Anaerobic metabolism of the explosive 2,4,6-trinitrotoluene (TNT)
by Pseudomonas sp. JLR11
Juan Luis Ramos
TNT team in DOT group:
Abraham Esteve-Núñez, postdoc
Antonio Caballero, Ph.D. student
Pieter van Dwilling, postdoc
M. Mar González-Pérez, postdoc
Natural nitroaromatic compounds
Choramphenicol
Nitropyoluteorin
Oxypyrrolnitrin
Phidolopin
NO2-
Microbial metabolism of nitroaromatic compounds
CH3
CH3
NO2
OH
O2
OH
NO2 -
CH3
COOH
O2
OH
NH4 +
NO2
OH
CH3
CH3
NO2
OH
O2
NO2 -
NO2
OH
OH
CO2 + H20
The explosive 2,4,6-trinitrotoluene
Reduction mechanisms of nitro groups
of nitroaromatic compounds
1e1e- / H+
2e- / H+
2e- / H+
Toxicity
2e- / H+
CH
NO2
3
NH 2
2,4-DANT
CH3
NH
NH2
CH 3
NO2
4-ADNT
NO
2
CH 3
OH
NO2
NO2
OH
OH
OH
2
+
Ar-N=N-Ar´
NO2
O
Azoxynitrotoluene
CH3
NHOH
NHCOCH
3
CH
3
NO
2
NO
2
NO
NO2
NH
NO 2
NO2
NO2
NO 2
CH3
OH
CH3
CH3
NH2
NO
NH +
4
OH
NO2
2
2-ADNT
NHOH NO 2
NO2
CH3
NHOH
NHOH
NO
Yellow product
TNT
NO2
NO2
H
H
CH3
NO 2
NO2
NO2
H
H
CH3
NO 2
NO2
NO2
H
H
Meisenheimer complex
NO2
TCA cycle
CH 3
NO 2
NO2
2
Proposed
mechanisms for
TNT aerobic
metabolism by
bacteria
Azoxynitrotoluenes accumulation in TNT cultures
Anoxic culture
A254 (AU)
0,2
0,1
2
4
6
8
10
Time (min)
A254 (AU)
Azoxinitrotoluenes
0,2
0,1
2
4
6
Time (min)
8
10
Aerobic culture
Proposed mechanisms
for TNT anaerobic
metabolism in bacteria
TNT
CH3
NO
NHOH
2
2-NHOH-4,6-DNT
NHOH
CH3
CH3
NH
OH
NO 2
NO2
2
NO
2
CH 3
NH2
NO 2
CH
3
NO2
2
NHOH
NH
NO 2
2
NO
NHOH
NO2
NO2
NO 2
2-ADNT
CH3
CH3
4-NHOH-2,6DNT
NO2
4-ADNT
NH2
NH 2
CH
CH 3
?
NH2
NH4+
NH
toluene
3
NH2
2
CH 3
CH 3
OH
OH
p-cresol
OH
TAT
OH
methylphloroglucinol
Isolation and characterization of microorganisms able to use
TNT as nitrogen source under anoxic conditions
Optimization of the parameters for TNT degradation by Pseudomonas
sp. JLR11 in anaerobiosis
Elucidation of the metabolic pathway used by Pseudomonas
sp. JLR11 to degrade TNT under anoxic conditions
Role of the TNT as terminal electron acceptor under anaerobic
conditions
Isolation and characterization of microorganisms
able to use TNT as nitrogen source under anoxic
conditions
Isolation of a Pseudomonas strain able to use TNT
as sole nitrogen source
Wastewater treatment
plant of Granada
Argon
atmosphere
Pseudomonas sp.
JLR11 strain
Sample
Enrichment in
anoxic
minimal medium
with glucose and
TNT
Ultrastructure of Pseudomonas sp. JLR11
Exponential phase growth
Glucose, ammonium
Exponential phase growth
Glucose, TNT
Nitroaromatic compounds as nitrogen source for
Pseudomonas sp. JRL11
Nitroaromatic
2-NT
3-NT
4-NT
2,3-DNT
2,4-DNT
2,6-DNT
TNT
Aerobiosis
Anaerobiosis
+
-
+
-
+
+
++
+
+
++
Optimization of the parameters for TNT
degradation by Pseudomonas sp. JLR11 in
anaerobiosis
TNT degradation in batch culture under
anoxic conditions
Parameters optimization
Temperature 30  2 ºC
pH
7  0.5
Stirring
600 100 rpm
Cosubstrate 0.1% glucose
Results of the biodegradation
process
Batch culture
99% TNT elimination
0.6 mol TNT/(mgProtein * day)
85% N-TNT in cellular protein
Low biomass production: 75 mg Protein/l culture.
10-15% accumulation of aminoaromatic compounds
TNT degradation in batch culture under
anoxic conditions
Cellular growth (Protein mg/ culture l)
100
300
50
200
100
10
0
100
200
300
Time (h)
TNT (M)
Protein (mg/l)
Batch culture
Nitrite (M)
400
500
600
Elucidation of the metabolic pathway used by
Pseudomonas sp. JLR11 to degrade TNT under anoxic
conditions
Spectroscopic analysis of TNT metabolites from
culture supernatants
Supernatant extraction
Ethyl acetate
Hexane
MeOH
Liquid-Liquid
Extraction
Solid Phase
Extraction (SPE)
HPLC-DAD, GC-MS
1H-NMR
Structural
elucidation
Copolimer HLB
Catabolic pathway proposed for TNT anaerobic metabolism
by Pseudomonas sp. JLR11
Respiratory
branch
3
TNT
NO
Assimilatory
branch
Biomass
N-TNT assimilatory metabolism by Pseudomonas sp. JLR11
Distribution of nitrite reductase activity
in cell-free extracts
Fraction
Sp. Activity (U/mg Protein)
Crude Extract
Periplasmic Fr.
Cytoplasmic Fr.
Membrane Fr.
Substrate
Growth medium
39
36
3
Vol. Activity (%)
100
90
10
NO2NO3-
NO3-
Native
PAGE
Isolation of a mutant unable to use TNT as
nitrogen source
kilAtelAB
Triparental mating
Donor
Host
Helper
E.coli CC118pir (pJMT4)
Pseudomonas sp. JLR11
E. coli HB101 (pRK600)
miniTn5-tellurite
Pseudomonas sp.
JLR11-P12E2
Glucose + NH4+
Glucose + TNT
Isolation of a mutant unable to use TNT
as nitrogen source
Dig

6,6
4,4
Mutant
6 kb
Nitrogen source
TNT
NO2
TNT + NH4+
NO2 + NH4+
Southern hybridization
Probe: tellurite genes
Growth
+
+
Resting cell assays with mutant and wild type
strains
Nitrite medium
120
TNT medium
50
300
100
40
200
80
30
60
20
40
100
10
20
0
0
0
50
100
150
Time (min)
[NO2-] WT
[NO2-] mutant
[NO2-] control
200
0
50
100
150
Time (min)
[TNT] WT
[TNT] mutant
200
Nitrite reductase
activity in native
PAGE
WT
TNT
NO2
mutant
-
TNT
NO2
-
gnrA is involved in nitrite metabolism but it is not a nitrite reductase
MiniTn5 insertion: gnrA gene
orf
gnrA
1 kb
aa
% identity
NADPH reductase E. coli
376
43% (145)
Oxidoreductase E. coli
353
43% (145)
Oxidoreductase Sp. coelicolor
364
44% (148)
Protein and microorganism
The role of GNRA
Electron donor
GNRA
e-
in vivo
-
NO2
Nitrite reductase
+
in vitro
e-
Methylviologen
NH4
Organic
nitrogen
How Pseudomonas sp. JLR11 release nitrite from TNT?
Isolation of genes involved in TNT denitration
3
3
TCA
DNT
TNT
NO
Genes from
Pseudomonas
NO
Burkholderia R34
Isolation of a cosmid involved in TNT denitration
Triparental mating
Donor
Host
Helper
E.coli HB101pir (gene library from Pseudomonas sp. JLR11)
Burkholderia R34
E. coli HB101 (pRK600)
Burkholderia R34 pCJL1.82
Minimal medium, glucose, TNT
orf1
orf2
orf3 orf4
orf5
orf12
cluster
No match with other known proteins
pCJL1.82
Nitrite release activity from TNT
40
E. coli pCJLl1.82
35
Nitrite (M)
30
E. coli
25
20
15
10
5
0
0
20
40
60
time (h)
80
100
120
Isolation of a cosmid
with Meisenheimer complex
forming activity
Pseudomonas sp. JLR11 gene library
was screened for TNT aromatic ring
reduction
E. coli pTMF
Meisenheimer complex detected in resting cells assays
Nitro group reduction of TNT
Cytoplasmic associated
reduction
NO2
No apparent metabolic benefit
for the cell
Membrane associated
reduction
Involved in anaerobic
respiratory processes
NH2
Cytoplasmic nitro reduction by Pseudomonas sp. JLR11
TNT nitroreductase: PNRA
kD
NADPH as electron donor
66
Km=0.027
Vmax=111.210
41
1
2
3
31
1. Crude extract
2. Anionic chromat.
3. Hydrophobic chromat.
4. Affinity chromat.
4
Future:
knock-out of pnrA to achieve a more efficient TNT removal
pnrA
NH2
NO2
assimilatory metabolism
NO2-
Role of the TNT as terminal electron acceptor under
anaerobic conditions
Role of TNT as terminal electron acceptor in
Pseudomonas sp. JLR11
Denitrification
TNT respiration
NO2
NO
NHOH
NH2
TNT respiration with acetate as cosubstrate
NH2
NO2
CH3COOH
Growth (DO660)
Substrate
Ammonium
CO2 + H2O
TNT
Initial
Final
+
-
0,02
0,02
+
+
0,02
0,45
-
+
0,02
0,32
Extracellular
medium
Positively
charged
Cytoplasm
eNO2
H+
H+
electron transport
complex
e-
H+
NH2
H+
ATP
ATP-Synthetase
ADP + Pi
Cytoplasmic
membrane
Negatively
charged
Model for TNT respiration
in Pseudomonas sp.
JLR11
Nitroaromatic reductase activity in Pseudomonas sp. JLR11
Distribution of nitroaromatic reductase activity in cell-free extracts
Fraction
Sp. Activity (U/mg Protein)
Crude Extract
Cytoplasmic Fr.
Membrane Fr.
41.3
57.2
23.7
100
25
75
TNT
TNT, ADNTs (M)
3 00
ADNT
Resting cell assay
2 00
1 00
00
Vol. Activity (%)
20
40
60
Time (min)
80
Proton extrusion coupled to TNT reduction
N2
Septum
N2, cells, substrates
Needles
Incubation
chamber
pH electrode
Water stream at 30 ºC
Magnetic bar
stirring
In situ proton extrusion in response to different
electron acceptors
6.8
TNT
cells grown in NO3-
NO3-
6.6
6.4
0
6.8
TNT
6.6
NO2-
6.4
0
0
2
4
Time (min)
6
cells grown in NO2-
ATP synthesis coupled to proton extrusion
in membranes vesicles
ATP
Synthetase
Membrane
vesicles
6-P-Gluc
Acid
NADPH
Ar-NO2
Ar-NH2
Respiratory
metabolism
NO2NH4+
Biomass
Assimilatory
metabolism
Anaerobic metabolism of the explosive 2,4,6-trinitrotoluene (TNT)
by Pseudomonas sp. JLR11
Juan Luis Ramos
TNT team in DOT group:
Abraham Esteve-Núñez, postdoc
Antonio Caballero, Ph.D. student
MªÁngeles Guerrero, technician
Isolation of a mutant unable to use TNT as
nitrogen source
Triparental mating
Donor
Host
Helper
E.coli CC118pir (pJMT4)
Pseudomonas sp. JLR11
E. coli HB101 (pRK600)
Nitrogen source
TNT
NO2
TNT + NH4+
NO2 + NH4+
Glucose + NH4+
Glucose + TNT
Pseudomonas sp.
JLR11-P12E2
Growth
+
+