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


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


2. GLOBAL REGULATION OF NITROGEN AND CARBON

METABOLISM IN Pseudomonas putida KT2440


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


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


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


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


• 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


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


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



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?)


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.


COMPANIES (Art. 68/83 Contracts):

Biomedal, S. L.

Canagrosa S. L.

Abengoa Research

microbiology at cabd - instituto de la grasa · microbiology at cabd contaminants biodegradation...

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