Transcriptome and regulatory network analysis of the response to glucose and catabolite repression in

Escherichia coli

Escherichia coli

Nutrients

pHO2

Osmolarity

Objective

Glucose

Sugar B

Sugar C

Carbon Catabolite Repression in Escherichia coli

Biomass

Time

Transcriptional control

Adenilate cyclase

ATP

AMPc

-10 -35

RNA Pol. CRP AMPc

+

Periplasm

Cytoplasmic membrane

Cytoplasm

IICIIB

PEP

Pyruvate

EI

EI~P

Hpr~P

Hpr

IIAGlc

IIAGlc~P Glucose

Glucose 6-P

P

Lac

Y

Lactose H+

Lactose H+

Inducer exclusion

-

IIC (20)

IIA IIB

A (17)

A~P

Inducer exclusion

(21)(19)

IIAGlc

IIBCGlc

PEP

PIR

PTS -P

AC

ATP

AMPc

?

AMPc

CPD

AMP

P-

P

Absence of PTS sugarsPTS sugars: Galactosamine, N-acetylgalactosamine,arbutin, cellobiose, salicin, Di-N-acetylchitobiose, dihydroxyacetone, fructose, galactitol, glucitol,maltose, mannose, glucose, glucosamine, mannitol,acetylglucosamine and trehalose.

Glucose-6-P

IIAGlc

IIBCGlc

PEP

PIR

PTS -P

AC

ATP

AMPc

?

AMPc

CPD

AMP

Glucose

>99.9%

-Catabolic repression.-Gluconeogenesis.-Flagellum synthesis.-Coordination of DNA replication and cell division.-Glycogen metabolism.-Antibiotic resistance.-Toxin production.

Steve Busby and Richard Ebright

cAMP receptor protein (CRP)

TGTGAGTTAGCTCACT

Complex medium Complex medium+

Glucose

Glucose

Cells grow faster and they secrete acetate

Use genome-wide transcriptome data and regulatory network analysis to determine the cellular functions responding to the presence of glucose

and the transcriptional factors controlling this response.

CRP AMPc

-10 -35

-10 -35

-10 -35

-10 -35

-10 -35

-10 -35Glucose

+

++

+

+

-

-10 -35

-10 -35

-10 -35

-10 -35

-10 -35

TF??

?

?

?

?

--

Metabolite?

??

Cellfunctions

Análisis de transcriptoma

GENOMA PROTEINAS METABOLISMO

Transcriptoma Proteoma MetabolomaFluxoma

RNA

Microarreglos de DNA

Cromosoma

Cultivo bacteriano

Extracción de RNASíntesis de cDNA y marcado

[RNA]SíntesisPromotor Degradación

Component LB LB+G

Tryptone 10 g/L 10 g/LYeast Extract 5 5 NaCl 10 10 Glucose - 4

Escherichia coli BW25113 (WT)Escherichia coli BW25113 crp- (CRP)

AminoacidsNucleic acids

VitaminsCarbohydrates (5-10%)

No lipidsGlucose (0.003%)

Luria Bertani medium (LB)

WT WTg CRP CRPg

1 2 3 4

LB + glucose

0 1 2 3 4 5 6 7 80.0625

0.125

0.25

0.5

1

2

4wtcrp

Time (h)

OD

600

LB

0 1 2 3 4 5 6 7 8

0.0625

0.125

0.25

0.5

1

2

4 wtcrp

Time (h)

OD

600 25 ml cultivo OD 0.5

Filtrar

N2 líquido

Rompimiento

Extracción con fenol

RNA crudo

Kit Stratagene

RNA puro

Experimentos por triplicado

E. coli BW25113 37 min 35 min

E. coli BW25113 crp- 43 min 41 min

5%

5%

LB LB+G

Microarreglos Afymetrix

Nucleic Acids Research, 2002, Vol. 30, No. 17 3732-3738

Oligonucleótidos de 25 bases (296,936 ).

11-20 oligos /gene

2 tipos de oligos:

Perfect Match (PM)

MissMatch (MM)

4,327 ORF

2,885 intergénicos

AvgDiff = N PM - MM

N

PM

MM

Microarreglos Afymetrix

WT1

WT2 WT3 WT3

WT2

Pair-wise comparison of triplicate data sets

1,908 1,910 3,083 1,910

4,327 ORFs.

WT WTg CRP CRPg

WTg/WT CRP/WT CRPg/CRP CRPg/WTg

WTg/WT CRP/WT CRPg/CRP CRPg/WTg 380 333 271 298 8.8% 7.7% 6.3% 6.9%

Outlier iteration method

Affymetrix data reliability filter

0

2

4

6

8

10

12

0 20 40 60 80 100 120 140 160 180

Number of genes

WTg/W

t ra

tio

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 20 40 60 80 100 120 140 160 180 200

Number of genes

WTg/W

t ra

tio

glpF (0.04)tnaL (0.01)

spf (11.2)

fis (6.9)

Expression ratios for genes responding to glucose in WTg/Wt

Functional class

Number of

genes

induced

Number of

genes

repressed

Adaptation - 7

Amino acid biosynthesis 1 8

Biosynthesis of cofactors, carriers 4 5

Cell division - 5

Cell envelope 3 5

Central intermediary metabolism 8 19

Chromosome replication 3 2

Degradation of small molecules 1 10

Energy metabolism, carbon 3 25

Fatty acid biosynthesis 2 1

Folding and ushering proteins - 2

Global regulatory functions - 9

Laterally acquirred elements - 5

Macromolecule degradation - 9

Macromolecule synthesis, modification 18 10

Nucleotide biosynthesis 3 -

Protection responses 1 4

Ribosome constituents 35 -

Some information, but not classifiable 7 25

Transport/binding proteins 12 20

Unexpressed functions 2 -

Unknown proteins, no known homologs 77 29

180 200

Glucose

IIBGlc ptsG

G6P

F6P

F1,6BPHpr~P ptsH

IIAGlc ~P crr

IICGlcpotD

Spermidine/ putrescine

Phosphate

zntAZinc

mgtAMg2+

ompF

Trp tnaB TrptnaCA

Indole + PYR + NH3

lamBMaltose

Heat shock proteins and chaperones: ibpA ibpB hslU hslV htpX dnaK grpE groE mopA hslONucleotide biosynthesis: adk guaA pyrL

Acetate

Lactate

malE

dppA

hisJ

rbsB

gltL

Dipeptides

Histidine

Ribose

Glutamate

cycA

Serine/alanine/glycine

glnH

Glutamine

glpF

Glycerol

tsx

Nucleoside channel

putP

Proline

sdaC

Serine

IIABMan

IICMan

manX

man

Y

Mannose/glucose/glucosamine/fructose

IIBMal

IICMal

malX

Maltose/glucose

ompX

potA

PEP

PYR

EI~P ptsIppsA

AcCoA

aceE aceF

pckApta

Ac~PackA

Acetate

OAA

MAL

CIT

AKG

SUCCoA

SUC

FUM

ICITgltA

ldhA

sucB

sdhADC

mdh

fumA

acnB

icdA

aceA

maeB

Salvage pathway of purine and pyrimidine: deoB deoD hpt gpt upp apt tdk

glpXRegulatory proteins: dps lon uspA cytR crp ykgA hcaR fis glnB marA

IIC1Gut

IIAGut

Glucitol

Gut6P

srlD

srlB

pitA

gatY gatZ

Tag1,6BP

DHA + GAP

Lactate

lctP

proX

Glycine/betaine/proline

Amino acid biosynthesis: aspC cysK aroD pheL thrL ivbL ilvB ilvC

Biosynthesis of cofactors: entC ispA moaB nrdH gshA bioH folC menE trxA

Transcription functions: rpoE rpoS rpoD nusB mfd greA rpoB rpoA nusA nusG

IIAGat

gatA

Galactitol

Gat1P

Ribosome constituents: rplB rplC rplD rplI rplK rplM rplP rplR rplS rplV rplW rplY rpmA rpmC rpmD rpmE rpmF rpmG rpmH rpmI rpsB rpsC rpsD rpsE rpsF rpsG rpsI rpsJ rpsN rpsO rpsP rpsQ rpsR rpsS rpsT rpsU

Degradation of proteins: prlC hflX clpA hflB hflK pepD pepN clpX

Protein translocation: prlA secE secG yidC

Proteins - translation and modification: ppiC efp infA infB infC tsf

fliO

Cell division: ftsJ minC minD

Multipurpose conversions of intermediates: aspA gcvH gcvP gcvT gloB kbl sufB

mgsA

MG

Degradation of small molecules: eutB sdaA tdh galK galT malM

fimA

cstAPeptides

potB

IICGat

srlA

gpsAG3P

Transfer RNA: alaT alaU alaW alaX argQ argV argX argZ cysT glnV glnW glnX glyT glyU glyV glyW glyX glyY ileT ileU leuQ leuV leuW leuX leuZ lysT lysW lysY lysZ metT metU proL serT serU serV thrV tyrT tyrU tyrV thrW valT

valV valW valX valY valZ

Carbon source Strain

wt crp Acetate – Fumarate + – D,L-malate + – Succinate + – Formate* + – Galactitol* + – D--ketoglutarate + – D,L-lactate + – Glycerol + – D,L--glycerol phosphate + – Maltose + – Maltotriose + – Mannose* + Galactose* + D-glucitol + – Trehalose* + L-aspartate + – Glycyl-L-aspartate + – Glycyl-L-glutamate + – L-proline – Tryptophan* + – D-serine +

Oxidation or utilization of various carbon by wild type or crp- E. coli strains

gat (0.2) (0.1)

glp (0.2) (0.2)lct (0.2) (0.1)

mal (0.3) (0.1)

fdo (0.2) (0.4)

man (0.5) (0.3)

mgl (0.2) (0.1)

srl (0.3) (0.2)tre (0.2) (0.1)

aspA (0.4) (0.3)

dsdA (0.2) (0.1)tna (0.05) (0.01)putP (0.4) (0.3)

glp (0.2) (0.2)

crp/wt wtg/wt

mal (0.3) (0.1)

Krebs cycle (0.3) (0.2)

Krebs cycle (0.3) (0.2)

prlC (0.3) (0.4)prlC (0.3) (0.4)

Amino acid biosynthesis:

aspC (0.6) AspartatecysK (0.6) CysteinearoD (0.4) Aromatic amino acids pheL (0.5) PhenylalaninethrL (0.6) Threonineivb ilvB ilvC (0.4) Isoleucine-valine

Multipurpose conversions of intermediates:

aspA (0.3) aspartate ammonia-lyase gcvH gcvP gcvT (0.3) glycine cleavage enzyme complex gloB (0.4) glyoxalase II kbl (0.6) 2-amino-3-ketobutyrate CoA ligasesufB (0.4) cysteine desulfurase activator complex

Degradation of small molecules:

eutB (0.5) ethanolamine ammonia-lyase sdaA (0.6) L-threonine deaminase tdh (0.6) L-threonine dehydrogenase galK (0.5) galactokinase galT (0.4) galactose-1-P uridylyltransferase

malM (0.3) periplasmic protein of mal regulon

Amino acid metabolism

Amino acid import:

cstA (0.4) PeptidesdppA (0.2) DipeptideshisJ (0.4) Histidine gltL (0.6) GlutamatecycA (0.6) Serine, alanine, glycineglnH (0.4) GlutamineproX (0.6) Glycine, betaine, proline putP (0.3) ProlinesdaC (0.6) Serine

Nucleotide biosynthesis: adk (2.2) adenylate kinase (AMP + ATP <=> ADP + ADP)guaA (4.1) GMP synthetasepyrL (3.4) pyrB operon leader peptide

pyrB catalytic subunit of aspartate carbamoyltransferase

Salvage pathway of purine and pyrimidine: deoB (0.4) phosphopentomutase deoD (0.4) purine nucleoside phosphorylase hpt (2.1) guanine phosphoribosyltransferasegpt (2.9) xanthine phosphoribosyltransferase apt (2.9) adenine phosphoribosyltransferaseupp (2.5) uracil phosphoribosyltransferasetdk (3.0) thymidine kinase / deoxyuridine kinase

Nucleic acid metabolism

Purine nucleotides de novo biosynthesis

Pyrimidine nucleotides de novo biosynthesis

Nucleoside import:

tsx (0.3) protein involved with the permeation of ribo- and deoxy-nucleosides

salvage pathways of pyrimidine ribonucleotides

(deoxy)ribose phosphate degradation

salvage pathways of guanine, xanthine, and their nucleosides

salvage pathways of pyrimidine deoxyribonucleotides

Degradation of proteins: prlC (0.4) oligopeptidase AhflX (0.4) possible regulator of HflKC clpA (0.5) ATP-binding component of serine protease hflB (0.5) peptidase that degrades sigma 32 hflK (0.5) regulator of FtsH protease pepD (0.6) peptidase DpepN (0.6) aminopeptidase NclpX (0.6) component of ClpP serine protease

Heat shock proteins and chaperones:ibpA, ibpB (0.07) small heat shock proteins hslU, hslV (0.3) HslVU proteasehtpX (0.4) integral membrane heat shock protein ftsJ (0.3) heat shock protein RrmJ dnaK, grpE (0.3) components of the DnaJ/DnaK/GrpE chaperone systemgroE (0.3) GroES chaperone mopA (0.3) GroEL chaperonehslO (0.6) chaperone Hsp33

Protein metabolism

Transcription functions:

rpoE (0.6) sigma E rpoS (0.4) sigma 38 rpoD (0.5) sigma 70 nusB (0.6) transcription termination factor mfd (0.5) transcription-repair coupling factor greA (3.4) transcription elongation factor rpoB, rpoA (2.3) α and β subunits of RNA Pol. nusA (2.7) transcription pausing factornusG (2.4) component in transcription

antitermination

Cell division: minC, minD (0.4) cell division inhibitor and

membrane ATPase of the MinC-MinD-MinE and DicB-MinC pathways of inhibition of cell division

Cell division and transcription

Proteins - translation and modification: ppiC (2.2) peptidyl-prolyl cis-trans isomerase C efp (2.5) elongation factor P infA, infB, infC (2.4) protein chain initiation factors IF 1-3 tsf (2.7) protein chain elongation factor EF-Ts

Transfer RNA: alaT alaU alaW alaX (3.5)argQ argV argX argZ (2.5)cysT (2.2)glnV glnW glnX (2.4)glyT glyU glyV glyW glyX glyY (3.2)ileT ileU (2.2)leuQ leuV leuW leuX leuZ (2.2)lysT lysW lysY lysZ (2.2)metT metU (2.4)proL (2.1)serT serU serV (3.0)thrV thrW (2.3)tyrT tyrU tyrV (2.5)valT valV valW valX valY valZ (2.7)

Ribosome constituents: rplB rplC rplD rplI rplK rplM rplP rplR rplS rplV rplW rplY rpmA rpmC rpmD rpmE rpmF rpmG rpmH rpmI (2.7)

rpsB rpsC rpsD rpsE rpsF rpsG rpsI rpsJ rpsN rpsO rpsP rpsQ rpsR rpsS rpsT rpsU (2.9)

50S ribosomal subunit proteins

30S ribosomal subunit proteins

Protein synthesis

Aerobic respiration

Aerobic respiration: nuoABCEFHIJKLN (0.4) NADH dehydrogenase I

AcCoApta

Ac~PackA

AcetateADP ATP

LB medium LB medium + glucose

Import of a wide variety of carbon sources and small molecule degradation

Nucleic acids and amino acids are imported and used as carbon sources and building blocks

Active gluconeogenesis

Protein degradation and refolding

Partial heat shock response

Catabolic repression of small molecule import and degradation

Repression of protein degradation

Nucleic acids and amino acids are synthesized from glucose

Active glycolysis

Increased RNA synthesis capacity

Increased protein synthesis capacity

What transcriptional factors are controlling this response?

Transcriptional factors involved in the response to glucose in Escherichia coli

Of 380 genes responding to glucose,133 have detailed regulatory information.

37 different transcripcional factors are involved.

Cluster analysis

Comparison of WTg/WT vs CRP/WT ratios

crp/wt wtg/wt

Regulatory network analysis

133 genes37 TFs

FIS

IHF

CRP

RPOH

FNR

ARCA

NARL

Modular organization of the RN

A(i,j)=1/M(i,j)2

Network clustering

dusB-fis

PdhR + pyruvate = PdhR-pyruvate

FruR + fructose-1-6-bisP =

FruR-fructose-1-6-bisP

CRP

Sigma32

IHF

NtrC

OxyR

ArcA

PdhR

FNR

Fur

SoxS

H-NS

FlhD

OmpR

Fis

MarA

Rob

SoxS

MarR

Mlc

FruR

GLUCOSE

Transport

Metabolism(pyruvate,

fructose-1-6-bisP)

cAMP

-

PTS

-

Increased growth rate

+

How does the RN senses glucose?

-

+

pyruvatefructose-1-6-bisP

--

+

Glucose-6-P

Is the observed response conserved in other organisms?

What would be the response to non-PTS sugars?

Are the properties of the RN involved in glucose response different from the complete RN?

Can this analysis help in finding the functions of the hypotetical genes (77 29 )?

Can this information be used for the improvement of industrial production strains?

QUESTIONS

GlucoseInformation transfer --> Protein related --> Translation

Information transfer --> RNA related --> tRNA

Information transfer --> Protein related --> Chaperone, folding

Metabolism --> Energy metabolism (carbon) --> Tricarboxylic acid cycle

Metabolism --> Carbon compound utilization --> Carbohydrate transport Metabolism --> Energy metabolism (carbon) --> Pentose phosphate shuntMetabolism --> Building block biosynthesis --> Amino acid biosynthesis --> Glutamate

Metabolism --> Macromolecule degradation --> Protein/peptide/glycopeptide

Cell processes --> Adaptation to stress --> Temperature extremes

Glucose

Global analysis of nutrient control of gene expression in Saccharomyces cerevisiae during growth and starvation

Wu et al. PNAS, 2004, 101:3148–3153

Transport --> Substrate transported --> Glucose

Osbaldo Resendiz (CCG-UCSD)

PARTICIPANTES

Julio Collado (CCG)

Julio Freyre (CCG)

Milton H. Saier (UCSD)

Guillermo Gosset (IBT)

Rosa María Gutiérrez (IBT)

Zhongge Zhang (UCSD)

Gracias