kitp forum microbial metabolism 2021 microbial metabolism
TRANSCRIPT
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
KITP Forum Microbial Metabolism 2021
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)
KITP Forum Microbial Metabolism 2021
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
KITP Forum Microbial Metabolism 2021
S1
P2P1
n[H]
ATP
Anabolic Substrates(C, N, S..)S2
[H]
Inter-mediates
ATP
General
Catabolic and Anabolic PathwaysYields:
ATP/mol catabolic substrate
YATP = 10 !"#$%& '()
FractionofcarbonSincatabolism andanabolismasafunctionofcatabolicDG
1-
DG/mol<<<0 DG/mol<0
KITP Forum Microbial Metabolism 2021
Molecular Crowding in a Microbial Cell
(Yu et al 2016 eLife
After McGuffee and Elcock 2009
KITP Forum Microbial Metabolism 2021
Types of Microbial Catabolism
S1
P2P1
n[H]
ATP
Anabolic Substrates(C, N, S..)S2
[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-
KITP Forum Microbial Metabolism 2021
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
KITP Forum Microbial Metabolism 2021
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-
KITP Forum Microbial Metabolism 2021
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-
KITP Forum Microbial Metabolism 2021
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
KITP Forum Microbial Metabolism 2021
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
KITP Forum Microbial Metabolism 2021
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
KITP Forum Microbial Metabolism 2021
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
KITP Forum Microbial Metabolism 2021
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,
KITP Forum Microbial Metabolism 2021
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
KITP Forum Microbial Metabolism 2021
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+
KITP Forum Microbial Metabolism 2021
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.
KITP Forum Microbial Metabolism 2021
Glucose(((((((((((((((((((((((((((((((((((((Pyruvate((((((((Acetate/CoA(((((((((((((((((((((((((((((((((((((((((((((((((CO2
Ancestral*wild*type
Glucose(((((((((((((((((((((((((((((((((((((Pyruvate((((((((Acetate/CoA(((((((((((((((((((((((((((((((((((((((((((((((((CO2
Small*colony*variant
Glucose(((((((((((((((((((((((((((((((((((((Pyruvate((((((((Acetate/CoA(((((((((((((((((((((((((((((((((((((((((((((((((CO2
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)
KITP Forum Microbial Metabolism 2021
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+
KITP Forum Microbial Metabolism 2021
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
KITP Forum Microbial Metabolism 2021
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
KITP Forum Microbial Metabolism 2021
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
KITP Forum Microbial Metabolism 2021
Fitne
ss
Environment 1
Genotype/phenotype
Fitne
ssGenotype/phenotype
Fitne
ss
Genotype/phenotype
SelectionFitness landscape
Ecological Selection
Evolutionary Selection
Genotype/phenotype
KITP Forum Microbial Metabolism 2021
….
….
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
KITP Forum Microbial Metabolism 2021
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)
KITP Forum Microbial Metabolism 2021
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
KITP Forum Microbial Metabolism 2021
Glucose(((((((((((((((((((((((((((((((((((((Pyruvate((((((((Acetate/CoA(((((((((((((((((((((((((((((((((((((((((((((((((CO2
Ancestral*wild*type
Glucose(((((((((((((((((((((((((((((((((((((Pyruvate((((((((Acetate/CoA(((((((((((((((((((((((((((((((((((((((((((((((((CO2
Small*colony*variant
Glucose(((((((((((((((((((((((((((((((((((((Pyruvate((((((((Acetate/CoA(((((((((((((((((((((((((((((((((((((((((((((((((CO2
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
KITP Forum Microbial Metabolism 2021
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