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1
Strassmann/ Queller lab group
Guns and butter in microbial farming
interactions
Joan Strassmann
strassmann@wustl.edu, ht...
2
Dicty life cycle
Test questions on the talk
you heard yesterday,
Cooperation and conflict
in the social amoeba
Dictyostelium discoideum
Dav...
1. Why study Dictyostelium? Which
of these reasons is NOT true?
1. To test social evolution theory in a completely differe...
SM plate SM plate
1 round
2. Selection to find cheater mutations
1. was impossible
2. used wild clones
3. identified many ...
3. Which of the following is NOT true about genetic
relatedness in wild fruiting bodies?
1. Wild fruiting bodies are
clona...
4. Do obligate stalkless cheaters occur in nature?
1. Yes. About 50% of clones
cannot make stalks on their
own.
2. Yes. Ab...
SM plate SM plate
1 round
5. Under experimental evolution
conditions of very low relatedness:
1. Some mutants lost the abi...
…and the answers are….
1. Why study Dictyostelium? Which
of these reasons is NOT true?
1. To test social evolution theory in a completely differe...
2. Selection to find cheater mutations
1. was impossible
2. used wild clones
3. identified many genes
4. was impeded by pl...
3. Which of the following is NOT true about genetic
relatedness in wild fruiting bodies?
1. Wild fruiting bodies are
clona...
4. Do obligate stalkless cheaters occur in nature?
1. Yes. About 50% of clones
cannot make stalks on their
own.
2. Yes. Ab...
SM plate SM plate
1 round
5. Under experimental evolution
conditions of very low relatedness:
1. Some mutants lost the abi...
Why and how to make a huge scientific
transition
Enormity of the
transition
Different natural history
Different kingdom
Different techniques
Different colleagues
Different...
Enormity of the transition
About 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 new
B. Student needing help with choosing
the ...
3. We needed a hook that made the
new field enticing
• The genome meant we could get genetic
markers – microsatellites- ea...
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...
What kept us going?
1. Continuing interesting
questions
2. Students interested in
the work
What kept us going?
1. Continuing interesting questions
2. Students interested in the work
3. Great mentors – Richard Kess...
What kept us going?
1. Continuing interesting questions
2. Students interested in the work
3. Great mentors
4. Great colla...
What kept us going?
1. Continuing interesting questions
2. Students interested in the work
3. Great mentors
4. Great colla...
Funding
Collaborators
Mentors
Students
Questions
Each other, continuing scientific risk
taking, and an eye for the big
questions
A true love of the organism
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...
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 geneticall...
Some clones transport bacteria; some do not
Micrographs of dispersed slug amoeba
Farmer Non-farmer
Nuclei
Nuclei
Bacteria
...
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
%oc...
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...
Host amoebae have greater
proliferation in host bacteria
0
500
1000
1500
2000
2500
Pf3 Kp
Totalamoebaex10⁴
D. discoideum a...
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 ...
Totalsporesx104
B)
0
50
100
150
Non-farmer Farmer
Non-farmer Farmer
A)
0
50
100
150
200
Non-farmer Farmer
Totalsporesx106
...
Tradeoff:
Advantage to farmers where
delicious bacteria are sparse.
Disadvantage to farmers for
prudent eating.
Brock et a...
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 inna...
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...
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 % Identi...
Carried Burkholderia xenovorans is a poor food -
but could be a weapon
Bs = Burkholderia xenovorans, Kp= Klebsiella pneumo...
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 n...
Besides food bacteria, farmers carry
bacteria for defense
D.discoideum
farmer clones
Location
collected
Closest relative i...
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
Far...
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 ...
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
• ...
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
76
Who knew small molecules are so
cool?
• Low molecular weight (<900 daltons) organic compound
• Size allows rapid diffusion...
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 ...
GacS/GacA (global activator)
two component system
• Highly conserved in Gram-negative bacteria
• GacS sensor kinase autoph...
Did Pf2 evolve into edible Pf3 by losing
the GacA function?
∆gacA knockout has same spectra
as Pf3 food bacteriaCurrent Chromatogram(s)
min5 10 15 20
mAU
0
100
200
300
400
500
DAD1 A...
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 woul...
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 i...
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 ...
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/photo...
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 remo...
© 2005 Tree of Life Web Project
A little
taxonomic
adventure
is fun!
John Templeton
Foundation
Guns and butter in social amoeba bacteria interactions
Guns and butter in social amoeba bacteria interactions
Guns and butter in social amoeba bacteria interactions
Guns and butter in social amoeba bacteria interactions
Guns and butter in social amoeba bacteria interactions
Guns and butter in social amoeba bacteria interactions
Guns and butter in social amoeba bacteria interactions
Guns and butter in social amoeba bacteria interactions
Guns and butter in social amoeba bacteria interactions
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Guns and butter in social amoeba bacteria interactions

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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.

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Guns and butter in social amoeba bacteria interactions

  1. 1. 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
  2. 2. 2
  3. 3. Dicty life cycle
  4. 4. Test questions on the talk you heard yesterday, Cooperation and conflict in the social amoeba Dictyostelium discoideum David Queller
  5. 5. 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
  6. 6. 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
  7. 7. 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
  8. 8. 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.
  9. 9. 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
  10. 10. …and the answers are….
  11. 11. 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
  12. 12. 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
  13. 13. 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
  14. 14. 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
  15. 15. 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
  16. 16. Why and how to make a huge scientific transition
  17. 17. Enormity of the transition Different natural history Different kingdom Different techniques Different colleagues Different scientific societies Different hurdles Different opportunities
  18. 18. Enormity of the transition About two years spent studying the biology of dying cells.
  19. 19. Why did we do it? How could we do it?
  20. 20. Joint crazy risk taking!
  21. 21. 1. We had to know about the new system and have some idea of its potential
  22. 22. 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
  23. 23. 3. We needed a hook that made the new field enticing • The genome meant we could get genetic markers – microsatellites- easily.
  24. 24. 4. When we began exploring, we found a friendly community
  25. 25. 5. When we got close we got both an offer and a push Dennis Welker, Utah State University
  26. 26. Push Community Hook Impetus Knowledge
  27. 27. 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?
  28. 28. What kept us going? 1. Continuing interesting questions 2. Students interested in the work
  29. 29. What kept us going? 1. Continuing interesting questions 2. Students interested in the work 3. Great mentors – Richard Kessin
  30. 30. What kept us going? 1. Continuing interesting questions 2. Students interested in the work 3. Great mentors 4. Great collaborators
  31. 31. What kept us going? 1. Continuing interesting questions 2. Students interested in the work 3. Great mentors 4. Great collaborators 5. Funding
  32. 32. Funding Collaborators Mentors Students Questions
  33. 33. Each other, continuing scientific risk taking, and an eye for the big questions
  34. 34. A true love of the organism
  35. 35. 41 Strassmann/ Queller lab group How about social amoeba mutualisms? Pierre Stallforth Jon ClardyDebbie Brock David Queller
  36. 36. What are the competitive and cooperative interactions of D. discoideum?
  37. 37. D. Discoideum eats bacteria during the amoeba stage, then apparently clears it for the social stage when it is not feeding Kessin 2000
  38. 38. Debbie noticed that some fruiting bodies looked different. Debbie Brock Brock et al 2011 Nature
  39. 39. 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:
  40. 40. 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)
  41. 41. 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%
  42. 42. Farmers are not a separate species 48
  43. 43. Farmers are not sick: No difference observed in solitary proliferation rates
  44. 44. a) NF NFNF F F F Does carrying your own bacteria increase proliferation on natural soils?
  45. 45. 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
  46. 46. 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
  47. 47. 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
  48. 48. 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
  49. 49. Tradeoff: Advantage to farmers where delicious bacteria are sparse. Disadvantage to farmers for prudent eating. Brock et al 2011 Nature
  50. 50. There could be other costs to farmers
  51. 51. Are farmers immune compromised because they are nice to their bacteria? Kessin 2000
  52. 52. 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
  53. 53. Farmers have fewer sentinel cells than non-farmers Figure removed, not yet published.
  54. 54. 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?
  55. 55. Turn to some other questions Look harder at the bacteria farmers carry.
  56. 56. 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?
  57. 57. Carried Burkholderia xenovorans is a poor food - but could be a weapon Bs = Burkholderia xenovorans, Kp= Klebsiella pneumoniae
  58. 58. Do farmers use bacteria as weapons against other Dicty clones? Kessin 2000
  59. 59. 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
  60. 60. 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
  61. 61. Are farmer-associated bacteria directly implicated in defense of public goods? 67
  62. 62. 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.
  63. 63. Farmers have ways of protecting their crop against other D. discoideum clones, partly using their bacterial weapons.
  64. 64. What is in that supernatant?
  65. 65. A story about one clone of D. discoideum, clone QS160, from Mountain Lake Biological Station which is a farmer Stallforth et al. PNAS, 2013
  66. 66. Debbie noticed two different bacteria colony morphologies from QS161. Both turned out to be Pseudomonas fluoresens. Pf2 Pf3
  67. 67. 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)
  68. 68. Pf2 alone is not a food for D. discoideum; we’ll show it is a weapon Kp control Pf2 alone 74
  69. 69. Pf3, the other bacterium from QS161 works well as food Kp control Pf3 Pf2 75
  70. 70. 76
  71. 71. 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
  72. 72. Are there small molecule differences that make Pf2 inedible and Pf3 edible?
  73. 73. Pf2 Inedible Pf2 makes pyrrolnitrin and chromene
  74. 74. Edible Pf3 makes the iron-chelating siderophore, pyochelin Pf3 Pf2 Inedible Pf2 makes pyrrolnitrin and chromene
  75. 75. Are chromene and pyrrolnitrin weapons farmer Dicty can use against non-farmers?
  76. 76. Chromene diminishes non-farmer growth, augments farmer growth Stallforth et al. PNAS 2013
  77. 77. Pyrrolnitrin diminishes non-farmer growth, augments farmer growth
  78. 78. Are chromene and pyrrolnitrin weapons farmer Dicty can use against non-farmers? YES!
  79. 79. How do Pf2 and Pf3 differ? They were isolated from the same clone of D. discoideum, after all.
  80. 80. 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
  81. 81. 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 *
  82. 82. Did Pf2 evolve into edible Pf3 by losing the GacA function?
  83. 83. ∆gacA knockout has same spectra as Pf3 food bacteriaCurrent Chromatogram(s) min5 10 15 20 mAU 0 100 200 300 400 500 DAD1 A, Sig=254,4 Ref=360,100 (PIERRE2-3-KO 2013-03-03 17-03-233.D) min5 10 15 20 mAU 0 100 200 300 400 500 DAD1 A, Sig=254,4 Ref=360,100 (PIERRE2-3-KO 2013-03-03 17-03-233.D) min5 10 15 20 mAU 0 500 1000 1500 2000 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 Current Chromatogram(s) min5 10 15 20 mAU 0 100 200 300 400 500 DAD1 A, Sig=254,4 Ref=360,100 (PIERRE2-3-KO 2013-03-03 17-03-233.D) min5 10 15 20 mAU 0 100 200 300 400 500 DAD1 A, Sig=254,4 Ref=360,100 (PIERRE2-3-KO 2013-03-03 17-03-233.D) min5 10 15 20 mAU 0 500 1000 1500 2000 DAD1 A, Sig=254,4 Ref=360,100 (PIERRESC-3 2011-08-29 13-22-5108-29-11-PF2-50.D) ===================================================================== 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 Current Chromatogram(s) min5 10 15 20 mAU 0 100 200 300 400 500 DAD1 A, Sig=254,4 Ref=360,100 (PIERRE2-3-KO 2013-03-03 17-03-233.D) min5 10 15 20 mAU 0 100 200 300 400 500 DAD1 A, Sig=254,4 Ref=360,100 (PIERRE2-3-KO 2013-03-03 17-03-233.D) min5 10 15 20 mAU 0 500 1000 1500 2000 DAD1 A, Sig=254,4 Ref=360,100 (PIERRESC-3 2011-08-29 13-22-5108-29-11-PF2-50.D) Instrument 1 3/5/2013 2:09:25 PM Tim Page 1 of 1 5 10 15 20 min Current Chromatogram(s) min5 10 15 20 mAU 0 100 200 300 400 500 DAD1 A, Sig=254,4 Ref=360,100 (PIERRE2-3-KO 2013-03-03 17-03-233.D) min5 10 15 20 mAU 0 100 200 300 400 500 DAD1 A, Sig=254,4 Ref=360,100 (PIERRE2-3-KO 2013-03-03 17-03-233.D) min5 10 15 20 mAU 0 500 1000 1500 2000 DAD1 A, Sig=254,4 Ref=360,100 (PIERRESC-3 2011-08-29 13-22-5108-29-11-PF2-50.D) Data File : D:DATAPIERRE2-3-KO 2013-03-03 17-03-233.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 Current Chromatogram(s) min5 10 15 20 mAU 0 100 200 300 400 500 DAD1 A, Sig=254,4 Ref=360,100 (PIERRE2-3-KO 2013-03-03 17-03-233.D) min5 10 15 20 mAU 0 100 200 300 400 500 DAD1 A, Sig=254,4 Ref=360,100 (PIERRE2-3-KO 2013-03-03 17-03-233.D) mAU 2000 DAD1 A, Sig=254,4 Ref=360,100 (PIERRESC-3 2011-08-29 13-22-5108-29-11-PF2-50.D) Print of window 38: Current Chromatogram(s) Data File : D:DATAPIERRE2-3-KO 2013-03-03 17-03-233.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) Current Chromatogram(s) min5 10 15 20 mAU 0 100 200 300 400 500 DAD1 A, Sig=254,4 Ref=360,100 (PIERRE2-3-KO 2013-03-03 17-03-233.D) mAU 300 400 500 DAD1 A, Sig=254,4 Ref=360,100 (PIERRE2-3-KO 2013-03-03 17-03-233.D) Print of window 38: Current Chromatogram(s) Data File : D:DATAPIERRE2-3-KO 2013-03-03 17-03-233.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) 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
  84. 84. Inedible Pseudomonas fluorescens with GacA knocked out becomes edible Kp control Pf3 Pf2Pf2-∆gacA
  85. 85. The edible and inedible Pf strains are each other’s closest relative
  86. 86. In the environment of D. discoideum, this Pseudomonas fluorescens clone evolved edibility with a single mutation that would be disabling in nature.
  87. 87. This is a super amazing result!!!!!!
  88. 88. How does Dicty choose what bacteria to associate with?
  89. 89. 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.
  90. 90. It is not easy to collect feces samples! http://www.flickr.com/photos/hbarrison/2874265346/in/photostream/
  91. 91. Fisher Brodie to the rescue!
  92. 92. 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.
  93. 93. What did we find? http://www.flickr.com/photos/hbarrison/2874265346/in/photostream/
  94. 94. More Dicty in soil than feces Dicty No Dicty soil 12 17 feces 3 26 Chi Square, p< 0.016
  95. 95. How about farmers? Farmer clone Not a farmer clone Soil 0 12 feces 0 3
  96. 96. Did not find more farmers in soil. Did not find farmers at all! http://www.flickr.com/photos/hbarrison/2874265346/in/photostream/
  97. 97. Possible problems • Wrong time of year • Sat too long before plating • Did not plate out enough sample
  98. 98. Can’t we do some of that sexy microbiome stuff to see how Dicty chooses what it picks up?
  99. 99. 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
  100. 100. © 2005 Tree of Life Web Project A little taxonomic adventure is fun! John Templeton Foundation

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