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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…. CH 3 -R. HCHO. CO 2. biomass.

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Project 1 experimental evolution
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

CH3-R

HCHO

CO2

biomass

Methylotrophy (aerobic)

  • Methylotrophy (growth on C1)

    • C1 compounds oxidized to formaldehyde

    • Oxidation of formaldehyde to CO2

    • Assimilation of formaldehyde into cell material

  • 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


Methylotrophy aerobic1

CH3-R

HCHO

CO2

biomass

Methylotrophy (aerobic)

  • Methylotrophy (growth on C1)

    • C1 compounds oxidized to formaldehyde

    • Oxidation of formaldehyde to CO2

    • Assimilation of formaldehyde into cell material

  • 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

“If the consumption of cytoplasmic formaldehyde were inhibited, the cytoplasmic formaldehyde concentration would increase to about 100 mM in less than 1 min.” (Vorholt et al., 2000, J Bacteriol)


Methylotrophy and hgt

Tree of Bacteria

Gram+

Methylotrophs

a

b

16S rDNA

g

d

Proteo-bacteria

e

Methylotrophy and HGT

  • Methylotrophy has arisen multiple, independent times in different lineages

  • HGT major force in enabling this specialized metabolism

(Kalyuzhnaya et al., 2005, J Bacteriol)


Multiple c 1 modules for each role
Multiple C1 modules for each role


Methylotrophs possess multiple combinations of c 1 modules
Methylotrophs possess multiple combinations of C1 modules

Xanthobacter autotrophicus

Methylobacillus flagellatus KT

CH3OH

CH3OH

CH3NH2

MDH

MaDH

MDH

HCHO

RuMP

assim.

HCHO

H4MPT

HCOOH

H4MPT

Oxidation

FDH1

FDH2

HCOOH

CO2

FDH1

FDH2

CO2

CBB

Methylococcus capsulatus Bath

Methylobacterium extorquens AM1

CH4

CH3OH

CH3NH2

pMMO2

sMMO

pMMO1

CH3OH

MDH

MaDH

RuMP

assim.

MDH

PHB

HCHO

HCHO

serine

cycle

Glyoxylate

regeneration

CH2=H4F

H4MPT

HCOOH

TCA

H4MPT

H4F

FDH1

FDH2

CO2

HCOOH

FDH3

FDH1

FDH2

CBB

CO2


Model system methylobacterium

succinate

methanol

succinate

methanol

C1 transfers

C1 transfers

TCA cycle

TCA cycle

serine cycle

serine cycle

energy

energy

biomass

biomass

CO2

CO2

Model system: Methylobacterium

  • a-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:


CH3OH

CH3NH2

CH3NH2

CH3OH

Methylotrophy in M. extorquens AM1

MDH

MaDH

HCHO

periplasm

H2O, 2e-

H2O, NH3, 2e-

cytoplasm

H4MPT

HCHO

H4F

Fae

serine

cycle

1. Oxidation of C1 substrates to formaldehyde

spont.

spont.

H2O

H2O

CH2=H4F

CH2=H4MPT

MtdA

MtdA, MtdB

NAD(P)H

NADPH

BIOMASS

CH=H4F

CH=H4MPT

H2O

H2O

Mch

Fch

CHO-H4F

CHO-H4MPT

H2O

FtfL

H4MPT

H2O

Fhc

H4F, ATP

HCOOH

FDHs

NADH

CO2


Methylotrophy in M. extorquens AM1

CH3OH

CH3NH2

MDH

MaDH

HCHO

CH3NH2

CH3OH

periplasm

H2O, 2e-

H2O, NH3, 2e-

cytoplasm

H4MPT

HCHO

H4F

Fae

spont.

spont.

serine

cycle

2. Condensation of formaldehyde with H4F or H4MPT

H2O

H2O

CH2=H4F

CH2=H4MPT

MtdA

MtdA, MtdB

NAD(P)H

NADPH

BIOMASS

CH=H4F

CH=H4MPT

H2O

H2O

Mch

Fch

CHO-H4F

CHO-H4MPT

H2O

FtfL

H4MPT

H2O

Fhc

H4F, ATP

HCOOH

FDHs

NADH

CO2


Methylotrophy in M. extorquens AM1

CH3OH

CH3NH2

MDH

MaDH

HCHO

CH3NH2

CH3OH

periplasm

H2O, 2e-

H2O, NH3, 2e-

cytoplasm

H4MPT

HCHO

H4F

Fae

serine

cycle

3. Oxidation of CH2=H4MPT to formate

spont.

spont.

H2O

H2O

CH2=H4F

CH2=H4MPT

MtdA, MtdB

MtdA

NAD(P)H

NADPH

BIOMASS

CH=H4F

CH=H4MPT

H2O

H2O

Mch

Fch

CHO-H4F

CHO-H4MPT

H2O

FtfL

H4MPT

Fhc

H2O

H4F, ATP

HCOOH

FDHs

NADH

CO2


Methylotrophy in M. extorquens AM1

CH3OH

CH3NH2

MDH

MaDH

HCHO

CH3NH2

CH3OH

periplasm

H2O, 2e-

H2O, NH3, 2e-

cytoplasm

H4MPT

HCHO

H4F

Fae

serine

cycle

4. Oxidation of formate to CO2

spont.

spont.

H2O

H2O

CH2=H4F

CH2=H4MPT

MtdA

MtdA, MtdB

NAD(P)H

NADPH

BIOMASS

CH=H4F

CH=H4MPT

H2O

H2O

Mch

Fch

CHO-H4F

CHO-H4MPT

H2O

FtfL

H4MPT

H2O

Fhc

H4F, ATP

HCOOH

FDHs

NADH

CO2


serine

cycle

Methylotrophy in M. extorquens AM1

CH3OH

CH3NH2

MDH

MaDH

HCHO

CH3NH2

CH3OH

periplasm

H2O, 2e-

H2O, NH3, 2e-

cytoplasm

H4MPT

HCHO

H4F

Fae

5. Assimilation of CH2=H4F by serine cycle

spont.

spont.

H2O

H2O

CH2=H4F

CH2=H4MPT

MtdA

MtdA, MtdB

NAD(P)H

NADPH

BIOMASS

CH=H4F

CH=H4MPT

H2O

H2O

Mch

Fch

CHO-H4F

CHO-H4MPT

H2O

FtfL

H4MPT

H2O

Fhc

H4F, ATP

HCOOH

FDHs

NADH

CO2


Methylotrophy in M. extorquens AM1

CH3OH

CH3NH2

MDH

MaDH

HCHO

CH3NH2

CH3OH

periplasm

H2O, 2e-

H2O, NH3, 2e-

cytoplasm

H4MPT

HCHO

H4F

Fae

serine

cycle

6. Interconversion of CH2=H4F and formate

spont.

spont.

H2O

H2O

CH2=H4F

CH2=H4MPT

MtdA

MtdA, MtdB

NAD(P)H

NADPH

BIOMASS

CH=H4F

CH=H4MPT

H2O

H2O

Fch

Mch

CHO-H4F

CHO-H4MPT

H2O

FtfL

H4MPT

H2O

Fhc

H4F, ATP

HCOOH

FDHs

NADH

CO2


Primary hub of c 1 metabolism

CH3-R

HCHO

CO2

biomass

Primary hub of C1 metabolism:

What happened to simplicity???:


Model system c 1 metabolism in methylobacterium
Model system: C1 metabolism in Methylobacterium

Topologically, any 2 of the 3 pathways leading to biomass or CO2 should be sufficient…

1.

2.

3.

Mutants defective in pathway 2. or 3. are C1-


Why are both c 1 transfer pathways needed
Why are both C1 transfer pathways needed?

1. & 3. “redundant” for assimilation?

2. & 3. “redundant” for dissimilation?

1.

2.

3.

3.

assimilation

dissimilation


Measured fluxes through hub
Measured fluxes through hub

nmol min-1 mL-1 OD600

nmol min-1 mL-1 OD600

nmol min-1 mL-1 OD600

  • Dynamics of transition from S to M

(Marx et al., 2005, PLoS Biology)


Do we really understand this
Do we really understand this?

nmol min-1 mL-1 OD600

?

nmol min-1 mL-1 OD600

nmol min-1 mL-1 OD600

  • Developed kinetic model of central C1 hub

(Marx et al., 2005, PLoS Biology)


Switch from long to direct assimilation
Switch from long to direct assimilation

Experimental data

Model predictions

  • Model prediction qualitatively recapitulated the phenomenon…

(Marx et al., 2005, PLoS Biology)


Experimental evolution of laboratory populations

growth

transfer

growth

transfer

growth

transfer

growth

transfer

growth

transfer

growth

transfer

ancestor

-80°C

Experimental evolution of laboratory populations

  • Living fossil record

    • Examine through time & across replicates

  • Assay competitive fitness:

day 0

day 1

acclimate

Ww > Wp

mix

growth

Competitor #1

Competitor #2

W > P

W = P


growth

transfer

growth

transfer

growth

transfer

growth

transfer

growth

transfer

growth

transfer

ancestor

-80°C

No Venus

Venus (fancy YFP)

Relative fitness of Venus/no Venus:

WM = 1.00001 ± .000352

WS = 1.00016 ± .000154

Experimental evolution of laboratory populations

What this looked like before…

What it looks like now…

  • Living fossil record

    • Examine through time & across replicates

  • Assay competitive fitness:

Average CV: 5.7 ± 3.1%

day 0

day 1

acclimate

Ww > Wp

mix

growth

Competitor #1

Competitor #2

W > P

W = P

(David Chou)


Project 1 experimental evolution1
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 evolution2
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
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
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

ethanol

methanol

acetate

succinate

C1 transfers

TCA cycle

serine cycle

energy

biomass

CO2

formate

glycerol

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)…


Option 4 adaptation to rich medium

Rich medium

C1 transfers

TCA cycle

serine cycle

energy

biomass

CO2

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


Option 5 evolve on formaldehyde
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…

???


Option 6 evolve on increasing concentrations of methanol
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…

???


Option 7 alternate between media lacking c or n

C methanol

transfer

N

transfer

C

transfer

N

transfer

C

transfer

N

transfer

Option #7 – Alternate between media lacking C, or N

ancestor

  • Make PHB (a biodegradable plastic) as storage product

  • Force storage and efficient reutilization?

  • Tradeoffs with normal growth?


Option 8 select for growth upon a novel substrate

glucose, fructose methanol

citrate

C1 transfers

TCA cycle

serine cycle

energy

biomass

CO2

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?


Option 9 long term incubation for g rowth a dvantage in s tationary p hase
Option #9 – Long-term incubation for methanolgrowth 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
Option #10 – Evolve new, compensatory functions methanol

  • 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’)


Many possibilities
Many possibilities… methanol

  • Diversification in still medium

  • Adaptation on solid surface

  • Adaptation to poor substrates

  • Adaptation to rich medium

  • Evolve on formaldehyde

  • Evolve on increasing concentrations of methanol

  • Alternate between medium lacking C, or N

  • Select for growth on a novel substrate

  • Long-term incubation for GASP

  • Evolve new, compensatory functions


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