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