kitp forum microbial metabolism 2021 microbial metabolism

Microbial Metabolism

• Metabolism in Cellular Context

• Types of Microbial Metabolism (Catabolism)

• How Microbes use Energy

• Link between Thermodynamics and Kinetics

• Speciation of Metabolic pathways

• Metabolism in Natural Selection and Isolation of Microbes

What constrains microbial metabolism?

Today

Next Thursday

• Principles of Metabolic Transformations

• Patterns of MetabolismChemical constraints microbial metabolism

KITP Forum Microbial Metabolism 2021

What of metabolism is it that we want to understand?

Metabolism

Physiology Ecology


Nothing in biology makes sense except in light of evolution

Theodosius Dobzhansky, 1973

Life is nothing but an electron looking for a place to rest.

Albert Szent-Gyorgi

Selection is everywhereJohn Roth (paraphrased)


It’s not!!!

Why is metabolism complicated?

We need some order!!

… but it looks complicated?

R2

R1 COH

H R2R1 CO

HCO

OCO

R2R1R1

C CO

CRH

H

H

HS-CoA C C

OCRH

HS-CoA

Alcohol oxidation

Aldehyde oxidation

Acyl-CoA oxidation

Oxygenation

CH

HR1 H + 2[H]

H

CO

HR1 H

O2

+ H2O

CCH

H

H

HCH

HR1 H CC

H

H HCH

R1 H

OH OHO2

Methyl-CoM reduction

CH3-S-CoM + H-S-CoB CoM-S-S-CoB + CH4

S-CoAOS-CoAOFd2 + Energy (ATP or via electron bifurcation)

Benzoyl-CoA reduction (dearomatizing)

Oxidations/reductions

+ H- + H+

+ H- + H+

R1R2 CO H

H

R1CO

Hemiacetal oxidation

+ H- + H+O R2 O

R CC

H O

H

H

H

HCO

CO

H

CH

HH+ HR

R CH

H

H

HC H+H

H

CH

CO

H O

CO

CO

H

R CO

Aldol cleavage

Ketol cleavage

Fumarate additionCOO-

-OOC CCH

H

R

CH

H

COO--OOC CCH

H

RC RH

H+ R

CO + HS-CoA + CH3 - H4F CH3CO-S-CoA + H4F

CO2 + Fd2- +2H+ Fd + H2O + CO

De/carbonylation

C-C cleavage/condensations

1

2

1

2

CR CCH

HR2

OCCR

H

H

H

NuNu-

CO

R2

CC

H

H H

R2R1

R3

CC

H

R2R1

R3

H H

Rearrangement

Nucleophilic addition/elimination

C COH

S-CoA

OH

CR

H

H

C CO

CH

HS-CoAR

H2O

a hydroxy elimination

Auxiliary reactions

Hydrogen oxidation

H2 2 e- + 2 H+

Com

plex

ity o

f org

anic

subs

trate

Module (more than one prototypic enzymatic reaction)

Single, less common enzymatic reaction

= Module

Pathway

A

B

Single prototypic enzymatic reaction

o Pathway node

o

o

o

oo Com

plex

ity o

f org

anic

subs

trate

Module (more than one prototypic enzymatic reaction)

Single, less common enzymatic reaction

= Module

Pathway

A

B

Single prototypic enzymatic reaction

o Pathway node

o

o

o

oo

Modularity of Catabolic PathwaysKITP Forum Microbial Metabolism 2021

S1

P2P1

n[H]

ATP

Anabolic Substrates(C, N, S..)S2

[H]

Inter-mediates

ATP

General

Catabolic and Anabolic PathwaysKITP Forum Microbial Metabolism 2021

S1

P2P1

n[H]

ATP

S2

[H]

Inter-mediates

ATP

Monomers(amino acids, nucleotides, long

chain fatty acids, carbohydrates,…)

Polymers(proteins, RNA, DNA,

lipids, peptidoglycan,…)

Anabolic Substrates(C, N, S..)

Transport, Adjusting oxidation state,Forming covalent C-C, C-N, etc. bonds

ATP-dependent polymerizations via H2O abstraction

Folding, transport/export

Catabolic and Anabolic PathwaysKITP Forum Microbial Metabolism 2021

Biosynthetic precursors of amino acids,nucleic acids, and cofactors


S1

P2P1

n[H]

ATP


[H]

Inter-mediates

ATP

General

Catabolic and Anabolic PathwaysYields:

ATP/mol catabolic substrate

YATP = 10 !"#$%& '()

FractionofcarbonSincatabolism andanabolismasafunctionofcatabolicDG

1-

DG/mol<<<0 DG/mol<0


Molecular Crowding in a Microbial Cell

(Yu et al 2016 eLife

After McGuffee and Elcock 2009


Types of Microbial Catabolism

S1

P2P1

n[H]

ATP


[H]

Inter-mediates

ATP

General

NH3

H2ONO2-

n[H]

ATP

Anabolic Substrates(N, S..)O2 CO2

<CH2O>

Lithoautotrophic

m[H]

ATP

Anabolic Substrates(N, S..)CO2

<CH2O>

A[H] A

hnPhotoautotrophic

m[H]

Organic compound 1

Organic acids,Alcohols,H2

n[H]

ATP

Anabolic Substrates(C, N, S..)

m[H]

Inter-mediates

ATP

Fermentative

Organic compound 2

Organic acids,CO2

Features and Environmental Footprints of Major Types of Microbial Catabolic and Anabolic Pathways

Carbon source Anabolism> 50% CO2 autotroph

organic (<>0% ) heterotroph

S1 S2 Catabolismorganic Organic/inorganic chemoorganotroph

inorganic organic chemoorganotroph

inorganic inorganic chemolithotroph

=O2 aerobic

=O2 anaerobic

=O2 or =O2 facultative

Metabolic Definitions of Microbes

Energy source Catabolic Electron Donor/Acceptor

Carbon source Microorganism

Photo- Litho (S1 AND S2) auto-

troph

Chemo- organo (S1 OR S2) hetero-


How Microbes use Energy

Use of Gibbs Free Energy as a Resources

Energy(DG) ADP + Pi

ATP + H2O

Heat

WorkBiosynthesisTransportMovementetc

< -50kJ/mol -5kJ/mol

<0kJ/mol -45kJ/mol

Catabolism Anabolism


S P DGS->P<0

S I1 I2 I3 I4 I5 PΔ)*+→-Δ).→*/ Δ)*/→*0 Δ)*0→*1 Δ)*1→*2 Δ)*2→*+

Δ)3→- = ΣΔ)*/67

A

B

DG-

+

DG-


ATP ATP ATP

nATPC S I1 I2 I3 I4 I5 P

ATP

DG-

+

DGS->P=nATPDGATP +DGheat

DGS->P,coupled=DGheatS + nADP + nPi P + nATPD

DG-

DG-


pH2 [Pa]

-140

-120

-100

-80

-60

-40

-20

0

20

DG’

[kJ/

mol

]

4 H2 + CO2 + nATP ADP + nATP Pi CH4 + nATP ATP + 3 H2ODG’nATP=0 - 131 kJ/mol

nATP = 0

nATP = 0.5

nATP = 1.0

DGS->P=nATPDGATP +DGheat

ATP ATP ATP

nATPS I1 I2 I3 I4 I5 PATP


A link between thermodynamics (nATP) and kinetics (flux JS)

Ecological success and microbial growth- Relationship between growth, available energy, and growth rate -

In thermodynamic equilibrium, 0, and

Available energy:

S … PATP ATP ATP

1 2 nATP

Pfeiffer & Bonhoeffer 2002, Costa et al 2006


Ecological success and microbial growth- Relationship between growth, available energy, and growth rate -

Fluxes (rates):

In thermodynamic equilibrium, JS and JATP = 0However at yields nATP lower than nmax, <0 and drives the reaction, and consequently, JS and JATP become positive.

In thermodynamic equilibrium, 0, and

Available energy:

S … PATP ATP ATP

1 2 nATP

Pfeiffer & Bonhoeffer 2002, Costa et al 2006


Rate versus yield tradeoff- for fundamental thermodynamic reasons, irrespective of the pathway -

ATP yield

Rate

of s

ubst

rate

util

izatio

nAT

P pr

oduc

tion

Pfeiffer & Bonhoeffer 2002


Competition favors rate over yield

P = pay-off, total amount of ATP produced from a given amount of resource S for a given strategy nATP

P under non-competitive conditions

Pay-off for a given strategy is determined only by its yield but not its rate of ATP production

P under competitive conditions

If JC is small, P is large if JS is small; i.e., if competition is small, a slow strategy for efficient ATP formation is advantageous.

If JC is large, a more efficient pathway (nATP) will not significantly reduce JS, and strategies with high rates but lower yields will result in higher pay-off.

Size of a population is determined by yield; outcome of competition is determined by rate of ATP production

Pfeiffer & Bonhoeffer 2002,


1 ATP

2 Pyruvate-

Glucose

Lactate dehydrogenase

2 NADH

2 Lactate-

2 Acetyl-CoA

1 Ethanol

Pyruvate:Formatelyase

2 HCOO-

1 Acetate-

High glucose flux(Rate mode)

Low glucose flux(Yield mode)

4 ATP

OH

O

O-

GAP->Pyruvate Module

2 ATP

Fructose-1,6-bisphosphate aldolase

Lactococcus lactis

2 NADH


in (-) out (+)

Ubiquinol oxidase

m H+

cyt c

2H+ + Q QH2

4 H+

NADHdehydrogenase

NADH

NAD+

3.3 H+ADP + Pi

ATP

Cytochrome coxidase

2H+

½ O2

H2O

DG0’ = - 61kJ/mol NADHNADH + ubiquinone → NAD+ + Ubiquinol

NDH 1 m = 3-4 H+

NDH 2 m = 0 H+

Nqr m = 2 Na+


Q

4H+ 4H+ 2H+ 3-4H+

Cyt c

NADH NAD+ Succinate Fumarate 1/2O2 H2OADP ATP

In (-)

Out (+)

NADH dehydrogenase(complex I)

Succinate dehydrogenase(complex II)

Ubiquinol dehydrogenase(bc1 complex, III)

Cytochrome c oxidase(complex IV)

ATP synthase

S= 10H+/2e-

Paracoccus sp.Pseudomonas sp.Mitochondria

E. coli S= 8H+/2e-

Cytochrome bo oxidase

Cytochrome bd oxidase

Q

4H+ 4H+ 3-4H+

NADH NAD+ Succinate Fumarate1/2O2 H2OADP ATP

Q

4H+ 3-4H+

NADH NAD+ Succinate Fumarate ADP ATP 1/2O2 H2O

E. colilow O2

S= 4H+/2e-

Q

4H+ 4H+ 1H+ 3-4H+

Cyt c

NADH NAD+ Succinate Fumarate 1/2O2 H2OADP ATP

Helicobacter pylori

S= 9H+/2e-

Protons pumped

Speciation of Pathways (i. e., metabolic interactions)

Julian Adams experiment: E. coli in a glucose-limited chemostat

800 generationsHelling, Vargas, Adams 1987

SCV

LCV

(simplified)

Metabolism and Ecological PatternsKITP Forum Microbial Metabolism 2021

Julian Adams experiment: E. coli in a glucose-limited chemostat

Helling, Vargas, Adams 1987

Higher relative growth yield

Lower relative growth yield Higher max growth rate, Lower Km for glucose,

Growth characteristics

Both populations are stably

maintained

Ancestral strain

Small Colony Variant

Large Colony Variant

Julian Adams experiment: E. coli in a glucose-limited chemostatKITP Forum Microbial Metabolism 2021

Helling, Vargas, Adams 1987

Spent supernantant

Tester strains

Ancestral strain Small Colony Variant Large Colony Variant

Large Colony Variant

Small Colony Variant

Ancestral strain

Growth on spent media supernatant: Metabolic cross-feeding, niche creation, and speciation in the evolved E. coli variants.


Glucose(((((((((((((((((((((((((((((((((((((Pyruvate((((((((Acetate/CoA(((((((((((((((((((((((((((((((((((((((((((((((((CO2

Ancestral*wild*type


Small*colony*variant


Large*colony*variant

Acetate

23 steps, 24 ATP

15 steps, 12 ATP

11 steps, 5 ATP

What the outcome of the Julian Adams experiment means:

•Small colony variants appear to be a predictable feature of adaptation to a glucose-limited environment.

•Stable (predictable) environments lead to speciation.

•SCV has superior glucose uptake but also utilizes glucose inefficiently.

•Some evolved organisms are more successful because they use the substrate less well.

•The less efficient types are maintained because they do not compete for glucose but rather grow on the excreted metabolite.

Wild type

Small colony variant (SCV)

Large colony variant (LCV)


Glucose Propionate-, Acetate-

Glucose Propionate-, Acetate-Lactate-Lactic acid bacterium

Propionic acid bacterium

Propionic acid bacteriumLow flux, low competition:

High flux, high competition:

Anoxic organic-rich environments:

Real World Examples of Pathway Speciation

Glucose 2 Lactate- + 2 H+S. gordonii

2 Lactate- 2 Propionate- + 1 Acetate- + CO2V. atypica

Glucose 2 Lactate- 2 Propionate- + 1 Acetate- + CO2S. gordonii V. atypica

amyB+


Glucose + 2 H2O 2 Acetate-+ 2 H++ 2 CO2 + 4 H2

3.75 ATP/Glucose

Ruminococcus alba

Low pH2 (10 Pa)

2 Pyruvate

Glucose

2 CO2

4 H2

2 Acetyl-CoA

2 Acetate

2 ATP

4 ATP

Pyruvate:Ferredoxin

oxidoreductase

2 NADH

Confurcatinghydrogenase

DG0’= - 197 kJ/mol glucose

2 H2

1 Acetate

1 ATP

1 Ethanol

Bifunctional acetaldehyde/ethanol dehydrogenase

High pH2 (101 kPa)

2 Fd2-

GAP->Pyruvate module

Fructose-1,6-bisphosphate aldolase

2 ATP

1 H+

0.25 ATP

ATPase

1 H+

Glucose + 1 H2O 1 Acetate-+ 1 Ethanol + 1 H+ + 2 CO2 + 2 H2

DG0’= - 207 kJ/mol glucose2.75 ATP/Glucose

-420

-390

-360

-330

-300

-270

-240

-210

pH2 [Pa]

Redu

ctio

n po

tent

ial [

mV]

2 e- + 2 H+ ⇌ H2

H2 Partial Pressure (pH2) and the Redoxpotential of H2

Butyrate-

Acetate-

H2

CO2

Butyrate-

H2

CO2

CH4

Clostridium

Clostridium

Monoculture

DDefined mixed culture

Hydrogenotrophic

Methanogen

‘nATP’

CH4Acetate-Acetoclastic Methanogen

‘nATP’

Carbohydrates

Carbohydrates

pH2 = 101 kPa

pH2 = 10 Pa


Polymeric Organic Matter

Carbohydrates, Nucleic acids, Amino acids, Lipids

Lactate, Propionate, Butyrate,

Higher fatty acids,Alcohols

Aromatic compounds,…..

AcetateH2, HCO2-, CO2

CH4, CO2

11

2

3

4


Flow of Electrons and Carbon under Methanogenic Conditions

Selection, Isolations, and Metabolism

µm

Js = !"!# JP = !$,&'(() '#&!#S P

A Microbe’s Environment consists of:- A spatial (µm) component- At least one catabolic resource- A flux of resources

A microbe’s environmentKITP Forum Microbial Metabolism 2021

Flux of Resources

FluctuatingConstant

Predictably fluctuating Unpredictably fluctuating

Physical-Chemical Environment


Fitne

ss

Environment 1

Genotype/phenotype

Fitne

ssGenotype/phenotype

Fitne

ss

Genotype/phenotype

SelectionFitness landscape

Ecological Selection

Evolutionary Selection

Genotype/phenotype


….

….

time

. ...

..

. . .. .. . .. . .. .

time

. ...Competition is global;Selection under competition favors fast growing microbes(high JS, high JATP)

Competition is local (initially)Effect of Drift

Isolation of Microbes and SelectionLiquid enrichments Direct isolations


lgce

ll num

ber/

ml

Time

Lag phase

log phase

Stationary phase

Death phaseLate log phase

µmax

Grow

th ra

te [µ

] Substrate concentration

µmax

Time

A

B

Growth in Batch

Growth in batch selects for the fastest microbes (dATP/dt)


Slow growing

Anabolic(includingribosomes)

Proteome allocation

Fast growing cell JATP f

JATP f > JATP s

A smaller catabolic proteome needs to mediate a higher ATP flux

JATP sSlow growing cell

Anabolic (including ribosomes)The proteome dedicated to biosynthesis is linearly proportional to growth rate.

CatabolicThe proteome dedicated to energy conservation for the anabolic demand, i.e., growth rate

E. coli: Glucose + 6 O2 → 6 CO2 + 6 H2O DG0’ ~ 2800 kJ/mol

Terry Hwa & coworkers



Ancestral*wild*type


Small*colony*variant


Large*colony*variant

Acetate

23 steps, 24 ATP

15 steps, 12 ATP

11 steps, 5 ATP

Acetate Overflow Metabolism in E. coli

Basan et al 2015


The proteome dedicated to biosynthesis is linearly proportional to growth rate.

The proteome dedicated to energy conservation for the anabolic demand, i.e., growth rate

Anabolic (including ribosomes) Catabolic

Fast growing JATP f

FermentationRespiration

Slow growingJATP sSlow growing

Terry Hwa & coworkers

Proteome allocationKITP Forum Microbial Metabolism 2021

Collectingvessel

Pump

Gas as needed

Pump

Sreservoir

P, Sunused, cells

fS

V

lgce

ll num

ber/

ml

Time

µmax

Grow

th ra

te [µ

] Substrate concentration

Time

A

B

! = #$% = &

Growth in Chemostat

Growth in chemostat selects for the ‘efficient’ microbes


kitp forum microbial metabolism 2021 microbial metabolism

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