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Escherichia coli

Transcriptome and regulatory network analysis of the response to glucose and catabolite repression in Escherichia coli. Escherichia coli. Osmolarity. Nutrients. O 2. pH. Objective. Carbon Catabolite Repression in Escherichia coli. Sugar C. Sugar B. Biomass. Glucose. Time. A (17).

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Escherichia coli

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

  2. Escherichia coli

  3. Osmolarity Nutrients O2 pH Objective

  4. Carbon Catabolite Repression in Escherichia coli Sugar C Sugar B Biomass Glucose Time

  5. A (17) Lactose H+ Inducer exclusion IIC (20) Inducer exclusion LacY IIB (21) IIA + (19) Lactose H+ A~P - ATP -10 -35 AMPc AMPc RNA Pol. Transcriptional control CRP Adenilate cyclase IIAGlc EI EI~P Hpr~P Hpr PEP Pyruvate P IIB IIC Glucose IIAGlc~P Glucose 6-P Cytoplasm Cytoplasmic membrane Periplasm

  6. P -P -P PTS PTS PEP PEP PIR PIR CPD AC AC CPD AMP AMP 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 IIBCGlc IIAGlc IIAGlc ATP ATP AMPc AMPc ? ? >99.9% AMPc AMPc

  7. 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

  8. Complex medium + Glucose Glucose Complex medium 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.

  9. + -10 -35 -10 -35 -10 -35 -10 -35 -10 -35 -10 -35 -10 -35 -10 -35 -10 -35 -10 -35 -10 -35 + + + + - - - AMPc CRP Cell functions Glucose ? ? ? ? ? TF? Metabolite? ? ?

  10. Análisis de transcriptoma RNA GENOMAPROTEINAS METABOLISMO Metaboloma Fluxoma Transcriptoma Proteoma

  11. Microarreglos de DNA Cromosoma

  12. Cultivo bacteriano Extracción de RNA Síntesis de cDNA y marcado

  13. Promotor Síntesis [RNA] Degradación

  14. Escherichia coli BW25113 (WT) Escherichia coli BW25113 crp- (CRP) Luria Bertani medium (LB) Component LB LB+G Tryptone 10 g/L 10 g/L Yeast Extract 5 5 NaCl 10 10 Glucose - 4 Aminoacids Nucleic acids Vitamins Carbohydrates (5-10%) No lipids Glucose (0.003%) 1 2 3 4 WT WTg CRP CRPg

  15. 25 ml cultivo OD 0.5 Filtrar N2 líquido Rompimiento Extracción con fenol RNA crudo Kit Stratagene RNA puro Experimentos por triplicado LB LB+G E. coli BW25113 37 min 35 min E. coli BW25113 crp- 43 min 41 min 5% 5%

  16. Microarreglos Afymetrix

  17. N PM - MM N AvgDiff = Microarreglos Afymetrix 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 PM MM Nucleic Acids Research, 2002, Vol. 30, No. 17 3732-3738

  18. Pair-wise comparison of triplicate data sets WT1 WT2 WT2 WT3 WT3

  19. 4,327 ORFs. Affymetrix data reliability filter WT WTg CRP CRPg 1,908 1,910 3,083 1,910 WTg/WT CRP/WT CRPg/CRP CRPg/WTg Outlier iteration method WTg/WT CRP/WT CRPg/CRP CRPg/WTg 380 333 271 298 8.8% 7.7% 6.3% 6.9%

  20. Expression ratios for genes responding to glucose in WTg/Wt spf (11.2) fis (6.9) glpF (0.04) tnaL (0.01)

  21. 180 200

  22. Maltose/ glucose Galactitol fimA ompX IICMal IIBMal IIAGat gatA malX Gat1P tnaCA Indole + PYR + NH3 lamB dppA malE glpF Glycerol Glucose Mannose/glucose/ glucosamine/fructose Spermidine/ putrescine Glucitol ompF IICMan IIC1Gut IICGat potD IICGlc potB manY srlA IIBGlc IIABMan potA ptsG IIAGut Gut6P manX srlB G6P srlD IIAGlc~P crr Transcription functions: rpoE rpoS rpoD nusB mfd greA rpoB rpoA nusA nusG Amino acid biosynthesis: aspC cysK aroD pheL thrL ivbL ilvB ilvC pitA Phosphate G3P F6P Tag1,6BP gpsA glpX Regulatory proteins: dps lon uspA cytR crp ykgA hcaR fis glnB marA F1,6BP Biosynthesis of cofactors: entC ispA moaB nrdH gshA bioH folC menE trxA Hpr~P ptsH gatY gatZ zntA Zinc DHA + GAP Heat shock proteins and chaperones: ibpA ibpB hslU hslV htpX dnaK grpE groE mopA hslO Nucleotide biosynthesis: adk guaA pyrL mgsA PEP EI~P ptsI Salvage pathway of purine and pyrimidine: deoB deoD hpt gpt upp apt tdk Cell division: ftsJ minC minD ppsA mgtA Mg2+ MG ldhA Lactate PYR aceE aceF pta ackA AcCoA Acetate Ac~P Acetate pckA tnaB Trp Trp maeB 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 CIT acnB Degradation of small molecules: eutB sdaA tdh galK galT malM gltA ICIT OAA Maltose icdA Multipurpose conversions of intermediates: aspA gcvH gcvP gcvT gloB kbl sufB Proteins - translation and modification: ppiC efp infA infB infC tsf mdh aceA AKG cstA MAL Peptides sucB 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 Degradation of proteins: prlChflXclpAhflBhflKpepDpepNclpX fumA Dipeptides FUM SUCCoA sdhADC SUC Histidine hisJ Protein translocation:prlA secE secG yidC rbsB Ribose fliO putP lctP sdaC gltL cycA proX glnH tsx Glutamate Lactate Serine/ alanine/ glycine Glutamine Proline Serine Glycine/ betaine/ proline Nucleoside channel

  23. 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)

  24. Amino acid metabolism Amino acid import: cstA (0.4)Peptides dppA (0.2)Dipeptides hisJ (0.4)Histidine gltL (0.6)Glutamate cycA (0.6)Serine, alanine, glycine glnH (0.4)Glutamine proX (0.6) Glycine, betaine, proline putP (0.3)Proline sdaC (0.6)Serine 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 biosynthesis: aspC (0.6)Aspartate cysK (0.6)Cysteine aroD (0.4)Aromatic amino acids pheL (0.5)Phenylalanine thrL (0.6)Threonine ivb 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 ligase sufB (0.4)cysteine desulfurase activator complex

  25. 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

  26. 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

  27. Cell division and transcription 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 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 factor nusG (2.4)component in transcription antitermination

  28. 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 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

  29. ackA pta AcCoA Ac~P Acetate ADP ATP Aerobic respiration Aerobic respiration: nuoABCEFHIJKLN (0.4)NADH dehydrogenase I

  30. 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

  31. What transcriptional factors are controlling this response?

  32. 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.

  33. Cluster analysis Comparison of WTg/WT vs CRP/WT ratios

  34. crp/wt wtg/wt

  35. Regulatory network analysis

  36. 133 genes 37 TFs

  37. FIS

  38. FNR ARCA RPOH CRP NARL IHF

  39. Modular organization of the RN

  40. Network clustering A(i,j)=1/M(i,j)2

  41. dusB-fis

  42. PdhR + pyruvate = PdhR-pyruvate FruR + fructose-1-6-bisP = FruR-fructose-1-6-bisP

  43. FNR - - - - - + + + How does the RN senses glucose? fructose-1-6-bisP pyruvate GLUCOSE FruR Transport PTS PdhR Glucose-6-P ArcA Mlc SoxS OmpR OxyR cAMP NtrC Fur CRP FlhD Metabolism (pyruvate, fructose-1-6-bisP) Sigma32 Fis SoxS IHF H-NS Increased growth rate MarR MarA Rob

  44. 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?

  45. Global analysis of nutrient control of geneexpressionin Saccharomyces cerevisiae during growthand 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 Metabolism --> Carbon compound utilization --> Carbohydrate transport Metabolism --> Energy metabolism (carbon) --> Pentose phosphate shunt Metabolism --> Building block biosynthesis --> Amino acid biosynthesis --> Glutamate Glucose Metabolism --> Macromolecule degradation --> Protein/peptide/glycopeptide Cell processes --> Adaptation to stress --> Temperature extremes Information transfer --> Protein related --> Chaperone, folding

  46. PARTICIPANTES Julio Collado (CCG) Julio Freyre (CCG) Guillermo Gosset (IBT) Rosa María Gutiérrez (IBT) Milton H. Saier (UCSD) Osbaldo Resendiz (CCG-UCSD) Zhongge Zhang (UCSD) Gracias

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