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Lecture 3: Bacterial Fueling Metabolism

Lecture 3: Bacterial Fueling Metabolism. Reading assignments in Text: Lengeler et al. 1999 Text: pages 114-116, 123-128 Central metabolism Text: pages 52-58 Substrate level phosphorylation Text: pages 62-67 Electrontransport-coupled phosphorylation Text: pages 296-307 Fermentation

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Lecture 3: Bacterial Fueling Metabolism

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  1. Lecture 3: Bacterial Fueling Metabolism Reading assignments in Text: Lengeler et al. 1999 Text: pages 114-116, 123-128 Central metabolism Text: pages 52-58 Substrate level phosphorylation Text: pages 62-67 Electrontransport-coupled phosphorylation Text: pages 296-307 Fermentation Text: pages 263-266 Oxygen and metabolism Text: pages 524 Energy generation Lecture 2 Text: pages 343-352 DNA replication Text: pages 362-368, 441 RNA transcription Text: pages 369-376 Translation Lecture 1 Text: pages 110-113 Metabolic overview Text: pages 568-569, 573-574 Pili (Fimbriae) and flagella Text: pages 825-829 Surface virulence factors

  2. Recap Replication RNA Protein Transcription Translation Fuelling Biosyn. Polymer. Assembly DNA Streptomycin Tetracycline Nalidixic acid Chloramphenicol Novobiocin Erythromycin Metabolism = Lecture 1 Lecture 2 Pili Flagella Rifampicim

  3. What makes a good Antibiotic ? Good Great Target heirarchy 1 Distinguish 2 Effective Block ? 3 Cause danger Bacteristatic Bactericidal Gene families (PBP’s, Topo’s) Major assemblies Minor assemblies Unique genes (most, e.g. biosyn.) General Biosyn.

  4. RNA and Protein synthesis, “run out experiment” RNA Protein E. coli Add radio-label cpm RNA Protein cpm 0 time 3-H radio-label uridine 14-C radio-label Amino acids Measurements: Sample culture Precipitate polymer (TCA) Collect, place in fluoro-phore Scintillation counter

  5. RNA and Protein synthesis, “run out experiment” RNA Protein Add radio-label E. coli Plus Rifampicin Protein uses mRNA RNA Un-stable RNA Protein Rapid 1-2 min half-lives cpm Stable RNA ~ 50% rRNA, tRNA 3-H radio-label uridine 14-C radio-label Amino acids 0 5 10 15 min

  6. Polymerization without a nucleus protein Rapid response membrane DNA RNA Who needs a nucleus ? “prokaryotes” vs “eukaryotes”

  7. Overview of Metabolism Lecture 1 Lecture 2 Fuelling Pili Flagella DNA, RNA Protein 12 MP’s Met. Precursors ( 2C - 6C units) eTS (electron- Transp. Sys.) Reducing Power NADPH NADH Foods: glucose, ribose, acetate, ... Lectures 3,4 ATP F0F1 ATPase Biosyn. Polymer. Assembly PMF (Proton Motive Force) CM Central Metabolism PM Peripheral Met. Strange foods: oils, benzene, pesticides, ... “oxidative phosphorylation” “substrate-level” = Glycolysis (EMP Pw) Pentose phosphate cycle (PPC) Citric acid [Kreb’s] cycle (TCA)

  8. “Strange food” 1,2-propane-diol B12-dependent degradation Protein shells encasing four enzymes Possibly sequester toxic propion-aldehyde intermediate Found in 30 of 209 sequenced bacterial genomes “Polyhedral organelles” Salmonella enterica 150 nm

  9. Central Metabolism / 12 Metabolic Precursors Pentose phosphate cycle Pentose 5-P (5C) Erythrose 4-P (4C) Fumarate Succinate Malate Succinyl~CoA Oxaloacetate a-Ketoglutarate Acetyl~CoA Citrate Isocitrate Biosynthesis (see Text: page 115) Glycolysis (EMP Pw) Glucose Glucose-6-P (6C) Fructose-6-P Fructose 1,6-P Triose 3-P (2x 3C) 1, 3-Diphosphoglycerate Citric acid cycle (TCA) 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate Pyruvate

  10. Central Metabolism / ATP and Reducing Power ATP Biosynthesis Glycolysis (EMP) Glucose Pentose phosphate cycle Glucose-6-P (6C) -ATP Fructose-6-P Pentose 5-P (5C) Fructose 1,6-P Erythrose 4-P (4C) ATP Triose 3-P (2x 3C) F0F1 Citric acid cycle (TCA) 1, 3-Diphosphoglycerate +ATP Fumarate Malate 3-Phosphoglycerate Succinate 2-Phosphoglycerate Succinyl~CoA Oxaloacetate Phosphoenolpyruvate +ATP a-Ketoglutarate Pyruvate Acetyl~CoA Citrate Isocitrate mitochondria “substrate-level” NADH NADPH NADH NADH FADH 2 +GTP NADH NADH NADH NADH

  11. electron Transport Systems (eTS) Generate Proton Motive Force (PMF) H+ 2e- NAD+ DG acceptor = O2 H2O 2 [H+ e- ] 2 H+ 2 H+ out PMF E. coli 2e- in H2O 2 H+ 2 H+ 1/2 O2 NAD+ PMF NADH Anaerobic respiration eTS Nitrate, sulfate, fumerate, dyes, legnins, etc... eTS = Flavine proteins, FeS-proteins, Cytochromes /heme-proteins, Quinones NADH H+

  12. ATP from PMF F0 eTS ATP Rotary engine F1 ADP + P i H+ H+ out PMF in

  13. Metabolic flexibility Ability to grow on just one C-source. Ability to grow without oxygen. “Anaerobic respiration” “Fermentation”

  14. E. coli growth on Glucose as sole C-source plus oxygen NADPH eTS NADH ? +ATP A NADH NADH NADH eTS eTS eTS +ATP CO2 ATP Phosphoenolpyruvate Oxaloacetate A = anapleurotic Rxn “PEP carboxylase” Pentose phosphate cycle Glucose-6-P (6C) Pentose 5-P (5C) Fructose-6-P Erythrose 4-P (4C) Fructose 1,6-P Triose 3-P (2x 3C) 1, 3-Diphosphoglycerate Citric acid /Kreb’s “cycle” ? 3-Phosphoglycerate Fumarate Succinate Malate 2-Phosphoglycerate Succinyl~CoA Oxaloacetate Phosphoenolpyruvate a-Ketoglutarate Pyruvate Acetyl~CoA Citrate Isocitrate

  15. Fueling without oxygen / Fermentation Bug PH2 ATP NADH acid / alcohol Often: S = sugar P = [ pyruvate] Simplest: EMP Pw glucose pyruvate EMP Pw Ethanol glucose pyruvate Beer/bread CO2 poor energy poor growth yield More process than consumption (Food/ Industrial applications) (Abstract) ? NAD+ P S Lactate dehydrogenase Lactic acid Yogurt Points: ATP “substrate level” ~redox balance, C e-donors / C e-acceptors Most C passes through cell without incorporation

  16. E. coli growth on Glucose as sole C-source but No oxygen No electron acceptors Fermentation Anapleurotic reaction strategies: Reach all 12 Metabolic precursors Produce less NADH Balance NADH production with consumption

  17. E. coli growth on Glucose as sole C-source but No oxygen Pentose phosphate cycle Glucose-6-P (6C) Pentose 5-P (5C) Fructose-6-P Erythrose 4-P (4C) Fructose 1,6-P Triose 3-P (2x 3C) NADH Citric acid Kreb’s “cycle” 1, 3-Diphosphoglycerate A5 A4 3-Phosphoglycerate Fumarate Aspartate Succinate A3 Malate 2-Phosphoglycerate Succinyl~CoA A1 Oxaloacetate Phosphoenolpyruvate A2 a-Ketoglutarate NADH Pyruvate Acetyl~CoA Citrate Isocitrate A3 = Asp synthetase Anapleurotic reactions: A1 = Pep carboxylase (as before) A4 = Asp deaminase A2 = Pyruvate formate lyase (no NADH) A5 = Fumarate reductase No synthetase, no NADH

  18. Non-fermentable C-sources Fermentable C-sources Non-fermentable C-sources Other anaerobic metabolisms CM E.g. fermentation products E.g. methanogens

  19. Dumping excess electrons (NAD+ from NADH) Bug PH2 ATP NADH CO2 eTS H2 Dissimulatory reduction Assimilatory reduction ? NAD+ Fermentation P S = anaerobic respiration e.g. nitrate > nitrite > ammonium > nitrogen gas

  20. Overview of Metabolism Lecture 1 Lecture 2 Fuelling Pili Flagella DNA, RNA Protein Lectures 3,4 ATP Biosyn. Polymer. Assembly [1-C units] 12 MP’s Met. Precursors ( 2C - 6C units) eTS (electron- Transp. Sys.) Reducing Power NADPH NADH Foods: glucose, ribose, acetate, ... F0F1 ATPase PMF (Proton Motive Force) Strange foods: oils, benzene, pesticides, ... “oxidative phosphorylation” “substrate-level” = Glycolysis (EMP Pw) CM Central Metabolism Pentose phosphate cycle (PPC) PM Peripheral Met. Citric acid [Kreb’s] cycle (TCA)

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