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Transcriptome and regulatory network analysis of the
response to glucose and catabolite repression in
Escherichia coli
Escherichia coli
Osmolarity
O2
Nutrients
pH
Objective
Carbon Catabolite Repression in Escherichia coli
Sugar C
Sugar B
Biomass
Glucose
Time
A (17)
Lactose H+
Inducer
exclusion
IIC (20)
Inducer
exclusion
(19)
(21)
IIA IIB
Lactose H+
-
A~P
PEP
Pyruvate
EI
Hpr~P
IIAGlc
EI~P
Hpr
IIAGlc~P
P
IIB
IIC
Glucose
Glucose 6-P
+
-10
ATP
-35
RNA Pol. CRP
Cytoplasm
Cytoplasmic
membrane
Periplasm
AMPc
AMPc
Adenilate
cyclase
Transcriptional
control
PTS sugars: Galactosamine, N-acetylgalactosamine,
arbutin, cellobiose, salicin, Di-N-acetylchitobiose,
dihydroxyacetone, fructose, galactitol, glucitol,
maltose, mannose, glucose, glucosamine, mannitol,
acetylglucosamine and trehalose.
Absence of PTS sugars
Glucose
Glucose-6-P
P- IIBCGlc
PTS -P
PEP
IIBCGlc
PTS -P
PEP
P
PIR
ATP
IIAGlc
IIAGlc
PIR
ATP
AC
CPD
AMPc
AC
CPD
AMP
AMPc
AMP
?
>99.9% AMPc
?
AMPc
cAMP receptor protein (CRP)
TGTGAGTTAGCTCACT
-Catabolic repression.
-Gluconeogenesis.
-Flagellum synthesis.
-Coordination of DNA replication and cell division.
-Glycogen metabolism.
-Antibiotic resistance.
-Toxin production.
Steve Busby and Richard Ebright
Glucose
Complex medium
Complex medium
+
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.
+
+
+
+
+
-
-10 -35
-10 -35
-10 -35
-10 -35
Cell
functions
-10 -35
-10 -35
-10 -35
?
-10 -35
?
-10 -35
?
-10 -35
?
-10 -35
?
CRP
AMPc
-
-
Glucose
?
?
TF? Metabolite?
Análisis de transcriptoma
GENOMA
RNA
Transcriptoma
PROTEINAS
METABOLISMO
Proteoma
Metaboloma
Fluxoma
Microarreglos de DNA
Cromosoma
Cultivo bacteriano
Extracción de RNA
Síntesis de cDNA y marcado
Promotor
Síntesis
[RNA]
Degradación
Escherichia coli BW25113
Escherichia coli BW25113 crp-
(WT)
(CRP)
Luria Bertani medium (LB)
Component
Aminoacids
Nucleic acids
Vitamins
Carbohydrates (5-10%)
No lipids
Glucose (0.003%)
LB
Tryptone
Yeast Extract
NaCl
Glucose
1
WT
LB+G
10 g/L
5
10
-
2
3
4
WTg
CRP
CRPg
10 g/L
5
10
4
LB
Experimentos por triplicado
4
wt
2
c rp
25 ml cultivo OD 0.5
O D 600
1
0 .5
Filtrar
0 .2 5
0 .1 2 5
0 .0 6 2 5
N2 líquido
0
1
2
3
4
5
6
7
8
T im e ( h )
Rompimiento
Extracción con fenol
L B + g lu c o s e
wt
4
RNA crudo
c rp
O D 600
2
1
Kit Stratagene
0 .5
0 .2 5
RNA puro
0 .1 2 5
0 .0 6 2 5
0
1
2
3
4
5
6
7
8
T im e ( h )
LB
LB+G
E. coli BW25113
37 min
35 min
5%
E. coli BW25113 crp-
43 min
41 min
5%
Microarreglos Afymetrix
Microarreglos Afymetrix
Oligonucleótidos de 25 bases (296,936 ).
11-20 oligos /gene
AvgDiff =
2 tipos de oligos:
N PM - MM
N
Perfect Match (PM)
MissMatch (MM)
4,327 ORF
2,885 intergénicos
PM
MM
Nucleic Acids Research, 2002, Vol. 30, No. 17 3732-3738
Pair-wise comparison of triplicate data sets
WT1
WT2
WT2
WT3
WT3
4,327 ORFs.
Affymetrix data reliability filter
WT
WTg
1,908
WTg/WT
1,910
CRP/WT
CRP
CRPg
3,083
1,910
CRPg/CRP
CRPg/WTg
Outlier iteration method
WTg/WT
380
8.8%
CRP/WT
333
7.7%
CRPg/CRP
271
6.3%
CRPg/WTg
298
6.9%
Expression ratios for genes responding to glucose in WTg/Wt
12
spf (11.2)
8
6
4
2
0
0
20
40
60
80
100
120
140
160
180
Number of genes
0.7
0.6
WTg/Wt ratio
fis (6.9)
WTg/Wt ratio
10
0.5
0.4
0.3
0.2
0.1
0
0
20
40
60
80
100
120
Number of genes
140
160
180
200
glpF (0.04)
tnaL (0.01)
Number of Number of
Functional class
genes
genes
induced
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
Glucitol
IIC1Gut
fimA
IIAGat
potA
gatA
IIBGlc ptsG
IIAGut
srlB Gut6P
Gat1P
G6P
IIAGlc ~P crr
srlD
Amino acid biosynthesis: aspC cysK
aroD pheL thrL ivbL ilvB ilvC
pitA
Phosphate
zntA
Zinc
G3P
gatY
gatZ
Nucleotide biosynthesis: adk guaA pyrL
tnaB
Trp
Trp
tnaCA
Indole
glpX
F1,6BP
+ PYR + NH3
lamB
ICIT
dppA
Histidine
hisJ
mdh
Degradation of proteins: prlC hflX clpA hflB hflK pepD pepN
clpX
aceA
Proteins - translation and modification: ppiC efp infA infB infC tsf
AKG
sucB
fumA
FUM
sdhADC
Acetate
Acetate
icdA
MAL
cstA
Dipeptides
Ribose
Multipurpose conversions of intermediates: aspA gcvH gcvP gcvT
gloB kbl sufB
ackA
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
acnB
OAA
malE
Ac~P
SUCCoA
SUC
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
Protein translocation: prlA secE secG yidC
rbsB
gltL
Glutamate
putP
Peptides
gltA
malX
Lactate
cycA
glnH
proX
Serine/
alanine/
glycine
Glutamine
Glycine/
betaine/
proline
Lactate
fliO
tsx
Maltose
pta
CIT
Degradation of small molecules: eutB sdaA tdh galK galT
malM
manX
Cell division: ftsJ minC minD
ldhA
PYR
aceE
aceF
AcCoA
maeB
IIBMal
EI~P ptsI
ppsA
pckA
IIABMan
Heat shock proteins and chaperones: ibpA ibpB hslU hslV htpX
dnaK grpE groE mopA hslO
PEP
MG
IICMal
Regulatory proteins: dps lon uspA cytR crp ykgA hcaR
fis glnB marA
Hpr~P ptsH
DHA + GAP
mgsA
Salvage pathway of purine and pyrimidine:
deoB deoD hpt gpt upp apt tdk
mgtA
Mg2+
gpsA
Maltose/
glucose
Transcription functions: rpoE rpoS rpoD nusB mfd
greA rpoB rpoA nusA nusG
F6P
Tag1,6BP
Biosynthesis of cofactors: entC ispA
moaB nrdH gshA bioH folC menE trxA
IICMan
IICGlc
srlA
potB
IICGat
Mannose/glucose/
glucosamine/fructose
manY
Galactitol
ompF
Spermidine/
putrescine
Glycerol
Proline
Nucleoside
channel
Serine
Oxidation or utilization of various carbon by wild type or crp- E. coli strains
crp/wt wtg/wt
Krebs cycle (0.3) (0.2)
fdo (0.2) (0.4)
gat (0.2) (0.1)
Krebs cycle (0.3) (0.2)
lct (0.2) (0.1)
glp (0.2) (0.2)
glp (0.2) (0.2)
mal (0.3) (0.1)
mal (0.3) (0.1)
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)
prlC (0.3) (0.4)
prlC (0.3) (0.4)
putP (0.4) (0.3)
tna (0.05) (0.01)
dsdA (0.2) (0.1)
Carbon source
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
Strain
wt

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
crp
–
–
–
–
–
–
–
–
–
–
–
–


–

–
–
–
–
–

Amino acid metabolism
Amino acid import:
Degradation of small molecules:
cstA (0.4)
dppA (0.2)
hisJ (0.4)
gltL (0.6)
cycA (0.6)
glnH (0.4)
proX (0.6)
putP (0.3)
sdaC (0.6)
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
Peptides
Dipeptides
Histidine
Glutamate
Serine, alanine, glycine
Glutamine
Glycine, betaine, proline
Proline
Serine
Multipurpose conversions of intermediates:
Amino acid biosynthesis:
aspA (0.3)
gcvH gcvP gcvT (0.3)
gloB (0.4)
kbl (0.6)
sufB (0.4)
aspC (0.6)
cysK (0.6)
aroD (0.4)
pheL (0.5)
thrL (0.6)
ivb ilvB ilvC (0.4)
aspartate ammonia-lyase
glycine cleavage enzyme complex
glyoxalase II
2-amino-3-ketobutyrate CoA ligase
cysteine desulfurase activator complex
Aspartate
Cysteine
Aromatic amino acids
Phenylalanine
Threonine
Isoleucine-valine
Nucleic acid metabolism
Nucleoside import:
tsx (0.3) protein involved with the permeation of ribo- and deoxy-nucleosides
Salvage pathway of purine and pyrimidine:
deoB (0.4) phosphopentomutase
deoD (0.4) purine nucleoside phosphorylase
hpt (2.1) guanine phosphoribosyltransferase
gpt (2.9) xanthine phosphoribosyltransferase
apt (2.9) adenine phosphoribosyltransferase
upp (2.5) uracil phosphoribosyltransferase
tdk (3.0) thymidine kinase / deoxyuridine kinase
(deoxy)ribose phosphate degradation
salvage pathways of guanine, xanthine, and their nucleosides
salvage pathways of pyrimidine ribonucleotides
salvage pathways of pyrimidine deoxyribonucleotides
Nucleotide biosynthesis:
adk (2.2) adenylate kinase (AMP + ATP <=> ADP + ADP)
guaA (4.1) GMP synthetase
pyrL (3.4) pyrB operon leader peptide
pyrB catalytic subunit of aspartate carbamoyltransferase
Purine nucleotides
de novo biosynthesis
Pyrimidine nucleotides
de novo biosynthesis
Protein metabolism
Degradation of proteins:
prlC (0.4)
oligopeptidase A
hflX (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 D
pepN (0.6)
aminopeptidase N
clpX (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 protease
htpX
(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 system
groE
(0.3) GroES chaperone
mopA
(0.3) GroEL chaperone
hslO
(0.6) chaperone Hsp33
Cell division and transcription
Cell division:
Transcription functions:
minC, minD (0.4) cell division inhibitor and
membrane ATPase of the
MinC-MinD-MinE and
DicB-MinC pathways of
inhibition of cell division
rpoE (0.6)
rpoS (0.4)
rpoD (0.5)
nusB (0.6)
mfd (0.5)
greA (3.4)
rpoB, rpoA (2.3)
nusA (2.7)
nusG (2.4)
sigma E
sigma 38
sigma 70
transcription termination factor
transcription-repair coupling factor
transcription elongation factor
α and β subunits of RNA Pol.
transcription pausing factor
component in transcription
antitermination
Protein synthesis
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
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)
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
Aerobic respiration
Aerobic respiration:
nuoABCEFHIJKLN (0.4)
NADH dehydrogenase I
AcCoA
pta
Ac~P
ackA
Acetate
ADP ATP
LB medium
Import of a wide variety of carbon
sources and small molecule
degradation
LB medium + glucose
Catabolic repression of small molecule
import and degradation
Repression of protein degradation
Nucleic acids and amino acids are
imported and used as carbon sources
and building blocks
Nucleic acids and amino acids are
synthesized from glucose
Active gluconeogenesis
Active glycolysis
Protein degradation and refolding
Increased RNA synthesis capacity
Partial heat shock response
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 genes
37 TFs
FIS
FNR
ARCA
RPOH
CRP
NARL
IHF
Modular organization of the RN
Network clustering
A(i,j)=1/M(i,j)2
dusB-fis
PdhR + pyruvate = PdhR-pyruvate
FruR + fructose-1-6-bisP =
FruR-fructose-1-6-bisP
How does the RN senses glucose?
fructose-1-6-bisP
GLUCOSE
pyruvate
-
FruR
Transport PTS
PdhR
FNR
+
Glucose-6-P
-
Mlc
cAMP
ArcA
SoxS
OmpR
OxyR
NtrC
Fur
CRP
Metabolism
(pyruvate,
fructose-1-6-bisP)
+
Fis
FlhD
+
Sigma32
SoxS
IHF
H-NS
Increased growth rate
MarR
Rob
MarA
QUESTIONS
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?
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
Glucose
Information transfer --> Protein related --> Translation
Information transfer --> RNA related --> tRNA
Metabolism --> Energy metabolism (carbon) --> Tricarboxylic acid cycle
Glucose
Metabolism --> Carbon compound utilization --> Carbohydrate transport
Metabolism --> Energy metabolism (carbon) --> Pentose phosphate shunt
Metabolism --> Building block biosynthesis --> Amino acid biosynthesis --> Glutamate
Metabolism --> Macromolecule degradation --> Protein/peptide/glycopeptide
Cell processes --> Adaptation to stress --> Temperature extremes
Information transfer --> Protein related --> Chaperone, folding
PARTICIPANTES
Julio Collado
(CCG)
Julio Freyre
(CCG)
Osbaldo Resendiz
(CCG-UCSD)
Guillermo Gosset
(IBT)
Milton H. Saier
(UCSD)
Gracias
Rosa María Gutiérrez
(IBT)
Zhongge Zhang
(UCSD)