Transcript Ecological speciation model

Enterics
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Emphasize novel pyruvate enzymes
Example of free radicals involved in C-C bond
cleavage.
Gram negative bacteria that ferment sugars to acids
and gas. All use glycolysis
Mixed acid group:
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Escherichia, Salmonella, Shigella, Proteus.
Make lactic, acetic, succinic, formic acids
Butanediol fermentors:
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Enterobacter, Serratia, Erwinia
Mixed acid at neutral pH
Make 2,3-butanediol at low pH
Ways to cleave a C-C bond
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Carbonyl beta to another oxygen-containing
molecule
Alpha decarboxylation: use TPP to stabilize
carbanion
Rearrangements: use free radical
mechanism, free radical provided by 5deoxyadenosyl on B12
Other free radicals can be formed on
proteins: glycyl and tyrosyl free radicals
See how E. coli cleaves pyruvate by free
radical mechanism
Fermentation Products
Products
E. col i
For m ate
Acetate
La ctate
Su ccini c
Ethanol
Butandiol
C O2
H2
2.4
36.5
79.5
10.7
49.8
0.3
88
75
E. aerogenes
17
0.5
2.9
69.5
66.4
172
35.4
Mixed acid fermentation
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Glucose to pyruvate by glycolysis
New enzymes
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Pyruvate metabolism by pyruvate-formate lyase
Pyruvate + CoA --> acetyl-CoA + formate
(HCOOH)
No TPP (glycine free radical),
no NADH made
Still get high energy intermediate, but don’t have
to recycle NADH
Mechanism of Pyruvate-formate lyase
Free radical cleavage of C-C bond
Transfer stable free radical on glycine to
one of the sulfurs in cysteine at the active site.
cys418
cys419
S• HS
O
C
CH3
C
cys419
cys418
S
HS
•O
C
CH3
O
Ocys418
C
O
cys418
cys419
S• HS
O
CH3-C-S-CoA
O
O-
C
CH3
cys419
S
HS
C
S HS
CoA-S•
CoA
cys418
O
C
CH3
O
O•
HCOO-
cys419
O
C
CH3
cys418
cys419
S S•
Mixed Acid fermentation
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Succinate formation
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PEP carboxylase (heterotrophic CO2 fixation)
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PEP + CO2 --> oxaloacetate + Pi
ATP synthesis by electron transport
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Formate metabolism
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Formate: hydrogen lyase
Formate --> CO2 + H2
2 enzymes involved, formate dehydrogenase and
hydrogenase
Note: Enterobacter group also does mixed
acid fermentation at neutral pH
Mixed Acid Fermentation
Glucose
Pi
Oxaoloacetate
malate
NADH
dehydrogenase
PEP carboxylase
Pyruvate
CoA
fumarase
H O
2
Fumarate
ATP
PEP
NAD+
Malate
ADP
CO2
NADH
NAD +
fumarate
reductase
Succinate
Pyruvate-formate lyase
xNAD +
xNADH
xADP
x ATP
Glycolysis
(See previous notes
for complete pathway
Not balanced!)
ADP
ATP
Lactate
NAD+
NADH
Acety-CoA Formate
CoA
Pi
Acetyl-P
ADP
ATP
Acetate
Formate-hydrogen
lyase
+
Co + NAD
A
Acetaldehyde
NADH
NAD+
Ethanol
CO2
H2
Butandiol fermentation
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Switch to solvent production in low pH
New enzymes:
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Alpha-acetolactate synthase
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2 pyruvate --> acetolactate + CO2
TPP as cofactor
Butandiol fermentation
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Reduce acetolactate to acetoin and butandiol.
2,3-Butanediol Fermentation
Glucose
a -Acetolactate Synthase
[
CH 3
C
TPP
OH
PEP
ADP
ATP
Pyruvate
CO2
]
CoA
Pyruvate
OH O
CH 3
decarboxylase
Acetoin
2,3-Butanediol
dehydrogenase
C
CH 3
COOH
a -Acetolactate
CO 2
C
CH 3
OH
O
CH
C
CH 3
OH
OH
CH
CH
NADH
NAD +
2,3-Butanediol
CH 3
CH3
Glycolysis
See previous
handout for reactions.
(not balanced)
Lactate
Pyruvate-formate lyase
Acety-CoA
a -Acetolactate
xNAD +
xNADH
xADP
xATP
CO 2
+ Formate
NADH
CoA
+
NAD +
Acetaldehyde
NADH
NAD +
Ethanol
H2
The problem of food and
water pollution
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“..its waters returning, Back to the springs,
like the rain, Shall fill them full of refreshment,
that which the fountain sends forth returns
again to the fountain”
All the water on the planet is recycled.
Risks of fecal contamination differentiation
between fecal and non-fecal enterics is
critical
Shanks, O. C. et. al. (2006) Competitive Methagenomic DNA Hybridization identifies host-specific
microbial genetic markers in cow fecal samples. AEM V 72 N6 p. 4054 – 4060.
Simpson, J. M. et. al. (2004). Assessment of equine fecal contamination: the search for alternative
bacterial source-tracking targets. FEMS Microbiol. Ecol. V 47 p. 65-75.
Dick, L. K., et. al. (2005). Host distributions of uncultivated fecal Bacteriodes bacteria reveal
genetic markers for fecal source identification. AEM V71 N6 p. 3184- 3191.
Differentiation of Enterics
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Differentiation based on metabolic
characterization
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Mixed acid
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Enzyme analysis
Intermediate analysis
Gas: E. coli, Salmonella
No gas: Shigella, S. typhi
Butanediol (acetoin)
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Gas: Enterobacter
No gas: Erwinia, Serratia.
Summary
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Free radicals on proteins can also be
used to break C-C bonds.
Enterics are a good example of
reactions. They metabolize pyruvate to
most of the products we discussed.
ID of enterics critical to assess water
quality.
Alcohol fermentations
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Two possibilities: yeast and Zymomonas.
Yeast
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1815: Gay-Lussac found that yeast made 2 ethanols and 2 carbon
dioxides from glucose
Buchner: cell-free extract, beginnings of biochemistry
Uses glycolytic pathway to make pyruvate
Difference from Streptococcus is in what happens to pyruvate
New pyruvate enzyme:
References: Flores et al. FEMS Micro. Rev. 24: 507-529, 2000;
Conway, FEMS Micro. Rev. 103: 1-28, 1992.
Summary of the yeast
pathway
Glucose
Glycolytic
pathway
Net 2 ATP
2 NADH's made
2 pyruvates
Oxidative reactions:
3-phosphoglyceraldehyde + Pi + NAD+ ->
1,3-bisphosphoglycerate + NADH
Reductive reactions:
acetaldehyde + NADH -> ethanol + NAD+
Pyruvate decarboxylase Substrate-level phosphorylation:
PEP + ADP -> pyruvate + ATP
2 CO2
1,3-bisphosphoglycerate + ADP ->
3 phosphoglycerate + ATP
2 acetaldehyde H3C C O
H
Net ATP
2 NADH
use 2 ATP
make 4 ATP
+
net of 2 ATP
2 NAD
2 ethanol
Pyruvate decarboxylase
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Pyruvate decarboxylase
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Pyruvate -> acetaldehyde (CH3CHO) + CO2
Cofactor: thiamine pyrophosphate (TPP).
Thus, no oxidation/reduction and no high energy
intermediate is made
The “active aldehyde” rearranges and forms
acetaldehyde as one of the products
Function of TPP here is decarboxylation.
Zymomonas
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Natural agent of alcohol fermentations in
tropics, isolated from Mexican pulque.
Gram negative, motile, small rods, anaerobic
to microaerophilic
Usually make more than 2 mol ethanol per
mol glucose
Often more versatile than yeast in substrates
used
Organism of choice for bulk ethanol
production (gasohol)
Zymomonas
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Uses a new pathway for glucose
metabolism called Entner-Doudoroff
Oxidation of the number one carbon of
glucose as in Leuconostoc to form 6phosphogluconate
Followed by a dehydration to give a new
intermediate: 2-keto-3-deoxy-6phosphogluconate.
Glucose
ATP
ADP
Glucose-6-P
NADP+
NADPH
6-Phosphogluconate
ATP summary
used 1
made 2
net = 1
Reoxidation of NAD(P)H
H2O
6PG dehydratase
2-Keto-3-deoxy-6-phosphogluconate
KD6PG aldolase
pyruvate
3-phosphoglyceraldehyde
NAD+
Pi
NADH
1,3-bisphosphoglycerate
ADP
ATP
3-phosphoglycerate
Pyruvate
decarboxylase
2 CO2
2 acetaldehyde
Alcohol
dehydrogenase
pyruvate
ATP
H2O
2-phosphoglycerate
2 pyruvate
ADP
phosphoenol pyruvate
2 NAD(P)H
2 NAD(P)+
2 ethanol
Key enzymes and intermediates
CO OH
HC
HO
OH
CH
C
HO
HC
OH
HC
OH
H 2C
CO OH
H2O
O
6-Phosphogluconate
dehydratase
P
O
2-keto-3-deoxy6-phosphoglucon a te
(K D6PG)
CH
HC
OH
HC
OH
H 2C
O
P
KD6PG aldolase
CO OH
HC
O
C
HC
OH
O
CH 3
Pyruvate
H 2C
O
P
3-phosphoglyce raldehyde
Summary
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Pyruvate decarboxylase: uses TPP to decarboxylate
pyruvate but only makes acetaldehyde
ED pathway: only one G-3-P made, limits ATP
production
ED pathway oxidizes C-1 of glucose and makes new
intermediate, 2-keto-3-deoxy-6-phosphogluconate
Extra oxidative reaction in Zymomonas limits ATP
production compared to yeast.