Post on 13-Jan-2017
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 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.