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Warum ist Azoarcus
tolulyticus spannend?
•
Kann Toluol und Phenol abbauen•
Wurde aus einem kontaminierten Aquifer
isoliert in Michigan•
Denitrifizierer
sind praktisch alle fakultativ
anaerob und können aerob atmen!
Phylogenie von Azoarcus
tolulyticus
Domäne: BakterienPhylum: ProteobacteriaKlasse BetaproteobacteriaOrdnung RhodocyclalesFamilie RhodocyclaceaeGattung
Azoarcus
The uncultured majority
•
Black: 12 original Phyla (Woese
1987) many pure cultures
•
White: 14 new phyla since 1987 some isolates
•
Gray: 26 candidate phyla no isolates
Rappé
& Giovannoni
(Annu
Rev Microbiol, 2003)
Keller & Zengler
(Nat Rev Microbiol, 2004)
What are they all doing ?
1205
1367
220
1808
91
8
4
9
13
1124
25
n = published species
Anaerobic bacteria using aromatics as sole source of energy and cell carbon
Grampositive
Proteobacteria
Flavobacteria
Cyanobacteria
Aquifex
Green Sulfurbacteria
α
βγ
δ
ε
Green Nonsulfurbacteria
Thermotoga
Archaea Eukarya
RhodopseudomonasMagnetospirillum
Thauera aromaticaAzoarcus
Desulfococcus multivoransGeobacter metallireducensSynthrophobacterales
Desulfotomaculum
(Ferroglobus ?)
Facultative Anaerobes Obligate Anaerobes
Flavonoide
Phenole
Tannine
Lignane
Quinone
Abbau
durch Mikroorganismen
+ O2 - O2
CO2 CO2
Lignin
Aromaten in der Natur
Rohöl, KohleAminosäuren
CH2
CHCOOH
H2N
CH2
CHCOOH
H2N
OH
NH
CH2
CHCOOH
H2N
Prinzipielle Probleme des anaeroben Abbaus von Kohlenwasserstoffen
•
Aktivierung –
es fehlt der reaktive Sauerstoff
•
Resonanzenergie des aromatischen Ringes
•
Neue Chemie nötig
Anaerobic catabolism of toluene
C O-COSCoA
COO-
HO
COSCoA
COO-COSCoA
COSCoACOO-
Fumarate
CH3
Toluene
COO-O
Benzyl-succinate
Benzyl-succinyl-CoA
E-Phenylita-conyl-CoA
2-Carboxymethyl-3-Hydroxy-
Phenylpropionyl-CoA
O
COSCoACOO-
Benzoyl-Succinyl-CoA
Benzoyl-CoA
Succinyl-CoA Succinate 2[H]H2
O
2[H]CoASHSuccinyl-CoA
1
Anaerobic catabolism of toluene
C O-COSCoA
COO-COSCoA
COO-CH3
Toluene
COO-O
Benzyl-succinate
Benzyl-succinyl-CoA
E-Phenylita-conyl-CoA
2-Carboxymethyl-3-Hydroxy-
Phenylpropionyl-CoA
HO
COSCoA
COO-ACOSCo
Benzoyl-CoA
2
O
COSCoACOO-
Benzoyl-Succinyl-CoA
BenzylsuccinateSynthase
Benzylsuccinate-CoA Transferase
Benzylsuccinyl-CoADehydrogenase
Phenylitaconyl-CoA Hydratase
3-Hydroxyacyl-CoADehydrogenase
BenzoylsuccinylCoA Thiolase
Construction of the
multi-level
well
hochauflösendes Modul
4 Module vorgefertigt
Kabel- und Kapillarstränge
Bereit zur Abfahrt
Installation of a high resolution
multi-level well in Düsseldorf-Flingern
6
6,5
7
7,5
8
8,5
9
-5 0 5 106
6,5
7
7,5
8
8,5
9
0 10 20 30 40 506
6,5
7
7,5
8
8,5
9
0 20 40 606
6,5
7
7,5
8
8,5
9
0 100 200 300Uns
atur
ated
zone
Satu
rate
dzo
ne
Dep
th[m
bls
]
Sulfate + Toluene Sulfide [mg l-1] δ18O / δ34S [‰]
δ18O
δ34S
Sulfate Isotope Analysis
1)
The
plume
fringe
concept
holds!
2)
Steep
geochemical
gradients
at the
fringes
3)
Biodegradation and sulfate
reduction
take
place
in the
sulfidogenic
zone
of overlapping
gradients
of
toluene
and sulfate
Tolueneδ
13C Toluene
0 5 10 15 20 25 30 35 40 45 506
6,5
7
7,5
8
8,5
Dep
th[m
bls
]
Toluene
[mg l-1]
-25,0-24,5-24,0-23,5-23,0-22,5-22,0-21,5-21,0-20,5
δ
13C [‰]
-21.8 ‰ (7.1 m)
Toluene
Isotope Analysis
-24.5 ‰
(6.9 m)
Δ13C = -3.2 ‰
±
0.5
Significant
fractionationat plume
fringes!
February
2006
▼GW tableplume
core
sulfidogenic
gradient
zone
lower
contaminated
zone
deep
zone
103 105 107 109
5
6
7
8
9
10
11
12
13
0.0 0.5 1.0 1.5
Bacterial
16S rRNA genes [cp
g-1]
F1 cluster
bssA genes [cp
g-1]
Ratio bssA/16S rRNA genes
Dep
th[m
]• Highly specialized
degrader community in sulfidogenic zone
• Distribution correlates to different zones
• Biomass does not reflect specific degraders
[Winderl et al., in prep.]
Quantitative distribution
of bacterial
16S rRNA and bssA genes
150 300 450 600 750 900
▼GW tableplume
coresulfidogenic
gradient
zone
lower
contaminated
zone
deep
zone
1 2 3 45
6
7
8
9
10
11
12
13
Shannon index
(H‘)
Dep
th[m
]
A B
* 6.3 m
6.65 m
7.2 m
* 7.6 m
8.7 m
9.8 m
* 11.7 m
* 6.8 m
T-RF length
(bp)
130
228137159
228159
130 149
177
Depth-resolved bacterial community shifts
[Winderl et al., in prep.]
Sulfidogenic zone:
• 130
• 137
• 149
• 159
• 177
• 228 bp T-RFs
* = cloned
Frage!
•
Wie würden Sie Crotonyl-CoA
weiter abbauen?
•
Antwort: Beta-Oxidation der Fettsäuren–
Hydratisierung
zum Alkohol
–
Dehydrogenase
zum Keton–
Spaltung mit HS-CoA
zu zwei Acetyl-CoA
Die Schlüsselenzyme:
ΔG<<0
Monooxygenasen
C H 3 C H 2 O H
O2
+2 [H]
+ H2
O
DioxygenasenOH
OHC O O H
C O O HO H
O H
O2 O2
ΔG<<0
+2 [H] -2 [H]
Aromatenstoffwechsel von Aerobiern
Anaerobe Aromatenabbauer
Grampositive
Proteobacteria
Flavobacteria
Cyanobacteria
Aquifex
Green Sulfurbacteria
α
βγ
δ
ε
Green Nonsulfurbacteria
Thermotoga
Archaea Eukarya
PhototropheNitrat-Atmer
Nitrat-atmer
Sulfat-AtmerEisen AtmerFermentierer
Sulfat Atmer
Thauera aromatica
Fakultative Anaerobier Obligate Anaerobier
Anaerober Aromatenstoffwechsel: zentrale Rolle von Benzoyl-CoA
CO-SCoA
Benzoyl-CoA
Phenylalanin
Phenylacetat
Phenol
Ethylbenzol
Anthranilsäure
Benzoate
Toluol
Naphtalin
Xylole
Cresole
Salicylsäure
ReduktiveDearomatisierung
Ringöffnung
β-oxidation
Acetyl-CoA
CO2
?
e-
NO3 -
N2
SO42-
H2 S Fe(II)
Fe(III)
ATP ATP ATP
Die Birch-Reduktion von Aromaten
Chemie:
e-Donor: Na0
H-donor: X-OH
.-
e - H+, e-, H+
- 3 VH
H
CS C o AO AO
CS C o
-.e -
CS C o AO
H
H
Benzoyl-CoA Reduktase:
e-Donor: Ferredoxin (ATP)
H-donor: ?- 1.9 V
H+, e-, H+
Benzoyl-CoA Reduktase aus Thauera aromatica
2 NH3 + H2
Nitrogenase
8 H+, 8 e-
N N
16 ATP + 16 H2 O 16 ADP + 16 Pi
2 ATP / e-
Benzoyl-CoA Reduktase
C O S C oAC O S C o A2 ATP + 2 H2 O
2 Fd(red) 2 Fd(ox)
1 ATP / e-
2 ADP + 2 Pi
Energetics of benzoate degradation
Denitrifyer:C7
H6
O2
+ 6 HNO3 7 CO2 + 6 H2O + 3 N2ΔG’° = ~ -3000 kJ mol-1
Sulfate Reducer:C7
H6
O2
+ 4 H2
O + 3.75 SO42- 7 HCO3
- + 3.75 HS- + 3.25 H+
ΔG’° = -203 kJ mol-1
Fermenting bacteria:4 C7
H5
O2
+ 18 H2
O 12 C2H3O2+ CO2 + 3 CH4 + 8 H+
ΔG’° = -48,5 kJ mol-1
Iron reducer:C7
H6
O2
+ 19 H2
O + 30 Fe(III) 7 HCO3- + 30 Fe(II) + 36 H+
ΔG’° = <-1000 kJ mol-1
Central phloroglucinol/resorcinol pathways of anaerobic aromate degradation
OH O
O
COOH
O
OH
COOH
O O
OO
CO-SAoC
OH
OHOH H2
O
NADP+
NADPH
Acetyl-CoA
Acetate
2 CoASH
3 Acetyl-CoA
Phloroglucinol
O
COOH
O
O O
COOH
OH
OH
H2
O
2
Central phloroglucinol/resorcinol pathways of anaerobic aromate degradation
Resorcinol
H2
O2 [H]
2 Acetyl-CoA +1/2 Butyryl-CoA
3 Acetyl-CoA
Frage!
•
Welche Aktivierungsreaktionen für Kohlenwasserstoffe haben sie bis jetzt gelernt?
•
Welche Zentralen Metabolite?•
Welche Schlüsselreaktionen für den weiteren Abbau nach der Aktivierung?
Frage!
•
Welche Aktivierungsreaktionen für Kohlenwasserstoffe haben sie bis jetzt gelernt? –
Fumarataddition
radikalisch, direkte Oxidation,
Phosphorylierung, direkte Spaltung mit HS-CoA•
Welche zentralen Metabolite? –
Benzoat, Phloroplucinol, Resorcinol
•
Welche Schlüsselreaktionen für den weiteren Abbau nach der Aktivierung?–
Beta-Oxidation der Fettsäuren,
–
Ringreduktion durch Benzoyl-CoA-Reduktase
Anaerober Abbau von Naphthalinen
Meckenstock et al. (2000) Appl. Environ. Microbiol. 66, 2743-2747.
0 20 40 60 80 1000.0
0.5
1.0
1.5
2.0
2.5
Sulfi
de [m
M]
Time [d]
C O O H
Metabolites
in anaerobic
2- methylnaphthalene degradation
Annweiler et al. (2000) Appl. Environ. Microbiol. 66, 5329-5333.
50 100 150 200 250 300
115
141
167
195
226
286
VC OOC H3
C OOC H3
m/z
Inte
nsity
50 100 150 200 250 300
252
224
284
165
VIC OOC H3
C OOC H3
m/z
Inte
nsity
The
naphthylmethylsuccinate
synthase reaction
Annweiler et al. (2000) Appl. Environ. Microbiol. 66, 5329-5333.
0 1 2 3 4 5 6
0.0
0.2
0.4
0.6
0.8
Nap
htyl
-2-m
ethy
l-suc
cini
c ac
id [µ
M]
Time [hours]
+
COOH
COOH
HOOCCOOH
Activation
of naphthylmethylsuccinate
with co-enzyme
A
Safinowski and Meckenstock FEMS Microbiol. Lett. 2004
Succinyl-CoA
Succinate
COOH
CO-SCoA
COOH
COOH
0 10 20 30 400
100
200
300
400 Succinyl-CoA NMS-CoA NMS
Con
cent
ratio
n [µ
M]
Time [min]
β-Oxidation of naphthylmethylsuccinyl-CoA
2[H]
COOH
CO-SCoA
COOH
CO-SCoA
0 10 20 30 400.0
0.1
0.2
0.3
0.4
0.5
0.6
NMeS
NM
S-co
ncen
tratio
n [µ
M]
Time [min]
Safinowski and Meckenstock FEMS Microbiol. Lett. 2004
The
upper
2-methylnaphthaline degradation pathway
•
Addition of fumarate
•
β-Oxidation•
Central intermediate
2-
naphthoic acid
+1*2*
3*
4*
5*
Succinyl-CoA
2[H]
HS-CoA
H2O
2[H]
6
7
Succinyl-CoA
Succinate
8*CO-SCoA
COOH
CO-SCoA
COOH
CO-SCoA
COOH
COOH
HOOCCOOH
OH
COOH
CO-SCoAO
COOH
CO-SCoA
Safinowski and Meckenstock FEMS Microbiol. Lett. 2004
COOH
COOH
COOH
COOH
COOH
COOH
HOOCCOOH
+
COOHO
COOHOH
COOH
COOHor
COOH
COOH
CO2
Upper degradation pathways
to 2-
naphthoic acid Reduction
of 2- naphthoic acid
Ring cleavage
and degradation
via
cyclohexane
ring structures
CH3
How
is
naphthalene
activated?
Methylation
of an aromatic
hydrocarbon?
DD
DDDD
D
DD
D
DD D
D
DD
D
DD D
D
DCOOH
COOH
CH3
CH3
D
HOOCCOOH
Deuterated
naphthalene
as substrate
for
culture
N47
Product
should
have
the
mass of naphthyl-2-methylsuccinate
plus 7 mass
units
Postulated degradation
pathway
for
anaerobic naphthalene degradation
CO-SCoA
COOHCOOH
CH3
2*
3*
+HOOC COOH
COOH
CO-SCoA
4*
Succinyl-CoA
COOH
CO-SCoA
COOH
CO-SCoA
6
H2O
OH
2 [H]COOH
CO-SCoA
7 O
HS-CoA
5*
8*
1*
Succinat
COO-
?
?
CO2
[CH3]
[CoA] ?
9
10*
Succinyl-CoA
2 [H]
How
to assess
the
degradation
activity
in the environment
A B
-Adsorption
-Dilution/dispersion
-Microbial degradation
COOH
COOH
COOH
COOH
COOH
COOH
HOOCCOOH
+
COOHO
COOHOH
COOH
COOHor
COOH
COOH
Upper degradation pathways
to 2-
naphthoic acid Reduction
of 2- naphthoic acid
Ring cleavage
and degradation
via
cyclohexane
ring structures
CH3
Detection
of anaerobic
naphthalene degradation
in the
environment
Investigation area
?
N100 meter
Areas with NAPL-phase
wells
Groundwater flow
B 14
B 27B 28
B 29
B 42
B 44
B 47
B 48
B 49
B 53
B 54
B 55B 56
B 57
B85
A former
coal
gasification
site
near
Stuttgart, Germany
S1
S2
Contaminant source
1510501005001000500010000200003000040000500006000070000800008500090000
B 14
B 27B 28
B 29
B 42
B 44
B 47
B 48
B 49
B 53
B 54
B 55B 56
B 57
B85
Distribution of metabolites
on a contaminated gas work
site
naphthalene
1
5
10
50
100
250
500
1000
1500
2000
3000
4000
5000
6000
B 14
B 27B 28
B 29
B 42
B 44
B 47
B 48
B 49
B 53
B 54
B 55B 56
B 57
B85
2-methyl- naphthalene
S1
S2
[µg l-1] [µg l-1]
Griebler et al., Environ. Sci. Technol. 2004
1
5
10
50
100
250
500
1000
1500
2000
3000
4000
5000
6000
B 14
B 27B 28
B 29
B 42
B 44
B 47
B 48
B 49
B 53
B 54
B 55B 56
B 57
B85
Distribution of metabolites
on a contaminated gas work
site
2-methyl-naphthalene
COOH
COOH
COOH
COOH
[µg l-1]
Griebler et al., Environ. Sci. Technol. 2004
1
5
10
50
100
250
500
1000
1500
2000
3000
4000
5000
6000
B 14
B 27B 28
B 29
B 42
B 44
B 47
B 48
B 49
B 53
B 54
B 55B 56
B 57
B85
Distribution of metabolites
on a contaminated gas work
site
2-methyl-naphthalene
C O O H
C O O H
COOH
COOH
[µg l-1]
Griebler et al., Environ. Sci. Technol. 2004
1
5
10
50
100
250
500
1000
1500
2000
3000
4000
5000
6000
B 14
B 27B 28
B 29
B 42
B 44
B 47
B 48
B 49
B 53
B 54
B 55B 56
B 57
B85
Distribution of metabolites
on a contaminated gas work
site
2-methyl-naphthalene
COOH
COOH
C O O H
COOH
[µg l-1]
Griebler et al., Environ. Sci. Technol. 2004
1
5
10
50
100
250
500
1000
1500
2000
3000
4000
5000
6000
B 14
B 27B 28
B 29
B 42
B 44
B 47
B 48
B 49
B 53
B 54
B 55B 56
B 57
B85
Distribution of metabolites
on a contaminated gas work
site
2-methyl-naphthalene
COOH
COOH
COOH
C O O H
[µg l-1]
Griebler et al., Environ. Sci. Technol. 2004