Guns and butter in social amoeba bacteria interactions

1
Strassmann/ Queller lab group
Guns and butter in microbial farming
interactions
Joan Strassmann
strassmann@wustl.edu, http://strassmannandquellerlab.wordpress.com
Read my blog on how to become a professor!
http://sociobiology.wordpress.com

Test questions on the talk
you heard yesterday,
Cooperation and conflict
in the social amoeba
Dictyostelium discoideum
David Queller

1. Why study Dictyostelium? Which
of these reasons is NOT true?
1. To test social evolution theory in a completely different
system
2. To find the genes underlying cooperation and conflict
3. To find quantitative trait loci in sexual recombinants
4. To do experimental evolution
5. To use genomics and molecular evolution

SM plate SM plate
1 round
2. Selection to find cheater mutations
1. was impossible
2. used wild clones
3. identified many genes
4. was impeded by pleiotropy
10,000 REMI generated
mutants

3. Which of the following is NOT true about genetic
relatedness in wild fruiting bodies?
1. Wild fruiting bodies are
clonal.
2. Relatedness in wild
fruiting bodies is over
0.86.
3. Wild fruiting bodies
have never been
discovered.
sometimes contain
multiple species

4. Do obligate stalkless cheaters occur in nature?
1. Yes. About 50% of clones
cannot make stalks on their
own.
cannot make stalks on
their own.
3. Yes. About one in a
thousand cannot make
stalks on their own.
4. No. All clones can make

SM plate SM plate
1 round
5. Under experimental evolution
conditions of very low relatedness:
1. Some mutants lost the ability to
form stalks.
2. All mutants retained the ability
to make stalks
Low relatedness – plate 106 spores

2. Selection to find cheater mutations
1. was impossible
2. used wild clones
3. identified many genes
4. was impeded by pleiotropy
Santorelli et al. 2008 Nature
Lorenzo
Santorelli

3. Which of the following is NOT true about genetic
relatedness in wild fruiting bodies?
1. Wild fruiting bodies are
clonal.
2. Relatedness in wild
fruiting bodies is over
0.86.
have never been
discovered.
sometimes contain
multiple species
Gilbert et al. 2007 PNAS
Owen Gilbert

4. Do obligate stalkless cheaters occur in nature?
cannot make stalks on their
own.
cannot make stalks on
their own.
3. Yes. About one in a
thousand cannot make
4. No. All clones can make
stalks on their own. We
looked at 3316 clones from
95 fruiting bodies. Gilbert et al. 2007 PNAS
Owen Gilbert

SM plate SM plate
1 round
5. Under experimental evolution
conditions of very low relatedness:
1. Some mutants lost the ability to
form stalks, making them cheaters,
obligate social parasites.
2. All mutants retained the ability to
make stalks
Low relatedness – plate 106 spores
Kuzdzal-Fick et al Science 2010
Jennie Kuzdzal-Fick

Why and how to make a huge scientific
transition

Enormity of the
transition
Different natural history
Different kingdom
Different techniques
Different colleagues
Different scientific
societies
Different hurdles
Different opportunities

Enormity of the transition
About two years spent studying the
biology of dying cells.

Why did we do it? How could we do it?

1. We had to know about the new
system and have some idea of its
potential

2. We had to have some impetus to
explore
A. Feeling a desire for something new
B. Student needing help with choosing
the next project

3. We needed a hook that made the
new field enticing
• The genome meant we could get genetic
markers – microsatellites- easily.

4. When we began
exploring, we found a
friendly community

5. When we got close we got both an
offer and a push
Dennis Welker, Utah State University

Push
Community
Hook
Impetus
Knowledge

What kept us going?
1. Continuing interesting questions
1. Is there conflict in chimeras?
2. Do they recognize kin?
3. Can we change their social structure under
experimental evolution?

What kept us going?
1. Continuing interesting
questions
2. Students interested in
the work

What kept us going?
2. Students interested in the work
3. Great mentors – Richard Kessin

What kept us going?
3. Great mentors
4. Great collaborators

What kept us going?
3. Great mentors
4. Great collaborators
5. Funding

Funding
Collaborators
Mentors
Students
Questions

Each other, continuing scientific risk
taking, and an eye for the big
questions

41
Strassmann/ Queller lab group
How about social amoeba mutualisms?
Pierre Stallforth Jon ClardyDebbie Brock David Queller

What are the competitive and cooperative
interactions of D. discoideum?

D. Discoideum eats bacteria during the amoeba
stage, then apparently clears it for the social
stage when it is not feeding
Kessin 2000

Debbie noticed
that some fruiting
bodies looked
different. Debbie Brock
Brock et al 2011 Nature

Some clones carry bacteria through the social stage
Micrographs of sorus contents
Spores
Spores
Bacteria
5µm
12 genetically-distinct clones collected from a small transect in Va.
Experienced same environment; access to same potential food
Study population:

Some clones transport bacteria; some do not
Micrographs of dispersed slug amoeba
Farmer Non-farmer
Nuclei
Nuclei
Bacteria
Amoeba stained with DAPI (DNA stain) and Baclight Red (live bacteria specific stain)

About a third of clones are farmers.
MinnesotaVirginia
0
20
40
60
80
100
120
1 2 3 4
5/14
3/9
1/3
4/9
FarmerNon-farmer
%occurrence
Average
proportion
of farmers
35.5%

Farmers are not a separate species
48

Farmers are not sick: No difference
observed in solitary proliferation rates

a)
NF
NFNF
F F F
Does carrying your own bacteria
increase proliferation on natural soils?

Non-
farmer
Farmer
0
10
20
30
40
Soil 1 Soil 2
Foldincreasein
spores
Farmer
Farmer clones eat better than
nonfarmers
1.3-2.2 x 108
CFU’s/gm soil
0.6-0.64 x 108
CFU’s/gm soil

Host amoebae have greater
proliferation in host bacteria
0
500
1000
1500
2000
2500
Pf3 Kp
Totalamoebaex10⁴
D. discoideum amoebae proliferation at 24 hours
N=3
Host farmer
Non-farmer

0
0.5
1
1.5
2
2.5
3
3.5
Day1 Day3 Day5 Day7
Absorbance(A600)
Solitary Social
Farmers are prudent; they do not eat
all the bacteria present
Bacteria
Bacteria w/ Non-farmers
Bacteria w/ Farmers
Non-farmer
Fb’s:
3
mm
Farmer
Fb’
s:
Bacteria
5 days after beginning

Totalsporesx104
B)
0
50
100
150
Non-farmer Farmer
Non-farmer Farmer
A)
0
50
100
150
200
Non-farmer Farmer
Totalsporesx106
Carrying food is advantageous when delicious bacteria
are absent at new site,
but disadvantageous when they are abundant
Benefit Cost
Bacteria absent Bacteria present

Tradeoff:
Advantage to farmers where
delicious bacteria are sparse.
Disadvantage to farmers for
prudent eating.
Brock et al 2011 Nature

There could be other costs to
farmers

Are farmers immune compromised
because they are nice to their bacteria?
Kessin 2000

Sentinel cells are less adhesive cells that pass
through the multicellular body, accumulating
toxins and bacteria, an innate immune system,
and a liver. Then they are sloughed off the tail end
of the slug.
Chen et al. 2007

Farmers have fewer sentinel cells
than non-farmers
Figure removed, not yet published.

1. About a third of clones are farmers.
2. Farmers carry bacteria in the social stage.
3. Farmers prudently do not eat all the
bacteria.
4. Farmers proliferate more than non-
farmers on soil if no bacteria are added.
5. Farmers have fewer sentinel cells.
6. Farmers form more spores on toxic
media.
7. Farmers are not sick.
What do we know so far?

Turn to some other questions
Look harder at the bacteria farmers carry.

Some carried bacteria are not good food
D.discoideum
farmer clones
Location
collected
Closest relative in GenBank % Identity
5 clones Mt. Lake, VA Burkholderia xenovorans LB400 98
2 clones Mt. Lake, VA Stenotrophomonas maltophilia K279a 98
2 clones Mt. Lake, VA Enterobacter sakazakii ATCC BAA-894 98
3 clones Mt. Lake, VA Pseudomonas fluorescens Pf-5 98
2 clones Mt. Lake, VA Burkholderia phytofirmans psJN 97
4 clones Lake Itaska, MN Flavobacterium johnsoniae UW101 93
Pathogens?
Weapons?

Carried Burkholderia xenovorans is a poor food -
but could be a weapon
Bs = Burkholderia xenovorans, Kp= Klebsiella pneumoniae

Do farmers use bacteria as weapons
against other Dicty clones?
Kessin 2000

0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 5 50 95 100
Percapitasporeproduction
% farmer clone
Non-farmers
Farmers
Farmers outcompete non-farmers
Type F3,30=18.71, p<0.0001; Significant
differences found between types, results of a
post-hoc Tukey HSD test
Brock et al. Nature Communications 2013

Besides food bacteria, farmers carry
bacteria for defense
D.discoideum
farmer clones
Location
collected
Closest relative in GenBank
%
Identity
5 clones Mt. Lake, VA Burkholderia xenovorans LB400 98
2 clones Mt. Lake, VA Stenotrophomonas maltophilia K279a 98
2 clones Mt. Lake, VA
Enterobacter sakazakii ATCC BAA-
894 98
3 clones Mt. Lake, VA Pseudomonas fluorescens Pf-5 98
2 clones Mt. Lake, VA Burkholderia phytofirmans psJN 97
4 clones
Lake Itaska,
MN Flavobacterium johnsoniae UW101 93

Are farmer-associated bacteria directly implicated in
defense of public goods?
67

Farmer-associated B. xenovorans isolate exudates
harm non-farmers and benefit host farmers
68
%changeinsporeproduction
FarmerNon-farmer
Box plots of combined data.
Change in spore production is strongly affected by farmer status,
with non-farmers decreasing spore production compared to controls
and farmers increasing spore production compared to controls.

Farmers have ways of protecting their
crop against other D. discoideum
clones, partly using their bacterial
weapons.

A story about one clone of D.
discoideum, clone QS160, from
Mountain Lake Biological
Station which is a farmer
Stallforth et al. PNAS, 2013

Debbie noticed two different bacteria colony
morphologies from QS161. Both turned out to
be Pseudomonas fluoresens.
Pf2
Pf3

Pseudomonas fluorescens
• Gram negative rod shaped bacteria that inhabit
soil, plants (rhizosphere), and water surfaces
• Nonpathogenic and optimum growth at 25°C
make it ideal for plant disease suppression
• Commercially important for antibiotics
(Mupirocin) and fungicides for crops
• Produces siderophores such as pyoverdin (chelates
iron)

Pf2 alone is not a food for D.
discoideum; we’ll show it is a weapon
Kp control Pf2 alone
74

Pf3, the other bacterium from
QS161 works well as food
Kp control Pf3 Pf2
75

Who knew small molecules are so
cool?
• Low molecular weight (<900 daltons) organic compound
• Size allows rapid diffusion across membranes to intracellular
sites of action
• May be an enzyme substrate or regulator of biological
processes
• Variety of biological functions such as cell signalling
molecules, drugs, and pesticides
• Can be natural (secondary metabolites) or derived (some
drugs-Ravindranathan et al 2013; Wang et al 2013)
• Very common in soil bacteria and fungi

Are there small molecule
differences that make Pf2
inedible and Pf3 edible?

Pf2
Inedible Pf2 makes pyrrolnitrin
and chromene

Edible Pf3 makes the iron-chelating
siderophore, pyochelin
Pf3
Pf2
Inedible Pf2 makes pyrrolnitrin
and chromene

Are chromene and
pyrrolnitrin weapons
farmer Dicty can use
against non-farmers?

Chromene diminishes non-farmer
growth, augments farmer growth
Stallforth et al. PNAS 2013

Pyrrolnitrin diminishes non-farmer
growth, augments farmer growth

Are chromene and
pyrrolnitrin weapons
farmer Dicty can use
against non-farmers?
YES!

How do Pf2 and Pf3 differ? They were isolated
from the same clone of D. discoideum, after all.

A single stop codon in Pf3 turns off GacA
pathway
Pf2
Pf3
Pf2
Pf3
Pf2
Pf3
Pf2
Pf3
A single stop codon appears to make Pf3 edible

GacS/GacA (global activator)
two component system
• Highly conserved in Gram-negative bacteria
• GacS sensor kinase autophosphorylates and activates GacA
response regulator
•
• Disruption of either gene produces identical phenotype
• Gene disruption leads to:
– Loss of production of positively regulated external products such
as exotoxins, exoproteases, antibiotics (pyrrolnitrin), and
quorum sensing signals
– Overproduction of negatively regulated secondary metabolites
such as siderophores
– Flagella are affected *

Did Pf2 evolve into edible Pf3 by losing
the GacA function?

∆gacA knockout has same spectra
as Pf3 food bacteriaCurrent Chromatogram(s)
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DAD1 A, Sig=254,4 Ref=360,100 (PIERRE2-3-KO 2013-03-03 17-03-233.D)
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DAD1 A, Sig=254,4 Ref=360,100 (PIERRESC-3 2011-08-29 13-22-5108-29-11-PF2-50.D)
Sample Name : 3
=====================================================================
Acq. Operator : Pierre Seq. Line : 2
Acq. Instrument : Instrument 1 Location : Vial 62
Injection Date : 3/3/2013 5:35:09 PM Inj : 1
Inj Volume : 5.0 µl
Different Inj Volume from Sequence ! Actual Inj Volume : 100.0 µl
Acq. Method : D:DATAPIERRE2-3-KO 2013-03-03 17-03-23DEFAULT_ACID.M
Last changed : 1/17/2013 6:51:31 PM by Pierre
Analysis Method : C:CHEM321METHODSSHUGENG20 MIN GRADIENT_W&E.M
Last changed : 3/5/2013 2:08:20 PM by Tim
(modified after loading)
Instrument 1 3/5/2013 2:09:25 PM Tim Page 1 of 1
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Print of window 38: Current Chromatogram(s)
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mAU
254 nm
* Pyochelin II
* Pyochelin I
* Pyrrolnitrin
Chromene *
PfA
PfA ΔgacA
PfB
0
0
0
2000
500
500
* Pyochelin I
* Pyochelin II
Food
Non-food
Non-food
mutagenized
to food
Pf3
Pf2

Inedible Pseudomonas fluorescens
with GacA knocked out becomes edible
Kp control Pf3 Pf2Pf2-∆gacA

The edible and inedible Pf strains are
each other’s closest relative

In the environment of D. discoideum, this
Pseudomonas fluorescens clone evolved edibility
with a single mutation that would be disabling in
nature.

This is a super amazing
result!!!!!!

How does Dicty
choose what
bacteria to
associate with?

Where are the farmer Dicty?
1. We hypothesized that there would be more
clones of Dictyostelium discoideum in feces than in
soil samples because of the higher numbers of
bacteria in feces
2. We hypothesized that there would be more
farmer D. discoideum carrying bacteria in the soil
samples than in the feces because we reasoned
there would be fewer kinds of delicious bacteria in
soil compared to feces.

It is not easy to collect feces samples!
http://www.flickr.com/photos/hbarrison/2874265346/in/photostream/

Fisher Brodie
• collected paired samples of feces and soil
delivered to us on three dates: 5 July, 27 July,
and 11 August 2013.
• There were 9 pairs at the first collection, 12 at
the second, and 8 at the third.
• In all there were 20 deer feces samples, and 9
bear feces samples.

What did we find?

More Dicty in soil than feces
Dicty No Dicty
soil 12 17
feces 3 26
Chi Square, p< 0.016

How about farmers?
Farmer
clone
Not a
farmer
clone
Soil 0 12
feces 0 3

Did not find more farmers in soil. Did
not find farmers at all!

Possible problems
• Wrong time of year
• Sat too long before plating
• Did not plate out enough sample

Can’t we do some of that sexy
microbiome stuff to see how Dicty
chooses what it picks up?

What is in the soil at MLBS, family
level, and how does it compare to
what is in the collected D. discoideum?
Figures removed work in progress

Guns and butter in social amoeba bacteria interactions

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