozoa
(Johansen et al. 2005)
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DEPARTMENT OF ENVIRONMENTAL SCIENCE
Rhizosphere of Arabidopsis thaliana: Amoebae decrease total abundance
and change diversity of bacteria
Rosenberg et al 2009
Interaction between bacteria and protozoa in soil
Anne Winding.
July 8th 2014
31
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DEPARTMENT OF ENVIRONMENTAL SCIENCE
Susana Santos unpubl.
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DEPARTMENT OF ENVIRONMENTAL SCIENCE
P. fluorescens CHA0 and CHA0 pME3424
in soil
› Isolated from tobacco rhizosphere
› P. fluorescens CHA0: DAPG, Plt, Prn, HCN
› P. fluorescens CHA0 pME3424: ++ prod. of Plt and DAPG
Interaction between bacteria and protozoa in soil
Anne Winding.
July 8th 2014
33
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109
harvest after 1 day
Total culturable bacteria
CFU g-1 dw
DEPARTMENT OF ENVIRONMENTAL SCIENCE
a
a
a
a
108
107
control
E. aerogenes
CHA0/gfp1
CHA0/pME3424
CFU g-1 dw
109
harvest after 7 days
a
a
b
c
108
107
109
CFU g-1 dw
harvest after 14 days
(Winding and Oberender 2012)
a
a
a
b
108
107
106
0
10
20
Time (days)
30
40
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Soil Protozoa
c
c
c
104
b
h
h
e
h
X
104
X
f
1
7
harvest time (days)
14
1
103
bd
nd
nd
nd
bd
bd
102
nd
nd
103
7
Total protozoa g-1 dry soil
Y
c
105
Y
g
Y
d
Y
a
nd
Fast-growing protozoa g-1 dry soil
105
102
14
harvest time (days)
control
E. aerogenes
control P. fluorescens CHA0/gfp1
Enterobacter aerogenes
P. fluorescens CHA0/pME3424
P. fluorescens CHA0 gfp
P. fluorescens CHA0 pME3424
(Winding and Oberender 2012)
Figure 4 Winding and Oberender
Interaction between bacteria and protozoa in soil
Anne Winding.
July 8th 2014
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Effects of P. fluorescens MPCA on soil protozoa
› Slight negative effect of DR54 and CHA0 on CFU, no effect
on soil respiration and bacterial diversity
› Positive effect of DR54 on the abundance of fast growing
and total soil protozoa.
› Negative effect of CHA0 on abundance of protozoa
Interaction between bacteria and protozoa in soil
Anne Winding.
July 8th 2014
36
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DEPARTMENT OF ENVIRONMENTAL SCIENCE
Protozoa increase abundance of MPCA in rhizosphere
With protozoa
No protozoa
Protozoa positively increase effect of MPCA in rhizosphere?
Müller et al. 2013
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Protozoa change the
physiological profile of
bacteria in soil
(Rønn et al. 2002)
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Variation in changes of bacterial community
depends on protozoan species
AARHUS UNIVERSITY
(Rønn et al. 2002)
DEPARTMENT OF ENVIRONMENTAL SCIENCE
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DEPARTMENT OF ENVIRONMENTAL SCIENCE
Effects of predation in rhizosphere
The percentage of Verrucomicrobia and Actinobacteria of eubacterial biomass as revealed by the
FISH method at the last harvest date (day 32). The relative abundance of these bacterial groups
was significantly higher when protozoa were present ( P = 0.033 and 0.005; respectively).
Ekelund et al. 2009.
Interaction between bacteria and protozoa in soil
Anne Winding.
July 8th 2014
40
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What is the diversity of protozoa?
How to determine it?
› Determination
– Traditional: Isolate and identify in microscope
– Molecular analysis of soil protozoa
− Isolate and extract DNA and use bar coding
− Extract DNA and use bar coding or DNA primers or NGS
or??
› Challenges:
– Many phylums – design of primers
– Extraction of DNA – trophozoites and cysts
– Databases
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Molecular techniques demonstrate protists diversity is higher
than morphological studies suggest
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Genetic diversity of Kinetoplastidae
1 day
E. aer. b
CHA0/gfp1 a
CHA0/gfp1 c
CHA0/gfp1 b
CHA0/pME3424 a
CHA0/pME3424 b
CHA0/pME3424 c
Control a
Control b
E. aer. c
Control c
E. aer. a
0.7 0.8 0.9 1.0 S
(Winding and Oberender 2012)
7 days
CHA0/gfp1 c
CHA0/gfp1 a
Control b
Control a
E. aer. b
Control c
E. aer. a
E. aer. a
CHA0/pME3424 c
E. aer. b
Control b
CHA0/gfp1 a
Control a
CHA0/gfp1 c
Control c
CHA0/pME3424 b
E. aer. c
E. aer. c
CHA0/gfp1 b
CHA0/pME3424 a
CHA0/pME3424 a
CHA0/gfp1 b
CHA0/pME3424 b
CHA0/pME3424 c
0.7 0.8 0.9 1.0 S
AB
14 days
AB
0.7 0.8 0.9 1.0 S
AB
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Genetic diversity of Kinetoplastidae
bands on DGGE gel
14
control
E. aerogenes
P. fluorescens CHA0/gfp1
P. fluorescens CHA0/pME3424
12
10
8
6
1
7
14
harvest time (days)
(Winding and Oberender 2012.)
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Letendu et al 2014
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Protozoa as Trojan horses
Potential impacts on survival and spreading of human
pathogens
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Protozoa as Trojan horses
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Fate of bacteria after phagocytosis
- Protozoan lysis of bacteria
- Bacterial lysis of protozoa
- Bacterial multiplication inside
amoebae
- Bacterial survival
- Increased gene transfer
- Increased virulence
- Increased antibiotic resistance
- Transcriptome changes
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Campylobacter jejuni and Acanthamoeba castellanii
- poor survival of bacteria inside amoebae
0h
5h
24 h
(Bui et al. 2012 Env Microb)
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A. castellanii increases growth of C. jejuni
+ amoebae, separated
+ amoebae
- amoebae, micro O
2
- amoebae
(Bui et al. 2012 Env Microb)
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Bacteria
packaging by
amoebae
Dig. B: digestible bacteria
Res. B: resistant to
lysosomal degradation
Legionella
pneumophila
in Multimellar
bodies (MLB)
Empty MLB
Denoncourt et al 2014
Interaction between bacteria and protozoa in soil
Anne Winding.
July 8th 2014
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Bacterial spores
› Resting stage
› Resistant to draught, low food concentration, adverse
temperatures etc.
› Resistant to predation?
› Germinate at high nutrient availability
› Spore germination inside protozoa?
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Tetrahymena and Bacillus
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Interactions between bacteria and protozoa in soil
› Tight interaction :
– Protozoa affect bacteria
– Bacteria affect protozoa
› Effect on plant growth and N-cycling
› Survival and transport of bacteria
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Acknowledgement
Co-authors: Susana Santos, Valentina Imparato,
Annette Pedersen, Karen S Andersen, Karen S
Jensen, Jana Oberender, Flemming Ekelund,
Anders Johansen,
Technicians: Anne-Grethe Holm-Jensen, Tina Thane
Funding: Danish Research Councils, EU FP7
EcoFINDERS, ITN-Trainbiodiverse
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