Presentation on theme: "Transcriptome and regulatory network analysis of the response to glucose and catabolite repression in Escherichia coli."— Presentation transcript:
Transcriptome and regulatory network analysis of the response to glucose and catabolite repression in Escherichia coli
Nutrients pH O2O2 Osmolarity Objective
Glucose Sugar B Sugar C Carbon Catabolite Repression in Escherichia coli Biomass Time
Transcriptional control Adenilate cyclase ATP AMPc 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)
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%
-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 + 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.
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 BW min 35 min E. coli BW25113 crp - 43 min 41 min 5% LB LB+G
Nucleic Acids Research, 2002, Vol. 30, No Oligonucleótidos de 25 bases (296,936 ) 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 WT2 Pair-wise comparison of triplicate data sets
Aerobic respiration Aerobic respiration: nuoABCEFHIJKLN (0.4)NADH dehydrogenase I AcCoA pta Ac~P ackA Acetate ADP 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 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
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
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
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