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.