mechanisms of solvent tolerance in pseudomonas putida
DESCRIPTION
Mechanisms of solvent tolerance in Pseudomonas putida. Juan L. Ramos & Ana Segura, Antonia Rojas, Wilson Terán, M. Trini Gallegos, Estrella Duque. Solvent-tolerant microbes are envisaged as powerful tools for:. Decontamination of sites heavily polluted with solvents - PowerPoint PPT PresentationTRANSCRIPT
Mechanisms of solvent tolerance in Pseudomonas putida
Juan L. Ramos&
Ana Segura, Antonia Rojas, Wilson Terán, M. Trini Gallegos, Estrella Duque
Solvent-tolerant microbes are envisaged as powerful tools for:• Decontamination of sites heavily polluted
with solvents• Biotransformations in double-phase systems• Biosensors
*Inoue and Horikoshi, 1991, Nature 338, 264-266Pseudomonas putida, toluene tolerant
*Cruden et al., 1992, Appl. Environ. Microbiol. 58, 2723-2729P. putida , p-xylene tolerant
*Weber et al., 1993, Appl. Environ. Microbiol. 59, 3502-3504P. putida, styrene tolerant
*Ramos et al., 1995, J. Bacteriol. 177, 3911-3916P. putida, toluene tolerant and able to use toluene as the only carbon source
X F C1 C2B A TSIGED H
CH3
ISPTOL
ISPTOL
(todC1C2)FerredoxinTOL
FerredoxinTOL
(todB)ReductaseTOL
ReductaseTOL
(todA)NAD+
NADH+H+
O2
CH3
OHOH
H
H
toluene
cis-toluene dihydrodiolNAD+
(todD)NADH+H+
CH3
OHOH
3-MethylcatecholO2 (todE)
Ring fission
Growth of P. putida DOT-T1E in the presence of organic solvents
Solvent log Pow Growth (OD660)n-Decanen-Octane
n-HeptanePropylbenzene
DiethylphthalateCyclohexane
Ethylbenzenep-xyleneStyreneToluene
1-HeptanolDimethylphthalate
BenzeneChloroform
Butanol
5.64.54.13.63.33.23.13.13.02.52.42.32.02.00.8
>2.0>2.0>2.0>2.0>2.0>2.0>2.0>2.0>2.0>2.0>1.0>1.0<0.1<0.1<0.1
Why are Pseudomonas DOT-T1E and other strains tolerant to
toluene?
• Physical barriers that increase membrane rigidity
• Biochemical barriers that involve removal of toluene by efflux pumps
PHYSICAL BARRIERS(cis -> trans isomerization,Cardiolipin biosynthesis,Fatty acid metabolism)
TtgJ
Constitutive and inducibleEfflux pumps
BIOCHEMICAL BARRIERS
LB LB+tol(g)
+0.3% tol -0.3% tol-0.3% tol +0.3% tol
10-1 10-3
10-510-7
.... .... .. ...
... ... .... .. ..... ..... .
..
10-1 10-3
10-510-7
.... .... .. ...
... ... .... .. ..... ..... ..
.
10-1 10-3
10-510-7
.... .... .. ...
... ... .... .. ..... ..... .
.
10-1 10-3
10-510-7
.... .... .. ...... ..
. ..... ... .
log
CFU
/ml
10
5
00 20 40 60
DOT-T1E (wild type)
time (minutes)
PHYSICAL BARRIERS
• Cis trans isomerization of unsaturated fatty acids
• Biosynthesis of cardiolipin
C14:0C16:1 cisC16:1 transC16:0C17:cyclopropaneC18:2 cis cisC18:1 cis olC18:1 cis vaC18:1 trans vaC18:0
cis/transsaturated/unsaturated
LB LB +1%(v/v) toluene 1.015.43.1
55.01.11.04.0
13.30.0
0.95
10.71.3
1.08.3
10.353.31.01.03.0
10.03.90.9
1.51.2
actctimetH182bp
5´-CATAGGAACTACCTTACCTGGTCGGGCGAATATCAGAAGGTGCCGAATCATAACAAA GCTGCGCGGTTTTTAGGCATGTCGCCCATTTGCATGAAAACTGCTCATGTTG GGCGGGTGGAGGCAGCGCAAGGCACCCAGGACGACCAGGCAACAAATCGTGA TGGCTTTCAAGAACCAGGACTTTCCGCACATG-3´
194bp
5´-TGATCGGGTTGGCTGACCTTTCCGAGTACCTTGCGGTCGGAATGGGTG GGTGGTCTTGATCGATTGCAAAGGGGGCTGCTTTGCAGCCCTTCGCGG GTGAACCCGCTCCTACAACAGGTACGGCGCTGCTCTGAAGGCTGGCGC TGGCCTCTGCACTCGATACGGGCCTCAATGCACCGCCAAGCGCAGGGT ATTCCATG-´3
BglII BglIISphI SphI2.9 kbp 2.4 kbp1.6 kbp
6.9 kbp
BglII BglIIBamHIBamHI BglII
0,57kbp
KpnI
0,8 kbp 1,5 kbp 1,6 kbp
ctiT1N-terminal region
ctiT1N-terminal region
DOT-T1E-P4
Fatty acidGrowth conditions
LB LB plus heptane
C14:0C16:1,9 cisC16:1,9 transC16:0C17:cyclopropaneC18:2C18:1,9 cis olC18:1,11 cis vacC18:1,11 trans vacC18:0
2805417111602
1100
50413
2608
0 2 4 6 8 10 12
0.2
0.4
0.6
0.8
Time (hours)
Turb
idity
(OD
660
nm)
Head group phospholipid composition of P. putida DOT-T1 growing in the absence and
in the presence of organic solvents
Organic solvent PE PG CLPE
PG+CL
None 78 10 12 3.5
Toluene (1% v/v) 65 9 26 1.8
CL BIOSYNTHESIS TAKES PLACE AT THE EXPENSE OF PG
• 32P incorporation assays indicates that in the presence of toluene the rate of CL synthesis is twice as high as in the absence of the solvent.
• The Pseudomonas putida cls gene has been cloned, mutated in vitro and inactivated in vivo by homologous recombination.
• The response of a cls mutant to toluene shocks has been analyzed. Under a any growth conditions a solvent shock resulted in a survival that is two orders of magnitud below that of the wild-type strain.
Conclusions
• The main alterations in response to toluene observed in Pseudomonas putida are: cis -> trans isomerization of unsaturated fatty acids and increase in the level of cardiolipin.
• A cti mutant of Pseudomonas putida DOT-T1 exhibited a delay in growth in response to solvents, but it was as tolerant as the wild-type to sudden solvent shocks.
• A cls mutant of Pseudomonas putida DOT-T1 is more sensitive to solvent shocks than the wild-type strain.
Solvents
Fatty acids
saturated cis-isomer trans-isomer
EFFLUX PUMPS
Incorporation of 1,2,4-[14C]-trichlorobenzene intomembranes of P. putida cells
Conditions 14C/mg cell protein
Untreated 20.000FCCP-treated 300.000
Solvent tolerance is anenergy-dependent process
Isolation of Tn5 solvent-sensitive mutants of Pseudomonas putida DOT-
T1E• 1) Mutants that simultaneously exhibited increased
sensitivity to solvents and antibiotics (ampicillin, chloramphenicol and tetracycline)
• 2) Mutants that exhibited increased sensitivity to solvents but retained the wild-type level of resistance to ampicillin, chloramphenicol and tetracycline
9
5
10 20 40 40 4020 200 0
log
CFU
ml-1
Time (min)
DOT-T1E KT-2440 DOT-T1E-18
Incorporation of1,2,4-[14C]-trichlorobenzene into
membranes of P. putida cells
CultureConditions
14C/mg cell proteinWild-type DOT-T1E18
LB
LB+ toluene
20.000
30.000
280.000
252.000
ttgV ttgG
ttgH
ttgIttgW
ttgT ttgD
ttgE ttgF
ttgR ttgA
ttgB
ttgC
A C G T -tol +tol
A G T C 1 2 3 4
TtgB
TtgC
TtgA
Outer membrane
Periplasmicspace
Inner membrane
CH3
CH3
CH3
CH3
CH3
CH3
?
ttgV ttgG
ttgH
ttgIttgW
ttgT ttgD
ttgE ttgF
ttgR ttgA
ttgB
ttgC
A C G T -tol +tol
A G T C 1 2 3 4
Via
ble
cells
(log
CFU
ml-1
)
Time (min)
9
5
DOT-T1E DOT-T1E-1
40200
DOT-T1E-18 DOT-T1E-28
40200 4020040200
TtgABC TtgDEF TtgGHIWild-type
ttgV ttgG
ttgH
ttgIttgW
ttgT ttgD
ttgE ttgF
ttgR ttgA
ttgB
ttgC
A C G T -tol +tol
A G T C 1 2 3 4
GGAATATACTTACATTCATGGTTGTTTGTAA
TTTACAAACAACCATGAATGTAAGTATATT
-10
-35PttgABC PttgR
+1
-10
0 1000TtgR (nM)
PttgABC-PttgR DNA (10nM)
U
B1
B2
Substrates TtgABC TtgDEF TtgGHI
TolueneStyrenem-xylenePropylbenzeneEthylbenzene
TetracyclineAmpicillinChloramphenicolGentamicinNalidixicCarbenicillin
+++++
+++++
++---
------
+++-++
+++-++
Via
ble
cells
(log
CFU
ml-1
)
Time (min)
9
5
DOT-T1E
40200
DOT-T1E-18
402040200 0
DOT-T1E-109
Expression of solvent-tolerant efflux pumps in the wild-type and the TtgJ mutant background
Strain Fusion -galactosidase (Units)-Toluene +Toluene
Wild-type PttgA:lacZ 50 70PttgD:lacZ 10 20PttgG:lacZ 400 1000
DOT-T1E-109 PttgA:lacZ 70 110PttgD:lacZ 210 1600PttgG:lacZ 1000 3750
Incorporation of1,2,4-[14C]-trichlorobenzene into
membranes of P. putida cells
CultureConditions
14C/mg cell protein DOT-T1-109Wild-type
LB
LB+ toluene
20.000
30.000
30.000
82.000
109 109+tolueno
T1E T1E +tol 109+tol109
1 2 3 4 5 6 7 8
Incorporation of 13C-acetate in fatty acidsR
elat
ive
incr
ease
of
13C
605 30 120 605 30 120 605 30 120 605 30 120
1 565198 272 433 467
Fatty acid Acyl-CoA synthetase motif
ATP-binding P-loop motif
The TtgJ protein
The TtgJ protein exhibits 38-45% similarity with the FadD protein of several microorganisms, i.e. Pseudomonas putida, Pseudomonas aeruginosa, Bacillus subtilis, Mycobacterium tuberculosis, etc
The TtgJ protein exhibits 42% identity with an orf of Pseudomonas aeruginosa that probably encodes for an acetyl-CoA synthetase
The TtgJ protein does not complement an E.coli fadD mutant
T1E T1E +tol 109+tol109
1 2 3 4 5 6 7 8
Incorporation of 13C in proteinsR
elat
ive
incr
ease
of
13C
605 30 120 605 30 120 605 30 120 605 30 120
Signal-CoA
Signal -X
TtgJ
Estimulates transcription of
ttgDEF/ttgGHI
Inhibits biosynthesisof
phospholipids
Conclusions
• Biosynthesis of fatty acids is essential for solvent tolerance. In a ttgJ mutant background in which fatty acid biosynthesis is impeded, blebs are formed and cells become extremely solvent sensitive.
• Three efflux pumps are involved in solvent tolerance. Two of the pumps (TtgDEF and TtgGHI) are overexpressed in a mutant background deficient in the TtgJ protein.
• The TtgJ protein, that exhibits features of acyl-CoA synthases, might function as a sensor system for alarmone molecules produced in response to the presence of solvents
Solvent-tolerant bacteria allowing a broader performance of
biotransformations of organic compounds in two-phases fermentation
systems
EEZ
X F C1 C2B A TSIGED H
CH3
ISPTOL
ISPTOL
(todC1C2)FerredoxinTOL
FerredoxinTOL
(todB)ReductaseTOL
ReductaseTOL
(todA)NAD+
NADH+H+
O2
CH3
OHOH
H
H
toluene
cis-toluene dihydrodiolNAD+
(todD)NADH+H+
CH3
OHOH
3-MethylcatecholO2 (todE)
Ring fission
EEZ
KF
B
TO
Tn4653
INX K
Tn4651
B H
FVD
xylRxylS
D
A
E
E
IJC
P
meta
AD
GQF upper
I
EH
A
G
SG
CJ
C
MR
J L BH
pWW0
XhoI
EcoRI
HindIII
Tra/Rep
tnpT
restnpS
tnpA(Tn4651 )
tnpA(Tn4653 )
117 kb
OperónOperón
H
N
Plasmid pWWO
xyl U W C M A B NPu
CH3
xylMA xylBR1
R2
CH2OH
R1R2
CHO
R1R2
xylC
COOH
R1R2
Pm xylX Y Z L E G F J Q K I H
COOH
R1R2
xylXYZ
HOOC
R1R2
OHOHH
OH
R1R2
xylL
OHCO2 OH
R1R2
COOHxylE
EEZ
OOH
R2
COOH
COOH
R2
COOH
COOH
xylG
xylH
xylI
R2
COOHOO
R2
COOH
O
CO2
R1COOH xylF
OH
xylJxylKCH3COCOOH
R2CH2CHO+
OHCOOHO
OH
OHOH
XYZ+L E F... H Krebs cycle
CH 3 COOH
MA B C
xyl S Ps2 Ps1
+
Pm xylX Y Z L E G F J Q K I H
+
++
3- Methyl-benzoate
xylene
-
-
IHF54 70/38
70
54HU
70
xyl U W C M A B NPu
-Pr1 Pr2
70
Rxyl
EEZ
Toluene degradation pathway encoded in plasmid pWWO
xyl U W C M A B NPu Pm xylX Y Z L E G F J Q K I H
EEZ
Catechol and methylcatechol bioproduction
CH 3
R1
R2
OH
R1
R2
OHxylCMABN xylXYZL xylE
pWW0
Sm
xyl U W C M A B NPu
CH3
R1R2
xylMA xylB
CH2OH
R1R2
CHO
R1R2
xylC
COOH
R1R2
Pm xylX Y Z L E G F J Q K I H
OH
R1R2
OH
xylE
EEZ
R1COOH
COOH
R1R2
xylXYZ
HOOC
R1R2
OHOHH
xylL
CO2 OH
R1R2
COOHO
OH
R2
COOH
COOH
R2
COOH
COOH
xylG
xylH
xylI
R2
COOHOO
CO2
xylF
R2
COOH
O
OH
xylJxylKCH3COCOOH
R2CH2CHO+
CH 3
R1
R2
COOH
R1
R2
Nitrobenzoates synthesis
EEZ
Nitrobenzoates synthesisCH3
xylMA xylB
CH2OH CHO
xylC
COOH
CH3
NO2
CH3
NO2
xylUWCMABNCOOH
NO2
COOH
NO2
xyl S Ps1-2Pm xylX Y Z L E G F J Q K I H xyl U W C M A B NPu Pr1-2 xylR
ToluenesBenzyl-alcohol
p-chlorobenzaldehydep-nitrotoluene
m-nitrotolueneSTOP
xyl U W C M A B NPu
CH3
xylMA xylBR1
R2
CH2OH
R1R2
CHO
R1R2
xylC
COOH
R1R2
Pm xylX Y Z L E G F J Q K I H
EEZ
COOH
R1R2
xylXYZ
HOOC
R1R2
OHOHH
OH
R1R2
xylL
OHCO2 OH
R1R2
COOHxylE O
OH
R2
COOHCOOH
R2
COOHCOOH
xylG
xylH
xylI
R2
COOHOO
R2
COOHO
CO2
R1COOH xylF
OH
xylJxylKCH3COCOOH
R2CH2CHO+
CH 3
R1
R2
CHO
R1
R2
p- and m-nitrobenzaldehydes synthesis
xyl U W C M A B NPu
CH3
xylMA xylBR1
R2
CH2OH
R1R2
CHO
R1R2
xylC
COOH
R1R2
EEZ
CH 3
R1
R2
CHO
R1
R2
p- and m-nitrobenzaldehydes synthesis
xylC
Ptrc xylMABMiniTn5
xyl U W C M A B NPu
CH3
xylMA xylBR1
R2
CH2OH
R1R2
CHO
R1R2
xylC
COOH
R1R2
Pm xylX Y Z L E G F J Q K I H
COOH
R1R2
xylXYZ
HOOC
R1R2
OHOHH
OH
R1R2
xylL
OHCO2
xylE
EEZ
OH
R1R2
COOHO
OH
R2
COOH
COOH
R2
COOH
COOH
xylG
xylH
xylI
O
R2
COOH
O
R2
COOH
O
CO2
R1COOH xylF
OH
xylJxylKCH3COCOOH
R2CH2CHO+
CH3
R2
R1
GOH
R1R2
COOHO
xylG
xylF
xyl U W C M A B NPu
CH 3
HR2
Pm xylX Y Z L E G F J Q K I H
EEZ
OH
HR2
COOHO
NH3
COOHNH
NH
CO2
Conclusions• Pseudomonas putida exhibits three efflux pumps involved in solvent
tolerance.
• Some of these pumps are expressed constitutively and other are regulated in response to the presence of solvents
• The level of expression of each of the efflux operons is regulated by a repressor that binds at the –10 region and prevents access of the RNA polymerase to the promoter.
• In addition to the repressor global and specific positive regulators are involved in the control of the expression of the efflux pump operons.