FAPESP-2012 1

Genome sequence of the polyhydroxybutyrate producer Pseudomonas extremaustralis a highly

stress resistant Antarctic bacterium

Departamento de Química Biológica

Facultad de Ciencias Exactas y Naturales

Universidad de Buenos Aires

Nancy I. López

IQUIBICEN

FAPESP-2012 2 2

• High stress resistance • High PHB production

Heat & Cold shock

H2O2

López et al., (2009) Curr Microbiol 59:514–519

López et al., (2010) Validation list N°132. IJSEM 60: 469-

472.

Fluorescens group 16S rRNA

Pseudomonas extremaustralis sp. nov. (= DSM 17835T = CIP 109839T)

Low DNA-DNA hybridization value with

P. veronii (35.1%)

FAPESP-2012 3 3

Looking for PHB genes Genetic studies without having the genome Approaches: Genomic library: phenotypic characteristics Complementation studies using a mutant strain of Cupriavidus necator (formely Ralstonia eutropha)

PCR strategies: primers designed based on sequences available in databases

FAPESP-2012 4

phb gene cluster

. Ayub et al., (2006) FEMS Microbiol Lett 264:125–131.

. Ayub et al., (2007) Plasmid 58:240–248.

. Ayub et al., (2009) Extremophiles 13:59-66.

. Tribelli and López (2011) Extremophiles 15:541–547.

Direct repeat

Direct repeat

PHB genes are included in a genomic island

Acquired by horizontal transfer

FAPESP-2012 5

Genes involved in medium chain length PHA Production

Pseudomonas extremaustralis

Pseudomonas putida GPp104

P. putida GPp104/pBBRMSC5-phaC2(mcl)P.extremaustralis

Complementation studies Both polymerases restored the mclPHA production in P. putida GPp104

Why we could not find phaFI genes?

FAPESP-2012 6

Why Pseudomonas extremaustralis as a model of study?

PHA Production

Surfactant production

High stress resistance

Genome sequence

INDEAR (Instituto de Agrobiotecnología de Rosario-Argentina)

Roche 454 GS Titanium

pyrosequencer

FAPESP-2012 7 7

2,884

3,050

Total: 5,934 CDS J. Bacteriol. 2012, 194(9):2381.

FAPESP-2012 8

Contig 4: Genomic island-PHB genes 389,240 bp

Other group of genes related to PHB metabolism

phbF phbP posible PHB depolimerasaPutative PHB depolymerase

PHB genes

FAPESP-2012 9

Comparison of phaC1ZC2D…………….…….…phaFI cluster

References in the picture (numbers and colors) 2. phaC1 and phaC2 polymerases 4. phaZ 1. phaD 3. phaF/phaI Be careful with PCR strategies!

FAPESP-2012 10

What are the proteins in the granules?

Real Time RT-PCR experiments

SDS-PAGE + high resolution

mass espectrometry

Instituto de Biotecnología-UNAM. México

CYTED Program

Real Time RT-PCR

0.00

0.50

1.00

1.50

2.00

2.50

3.00

phbC phaC1 phaC2

pha Sintasas

pm

ol g

en

de in

tere

s/ p

mo

l 16S

AR

Nr

0.5NE2 Cap

0.5NE2 Glu

Analysis of PHB granule-associated

proteins

Putative PHB depolymerase

PhaC1 PhbC

PhbP PhaI

pg p

olym

eras

e ge

ne/p

g 16

S r

RN

A g

ene

Accumulation Low-accumulation

kDa cap glu

FAPESP-2012 11

Biofilm lifestyle enhances diesel bioremediation and biosurfactant production in the

Antarctic polyhydroxyalkanoate producer Pseudomonas extremaustralis

P. extremaustralis and hydrocarbon bioremediation

Other compounds of biotechnological interest

P. extremaustralis synthesizes surfactants of lipopeptide nature

Genes responsible for the production of surfactants are unknown

in P. extremaustralis

Surfactants

Tribelli et al., 2012. Biodegradation. Feb 3. [Epub ahead of print].

FAPESP-2012 12

Screening of genes involved in biosurfactant production using a

Mini-Tn5 Insertion Library of Pseudomonas extremaustralis

Mutants affected in surfactant production in blood agar plates supplemented with 2%

glucose hemolytic activity 2 clones H and N

Drop-collapsed assay

Identification of the Tn-5 insertion site in the genome sequence

PCR strategy

For H clone a PCR fragment of 1200 bp.

Sequence analysis

Guess what?

FAPESP-2012 13

Mini-Tn5 Insertion

Decreased PHB production resulted in increased surfactant production

PHB genes

FAPESP-2012 14

Conclusions

• P. extremaustralis synthesizes PHB, but also has the genomic information to synthesize medium chain length PHA.

• This species is able to produce surfactants. Metabolism and/or carbon fluxes between PHB and surfactants seem to be related.

• The overall genome analysis revealed a high potential for adaptability to extreme environmental conditions.

FAPESP-2012 15 15

Dra. Beatriz S Méndez

Departamento de Química Biológica

Dra. M.Julia Pettinari

Dra. Paula Tribelli Lic. Mariela Catone

Dra. Nancy I. López Dra. Laura Raiger-Iustman

Lic. Carla Di Martino

Natural PHA producers

Bioremediation

FCEyN-UBA

Dra. Jimena Ruiz

Pseudomonas extremaustralis

Lic. José G. Ibarra

The true workers

Picture: Mercedes Berlanga Grants from CONICET, ANPCyT and UBA.

FAPESP-2012 16 16

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