1. Lecture 4: Bacterial Fueling and Assimilation Lecture 2 Text: pages 343-352 DNA replication Text: pages 362-368, 441 RNA transcription Text: pages 369-376 Translation Reading assignments in Text: Lengeler et al. 1999 Text: pages 116-122 Assimilation Text: pages 177-182 Assimilation reactions Text: pages 155-157 Storage compounds Lecture 3 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

2. Fermentation products Yeast:Bread, beer, wine... Bacteria:Rye breads Butter milk (Lactobacilli) Sourdough bread Yogurt OlivesSausages“Thousand year” eggs Coffee (husk removal) Sauerkraut Vegetables / mushrooms (Lactobacillus plantarum) Vinegar (acetic acid) Organic acids, ketones, alcohols,... Hydrogen gas

4. AssemblyFuelling Biosyn. Polymer. Lecture 1 Pili Flagella Lecture 2 Lectures 3,4 DNA, RNA Protein CM Central Metabolism PM Peripheral Met. Foods: glucose, ribose, acetate,... Strange foods: oils, benzene, pesticides,... 12 MP’s Met. Precursors ( 2C - 6C units) “substrate-level” ATP [1-C units] NADH = Glycolysis (EMP Pw) Pentose phosphate cycle (PPC) Citric acid [Kreb’s] cycle (TCA) Reducing Power NADPH eTS (electron- Transp. Sys.) PMF (Proton Motive Force) F 0 F 1 ATPase “oxidative phosphorylation” Overview of Metabolism Fermentation N, SP

6. 1-C unit metabolism, Tetra Hydofolate (THF) and sulfa antibiotics Serine (AA) | CH 2 OH + THFGlycine (AA) |H|H + THF || CH 2 “mobile 1-C carrier” Many Biosyn. Rxns Methionine (AA) Purines (A, G) Thiamine (Vit. B1) Thymine (dUMP > dTMP) Coenzyme A, etc... THF = (1 + 2 +3) 1 Pteridine ring +2 p-amino- benzoic acid +3 Glutamate (AA) N NH O || C--O _ NH COO - CH 2 Competitive inhibitor DHF NH 2 SO 2 NH 2 Sulfanilamide 1st antibiotics Folic acid (Food, Vitamin) 2nd path way people bacteria Benefits ? Problems ? Non-competitive inhibitor Trimethoprin

8. COO - H-C-OH HO-C-H 3-C P Entner-Doudoroff Pathway and glycolysis Glycolysis (EMP Pw) Glucose Glucose-6-P (6C) Fructose-6-P Triose 3-P (2x 3C) Fructose 1,6-P 3-Phosphoglycerate 2-Phosphoglycerate Phosphoenolpyruvate Pyruvate 1, 3-Diphosphoglycerate -2 ATP +4 ATP +6 ATP Net: +8 ATP plus 2x NAD(P)H 6-P Gluconate H C=O H-C-OH HO-C-H 3-C P COO - C=O H-C-H 3-C P 2-Keto, 3-deoxy- 6-P Gluconate -H 2 O -1 ATP +3 ATP NADH +3 ATP +2 ATP Net: +7 ATP plus 2x Pyruvate TCA Aerobic glucose utilization NADH 2x

10. Dumping excess electrons (NAD+ from NADH) S P ATPNADH NAD + PH 2 Bug ? eTS Dissimulatory reduction e.g. nitrate > nitrite >> ammonium > nitrogen gas Assimilatory reduction Fermentation H2H2 CO 2 = anaerobic respiration

11. Geobacter sulfurreducens Oxidize Organics CO 2 Fumarate, Malate, Sulfur, Fe(III), Mn(IV) (terminal electron acceptors, “ dissimulatory reduction” ) Dominate many subsurface environments Attach to, and reduce insoluble Sulfur, Fe(III) and Mn(IV) oxides Sulfur

13. N Assimilation NH 3  Ketoglutarate (MP) NADPH Glutamate (AA) GDH GDH = Glutamate Dehydrogenase Glutamine (AA) GS GS = Glutamine Synthetase Biosyn. (big pool) All N-compounds Direct organic sources:R-NH 2 (amino acids)R=N-R’(purines, etc...) ATP NH 3 ? No preferred N-sources: Assimilatory reductases Nitrate Nitrite Nitrous oxideNH 3 Dissimulatory reductases (anaerobic respiration) :N N: gas Fixation Lithotrophic oxidation Nitrogen cycle ~80% atmosphere ~20 Mega-year turnover

15. Nitrogen fixation, conflict with oxygen NH 3 :N N: gas Nitrogenase Proteins (FeS) (Mo) poison Air = ~80 nitrogen (food) ~20% oxygen Four fixation strategies: Cyanobacteria sps. Rhizobia sps. Clostridium pasteurianum (pages 8, 304) Strict anaerobe Azotobacter vinlandii (pages 8, 44) Obligate aerobe soil N2N2 O2O2 air N2N2 O2O2 Size, rapid respiration protect Nitrogenase PS II makes oxygen Cell differentiation into “Heterocyst” Cell differentiation into “Bacteroid” Symbiosis with plants, form nodule organ

16. S Assimilation NH 2 COO - CH 2 Cysteine (AA) SHSH O-Acetyl serine NH 2 COO - CH 2 O-Acetyl SH2SH2 Methionine Lipoic acid Coenzyme A Thiamine, FeS clusters, etc... Anaerobic respiration “Dissimulatory reduction” e.g. Geobacter sp. Geological / volcanic ? Sources: + O 2 SO42-SO42- “Assimilatory reduction” 3 ATP 4 NADPH [S-P-5’Adenosine 3’P] Adenosine phosphosulfate carrier

18. P Assimilation PO43-PO43- free R-O-PO 3 2 - esters IM PS OM(-) porins Alkaline Phosphatase 5’ Nucleosidases, etc... PO43-PO43- R-OH PO43-PO43- PO43-PO43- H+ + ADP ATP F0F1F0F1 EMP Rxn, etc... Poly-Phosphate n = 1 to 1,000 Storage Inclusion bodies “volutin” Pre-biotic Molecular cloning Many roles: O - P - (O -P- O) n - P - O OOO OOO Stress tolerance, virulence, etc...

20. Lecture Overview Metabolism (Inside and Out) Growth Survival strategies Diversity and environmental niches Cell differentiation Sporulation Symbiosis Adaptation mechanisms Rapid responses through altered enzyme activities Altering genetic expression