MICROBIOLOGY AT CABD - Instituto de la Grasa · MICROBIOLOGY AT CABD CONTAMINANTS BIODEGRADATION...
Transcript of MICROBIOLOGY AT CABD - Instituto de la Grasa · MICROBIOLOGY AT CABD CONTAMINANTS BIODEGRADATION...
MICROBIOLOGY AT CABD
CONTAMINANTS BIODEGRADATION
GENE REGULATION IN BACTERIA
BIOFILM DEVELOPMENT
APPLICATIONS IN BIOTECHNOLOGY
Report CABD. Microbiology. June 2016 UPO-CSIC-JA
RESEARCH LINES
1. -Tetralin biodegradation by Sphingopyxis granulii strain TFA: Specific and global regulation.
Belén Floriano and Francisca Reyes-Ramírez.
2.- Global regulation of nitrogen and carbon metabolism in Pseudomonas putida KT2440.
Inés Canosa.
3.- Pharmaceuticals biodegradation.
Eva Camacho and Amando Flores.
4.- Metagenomic analyses to identify novel activities of industrial interest.
Eduardo Santero.
5.- Aspirin-controlled synthesis of therapeutic proteins in vivo.
Amando Flores, Carlos Medina and Eva Camacho. 6.- Genetic regulation of biofilm development
Fernando Govantes and Aroa López-Sánchez
UPO-CSIC-JA Report CABD. Microbiology. June 2016
1. TETRALIN BIODEGRADATION BY Sphingopyxis granulii STRAIN TFA
UPO-CSIC-JA
Tetralin
Report CABD. Microbiology. June 2016
Tetralin biodegradation by Sphingopyxis granulii strain TFA
thnR thnY
LysR-type activator
Ferredoxin Reductase-like
-oxidation genes
UPO-CSIC-JA
O O H
COOH
ThnA1A2A3A4
Dioxygenase
H O H
H
O H
ThnB
Dehydrogenase O H
O H
ThnD
Hydrolase
ThnC Extradiol
Dioxygenase
OH
ThnE
Hydratase
ThnF
Aldolase TCA cycle
ThnG
Aldehyde Dehydrogenase
-oxidation
HOOC OH OH
COOH
HOOC
HOOC-CO-CH3
HOOC-(CH2)5-CHO HOOC-(CH2)5-COOH
COOH
C A3 A4 R Y
O N M L K J I H G A2 A1 F E D B to
nB
Pyruvate
Pimelate
Report CABD. Microbiology. June 2016
site C site B
PC PB PR
ThnR
Dynamic model for transcriptional activation of thn genes
site C site B
PC PB PR
COMPLEX I
+THN
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60 70 80 90 100 110
[ThnR] (nM)
%D
NA
in
co
mp
lex
wt b* c* b* c* b c
ThnR
I
COMPLEX II
CGCAGGACACAATAACGGCGGGGCAGAACGACGGAAGCGACCGCCGAAATATCCAGCATCATG
AAAATTG
ATAACGCCGACGAAGCCGCAGATC
TGGACGCAAGGCGGGCTCATC
ACCGAAAATG
ATAACCACTAGAGGACATCGTTATC
AGCGTTTTA
CTGCGCCGCCGCCACCTGGTGTGCCATGACTGCGGAA
II
UPO-CSIC-JA Report CABD. Microbiology. June 2016
2. GLOBAL REGULATION OF NITROGEN AND CARBON
METABOLISM IN Pseudomonas putida KT2440
UPO-CSIC-JA Report CABD. Microbiology. June 2016
Urease
Transport N compounds
Proteasome
Nitrogen metabolism
Nitrogen limitation
GlnK-UMP
GlnK
NtrC
Nitrogen excess
potI potH potG pot-F1 pot-F2
ureD ureA ureB ureC ureE ureJ ureF ureG
PP4841 PP4842 PP4843 PP4844 PP4845 PP4846
PP4863 PP4864 PP4865 PP4866 PP4867
aapJ aapQ aapM aapP
PP2685 PP2686 PP2687 PP2688
Group III Carbon storage
PHA synthesis
PP5003 PP5004 phaC PP5006 PP5007 PP5008
glpD glpKglpR glpF
Glycerol metabolism
amtB glnK
Group II
gap-1 edd glk gltR hexR zwf -1 pgl eda Sugar ABC transporter
gdhA PPP and glycolysis Glutamate metabolism
Glycogen synthesis
glgA PP4051 PP4052 PP4053 PP4055
PP4054 PP4056
AmtB
aap
bra
Group I
pot
Group IV
fleN flhF flhA
PP4362 PP4363PP4364 PP4374
fliJ fliI fliH fliG fliF fliE fleR fleS fleQ fliS
flgL flgK flgJ flgI flgH flgG flgF flgE flgD flgC flgB
PP4387
Flagellum synthesis
Slow growth
ribosomal proteins
chaperonines
stress-related
C and N metabolism
UPO-CSIC-JA
Global nitrogen regulation in Pseudomonas putida KT2442: The NtrBC system
Report CABD. Microbiology. June 2016
CbrB-
CbrA
cbrA cbrB pcnB
PP3538 mvaB PP3541 PP3537
crcZ
crcY
Crc
PP3539
• Utilisation of different C
sources including amino acids
• Chemotaxis
• Stress response
• Motility
P
CrcZ
CrcY
RNP complex
Carbon Catabolite Repressed Genes
UPO-CSIC-JA
The pleiotropic function of CbrAB regulatory system
Report CABD. Microbiology. June 2016
3.- PHARMACEUTICALS BIODEGRADATION
• Isolation of an Achromobacter sp. strain growing on Ibuprofen
Carbon backbones Energy + CO2
• Isolation of a metagenomic clone providing capability of growing on Naproxen
Carbon backbones Energy + CO2
Report CABD. Microbiology. June 2016 UPO-CSIC-JA
4. METAGENOMIC ANALYSES TO IDENTIFY NOVEL ACTIVITIES OF INTEREST
UPO-CSIC-JA Metagenomic analyses
MICROBIAL GENOMICS (≤ 1%)
MICROBIAL METAGENOMICS: Access to microorganisms genetic
resources independently of their culture
Identified activities of interest
• Carbenicillin resistance
ETN1 (27 kb)
psal pT7 blaC RND family efflux pump
5 kb
14 kb A B C C2
TN2 (38 kb)
psal pT7 blaB
6 kb
blaR
TN3 (35 kb)
psal pT7 RND family efflux pump
16 kb A B C
TN4 (26 kb)
psal pT7 RND family efflux pump
13 kb A B C
N5 (31 kb)
psal pT7 blaB
21 kb
R
N6 (30 kb)
psal pT7
11 kb
blaB R
• Ring-cleavage extradiol dioxygenases
• Cellulases. Abengoa Research. Patented Cel-98
• Naproxen utilisation
Report CABD. Microbiology. June 2016 UPO-CSIC-JA
• Antimicrobial agents for food preservation?
5. ASPIRIN-CONTROLLED SYNTHESIS OF THERAPEUTIC PROTEINS
IN VIVO WITH ATTENUATED Salmonella CELLS
UPO-CSIC-JA
Psal Pr
nahR xylS2
Regulatory module Pm
Expression module
COOH
OH
Salicylate
Report CABD. Microbiology. June 2016
N
H
O
O
H
N
Indigo
N
H
OH
OH
N
H
Tryptophan Indole
N
H
COOH
NH3
Dioxygenase Tryptophanase
O2
Biological production of indigo
Elimination of actin filaments in HeLa cells by bacterial SpvB production
- SpvB
Z=7 μm Z=9.8 μm
+ SpvB
UPO-CSIC-JA
Bacteria-Actin-eukaryotic nuclei
Report CABD. Microbiology. June 2016
PBS SL7207-4S2 [pMPO16] + salicylate + 5-FC
-3 2 50
10
20
30
40
50
60
70
80
90
PBS
SL7207-4S2 + 5-FC
SL7207-4S2 [pMPO16]+ salicylate + 5-FCSL7207-4S2 [pMPO17]+ tetracycline + 5-FC
Time (days)
Me
an
tu
mo
r s
ize
(m
m2)
-3 0 2 5 -5
Bacteria i.p.
5-FC
-14
Tumor challenge
+ Inducer
Time (days)
Effect of Salmonella producing cytosine deaminase on tumor development
UPO-CSIC-JA Report CABD. Microbiology. June 2016
Report CABD. Microbiology. June 2016
6.- Genetic regulation of biofilm development
Mutant hunt Phenotypic
characterization
Global gene expression
analysis
Characterization
of regulatory
mechanisms
Regulatory network of
biofilm development
Development of a biofilm-based platform
for biotechnological production
Breeding robust biofilm-forming strain Building synthetic regulatory circuit for
biofilm formation and dispersal
Repressor 1
Repressor 2 Signal 2
Signal 1 Biofilm
promoting factor
(GGDEF protein,
adhesin, etc.)
Dispersal
promoting factor
(EAL protein,
anti-adhesin,
etc.)
Breeding optimized producing strain Design of biofilm-based reactor (collaboration?)
Report CABD. Microbiology. June 2016
RECENT UNOFFICIAL COLLABORATORS:
Jose Luis Ríos (IG-CSIC)
Milagros Medina (U. Zaragoza)
Rita Bernhardt (U. Saarlandes, Saarbrücken)
Michael Hecker (U. Ernst Moritz Arndt, Greifswald)
Cynthia M. Sharma (U. Würzburg)
Ray Dixon (John Innes Centre, Norwich)
Karine Lapouge (U. Lausanne)
UPO-CSIC-JA
OFFICIAL COLLABORATIVE PROJECTS:
Consolider metagenomic project: 11 research groups + 4 companies Genomics and proteomics: 7 research groups + 1 company. EU Herbicides bioremediation: 9 research groups + 2 companies EU Stress adaptation of leguminous trees: 4 research groups.
Report CABD. Microbiology. June 2016
COMPANIES (Art. 68/83 Contracts):
Biomedal, S. L.
Canagrosa S. L.
Abengoa Research