Mechanisms of solvent tolerance in Pseudomonas putida

transcript

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

ISPTOL

(todC1C2)FerredoxinTOL

FerredoxinTOL

(todB)ReductaseTOL

ReductaseTOL

(todA)NAD+

NADH+H+

toluene

cis-toluene dihydrodiolNAD+

(todD)NADH+H+

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)

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

.... .... .. ...... ..

. ..... ... .

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

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´

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

0,8 kbp 1,5 kbp 1,6 kbp

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

0 2 4 6 8 10 12

Time (hours)

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

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

10 20 40 40 4020 200 0

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+ toluene

20.000

30.000

280.000

252.000

ttgV ttgG

ttgIttgW

ttgT ttgD

ttgE ttgF

ttgR ttgA

A C G T -tol +tol

A G T C 1 2 3 4

Outer membrane

Periplasmicspace

Inner membrane

ttgV ttgG

ttgIttgW

ttgT ttgD

ttgE ttgF

ttgR ttgA

A C G T -tol +tol

A G T C 1 2 3 4

Time (min)

DOT-T1E DOT-T1E-1

DOT-T1E-18 DOT-T1E-28

40200 4020040200

TtgABC TtgDEF TtgGHIWild-type

ttgV ttgG

ttgIttgW

ttgT ttgD

ttgE ttgF

ttgR ttgA

A C G T -tol +tol

A G T C 1 2 3 4

GGAATATACTTACATTCATGGTTGTTTGTAA

TTTACAAACAACCATGAATGTAAGTATATT

-35PttgABC PttgR

0 1000TtgR (nM)

PttgABC-PttgR DNA (10nM)

Substrates TtgABC TtgDEF TtgGHI

TolueneStyrenem-xylenePropylbenzeneEthylbenzene

TetracyclineAmpicillinChloramphenicolGentamicinNalidixicCarbenicillin

------

+++-++

Time (min)

DOT-T1E

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+ toluene

20.000

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

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

605 30 120 605 30 120 605 30 120 605 30 120

Signal-CoA

Signal -X

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

X F C1 C2B A TSIGED H

ISPTOL

(todC1C2)FerredoxinTOL

FerredoxinTOL

(todB)ReductaseTOL

ReductaseTOL

(todA)NAD+

NADH+H+

toluene

cis-toluene dihydrodiolNAD+

(todD)NADH+H+

3-MethylcatecholO2 (todE)

Ring fission

Tn4653

Tn4651

xylRxylS

GQF upper

J L BH

HindIII

Tra/Rep

restnpS

tnpA(Tn4651 )

tnpA(Tn4653 )

117 kb

OperónOperón

Plasmid pWWO

xyl U W C M A B NPu

xylMA xylBR1

Pm xylX Y Z L E G F J Q K I H

xylXYZ

OHCO2 OH

COOHxylE

COOHOO

R1COOH xylF

xylJxylKCH3COCOOH

R2CH2CHO+

OHCOOHO

XYZ+L E F... H Krebs cycle

CH 3 COOH

MA B C

xyl S Ps2 Ps1

3- Methyl-benzoate

xylene

IHF54 70/38

xyl U W C M A B NPu

-Pr1 Pr2

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

Catechol and methylcatechol bioproduction

OHxylCMABN xylXYZL xylE

xyl U W C M A B NPu

xylMA xylB

R1COOH

xylXYZ

CO2 OH

COOHOO

xylJxylKCH3COCOOH

R2CH2CHO+

Nitrobenzoates synthesis

Nitrobenzoates synthesisCH3

xylMA xylB

CH2OH CHO

xylUWCMABNCOOH

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

xylMA xylBR1

xylXYZ

OHCO2 OH

COOHxylE O

COOHCOOH

COOHOO

R1COOH xylF

xylJxylKCH3COCOOH

R2CH2CHO+

p- and m-nitrobenzaldehydes synthesis

xyl U W C M A B NPu

xylMA xylBR1

p- and m-nitrobenzaldehydes synthesis

Ptrc xylMABMiniTn5

xyl U W C M A B NPu

xylMA xylBR1

xylXYZ

R1COOH xylF

xylJxylKCH3COCOOH

R2CH2CHO+

xyl U W C M A B NPu

COOHNH

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.

Mechanisms of solvent tolerance in Pseudomonas putida

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