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1 Title: Microbial Metabolism Title: Instructor: What is Consetta the title Helmick of this lecture? Speaker: Amit Dhingra Created by: (remove if same as speaker) online.wsu.edu

2 Microbial Metabolism Microbial Metabolism Aerobic Cellular Respiration or in bacteria called the Embden-Meyerhof Pathway Alternate Pathway Pentose Phosphate Pathway Entner-Doudoroff Pathway

3 A Glimpse of History Biologists had noticed that in vats of grape juice, alcohol and are produced while yeast cells increase in number In 1850s, Louis Pasteur set out to prove Simplified setup: clear solution of sugar, ammonia, mineral salts, trace elements Added a few yeast cells as they grew, sugar decreased, alcohol level increased Strongly supported idea, but Pasteur failed to extract something from inside the cells that would convert sugar In 1897, Eduard Buchner, a German chemist, showed that crushed yeast cells could convert sugar to ethanol and ; awarded Nobel Prize in 1907

All cells need to accomplish two fundamental tasks Synthesize new parts Cell walls, membranes, ribosomes, nucleic acids Harvest energy to reactions Sum total of these is called

4 All cells need to accomplish two fundamental tasks Synthesize new parts Cell walls, membranes, ribosomes, nucleic acids Harvest energy to reactions Sum total of these is called metabolism Human implications Microbial Metabolism Used to make biofuels Used to produce food Important in laboratory Invaluable models for study Unique pathways potential drug targets

5 Principles of Metabolism Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Can separate metabolism into two parts Catabolism Processes that degrade compounds to release energy Cells capture to make Anabolism Biosynthetic processes Assemble subunits of macromolecules Use to drive reactions Processes intimately linked CATABOLISM Energy source (glucose) Waste products (acids, carbon dioxide) Catabolic processes harvest the energy released during the breakdown of compounds and use it to make. The processes also produce precursor metabolites used in biosynthesis. Cell structures (cell wall, membrane, ribosomes, surface structures) Energy Macromolecules (proteins, nucleic acids, polysaccharides, lipids) Energy Subunits (amino acids, nucleotides, sugars, fatty acids) Energy Precursor metabolites Nutrients ANABOLISM (source of nitrogen, sulfur, etc.) Anabolic processes (biosynthesis) synthesize and assemble subunits of macromolecules that make up the cell structures. The processes use the and precursor metabolites produced in catabolism.

Energy is the capacity to do work Two types of energy Energy Potential: stored energy (e.g., chemical bonds, rock on hill, water behind dam) Kinetic: energy of movement (e.

6 Energy is the capacity to do work Two types of energy Energy Potential: stored energy (e.g., chemical bonds, rock on hill, water behind dam) Kinetic: energy of movement (e.g., moving water) Energy in universe cannot be created or destroyed, but it can be converted between forms

Harvesting Energy Photosynthetic organisms harvest energy in sunlight Power synthesis of organic compounds from Convert kinetic energy of photons to potential energy of chemical bonds

7 Harvesting Energy Photosynthetic organisms harvest energy in sunlight Power synthesis of organic compounds from Convert kinetic energy of photons to potential energy of chemical bonds Chemoorganotrophs obtain energy from organic compounds Depend on activities of photosynthetic organisms Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Radiant energy (sunlight) Chemical energy (organic compounds) Photosynthetic organisms harvest the energy of sunlight and use it to the synthesis of organic compounds from. This converts radiant energy to chemical energy. Chemoorganotrophs degrade organic compounds, harvesting chemical energy. (top): Photodisc Vol. Series 74, photo by Robert Glusie; (bottom): Digital Vision/PunchStock

Components of Metabolic Pathways Metabolic pathways Series of chemical reactions that convert starting compound end product May be linear, branched, cyclical Starting compound Intermediate a

8 Components of Metabolic Pathways Metabolic pathways Series of chemical reactions that convert starting compound end product May be linear, branched, cyclical Starting compound Intermediate a Intermediate b End product (a) Linear metabolic pathway Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Starting compound Intermediate a Intermediate b1 End product 1 (b) Branched metabolic pathway Intermediate b2 End product 2 Starting compound Intermediate d End product Intermediate a Intermediate c (c) Cyclical metabolic pathway Intermediate b

9 Three types of Glucose metabolism Aerobic Respiration Cellular Respiration Anaerobic Respiration Fermentation

Overview of Metabolism in Bacteria Central metabolic pathways

outcomes Precursor metabolites 2 Pentose phosphate pathway Starts the

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Acetyl- by substrate-level Acetyl- 3b TCA cycle Incorporates an acetyl

Fermentation Reduces pyruvate or a derivative Acids, alcohols, and

10 Overview of Metabolism in Bacteria Central metabolic pathways Glycolysis Pentose phosphate pathway Tricarboxylic acid cycle Key outcomes Precursor metabolites 2 Pentose phosphate pathway Starts the oxidation of glucose Biosynthesis 3a Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Transition step 1 GLUCOSE Glycolysis Oxidizes glucose to pyruvate Acetyl- by substrate-level Acetyl- 3b TCA cycle Incorporates an acetyl group and releases (TCA cycles twice) X 2 + by substrate-level Fermentation Reduces pyruvate or a derivative Acids, alcohols, and gases Respiration Uses the electron transport chain to convert reducing to proton motive force by oxidative

Relative energy Components of Metabolic Pathways Role of Enzymes Biological catalysts: accelerate conversion of substrate into product by lowering activation energy Highly specific: one at each step

11 Relative energy Components of Metabolic Pathways Role of Enzymes Biological catalysts: accelerate conversion of substrate into product by lowering activation energy Highly specific: one at each step Reactions would occur without, but extremely slowly Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Energy of reactants Activation energy with an enzyme Activation energy without an enzyme Energy of products Progress of reaction (a) Starting compound Enzyme a Intermediate a Enzyme b Intermediate b Enzyme c End product (b)

Adenosine triphosphate () Energy currency of cell Three negatively charged phosphate groups repel Bonds inherently unstable, easily broken Releases energy to drive cellular processes High energy

12 Adenosine triphosphate () Energy currency of cell Three negatively charged phosphate groups repel Bonds inherently unstable, easily broken Releases energy to drive cellular processes High energy phosphate bonds denoted by ~ ADP + Pi Energy Molecule Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Adenosine NH 2 Phosphate groups N N O O O O P O O P O O P O O CH 2 O N N Adenine High-energy bonds OH OH Ribose

Role of Adenosine triphospate () is energy currency Composed of ribose, adenine, three phosphate groups Adenosine diphospate (ADP) acceptor of free energy Cells produce by adding P i to ADP using

13 Role of Adenosine triphospate () is energy currency Composed of ribose, adenine, three phosphate groups Adenosine diphospate (ADP) acceptor of free energy Cells produce by adding P i to ADP using energy Release energy from to yield ADP and P i Three processes to generate Substrate-level Exergonic reaction s Oxidative Proton motive force drives Photo Sunlight used to create proton motive force to drive Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. P i Energy used The energy comes from catabolic reactions. Unstable (high-energy) bonds P ~ P~ P P~ P ADP P i Energy released The energy drives anabolic reactions.

14 Role of Electron Carriers Energy harvested in stepwise process Electrons transferred to electron carriers, which represent reducing (easily transfer electrons to molecules) Raise energy level of recipient molecule NAD + /NADH, NADP + /NADPH, and FAD/FADH 2

15 Aerobic cellular respiration Bacteria produce Precursor Metabolites Serve as carbon skeletons for building macromolecules

16 Overview of Metabolism in Bacteria Central metabolic pathways Glycolysis Pentose phosphate pathway Tricarboxylic acid cycle Key outcomes Precursor metabolites 2 Pentose phosphate pathway Starts the oxidation of glucose Biosynthesis 3a Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Transition step 1 GLUCOSE Glycolysis Oxidizes glucose to pyruvate Acetyl- by substrate-level Acetyl- 3b TCA cycle Incorporates an acetyl group and releases (TCA cycles twice) X 2 + by substrate-level Fermentation Reduces pyruvate or a derivative Acids, alcohols, and gases Respiration Uses the electron transport chain to convert reducing to proton motive force by oxidative

17 Aerobic Cellular Respiration or Embden- Meyerhof Pathway in bacteria Requires oxygen as the final electron acceptor Produces 38 s in Bacterial and 36 s in Eukaryotic cells Prokaryotic cells produce precursor metabolites which become cellular components or macromolecules

2 Pentose phosphate pathway Starts the oxidation of glucose Biosynthesis 3a Transition step 1 3b GLUCOSE Glycolysis Oxidizes glucose to pyruvate CO2 x2 TCA cycle Incorporates an acetyl group and

the electron transport Chain to convert reducing to proton motive force ~ by oxidative P ~ P ~ P Glycolysis Converts 1 glucose to 2 pyruvates; yields net 2, 2 NADH Investment phase: 2 phosphate

18 2 Pentose phosphate pathway Starts the oxidation of glucose Biosynthesis 3a Transition step 1 3b GLUCOSE Glycolysis Oxidizes glucose to pyruvate CO2 x2 TCA cycle Incorporates an acetyl group and releases CO2 (TCA cycles twice) by substrate-level P ~ P ~ CO2 P CO2 CO2 + by substrate-level 5 4 P ~ P P Fermentation Reduces pyruvate or a derivative Acids, alcohols, and gases + Respiration Uses the electron transport Chain to convert reducing to proton motive force ~ by oxidative P ~ P ~ P Glycolysis Converts 1 glucose to 2 pyruvates; yields net 2, 2 NADH Investment phase: 2 phosphate groups added Glucose split to two 3- carbon molecules Pay-off phase: 3-carbon molecules converted to pyruvate Generates 4, 2 NADH total Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. ADP ADP Glucose ADP Glucose 6-phosphate Fructose 6-phosphate ADP Fructose 1,6-bisphosphate Dihydroxyacetone phosphate Glyceraldehyde 3-phosphate 3-phosphoglycerate 1,3-bisphosphoglycerate 2-phosphoglycerate Phosphoenolpyruvate NAD + NADH + H + ~ ~ H 2 O ~ ~ 1 is expended to add a phosphate group H 2 O A chemical rearrangement occurs. is expended to add a phosphate group. The 6-carbon molecule is split into two 3-carbon molecules. NAD + A chemical rearrangement of one of the molecules occurs. NADH + H + ~ 6 ~ 9 The addition of a phosphate group is coupled to a redox reaction, generating NADH and a high-energy phosphate bond. 7 is produced by substrate-level. A chemical rearrangement occurs. Water is removed, causing the phosphate bond to become high-energy. 10 is produced by substrate-level.

19 Bacterial Alternative Pathway Pentose Phosphate Pathway Also breaks down glucose Important in biosynthesis of precursor metabolites Ribose 5-phosphate, erythrose 4-phosphate Also generates reducing : NADPH Precursor metabolites Glucose molecules can have different fates Can be completely oxidized to for maximum Can be siphoned off as precursor metabolite for use in biosynthesis

6.10. Anabolic Pathways Synthesizing Subunits from Precursor Molecules Copyright The McGraw-Hill Companies, Inc.

Pentose phosphate pathway Glycolysis Ribose 5-phosphate Erythrose 5-phosphate Nucleotides amino acids (histidine) Glucose 6-phosphate

tyrosine) Lipids (glycerol component) 3-phosphoglycerate Amino acids (cysteine, glycine, serine) Phosphoenolpyruvate Amino acids

20 6.10. Anabolic Pathways Synthesizing Subunits from Precursor Molecules Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Pentose phosphate pathway Glycolysis Ribose 5-phosphate Erythrose 5-phosphate Nucleotides amino acids (histidine) Glucose 6-phosphate Fructose 6-phosphate Dihydroxyacetone phosphate Lipopolysaccharide (polysaccharide) Peptidoglycan Amino acids (phenylalanine, tryptophan, tyrosine) Lipids (glycerol component) 3-phosphoglycerate Amino acids (cysteine, glycine, serine) Phosphoenolpyruvate Amino acids (phenylalanine, tryptophan, tyrosine) Amino acids (alanine, leucine, valine) Acetyl- Acetyl- Lipids (fatty acids) Oxaloacetate Amino acids (aspartate, asparagine, isoleucine, lysine, methionine, threonine) X 2 - ketoglutarate Amino acids (arginine, glutamate, glutamine, proline) TCA cycle

2 Pentose phosphate pathway Starts the oxidation of glucose Biosynthesis 3a Transition step 1 3b Acetyl- GLUCOSE Glycolysis Oxidizes glucose to pyruvate CO2 x 2 TCA cycle Incorporates an acetyl group

21 2 Pentose phosphate pathway Starts the oxidation of glucose Biosynthesis 3a Transition step 1 3b Acetyl- GLUCOSE Glycolysis Oxidizes glucose to pyruvate CO2 x 2 TCA cycle Incorporates an acetyl group and releases CO2 (TCA cycles twice) by substrate-level Acetyl- CO2 CO2 CO2 + by substrate-level 5 4 Fermentation Reduces pyruvate or a derivative Acids, alcohols, and gases Respiration Uses the electron transport chain to convert reducing to proton motive force + by oxidative Transition Step is removed from pyruvate Electrons reduce NAD + to NADH + H + 2-carbon acetyl group joined to coenzyme A to form acetyl- Takes place in mitochondria in eukaryotes 8 7 A redox reaction generates NADH. Water is added. Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. NAD + H 2 O NADH + H + Malate Fumarate Oxaloacetate Acetyl- NAD + NADH + H + Transition step: is removed, a redox reaction generates NADH, and coenzyme A is added. Citrate 1 The acetyl group is transferred to oxaloacetate to start a new round of the cycle. 2 Isocitrate A chemical rearrangement occurs. -ketoglutarate NAD + 3 NADH + H + A redox reaction generates NADH and is removed. 6 A redox reaction generates FADH 2- FADH 2 FAD Succinate Succinyl- NAD + NADH + H + 4 A redox reaction generates NADH, is removed, and coenzyme A is added. 5 The energy released during removal is harvested to produce. ~ + P i ADP

22 2 Pentose phosphate pathway Starts the oxidation of glucose Biosynthesis 3a Transition step 1 3b Acetyl- GLUCOSE Glycolysis Oxidizes glucose to pyruvate CO2 x 2 TCA cycle Incorporates an acetyl group and releases CO2 (TCA cycles twice) by substrate-level Acetyl- CO2 CO2 CO2 + by substrate-level 5 4 Fermentation Reduces pyruvate or a derivative Acids, alcohols, and gases Respiration Uses the electron transport chain to convert reducing to proton motive force + by oxidative Tricarboxylic Acid (TCA) Cycle Completes oxidation of glucose Produces NADH 2 FADH 2 Precursor metabolites A redox reaction generates NADH. Water is added. 5 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. H 2 O A redox reaction generates FADH 2- NAD + NADH + H + Malate Fumarate FADH 2 The energy released during removal is harvested to produce. Oxaloacetate Acetyl- NAD + NADH + H + FAD Succinate Succinyl- ~ + P i ADP Transition step: is removed, a redox reaction generates NADH, and coenzyme A is added. Citrate 1 The acetyl group is transferred to oxaloacetate to start a new round of the cycle. 2 Isocitrate A chemical rearrangement occurs. -ketoglutarate NAD + NAD + NADH + H + 3 NADH + H + 4 A redox reaction generates NADH and is removed. A redox reaction generates NADH, is removed, and coenzyme A is added.

23 ETC located in Mitochondria GLUCOSE Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 2 Pentose phosphate pathway Starts the oxidation of glucose 1 Glycolysis Oxidizes glucose to pyruvate P ~ P ~ P + by substrate-level Biosynthesis 5 Fermentation Reduces pyruvate or a derivative Eukaryotic cell Acids, alcohols, and gases 3a Transition step Acetyl- Acetyl- x 2 3b TCA cycle Incorporates an acetyl group and releases (TCA cycles twice) by substrate-level 4 Respiration Uses the electron transport chain to convert reducing to proton motive force Inner mitochondrial membrane P P P by oxidative Electron Transport Chain Use of Proton Motive Force Complex I 4 4 H + 2 H + 10 H + H + Ubiquinone Complex III Cytochrome c Complex IV Proton motive force is used to drive: synthase ( synthesis) Intermembrane space Path of electrons 2 e NADH + H + NAD + Complex II 2 H + 1 / 2 H 2 O O 2 Terminal electron acceptor Mitochondrial matrix 3 3 ADP + 3 P i

24 The Electron Transport Chain Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Prokaryotic cell Cytoplasmic membrane Electron Transport Chain Uses of Proton Motive Force NADH dehydrogenase H + (0 or 4) synthase Ubiquinol veoxidase force ( synthesis) rive: H + (2 or 4) 10 H + Active transport (one mechanism) H + H + Rotation of a flagella Ubiquinone Proton motive force is used to drive: Transported molecule Outside of cytoplasmic membrane Path of electrons 2 e NADH + H + NAD + Succinate dehydrogenase 2 H + 1 / 2 O 2 Terminal electron acceptor H 2 O Cytoplasm 3 3 ADP + 3 P i

The Electron Transport Chain Electron transport chain is membrane-embedded electron carriers Pass electrons sequentially, eject protons in process Prokaryotes: in

to synthesize Prokaryotes can also transporters, flagella Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

25 The Electron Transport Chain Electron transport chain is membrane-embedded electron carriers Pass electrons sequentially, eject protons in process Prokaryotes: in cytoplasmic membrane Eukaryotes: in inner mitochondrial membrane Energy gradually released Release coupled to ejection of protons Creates electrochemical gradient Used to synthesize Prokaryotes can also transporters, flagella Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Electrons from the energy source 2 High energy Low energy e 2 H + Energy released is used to generate a proton motive force. 1 / 2 O 2 Electrons donated to the terminal electron acceptor. H 2 O

26 The Electron Transport Chain Yield of Aerobic Respiration in Prokaryotes Substrate-level : 2 (from glycolysis; net gain) 2 (from the TCA cycle) 4 (total) Oxidative : 6 (from reducing gained in glycolysis) 6 (from reducing gained in transition step) 22 (from reducing gained in TCA cycle) 34 (total) Total gain (theoretical maximum) = 38

2 Pentose phosphate pathway Starts the oxidation of glucose Biosynthesis 3a Transition step Yield 1 3b Acetyl- GLUCOSE Glycolysis Oxidizes glucose to pyruvate CO2 x 2 TCA cycle Incorporates an acetyl

27 2 Pentose phosphate pathway Starts the oxidation of glucose Biosynthesis 3a Transition step Yield 1 3b Acetyl- GLUCOSE Glycolysis Oxidizes glucose to pyruvate CO2 x 2 TCA cycle Incorporates an acetyl group and releases CO2 (TCA cycles twice) by substrate-level Acetyl- CO2 CO2 CO2 by substrate-level 5 4 Fermentation Reduces pyruvate or a derivative Acids, alcohols, and gases Respiration Uses the electron transport chain to convert reducing to proton motive force by oxidative Yield of Aerobic Respiration in Prokaryotes Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. GLUCOSE Glycolysis Oxidizes glucose to pyruvate 2 net gain = NADH Oxidative Substrate-level 6 2 Acetyl- Acetyl- 2 NADH Oxidative 6 x 2 6 NADH Oxidative 18 2 FADH 2 Oxidative 4 TCA cycle Incorporates an acetyl group and releases (TCA cycles twice) Substrate-level 2

28 Anaerobic environments Prokaryotes unique in ability to use reduced inorganic compounds as sources of energy E.g., hydrogen sulfide (H 2 S), ammonia (NH 3 ) Produced by anaerobic respiration from inorganic molecules (sulfate, nitrate) serving as terminal electron acceptors Important example of nutrient cycling Four general groups

29 The Electron Transport Chain Anaerobic respiration in E. coli Harvests less energy than aerobic respiration Lower electron affinities of terminal electron acceptors Some components different Can synthesize terminal oxidoreductase that uses nitrate as terminal electron acceptor Produces nitrite E. coli converts to less toxic ammonia Sulfate-reducers use sulfate (SO 4 2 ) as terminal electron acceptor Produce hydrogen sulfide as end product

Fermentation Fermentation end products varied; helpful in identification, commercially useful Ethanol Butyric acid Propionic acid 2,3-Butanediol Mixed acids Copyright The McGraw-Hill Companies, Inc.

30 Fermentation Fermentation end products varied; helpful in identification, commercially useful Ethanol Butyric acid Propionic acid 2,3-Butanediol Mixed acids Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Fermentation pathway Lactic acid Ethanol Butyric acid Propionic acid Mixed acids 2,3-Butanediol Microorganisms Streptococcus Lactobacillus Saccharomyces Clostridium Propionibacterium E. coli Enterobacter End products Lactic acid Ethanol Butyric acid Butanol Acetone Isopropanol H 2 Propionic acid Acetic acid Acetic acid Lactic acid Succinic acid Ethanol H 2 H 2 (yogurt, dairy, pickle), b (wine, beer), (acetone): Brian Moeskau/McGraw- Hill; (cheese): Photodisc/McGraw-Hill; (Voges-Proskauer Test), (Methyl-Red Test): The McGraw-Hill Companies, Inc./Auburn University Photographic Services

31 Aerobic Cellular Respiration (ACR) in Bacteria summary Glycolysis Glucose to pyruvate Produces: 2 NADH, 2 s and 6 precursor metabolites Acetyl Co A or Transitional step Produces: 2 NADH, 2 CO2 and 1 precursor metabolite TCA or Krebs cycle Produces: 6 NADH, 2 FADH, 2 s, 4 CO2, and 2 precursor metabolites Electron Transport Chain Produces: 38 s Oxygen required Examples of Bacteria: E. Coil and Staph species

32 Summary Aerobic Cellular Respiration (ACR) Or Embden-Meyerhoff Pathway in bacteria Pentose Phosphate Pathway Entener-Doudoroff Pathway

33 Pentose Phosphate Pathway Summary Glucose to pyruvate Produces: 5 carbon sugar to intermediate products Nucleic acids, nucleotides and amino acids 1 2 NADPH (Calvin cycle) Photosynthetic bacteria Products go ACR, Anaerobic respiration and Fermentation Example of bacteria are E. Coli and Bacillus Sp.

34 Entner-Doudoroff Pathway summary Glucose pyruvate Produces: 1 1 NADH 1 NADPH (Calvin cycle) Products can go to ACR, Anaerobic respiration or Fermentation Can process Glucose independent EX Pseudomonas Sp, E. Coli, Bacteroides Sp. Only Gram negative can use

Microbial Metabolism Principles of Metabolism. Harvesting Energy. All cells need to accomplish two fundamental tasks Synthesize new parts

Microbial Metabolism Principles of Metabolism. Harvesting Energy. All cells need to accomplish two fundamental tasks Synthesize new parts Microbial Metabolism All cells need to accomplish two fundamental tasks Synthesize new parts Cell walls, membranes, ribosomes, nucleic acids arvest energy to reactions Sum total of these is called metabolism