project 1: experimental evolution methylobacterium –non-pathogenic, easy to culture, genetics,...
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Project 1: Experimental evolution
• Methylobacterium– Non-pathogenic, easy to culture, genetics, genome,
metabolic & biochemical knowledge– Have fluorescence-based fitness assays– Transfers only every other day– My lab studies it – can lead to ‘real’ work…
Methylotrophy (aerobic)
• Key issue: Efficient growth requires high flux through formaldehyde while maintaining a pool below toxic concentrations – and partition carbon appropriately into assimilatory and dissimilatory metabolism
CH3-R
HCHOHCHO
CO2biomass
• Methylotrophy (growth on C1)– C1 compounds oxidized to formaldehyde– Oxidation of formaldehyde to CO2
– Assimilation of formaldehyde into cell material
Methylotrophy (aerobic)
• Key issue: Efficient growth requires high flux through formaldehyde while maintaining a pool below toxic concentrations – and partition carbon appropriately into assimilatory and dissimilatory metabolism
CH3-R
HCHOHCHO
CO2biomass
• Methylotrophy (growth on C1)– C1 compounds oxidized to formaldehyde– Oxidation of formaldehyde to CO2
– Assimilation of formaldehyde into cell material
““If the consumption of cytoplasmic If the consumption of cytoplasmic formaldehyde were inhibited, the formaldehyde were inhibited, the cytoplasmic formaldehyde cytoplasmic formaldehyde concentration would increase to concentration would increase to about 100 mM in less than 1 min.” about 100 mM in less than 1 min.” (Vorholt et al., 2000, J Bacteriol)(Vorholt et al., 2000, J Bacteriol)
Methylotrophy and HGT
1. Methylotrophy has arisen multiple, independent times in different lineages
2. HGT major force in enabling this specialized metabolism
Proteo-Proteo-bacteriabacteria
GramGram++
16S 16S rDNArDNA
Tree of BacteriaTree of Bacteria
MethylotrophsMethylotrophs
(Kalyuzhnaya et al., 2005, J Bacteriol)
Multiple C1 modules for each role
Dichloro-methane
Methanol MethylamineChloro-methane
Methane
Methane-SulfonicAcid
Formaldehyde
CH2=H4F
Formate
CO2
SerineCycle
RuMPCycle
CBBCycle
Dissimilation
pMMO sMMO
MDHMADH
N-MGPathway
CMUDMU
MSM
FDH1 FDH2 FDH3
OtherFlDHses
H4F-Pathway 1
H4F-Pathway 2
H4MPT-Pathway
GSH-Pathway
1. Primary oxidation2. Secondary oxidation3. Assimilation
Dichloro-methane
Methanol MethylamineChloro-methane
Methane
Methane-SulfonicAcid
Formaldehyde
CH2=H4F
Formate
CO2
SerineCycle
RuMPCycle
CBBCycle
Dissimilation
pMMO sMMO
MDHMADH
N-MGPathway
CMUDMU
MSM
FDH1 FDH2 FDH3
OtherFlDHses
H4F-Pathway 1
H4F-Pathway 2
H4MPT-Pathway
GSH-Pathway
1. Primary oxidation2. Secondary oxidation3. Assimilation
Methylotrophs possess multiple combinations of C1 modules
HCOOH
CO2
CH4
sMMO pMMO2pMMO1
Methylococcus capsulatus Bath
MDH RuMPassim.
H4MPT
FDH1 FDH2
CBB
CH3OH
HCHOHCHO
MDH
FDH2FDH1
CBB
Xanthobacter autotrophicus
H4MPT
HCOOH
CH3OH
HCHOHCHO
CO2
Methylobacterium extorquens AM1
Glyoxylateregeneration
serinecycle
PHB
TCA
MDH MaDH
CH3OH
HCHOHCHO
HCOOH
CO2
H4MPT
FDH1 FDH2 FDH3
H4F
CH2=H4F
CH3NH2
HCOOH
Methylobacillus flagellatus KT
MDH MaDH
H4MPT
FDH2FDH1
Oxidation
HCHOHCHO
CH3OH CH3NH2
CO2
RuMPassim.
Model system: Methylobacterium• -proteobacterium, plant epiphyte• Grows on limited number of multi-C compounds
– Of cultured methylotrophs, nearly all highly specialized– Suggest consistent tradeoff? Ecological or physiological?– Leading a consortium to analyze sequence of 6 more
Methylobacterium genomes (JGI)
• C1 and multi-C growth are fundamentally different:succinate methanol
TCA cycle
serine cycle
C1 transfers
CO2
succinate
biomass
energy
TCA cycle
serine cycle
C1 transfers
CO2
methanol
biomass
energy
Methylotrophy in M. extorquens AM1
1. Oxidation of C1 substrates to formaldehyde
CH3OH
CH3OH HCHOMDHMDH MaDHMaDH
H4FH4MPT
Fae
CH2=H4F CH2=H4MPT
MtdA, MtdBMtdA
CHO-H4F
HCOOH
Fch
Fhc
Mch
FtfL
spont.
CH3NH2
CH3NH2
HCHO
CH=H4F
CHO-H4MPT
CO2
spont.
BIOMASS
serinecycle
FDHs
CH=H4MPT
NADPH
H4F, ATP
H4MPT
H2O H2O
NADH
NAD(P)H
H2O, 2e- H2O, NH3, 2e-
cytoplasm
periplasm
H2O
H2O
H2OH2O
CH3OH
CH3OH HCHOMDH MaDH
H4FH4MPT
FaeFae
CH2=H4F CH2=H4MPT
MtdA, MtdBMtdA
CHO-H4F
HCOOH
Fch
Fhc
Mch
FtfL
spont.spont.
CH3NH2
CH3NH2
HCHO
CH=H4F
CHO-H4MPT
CO2
spont.spont.
BIOMASS
serinecycle
FDHs
CH=H4MPT
NADPH
H4F, ATP
H4MPT
H2O H2O
NADH
NAD(P)H
H2O, 2e- H2O, NH3, 2e-
cytoplasm
periplasm
H2O
H2O
H2OH2O 2. Condensation of formaldehyde with H4F or H4MPT
Methylotrophy in M. extorquens AM1
CH3OH
CH3OH HCHOMDH MaDH
H4FH4MPT
Fae
CH2=H4F CH2=H4MPT
MtdA, MtdBMtdA, MtdBMtdA
CHO-H4F
HCOOH
Fch
FhcFhc
MchMch
FtfL
spont.
CH3NH2
CH3NH2
HCHO
CH=H4F
CHO-H4MPT
CO2
spont.
BIOMASS
serinecycle
FDHs
CH=H4MPT
NADPH
H4F, ATP
H4MPT
H2O H2O
NADH
NAD(P)H
H2O, 2e- H2O, NH3, 2e-
cytoplasm
periplasm
H2O
H2O
H2OH2O 3. Oxidation of CH2=H4MPT to formate
Methylotrophy in M. extorquens AM1
CH3OH
CH3OH HCHOMDH MaDH
H4FH4MPT
Fae
CH2=H4F CH2=H4MPT
MtdA, MtdBMtdA
CHO-H4F
HCOOH
Fch
Fhc
Mch
FtfL
spont.
CH3NH2
CH3NH2
HCHO
CH=H4F
CHO-H4MPT
CO2
spont.
BIOMASS
serinecycle
FDHsFDHs
CH=H4MPT
NADPH
H4F, ATP
H4MPT
H2O H2O
NADH
NAD(P)H
H2O, 2e- H2O, NH3, 2e-
cytoplasm
periplasm
H2O
H2O
H2OH2O 4. Oxidation of formate to CO2
Methylotrophy in M. extorquens AM1
CH3OH
CH3OH HCHOMDH MaDH
H4FH4MPT
Fae
CH2=H4F CH2=H4MPT
MtdA, MtdBMtdA
CHO-H4F
HCOOH
Fch
Fhc
Mch
FtfL
spont.
CH3NH2
CH3NH2
HCHO
CH=H4F
CHO-H4MPT
CO2
spont.
BIOMASS
serineserinecyclecycle
FDHs
CH=H4MPT
NADPH
H4F, ATP
H4MPT
H2O H2O
NADH
NAD(P)H
H2O, 2e- H2O, NH3, 2e-
cytoplasm
periplasm
H2O
H2O
H2OH2O 5. Assimilation of CH2=H4F by serine cycle
Methylotrophy in M. extorquens AM1
CH3OH
CH3OH HCHOMDH MaDH
H4FH4MPT
Fae
CH2=H4F CH2=H4MPT
MtdA, MtdBMtdAMtdA
CHO-H4F
HCOOH
FchFch
Fhc
Mch
FtfLFtfL
spont.
CH3NH2
CH3NH2
HCHO
CH=H4F
CHO-H4MPT
CO2
spont.
BIOMASS
serinecycle
FDHs
CH=H4MPT
NADPH
H4F, ATP
H4MPT
H2O H2O
NADH
NAD(P)H
H2O, 2e- H2O, NH3, 2e-
cytoplasm
periplasm
H2O
H2O
H2OH2O 6. Interconversion of CH2=H4F and formate
Methylotrophy in M. extorquens AM1
Primary hub of C1 metabolism:
CH3OH
CH3OH HCHOMDH MaDH
H4FH4MPT
Fae
CH2=H4F CH2=H4MPT
MtdA, MtdBMtdA
CHO-H4F
HCOOH
Fch
Fhc
Mch
FtfL
spont.
CH3NH2
CH3NH2
HCHO
CH=H4F
CHO-H4MPT
CO2
spont.
(DmrA, Orf4)
BIOMASS
serinecycle
FDHs
CH=H4MPT
NADPH
H4F, ATP
H4MPT
H2O H2O
NADH
NAD(P)H
H2O, 2e- H2O, NH3, 2e-
cytoplasm
periplasm
H2O
H2O
H2OH2O
CH3OH
CH3OH
CH3OH
CH3OH HCHOMDH MaDH
H4FH4MPT
Fae
CH2=H4F CH2=H4MPT
MtdA, MtdBMtdA
CHO-H4F
HCOOH
Fch
Fhc
Mch
FtfL
spont.
CH3NH2
CH3NH2
CH3NH2
CH3NH2
HCHO
CH=H4F
CHO-H4MPT
CO2
spont.
(DmrA, Orf4)
BIOMASS
serinecycle
FDHs
CH=H4MPT
NADPH
H4F, ATP
H4MPT
H2O H2O
NADH
NAD(P)H
H2O, 2e- H2O, NH3, 2e-
cytoplasm
periplasm
H2O
H2O
H2OH2O
CH3-R
HCHOHCHO
CO2biomass
What happened to simplicity???:
Model system: C1 metabolism in Methylobacterium
1.1.
3.3.
2.2.
Topologically, any 2 of the 3 pathways leading to biomass or CO2 should be sufficient…
Mutants defective in pathway 2. or 3. are C1
-
Why are both C1 transfer pathways needed?
3.3.2.2.
2. & 3. “redundant” for dissimilation?
3.3.
1.1.
1. & 3. “redundant” for assimilation?
assimilationassimilationdissimilationdissimilation
0
20
40
60
80
100
A B C D
nmol
min
-1
nmol
min
-1
Dissimilatory
B.A.
0
20
E F
CH2=H4F formation
nmol
min
-1
C.
0
20
40
G H I J
nmol
min
-1
Assimilatory
D.
CH3OH
HCHO
CH2=H4F
HCOOH
CH2=H4MPT
CO2
CO2*CO2
biomass
A
B
C
D
E
F
G
HI
J
CH3OH
HCHO
CH2=H4F
HCOOH
CH2=H4MPT
CO2
CO2*CO2
biomass
A
B
C
D
E
F
G
HI
J
0
20
40
60
80
100
A B C D
nmol
min
-1
nmol
min
-1nm
olm
in-1
Dissimilatory
B.A.
0
20
E F
CH2=H4F formation
nmol
min
-1
C.
0
20
40
G H I J
nmol
min
-1
Assimilatory
D.
CH3OH
HCHO
CH2=H4F
HCOOH
CH2=H4MPT
CO2
CO2*CO2
biomass
A
B
C
D
E
F
G
HI
J
CH3OH
HCHO
CH2=H4F
HCOOH
CH2=H4MPT
CO2
CO2*CO2
biomass
A
B
C
D
E
F
G
HI
J
nm
ol m
in-1 m
L-1 O
D60
0n
mol
min
-1 m
L-1 O
D60
0
• Dynamics of transition from S to M
Measured fluxes through hubn
mol
min
-1 m
L-1 O
D60
0
(Marx et al., 2005, PLoS Biology)
0
20
40
60
80
100
A B C D
nmol
min
-1
nmol
min
-1
Dissimilatory
B.A.
0
20
E F
CH2=H4F formation
nmol
min
-1
C.
0
20
40
G H I J
nmol
min
-1
Assimilatory
D.
CH3OH
HCHO
CH2=H4F
HCOOH
CH2=H4MPT
CO2
CO2*CO2
biomass
A
B
C
D
E
F
G
HI
J
CH3OH
HCHO
CH2=H4F
HCOOH
CH2=H4MPT
CO2
CO2*CO2
biomass
A
B
C
D
E
F
G
HI
J
0
20
40
60
80
100
A B C D
nmol
min
-1
nmol
min
-1nm
olm
in-1
Dissimilatory
B.A.
0
20
E F
CH2=H4F formation
nmol
min
-1
C.
0
20
40
G H I J
nmol
min
-1
Assimilatory
D.
CH3OH
HCHO
CH2=H4F
HCOOH
CH2=H4MPT
CO2
CO2*CO2
biomass
A
B
C
D
E
F
G
HI
J
CH3OH
HCHO
CH2=H4F
HCOOH
CH2=H4MPT
CO2
CO2*CO2
biomass
A
B
C
D
E
F
G
HI
J
nm
ol m
in-1 m
L-1 O
D60
0
nm
ol m
in-1 m
L-1 O
D60
0n
mol
min
-1 m
L-1
OD
600
?
• Developed kinetic model of central C1 hub
Do we really understand this?
(Marx et al., 2005, PLoS Biology)
Switch from long to direct assimilation
• Model prediction qualitatively recapitulated the phenomenon…
Experimental data Model predictions
(Marx et al., 2005, PLoS Biology)
Competitor #1
acclimate
Competitor #2
mixday 0 day 1
W = P W > P
grow
thWw > Wp
ancestorgr
owth
trans
fer
grow
th
trans
fer
grow
th
trans
fer
grow
th
trans
fer
grow
th
trans
fer
grow
th
trans
fer
-80°C• Living fossil record
– Examine through time & across replicates
• Assay competitive fitness:
Experimental evolution of laboratory populations
Experimental evolution of laboratory populations
Competitor #1
acclimate
Competitor #2
mixday 0 day 1
W = P W > P
grow
thWw > Wp
ancestorgr
owth
trans
fer
grow
th
trans
fer
grow
th
trans
fer
grow
th
trans
fer
grow
th
trans
fer
grow
th
trans
fer
-80°C• Living fossil record
– Examine through time & across replicates
• Assay competitive fitness:
What this looked like before…
Relative fitness of Venus/no Venus:WM = 1.00001 ± .000352WS = 1.00016 ± .000154
No Venus Venus (fancy YFP)
What it looks like now…
Average CV: 5.7 Average CV: 5.7 ± ± 3.1%3.1%
(David Chou)
Project 1: Experimental evolution
• What we can assay:– Fitness– Growth– In selected and other environments…– Diversity in colony morphology– For some projects, sequence candidate loci
Project 1: Experimental evolution
• Project possibilities– Need to be relatively easy to passage, but hopefully
somewhat interesting…– Will present 10 projects – can pick one, modify one,
or come up with your own– Each group will write a brief description of plans
• Will discuss further on Wednesday (and due 2/12)• Next Monday we will discuss these further and groups will
revise plan and consult with David and I (before 2/14)• If all goes well, initiate transfers on Wednesday, 2/14, go
over protocol and sign-up for transfer days…
Option #1 – Diversification in still medium
• Similar adaptive diversification as seen w/ P. fluorescens?– Try more than one genotype (lab
strain, wild isolate, an evolved isolate)
– Try more than one medium (rich vs. minimal, different substrates)
– Tradeoff w/ growth in shaken medium?
– Assay both diversity in colony morphology and fitness
??
Option #2 – Adaptation to solid surface
• Tradeoffs with growth in liquid?• Diversity due to spatial heterogeneity?• Changes in biofilm structure? (Initiate with
fluorescent strains)
Option #3 – Adaptation to poor substrates
• Are either the dynamics of adaptation or tradeoffs experienced more extreme with poor substrates?– Try more than one genotype (lab strain, an evolved
isolate)– Try substrates such as formate, glycerol, ethanol,
acetate (compared to methanol or succinate)…
TCA cycle
serine cycle
C1 transfers
CO2
methanol
biomass
energy
formate
ethanolacetate
glycerol
succinate
Option #4 – Adaptation to rich medium
• Does adaptation to rich medium lead to a diverse community?– Look for potential diversity and frequency-dep. fitness
effects between community members– Also can look at tradeoffs in minimal medium
TCA cycle
serine cycle
C1 transfers
CO2biomass
energy
Rich mediumRich medium
Option #5 – Evolve on formaldehyde
• Can cells balance need to grow with toxicity?– Wild-type is very poor at
using formaldehyde directly
– May need to supplement early growth with methanol
– Another very poor substrate
– Look at tradeoffs w/ other C1 substrates
– May unlock secret of formaldehyde transport…
CH3OH
CH3OH HCHOMDH MaDH
H4FH4MPT
Fae
CH2=H4F CH2=H4MPT
MtdA, MtdBMtdA
CHO-H4F
HCOOH
Fch
Fhc
Mch
FtfL
spont.
CH3NH2
CH3NH2
HCHO
CH=H4F
CHO-H4MPT
CO2
spont.
(DmrA, Orf4)
BIOMASS
serinecycle
FDHs
CH=H4MPT
NADPH
H4F, ATP
H4MPT
H2O H2O
NADH
NAD(P)H
H2O, 2e- H2O, NH3, 2e-
cytoplasm
periplasm
H2O
H2O
H2OH2O
CH3OH
CH3OH
CH3OH
CH3OH HCHOMDH MaDH
H4FH4MPT
Fae
CH2=H4F CH2=H4MPT
MtdA, MtdBMtdA
CHO-H4F
HCOOH
Fch
Fhc
Mch
FtfL
spont.
CH3NH2
CH3NH2
CH3NH2
CH3NH2
HCHO
CH=H4F
CHO-H4MPT
CO2
spont.
(DmrA, Orf4)
BIOMASS
serinecycle
FDHs
CH=H4MPT
NADPH
H4F, ATP
H4MPT
H2O H2O
NADH
NAD(P)H
H2O, 2e- H2O, NH3, 2e-
cytoplasm
periplasm
H2O
H2O
H2OH2O
??????
Option #6 – Evolve on increasing concentrations of methanol
• Push boundary of physiological capacities
• Tradeoffs with normal concentration?
• Can try w/ multiple genotypes– pre-evolved to M– strain w/ engineered
foreign formaldehyde oxidation pathway
• Can step up concentration as they improve…
CH3OH
CH3OH HCHOMDH MaDH
H4FH4MPT
Fae
CH2=H4F CH2=H4MPT
MtdA, MtdBMtdA
CHO-H4F
HCOOH
Fch
Fhc
Mch
FtfL
spont.
CH3NH2
CH3NH2
HCHO
CH=H4F
CHO-H4MPT
CO2
spont.
(DmrA, Orf4)
BIOMASS
serinecycle
FDHs
CH=H4MPT
NADPH
H4F, ATP
H4MPT
H2O H2O
NADH
NAD(P)H
H2O, 2e- H2O, NH3, 2e-
cytoplasm
periplasm
H2O
H2O
H2OH2O
CH3OH
CH3OH
CH3OH
CH3OH HCHOMDH MaDH
H4FH4MPT
Fae
CH2=H4F CH2=H4MPT
MtdA, MtdBMtdA
CHO-H4F
HCOOH
Fch
Fhc
Mch
FtfL
spont.
CH3NH2
CH3NH2
CH3NH2
CH3NH2
HCHO
CH=H4F
CHO-H4MPT
CO2
spont.
(DmrA, Orf4)
BIOMASS
serinecycle
FDHs
CH=H4MPT
NADPH
H4F, ATP
H4MPT
H2O H2O
NADH
NAD(P)H
H2O, 2e- H2O, NH3, 2e-
cytoplasm
periplasm
H2O
H2O
H2OH2O
??????
Option #7 – Alternate between media lacking C, or N
• Make PHB (a biodegradable plastic) as storage product
• Force storage and efficient reutilization?• Tradeoffs with normal growth?
ancestor Ctra
nsfer N
trans
fer Ctra
nsfer N
trans
fer Ctra
nsfer N
trans
fer
Option #8 – Select for growth upon a novel substrate
• All internal pathways present – only transport appears to be missing…
• Supplement growth with another compound to get them started, then wean them off?
• Tradeoffs with current substrates?
TCA cycle
serine cycle
C1 transfers
CO2
glucose, fructose
biomass
energy
citrate
Option #9 – Long-term incubation for growth advantage in stationary phase
• Donner Party for microbes…• Can try both shaken and still environments• Tradeoffs between GASP and normal growth?• Same molecular targets (ex: rpoS) as seen in E.
coli?• Lead to cheating?
Option #10 – Evolve new, compensatory functions
• Start with cells lacking a key enzyme and re-evolve growth
• Supplement initially and then wean?
• Risky, but could be very interesting (start multiple genotypes and examine those that ‘work’)
CH3OH
CH3OH HCHOMDH MaDH
H4FH4MPT
Fae
CH2=H4F CH2=H4MPT
MtdA, MtdBMtdA
CHO-H4F
HCOOH
Fch
Fhc
Mch
FtfL
spont.
CH3NH2
CH3NH2
HCHO
CH=H4F
CHO-H4MPT
CO2
spont.
(DmrA, Orf4)
BIOMASS
serinecycle
FDHs
CH=H4MPT
NADPH
H4F, ATP
H4MPT
H2O H2O
NADH
NAD(P)H
H2O, 2e- H2O, NH3, 2e-
cytoplasm
periplasm
H2O
H2O
H2OH2O
CH3OH
CH3OH
CH3OH
CH3OH HCHOMDH MaDH
H4FH4MPT
Fae
CH2=H4F CH2=H4MPT
MtdA, MtdBMtdA
CHO-H4F
HCOOH
Fch
Fhc
Mch
FtfL
spont.
CH3NH2
CH3NH2
CH3NH2
CH3NH2
HCHO
CH=H4F
CHO-H4MPT
CO2
spont.
(DmrA, Orf4)
BIOMASS
serinecycle
FDHs
CH=H4MPT
NADPH
H4F, ATP
H4MPT
H2O H2O
NADH
NAD(P)H
H2O, 2e- H2O, NH3, 2e-
cytoplasm
periplasm
H2O
H2O
H2OH2O
Many possibilities…1. Diversification in still medium
2. Adaptation on solid surface
3. Adaptation to poor substrates
4. Adaptation to rich medium
5. Evolve on formaldehyde
6. Evolve on increasing concentrations of methanol
7. Alternate between medium lacking C, or N
8. Select for growth on a novel substrate
9. Long-term incubation for GASP
10. Evolve new, compensatory functions