Physiology of Desulfovibrio

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Physiology of Desulfovibrio from
Experimentation and Sequence
Judy D. Wall Huei-Che Yen Grant Zane
Biochemistry Department University of
Missouri-Columbia
DOE Genomes to Life Virtual Institute for
Microbial Stress and Survival
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Presentation Outline

• Energy generating mechanism deduced from
  sequencing
• Observations from mutants supporting the
  inferences
• Growth parameters (practical info)
• Pictures (for fun)
• Cautionary tale

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Modes of Energy Generation in Desulfovibrio

• Fermentation
• Respiration
• Metabolite cycling (formerly hydrogen
  cycling)

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Lactate metabolism by Desulfovibrio
Periplasm
CH3COHCOO-
Lactate
Substrate level phosphorylation
2e-
CH3COCOO-
Pyruvate
Electron dump
2e-
ATP
CH3COO- CO2
Acetate
or
CH3COO- HCOO-
SO4
S
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Lactate metabolism by Desulfovibrio
Periplasm
CH3COHCOO-
ATP
Lactate
Respiration
2e-
e-
CH3COCOO-
H
Proton Gradient
Electron transport chain
Pyruvate
2e-
e-
ATP
CH3COO- CO2
Acetate
or
CH3COO- HCOO-
SO4
S
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Lactate metabolism by Desulfovibrio
Periplasm
CH3COHCOO-
ATP
Lactate
H2 cycling
2e-
CH3COCOO-
Electron transport chain
H
Proton Gradient
Pyruvate
2H
2e-
H2
H2
ATP
CH3COO- CO2
2e-
Acetate
or
c-type cytochromes
CH3COO- HCOO-
Electron transport chain
SO4
S
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Lactate metabolism by Desulfovibrio
Periplasm
CH3COHCOO-
ATP
Lactate
Formate cycling
2e-
CH3COCOO-
Electron transport chain
H
Proton Gradient
Pyruvate
2H
2e-
HCOOH
ATP
H2
CH3COO- HCOO-
2e-
CO2
Acetate
Formate
c-type cytochromes
HCOOH
Electron transport chain
SO4
S
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Requirements for Metabolite Cycling

• Periplasmic and cytoplasmic hydrogenases
• Pyruvate formate lyase and periplasmic formate
  dehydrogenases
• Transmembrane electron carrier complexes
  connected to periplasmic enzymes

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Periplasmic hydrogenases of D. vulgaris
H2
Periplasm
2 e-
Fe-only Hase
NiFe Hase-1
NiFeSe Hase
Cytoplasm
NiFe Hase-2
H
ATP
H
ADP Pi
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Cytoplasmic hydrogenases of D. vulgaris
Periplasm
Ech-type
H
Cytoplasm
Sulfate reduction!
ATP
H
2
H
2 H

2 e
-
CO2
HCOOH
ADP Pi
b
Lactate
Pyruvate AcetylCoA
HCOO
-
H


a
a
d
c
AcetylCoA
CO
2e
-
2H

2
AcetylCoA
CO
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Periplasmic Formate Dehydrogenases of D. vulgaris
Periplasm
Ech-type
H
Cytoplasm
Sulfate reduction!
ATP
H
2
H
2 H

2 e
-
CO2
HCOOH
ADP Pi
b
Lactate
Pyruvate AcetylCoA
HCOO
-
H


a
a
d
c
AcetylCoA
CO
2e
-
2H

2
AcetylCoA
CO
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Periplasmic cytochrome c3 matrix of D. vulgaris
H2
Periplasm
2 e-
Fe-only Hase
Cyt c3
NiFe Hase-1
NiFeSe Hase
Cytoplasm
NiFe Hase-2
H
ATP
H
ADP Pi
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Periplasmic cytochrome matrix of D. vulgaris
H2
Periplasm
2 e-
Fe-only Hase
Cyt c3
NiFe Hase-1
Hmc Hexadecaheme c
Triheme c
Nonaheme c
NiFeSe Hase
NiFe-2 c3
Acidic c3
Cytoplasm
NiFe Hase-2
H
ATP
H
ADP Pi
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Periplasmic cytochrome matrix of D. vulgaris
H2S
H2
Periplasm
2 e-
Fe-only Hase
Cyt c3
NiFe Hase-1
Hmc Hexadecaheme c
Triheme c
Nonaheme c
NiFeSe Hase
Trans Membrane Complex
NiFe-2 c3
Trans Membrane Complex
Acidic c3
Cytoplasm
NiFe Hase-2
e-
e-
H
e-
e-
ATP
H
ADP Pi
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Hydrogen and
Formate
Cycling
Proton gradient!
Periplasm
2 H

2 e
-
c
-
type
cytochromes
Periplasmic
Hydrogenases
Cytoplasmic
membrane
NADPH-linked
Cytoplasmic
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Initial model of
Desulfovibrio
reduction of uranium
Uranium (VI)
ox
H
Cytochrome
c
Hydrogenase
2
3
Uranium (IV)
red
Lovley et al., 1993
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Initial model of
Desulfovibrio
reduction of uranium
Uranium (VI)
ox
H
Cytochrome
c
Hydrogenase
2
3
Uranium (IV)
red
Lovley et al., 1993
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Periplasmic cytochrome matrix of D. vulgaris
H2
Periplasm
2 e-
Fe-only Hase
NiFe Hase-1
Hmc Hexadecaheme c
Triheme c
Nonaheme c
NiFeSe Hase
Trans Membrane Complex
NiFe-2 c3
Trans Membrane Complex
Acidic c3
Cytoplasm
NiFe Hase-2
e-
e-
H
e-
e-
ATP
H
ADP Pi
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LDH
LDH
4 e
-
2
H2
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Growth of WT and CycA
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Su
Lactate/sulfate growth of D. desulfuricans G20
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Sulfide Production of WT and CycA
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Hydrogen Accumulation from WT and CycA
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    Specific hydrogenase activities of D.
vulgaris wild type or Fe-only hydrogenase
mutanta  
a Specific activities are relative units/mg
proteinml Data taken from Pohorelic et al., J.
Bacteriol. 184679-686, 2002.
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 Reduction of U(VI) by Desulfovibrio vulgaris
Hildenborough wild type and Fe-only hydrogenase
mutant A  
ARates are µmol U(VI) reduced hour mg whole
cell protein B Results are averages of three
replicates   CResults are averages of two
experiments with three replicates each
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Practical Considerations for Manipulating
Desulfovibrio vulgaris

• Reductant selection
• Antibiotic resistance selections
• Growth of colonies

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D. vulgaris CFU on Defined Medium
KmR exconjugate selection with G418
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• Desulfovibrio desulfuricans G20
• Complete LS medium 1 mM Uranyl Acetate
• Early stationary phase (overnight)
• Late stationary phase (four days)

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D. desulfuricans G20 Early Stationary LS Medium
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D. desulfuricans G20 Early Stationary LS 1mM
U Medium
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D. desulfuricans G20 Four Day Stationary LS
Medium
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D. desulfuricans G20 Four Day Stationary LS
1mM U Medium
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Growth of D. desulfuricans G20 with U(VI) to test
effect on cytochrome c3 expression
1.0
0.8
0.6
mM U(VI)
0.4
0.2
0.0
0
2
4
6
8
10
12
14
16
18
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Time (h)
LS G20 Lactate
LSU G20 Lactate
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Single-copy fusion of cycA promoter and ATG start
codon to ß-Galactosidase gene
 

Plasmid sequences
cycA promoter
cycA promoter
cycA
KmR
ATG
DNA Introduced
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ß-Galactosidase Activities of Fusion Constructs
Sp. Act. µmol/minmg protein
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Implication of translational fusions with cycA

• Strong evidence that cytochrome c3 is necessary
  for hydrogen supported uranium reduction
• No measurable effect of exposure of the cell to
  subinhibitory concentration of uranium on cycA
  expression
• Therefore, transcript and proteome analysis will
  not reveal all critical genes for bioremediation
  of toxic metals