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AARHUS UNIVERSITY DEPARTMENT OF ENVIRONMENTAL SCIENCE

VERSITET

Anne Winding, senior scientist Dept. of Environmental Science, Aarhus University, Denmark

AARHUS UNIVERSITY

DEPARTMENT OF ENVIRONMENTAL SCIENCE

July 8th 2014

UNI

Interaction between bacteria and protozoa

in soil


2

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


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

3


Distinct morphological groups

Several phylum

4

Tikhonenkov, 2010 Ekelund, 2002

Foissner and Al-Rasheid, 2007

Smirnov and Brown, 2004

• Flagellates,

1-few flagella, asexual reproduction

• Amoebae,

No flagella, asexual reproduction

(sexual reproduction rare)

• Ciliates,

Many tiny flagella, sexual and asexual

reproduction


Trophic interactions btw bacteria and predators

5

Bacteria

Bacterial-feeding

nematodes

Protozoa

Predatory

nematodes

Omnivorous

nematodes

Enchytraeids


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%).

6


Predator – prey interaction

7 Jousset 2012


Food uptake ›Phagocytosis

›Food vacuoles

›Enzymatic digestion

8


9

Protozoa affect abundance of bacteria

(Sinclair and Alexander 1989)

Old news


10

Avoidance of predation

(Pernthaler 2005)


11

In vitro studies: controlled model systems

Bodo designis

Neocercomonas jutlandica

Bodo caudatus Cercomonas longicauda


12

Microbial Pest Control Agents:

• antagonistic effects on fungi and insects • 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


13

Growth of amoebae and bacteria in vitro

(Andersen and Winding 2004)

Amoebae

Bacteria


14

(Pedersen et al. 2010)

pseudomonads

E. aerogenes

protozoa

B. caudatus

Days

0 2 4 6 8 10

C. longicauda

Days

0 1 2 3 4 5 6

CF

U m

l-1

101

102

103

104

105

106

107

108

109

Control

P. chlororaphis ATCC43928

P. fluorescens DR54

P. fluorescens CHA0

Figure 1: Pedersen et al.

Figure 2: Pedersen et al.

N. jutlantica

Days

0 2 4 6 8 10

Flagellates: different sensitivity

N. jutlandica


15

Spent bacterial growth media

show effects on C. longicauda

growth - depending on bacteria


CHA0

Time (days)

103

105

107

109

C. longicauda

P. fluorescens

E. aerogenes

DSM50090T

Fla

ge

llate

or

ba

cte

ria (

cells

ml-1

)

103

105

107

109

Time (days)

0 2 4 6 8 10

Washed

Not washed


16

Effects of secondary metabolites

Growth of soil protozoa inhibited by DR54 cell extract

(Andersen and Winding 2004)


Cercomonas longicauda swimming

17


18

9 protozoa grown on 7 bacteria:

- 4 secondary metabolite producing

- 3 non-producing

B. designis 23

Time (days)

Fla

gella

tes (

ce

lls m

l-1)

101

102

103

104

105

106

N. jutlantica

Fla

gella

tes (

ce

lls m

l-1)

101

102

103

104

105

106

107

Spumella sp.

0 2 4 6 8 10

Fla

gella

tes (

ce

lls m

l-1)

101

102

103

104

105

106

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


N. jutlandica


19

Resulting average growth ratewhen fed to nine different protozoa

Food bacterium

P. f

luor

esce

ns D

SM

5009

0

P. c

hlor

orap

his ATC

C43

928

Ent

erob

acte

r aer

ogen

es

P. f

luor

esce

ns D

R54

P. c

hlor

orap

his M

a342

Pse

udom

onas

sp.

DSS73

Pho

spha

te b

uffe

r, no

bac

teria

P. f

luor

esce

ns C

HA0

ave

rag

e g

row

th r

ate

(d

ay

-1)

0.5

1.0

1.5

2.0

2.5

a

e

a

b

c

d

g

f

Food quality



20

Ratio between growth rate on 4 metaboliteproducing and 3 non producing bacteria

Cer

com

onas

long

icau

da

Neo

cerc

omon

as ju

tland

ica

Het

erom

ita g

lobo

sa

Pha

lans

teriu

m soilitar

ium

Har

tman

ella v

erm

iform

is

Bod

o ca

udat

us

Bod

o de

sign

is 2

3

Bod

o de

sign

is U

J

Spu

mella sp.

Ratio

0.2

0.4

0.6

0.8

1.0

_________ ___

__

Rhizaria

(Cercomonadidae)

Amoebozoa

Excavata

(Bodonidae)

Chromalveolata

b b

bc bc

cdd

de

e

a

Dependence on type

of protozoa



21

Flow cytometry for counting

(Pedersen et al 2009)


22

Difference in food

selectivity by protozoa

and nematode:

protozoa select,

nematodes don’t

(Pedersen et al 2009)


23

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



Anne Winding.

July 8th 2014 Interaction between bacteria and protozoa in soil

Location in soil

24

Ciliates

Flagellates

Bacteria



Anne Winding.


Location in soil

25

1. Plenty of water: trophozoites and viable bacteria

2. Less water: cysts and viable bacteria

3. Little water: cysts and spores

1 2 3

soil pore



Anne Winding.


Biochar as protection against bacterial predators?

26

Tetrahymena Pseudomonas

Valentina Imparato


27

Soil food web

M. B

onkow

ski e

t al. /

Eur.

J. S

oil

Bio

l. 3

6 (

2000)

135–147

Protozoa

Microflora

Bacteria Fungi

Earthworms Nematodes

Nutrients and

hormones Root

exudates

Organic

matter

Up to 100,000 individuals/gram soil



Anne Winding.


Interactions in rhizosphere

28

Prashar et al. 2014



Anne Winding.


Interaction btw prey and predator

29

fenpropimorph

Thirup et al 2000


30

Soil and rhizosphere inoculated with P. fluorescens DR54: Positive effect on fast-responding protozoa

(Johansen et al. 2005)



Anne Winding.


Rhizosphere of Arabidopsis thaliana: Amoebae decrease total abundance

and change diversity of bacteria

31

Rosenberg et al 2009


32

Susana Santos unpubl.


Anne Winding.


33

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


34

Total culturable bacteria

harvest after 1 day

CF

U g

-1 d

w

107

108

109

control

E. aerogenes

CHA0/gfp1

CHA0/pME3424

harvest after 7 days

CF

U g

-1 d

w

107

108

109

harvest after 14 days

Time (days)

0 10 20 30 40

CF

U g

-1 d

w

106

107

108

109

Figure 3. Winding and Oberender

a

b

a

abc

aaa

a

a

a

(Winding and Oberender 2012)


Anne Winding.


35

Soil Protozoa

control

Enterobacter aerogenes

P. fluorescens CHA0 gfp

P. fluorescens CHA0 pME3424

harvest time (days)1 7 14

Fa

st-

gro

win

g p

roto

zo

a g

-1 d

ry s

oil

102

103

104

105

control

E. aerogenes

P. fluorescens CHA0/gfp1

P. fluorescens CHA0/pME3424

harvest time (days)

1 7 14

To

tal p

roto

zo

a g

-1 d

ry s

oil

102

103

104

105

bd

bd

bd

Y

h

h

h

Figure 4 Winding and Oberender

a

b

e

d

cc

c

c

f

X

Y Y

X

Y gnd

nd

nd

nd

nd

nd



Anne Winding.


36

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


Protozoa increase abundance of MPCA in rhizosphere

37

With protozoa

No protozoa

Müller et al. 2013

Protozoa positively increase effect of MPCA in rhizosphere?


38

Protozoa change the physiological profile of bacteria in soil

(Rønn et al. 2002)


39

Variation in changes of bacterial community depends on protozoan species

(Rø

nn e

t al. 2

002)



Anne Winding.


Effects of predation in rhizosphere

40

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.


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

41


42

Molecular techniques demonstrate protists diversity is higher

than morphological studies suggest


43

Genetic diversity of Kinetoplastidae

1 day 7 days 14 days 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 SAB

CHA0/gfp1 c

CHA0/gfp1 a

E. aer. b

E. aer. a

CHA0/pME3424 c

Control b

Control a

Control c

E. aer. c

CHA0/gfp1 b

CHA0/pME3424 a

CHA0/pME3424 b

0.7 0.8 0.9 1.0 SAB

0.7 0.8 0.9 1.0 SAB

Control b

Control a

Control c

E. aer. a

E. aer. b

CHA0/gfp1 a

CHA0/gfp1 c

CHA0/pME3424 b

E. aer. c

CHA0/pME3424 a

CHA0/gfp1 b

CHA0/pME3424 c



44

harvest time (days)

1 7 14

ba

nd

s o

n D

GG

E g

el

6

8

10

12

14control

E. aerogenes

P. fluorescens CHA0/gfp1

P. fluorescens CHA0/pME3424

Figure 6. Winding and Oberender

(Winding and Oberender 2012.)

Genetic diversity of Kinetoplastidae


45 Letendu et al 2014


46

Protozoa as Trojan horses

Potential impacts on survival and spreading of human pathogens


47

Protozoa as Trojan horses


Fate of bacteria after phagocytosis

48

- Protozoan lysis of bacteria

- Bacterial lysis of protozoa

- Bacterial multiplication inside

amoebae

- Bacterial survival

- Increased gene transfer

- Increased virulence

- Increased antibiotic resistance

- Transcriptome changes


49

Campylobacter jejuni and Acanthamoeba castellanii - poor survival of bacteria inside amoebae

(Bui et al. 2012 Env Microb)

0 h

24 h

5 h


50

A. castellanii increases growth of C. jejuni

(Bui et al. 2012 Env Microb)

+ amoebae, separated

+ amoebae

- amoebae, micro O2

- amoebae



Anne Winding.


Bacteria packaging by amoebae Dig. B: digestible bacteria Res. B: resistant to lysosomal degradation

51

Legionella

pneumophila

in Multimellar

bodies (MLB)

Empty MLB

Denoncourt et al 2014


›Resting stage

›Resistant to draught, low food concentration, adverse temperatures etc.

›Resistant to predation?

›Germinate at high nutrient availability

›Spore germination inside protozoa?

Bacterial spores

52


Tetrahymena and Bacillus

53


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

54


55

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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