1Strassmann/ Queller lab group

Guns and butter in microbial farming interactionsJoan Strassmannstrassmann@wustl.edu, 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