guns and butter in social amoeba bacteria interactions
DESCRIPTION
If you have property, others will want it, even if you are a simple amoeba. Here we show how the amoeba Dictyostelium discoideum protects the bacteria they farm with other bacteria they use as weapons. We also show how a food bacterium evolved from a weapon bacterium with a single stop codon. In the process of telling this amazing story, we also discuss the challenges of making a major transition in a research career.TRANSCRIPT
1Strassmann/ Queller lab group
Guns and butter in microbial farming interactionsJoan [email protected], http://strassmannandquellerlab.wordpress.com
Read my blog on how to become a professor! http://sociobiology.wordpress.com
2
Dicty life cycle
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 conflict3. To find quantitative trait loci in sexual recombinants4. To do experimental evolution5. To use genomics and molecular evolution
SM plate SM plate
1 round
2. Selection to find cheater mutations
1. was impossible2. used wild clones3. identified many genes4. 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.
4. Wild fruiting bodies sometimes contain multiple species
4. Do obligate stalkless cheaters occur in nature?
1. Yes. About 50% of clones cannot make stalks on their own.
2. Yes. About 7% of clones 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 stalks on their own.
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
…and the answers are….
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 conflict3. To find quantitative trait loci in sexual recombinants4. To do experimental evolution5. To use genomics and molecular evolution
2. Selection to find cheater mutations
1. was impossible2. used wild clones3. identified many genes4. was impeded by pleiotropy
Santorelli et al. 2008 Nature
LorenzoSantorelli
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.
4. Wild fruiting bodies sometimes contain multiple species
Gilbert et al. 2007 PNAS
Owen Gilbert
4. Do obligate stalkless cheaters occur in nature?
1. Yes. About 50% of clones cannot make stalks on their own.
2. Yes. About 7% of clones 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 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 historyDifferent kingdom
Different techniquesDifferent colleaguesDifferent scientific
societiesDifferent hurdles
Different opportunities
Enormity of the transitionAbout two years spent studying the
biology of dying cells.
Why did we do it? How could we do it?
Joint crazy risk taking!
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 newB. 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
Com
mun
ity
Hoo
k
Impe
tus
Know
ledg
e
What kept us going?
1. Continuing interesting questions1. 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
questions2. Students interested in
the work
What kept us going?1. Continuing interesting questions2. Students interested in the work3. Great mentors – Richard Kessin
What kept us going?
1. Continuing interesting questions2. Students interested in the work3. Great mentors4. Great collaborators
What kept us going?
1. Continuing interesting questions2. Students interested in the work3. Great mentors4. Great collaborators 5. Funding
Funding
Colla
bora
tors
Men
tors
Stud
ents
Que
stion
s
Each other, continuing scientific risk taking, and an eye for the big questions
A true love of the organism
41Strassmann/ 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
Bacteria5µm
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 45/1
4
3/9
1/3
4/9
Farm
er
Non
-farm
er
% o
ccu
rren
ce
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)
NFNFNF
F F F
Does carrying your own bacteria increase proliferation on natural soils?
Non-farmer
Farmer
Soil 1 Soil 205
101520253035
FarmerNon-farmer
Fold
incr
ease
in
spor
es
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
Pf3 Kp0
500
1000
1500
2000
2500
Host farmerNon-farmer
D. discoideum amoebae proliferation at 24 hoursN=3
Tota
l am
oeb
ae x
10
⁴
Day1 Day3 Day5 Day70
0.5
1
1.5
2
2.5
3
3.5
BB w/NF
Absorb
ance
(A
600)
Solitary Social
Farmers are prudent; they do not eat all the bacteria presentBacteriaBacteria w/ Non-farmersBacteria w/ Farmers
Non-farmer
Fb’s:
3 mm
Farmer
Fb’s:
Bacteria
5 days after beginning
Tota
l spo
res
x 10
4
B)
Non-farmer Farmer0
20406080
100120140
Non-farmer Farmer
A)
Non-farmer Farmer0
20406080
100120140160
To
tal
spo
res
x 10
6Carrying 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 5 50 95 1000.0
0.2
0.4
0.6
0.8
1.0
1.2
Non-farmers
Farmers
% farmer clone
Per c
apita
spor
e pr
oduc
tion
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, VAStenotrophomonas maltophilia
K279a 98
2 clones Mt. Lake, VAEnterobacter 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 clonesLake 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
-50
-25
0
25
50
75S
po
res
no yes
Farmer
All Pairs
Tukey-Kramer
0.05
% c
hang
e in
spo
re p
rodu
ction
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.
What is in that supernatant?
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)
74
Pf2 alone is not a food for D. discoideum; we’ll show it is a weapon
Kp control Pf2 alone
75
Pf3, the other bacterium from QS161 works well as food
Kp control Pf3 Pf2
76
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
Pf2Pf3
Pf2Pf3
Pf2Pf3
Pf2Pf3
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 bacteria
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 to the rescue!
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?
http://www.flickr.com/photos/hbarrison/2874265346/in/photostream/
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!
http://www.flickr.com/photos/hbarrison/2874265346/in/photostream/
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
© 2005 Tree of Life Web Project
A little taxonomic adventure is fun!
John Templeton Foundation