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

2. Escherichia coli

3. Nutrients pH O2O2 Osmolarity Objective

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

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

7. IIA Glc IIBC Glc PEP PIRPIR PTS -P-P AC ATP AMPc ? CPD AMP P-P- P Absence of PTS sugars PTS 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 IIA Glc IIBC Glc PEP PIRPIR PTS -P-P AC ATP AMPc ? CPD AMP Glucose >99.9%

8. -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

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

11. Análisis de transcriptoma GENOMA PROTEINASMETABOLISMO Transcriptoma Proteoma Metaboloma Fluxoma RNA

12. Microarreglos de DNA Cromosoma

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

14. [RNA]SíntesisPromotorDegradación

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

16. 25 ml cultivo OD 0.5 Filtrar N 2 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% LB LB+G

17. Microarreglos Afymetrix

18. 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

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

20. 1,908 1,9103,0831,910 4,327 ORFs. WTWTg CRP CRPg WTg/WTCRP/WTCRPg/CRP CRPg/WTg 380 333 271 298 8.8% 7.7% 6.3% 6.9% Outlier iteration method Affymetrix data reliability filter

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

22. 180 200

23. Glucose IIB Glc ptsG G6P F6P F1,6BP Hpr~P ptsH IIA Glc ~P crr IIC Glc potD Spermidine/ putrescine Phosphate zntA Zinc mgtA Mg 2+ ompF Trp tnaB Trp tnaCA Indole + PYR + NH 3 lamB Maltose Heat shock proteins and chaperones: ibpA ibpB hslU hslV htpX dnaK grpE groE mopA hslO Nucleotide 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 IIAB Man IIC Man manX manY Mannose/glucose/ glucosamine/fructose IIB Mal IIC Mal malX Maltose/ glucose ompX potA PEP PYR EI~P ptsI ppsA AcCoA aceE aceF pckA pta Ac~P ackA Acetate OAA MAL CIT AKG SUCCoA SUC FUM ICIT gltA ldhA sucB sdhADC mdh fumA acnB icdA aceA maeB Salvage pathway of purine and pyrimidine: deoB deoD hpt gpt upp apt tdk glpX Regulatory proteins: dps lon uspA cytR crp ykgA hcaR fis glnB marA IIC1 Gut IIA Gut 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 IIA Gat 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 cstA Peptides potB IIC Gat srlA gpsA G3P 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

24. 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) prlC (0.3) (0.4)

25. 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 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) 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

26. 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 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 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

27. 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 Protein metabolism

28. 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 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

29. 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

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

31. 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

32. What transcriptional factors are controlling this response?

33. 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.

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

37. crp/wt wtg/wt

38. Regulatory network analysis

39. 133 genes 37 TFs

40. FIS

41. IHF CRP RPOH FNR ARCA NARL

42. Modular organization of the RN

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

45. dusB-fis

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

47. 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? - + pyruvate fructose-1-6-bisP - - + Glucose-6-P

48. 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

49. Glucose Information 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 shunt Metabolism --> 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

50. 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