Week 3 LO4_Microbial metabolism (2) PDF

Introduction to Microbiology SCIMA2-B22 Eduvos (Pty) Ltd (formerly Pearson Institute of Higher Education) is registered with the Department of Higher Education and Training as a private higher education institution under th...

Introduction to Microbiology SCIMA2-B22 Eduvos (Pty) Ltd (formerly Pearson Institute of Higher Education) is registered with the Department of Higher Education and Training as a private higher education institution under the Higher Education Act, 101, of 1997. Registration Certificate number: 2001/HE07/008 Recap: What was covered in LO3? Prokaryotic and eukaryotic cell comparisons Prokaryotic cell shapes and arrangements Prokaryotic cells: External and internal features Modes of cellular transportation Eukaryotic cells: External and internal features 1. Assume you stain Bacillus by applying malachite green with heat and then counterstain with safranin. Through the microscope, the green structures are a. cell walls. b. capsules. c. endospores. d. flagella. 2. Three-dimensional images of live cells can be produced with a. darkfield microscopy. b. fluorescence microscopy. c. transmission electron microscopy. d. confocal microscopy. e. phase-contrast microscopy. 3. Carbolfuchsin can be used as a simple stain and a negative stain. As a simple stain, the pH is a. 2. b. higher than the negative stain. c. lower than the negative stain. d. the same as the negative stain. 1. Which of the following is not a distinguishing characteristic of prokaryotic cells? a. They usually have a single, circular chromosome. b. They have 70S ribosomes. c. They have cell walls containing peptidoglycan. d. Their DNA is not associated with 2. Which of the following is false about fimbriae? a. They are composed of protein. b. They may be used for attachment. c. They are found on gram-negative cells. d. They are composed of pilin. e. They may be used for motility. 3. You have isolated a motile, gram-positive cell with no visible nucleus. You can assume this cell has a. ribosomes. b. mitochondria. c. an endoplasmic reticulum. d. a Golgi complex.  What will be covered Week 3 in today’s lesson? Lesson 1 Big Picture: Metabolism Catabolic and Anabolic Reactions LO4: Microbial Enzymes and enzyme components Metabolism Energy Production Carbohydrate Catabolism Lipid and Protein Catabolism Biochemical Tests and Bacterial Identification Photosynthesis Resources for this lesson: Tortora et al. (Chapter 5) Lecture slides myLMS notes - Including associated videos Rate your Applied Science Lecturer Virtual Lecturer Led Session Q&A All E. coli look alike through a microscope; so how can E. coli O157:H7 be differentiated? 8  Big Picture: Metabolism Although microbial metabolism can cause disease and food spoilage, many microbes have beneficial metabolism that can be used in various applications. 9 Big Picture: Metabolism Metabolism is the buildup and breakdown of nutrients within a cell. These chemical reactions provide energy and create substances that sustain life. Defined: The sum of all chemical reactions within a living organism. Two key players: Enzymes and the molecule adenosine triphosphate (ATP). 10 Big Picture: Metabolism Two key players: Enzymes and the molecule adenosine triphosphate (ATP). 11 Big Picture: Metabolism Central metabolism of Thermovibrio ammonificans HB-1. 12 Catabolic and Anabolic Reactions Learning Objectives 5-1 Define metabolism and describe the fundamental differences between anabolism and catabolism. 5-2 Identify the role of ATP as an intermediate between catabolism and anabolism. 13 Catabolic and Anabolic Reactions Metabolism: The sum of the chemical reactions in an organism. Catabolism: Provides energy and building blocks for anabolism. Anabolism: Uses energy and building blocks to build large molecules. 14 Catabolic and Anabolic Reactions 15 Catabolic and Anabolic Reactions Metabolism: The sum of the chemical reactions in an organism. Catabolism: Provides energy and building blocks for anabolism. Degradative reactions. Generally hydrolytic reactions. Are exergonic. Anabolism: Uses energy and building blocks to build large molecules. Biosynthetic reactions. Involve dehydration synthesis. Are endergonic. 16 Role of ATP in Coupling Reactions 17 Figure 5.1  Catabolic and Anabolic Reactions A metabolic pathway is a sequence of enzymatically catalyzed chemical reactions in a cell Metabolic pathways are determined by enzymes. Enzymes are encoded by genes. https://ecampusontario.pressbooks.pub/bioc2580/chapter/introduction-to- 18 metabolism-metabolic-pathways/ Check Your Understanding Distinguish catabolism from anabolism. 5-1 How is ATP an intermediate between catabolism and anabolism? 5-2 19 Enzymes and Enzyme components Learning Objectives 5-3 Identify the components of an enzyme. 5-4 Describe the mechanism of enzymatic action. 5-5 List the factors that influence enzymatic activity. 5-6 Distinguish competitive and noncompetitive inhibition. 5-7 Define ribozyme. 20  Enzymes Enzymes are: Biological catalysts Speed up a chemical reaction without being permanently altered. Specific for a chemical reaction. 21 Figure 5.2 Enzymes 22 Figure 5.3a  Enzyme components Enzyme components Apoenzyme: Protein Cofactor: Nonprotein component Coenzyme: Organic cofactor Holoenzyme: Apoenzyme plus cofactor 23 Figure 5.4  Enzyme components Electron Carriers assist enzymes Nicotinamide adenine dinucleotide (NAD+) Nicotinamide adenine dinucleotide phosphate (NADP+) Flavin adenine dinucleotide (FAD) Coenzyme A https://www.facebook.com/BOGObiology/photos/a.232575 960587338/826059584572303/?type=3 24 Enzyme Classification Oxidoreductase: Oxidation-reduction reactions Transferase: Transfer functional groups Hydrolase: Hydrolysis Lyase: Removal of atoms without hydrolysis Isomerase: Rearrangement of atoms Ligase: Joining of molecules, uses ATP 25 Figure 5.2  Factors Influencing Enzyme Activity Temperature: Optimal Range: Enzymes function best at a specific temperature range. High Temperature: Increases activity up to a point, but extreme heat denatures enzymes. Low Temperature: Slows down enzyme activity without causing denaturation. pH: Optimal pH: Each enzyme has an optimal pH where it is most active. Deviation: Changes in pH can alter enzyme structure and function, potentially leading to denaturation if too extreme. 26  Factors Influencing Enzyme Activity Substrate Concentration: Low Concentration: Increasing substrate levels boost enzyme activity. Saturation Point: Beyond a certain concentration, enzyme activity plateaus as all active sites are occupied. 27  Factors Influencing Enzyme Activity Inhibitors: Competitive Inhibitors: Compete with the substrate for the active site; effect can be reduced by increasing substrate concentration. Non-Competitive Inhibitors: Bind elsewhere on the enzyme, changing its shape and reducing activity. Uncompetitive Inhibitors: Bind only to the enzyme-substrate complex, lowering both the reaction rate and substrate affinity. Allosteric Inhibitors: Bind to an allosteric site, causing conformational changes that decrease enzyme activity. 28  Factors Influencing Enzyme Activity Inhibitors: Competitive Inhibitors: Compete with the substrate for the active site; effect can be reduced by increasing substrate concentration. Non-Competitive Inhibitors: Bind elsewhere on the enzyme, changing its shape and reducing activity. Uncompetitive Inhibitors: Bind only to the enzyme-substrate complex, lowering both the reaction rate and substrate affinity. Allosteric Inhibitors: Bind to an allosteric site, causing conformational changes that decrease enzyme activity. 29  Factors Influencing Enzyme Activity Feedback Inhibition 30 Check Your Understanding What is a coenzyme? 5-3 Why is enzyme specificity important? 5-4 What happens to an enzyme below its optimal temperature? Above its optimal temperature? 5-5 Why is feedback inhibition noncompetitive inhibition? 5-6 What is a ribozyme? 5-7 31 Figure 5.2 Energy Production Learning Objectives 5-8 Explain the term oxidation-reduction. 5-9 List and provide examples of three types of phosphorylation reactions that generate ATP. 5-10 Explain the overall function of metabolic pathways. 32 Oxidation-Reduction Reactions Oxidation: Removal of electrons Reduction: Gain of electrons Redox reaction: An oxidation reaction paired with a reduction reaction 33 https://byjus.com/jee/redox-reactions/ The Generation of ATP Energy released during oxidation-reduction reactions is trapped within the cell by the formation of ATP. Organisms use three mechanisms of phosphorylation to generate ATP from ADP. Substrate-level phosphorylation Oxidative phosphorylation Photophosphorylation 34 https://byjus.com/jee/redox-reactions/ The Generation of ATP Outline the three ways that ATP is generated. 5-9 What is the purpose of metabolic pathways? 5-10 35 https://byjus.com/jee/redox-reactions/ Carbohydrate Catabolism LEARNING OBJECTIVES 5-11 Describe the chemical reactions of glycolysis. 5-12 Identify the functions of the pentose phosphate and Entner-Doudoroff pathways. 5-13 Explain the products of the Krebs cycle. 5-14 Describe the chemiosmotic model for ATP generation. 5-15 Compare and contrast aerobic and anaerobic respiration. 5-16 Describe the chemical reactions of, and list some products of, fermentation. 36 Carbohydrate Catabolism The breakdown of carbohydrates to release energy involves 3 stages Glycolysis: Oxidation of glucose to pyruvic acid with production of ATP and NADH. Krebs cycle: Oxidation of acetyl CoA to CO2 with ATP, NADH & FADH2 production. Electron transport chain: Oxidation of NADH and FADH2, to release electrons in a cascade of oxidation- reduction reactions. A large amount of ATP generated. 37 Carbohydrate Catabolism The oxidation of glucose to pyruvic acid produces ATP and NADH 38 Preparatory Stage of Glycolysis Hexokinase 2 ATP are used. Phosphoglucose Isomerase Glucose is split to form 2 glucose-3-phosphate Phosphofructokinase Fructose bisphosphate aldose Triose phosphate isomerase Figure 5.12, steps 1–5 39 Energy-Conserving Stage of Glycolysis Glyceraldehyde-3-phophosphate dehydrogenase 2 glucose-3-phosphate oxidized to 2 Phosphoglycerate pyruvic acid Phosphoglycerate mutase 4 ATP produced 2 NADH produced Enolase Pyruvate Kinase Figure 5.12, steps 6–10 40  Glycolysis Glucose + 2 ATP + 2 ADP + 2 PO4– + 2 NAD+ → 2 pyruvic acid + 4 ATP + 2 NADH + 2H+ 41  Alternatives to Glycolysis Own reading Pentose phosphate pathway Uses pentoses and NADPH Operates with glycolysis Bacillus subtilis, E.coli, Enterococcus faecalis Entner-Doudoroff pathway Produces NADPH and ATP Does not involve glycolysis Found in some G-ve bacteria (not common among G+ves) ‒ Pseudomonas, Rhizobium, Agrobacterium ‒ Used to identify Pseudomonas in the clinical lab 42  Intermediate Step Own reading Pyruvic acid (from glycolysis) is oxidized and decarboxylated. 43  The Krebs Cycle Own reading Also called the citric acid cycle. Oxidation of acetyl CoA produces NADH and FADH2 44  The Electron Transport Chain Own reading A series of carrier molecules that are, in turn, oxidized and reduced as electrons are passed down the chain. Energy released can be used to produce ATP by chemiosmosis. In eukaryotes – electron transport chain occurs in the mitochondria. In prokaryotes – plasma membrane. Aerobic respiration in prokaryotes: generates 38 ATPs per molecule of glucose. In eukaryotes: 36 ATPs per molecule of glucose. 45 Overview of Respiration and Fermentation Figure 5.11 46 Overview of Respiration and Fermentation https://bio.libretexts.org/Courses/Harrisburg_Area_Community_College/BIOL_101%3A_General_Biology_l_- 47 _Laboratory_Manual/01%3A_Labs/1.08%3A_Respiration_and_Fermentation  A Summary of Respiration Aerobic respiration: The final electron acceptor in the electron transport chain is molecular oxygen (O2). Anaerobic respiration: The final electron acceptor in the electron transport chain is not O2. Yields less energy than aerobic respiration because only part of the Krebs cycles operates under anaerobic conditions. 48 Anaerobic Respiration Electron Acceptor Products NO3– NO2–, N2 + H2O SO4– H2S + H2O CO32 – CH4 + H2O 49 Carbohydrate Catabolism Pathway Eukaryote Prokaryote Glycolysis Cytoplasm Cytoplasm Intermediate step Cytoplasm Cytoplasm Krebs cycle Mitochondrial matrix Cytoplasm ETC Mitochondrial inner membrane Plasma membrane 50  Fermentation Any spoilage of food by microorganisms (general use). Any process that produces alcoholic beverages or acidic dairy products (general use). Any large-scale microbial process occurring with or without air (common definition used in industry) 51  Fermentation List the 5 characteristics of fermentation process in science. 52  Fermentation Alcohol fermentation: Produces ethanol + CO2 Glucose is oxidized to pyruvic acid; pyruvic acid is converted to acetaldehyde and CO2; NADH reduces acetaldehyde to ethanol Lactic acid fermentation: Produces lactic acid Homolactic fermentation: Produces lactic acid only Heterolactic fermentation: Produces lactic acid and other compounds Glucose is oxidized to pyruvic acid, which is then reduced by NADH 53 End-Products of Fermentation Figure 5.18b 54 Industrial Uses for Different Types of Fermentations 55 Check Your Understanding What happens during the preparatory and energy-conserving stages of glycolysis? 5-11 What is the value of the pentose phosphate and Entner-Doudoroff pathways if they produce only one ATP molecule? 5-12 What are the principal products of the Krebs cycle? 5-13 How do carrier molecules function in the electron transport chain? 5-14 Compare the energy yield (ATP) of aerobic and anaerobic respiration. 5-15 List four compounds that can be made from pyruvic acid by an organism that uses fermentation. 5-16 56 Class Break QUIZ TIME!!! Lipid and Protein Catabolism Learning Objectives 5-17 Describe how lipids and proteins undergo catabolism. 5-18 Provide two examples of the use of biochemical tests to identify bacteria in the laboratory. 59 Lipid Catabolism Figure 5.20 60 Protein Catabolism Extracellular proteases Protein Amino acids Deamination, decarboxylation, dehydrogenation, desulfurization Organic acid Krebs cycle 61 Catabolism of Organic Food Molecules Figure 5.21 62  Biochemical tests Biochemical tests identify bacteria by detecting enzymes (e.g., those involved in decarboxylation and dehydrogenation). Use our knowledge of carbohydrate, protein and lipid metabolism to form basic tests in the lab to identify an unknown bacterium 63 Fermentation Test a. Uninoculated b. Inocuated with an organism c. Organism used d. Organism used mannitol fermentation tube which grew on the protein in mannitol (colour (colour change) and containing the media but did not use change) (S. aureus) produced gas from mannitol mannitol mannitol. (S. epidermidis) (bubble in Durham tube) 64 (carbohydrate) (E.coli)  Fermentation Test Fermentation test: bacteria that catabolize carbohydrate or protein produce acid, causing the pH indicator to change color E. coli ferments sorbitol Pathogenic E. coli O157 does not 65  Oxidase test Oxidase test: identifies bacteria that have cytochrome oxidase Neisseria gonorrhoeae positive for cytochrome oxidase. Cytochrome c oxidase is the enzyme which transfers electrons to O2 Distinguish between Gram-neg. bacilli Pseudomonas is oxidase positive E. coli is oxidase negative 66  Lactate Dehydrogenase Test Shigella cannot break down lactose within 2 days. Differentiated from E. coli which does. 67 https://marlerclark.com/foodborne-illnesses/shigella/about-shigella Protein Catabolism Yellow pH Purple (alkaline) indicator when products from bacteria decarboxylation produce acid from glucose 68 Figure 5.22 Protein Catabolism E. coli Salmonella Figure 5.24 69 Protein Catabolism Urea Urease NH3 + CO2 Clinical Focus Figure B 70 Identification tests to detect slow growing Mycobacteria 71 Check Your Understanding What are the end-products of lipid and protein catabolism? 5-17 On what biochemical basis are Pseudomonas and Escherichia differentiated? 5-18 72  Photosynthesis Learning Objectives Own reading 5-19 Compare and contrast cyclic and noncyclic photophosphorylation. 5-20 Compare and contrast the light-dependent and light-independent reactions of photosynthesis. 5-21 Compare and contrast oxidative phosphorylation and photophosphorylation. 73  Photosynthesis Oxygenic: (Plants, algae, cyanobacteria) 6 CO2 + 12 H2O + Light energy → C6H12O6 + 6 H2O + 6 O2 Anoxygenic: (purple sulphur and green sulphur bacteria) 6 CO2 + 12 H2S + Light energy → C6H12O6 + 6 H2O + 12 S 74  Photosynthesis Photo: Conversion of light energy into chemical energy (ATP) Light-dependent (light) reactions. Synthesis: Carbon fixation: Fixing carbon into organic molecules Light-independent (dark) reaction: Calvin-Benson cycle 75 Check Your Understanding How is photosynthesis important to catabolism? 5-19 What is made during the light-dependent reactions? 5-20 Summarize how oxidation enables organisms to get energy from glucose, sulfur, or sunlight. 5-22 76  Metabolic Diversity among Organisms Learning Objectives 5-23 Categorize the various nutritional patterns among organisms according to carbon source and mechanisms of carbohydrate catabolism and ATP generation. 77  Nutritional Classification of Organisms Depending on their energy source, microorganisms can be classified as Chemotrophs - inorganic or organic compounds for energy Phototrophs – light for energy Autotrophs – CO2 as principal carbon source Heterotrophs – organic source of carbon 78 Nutritional Classification of Organisms Figure 5.28 79  Nutritional Classification of Organisms Combining energy and carbon sources: Photoautotrophs Photoheterotrophs Chemoautotrophs Chemoheterotrophs 80  Phototrophs Use light energy Photoautotrophs use CO2 as the source of C ‒ Cyanobacteria, algae, plants (oxygenic – produce O2) ‒ Green and purple bacteria (anoxygenic - does not produce O2 ) Photoheterotrophs use organic compounds for C (alcohols, fatty acids) ‒ Green non-sulphur bacteria ‒ Purple non-sulphur bacteria 81  Nutritional Classification of Organisms Combining energy and carbon sources: Photoautotrophs Photoheterotrophs Chemoautotrophs Chemoheterotrophs 82  Chemotrophs Use energy from inorganic chemicals; CO2 as carbon source. Energy is used in the Calvin-Benson cycle to fix CO2 Fe: Thiobacillus ferrooxidans H2S: Beggiatoa NH3: Nitrosomonas 83  Chemotrophs Chemoheterotrophs Energy and carbon source often the same (glucose). Most bacteria, all fungi, protozoa and animals. Saprophytes: live on dead organic matter. Parasites: Obtain nutrients from living hosts. 84 Photosynthesis Compared in Selected Eukaryotes and Prokaryotes 85 Metabolic Diversity among Organisms Nutritional Type Energy Source Carbon Source Example Photoautotroph Light CO2 Oxygenic: Cyanobacteria plants Anoxygenic: Green, purple bacteria Photoheterotroph Light Organic compounds Green, purple nonsulfur bacteria Chemoautotroph Chemical CO2 Iron-oxidizing bacteria Chemoheterotroph Chemical Organic compounds Fermentative bacteria Animals, protozoa, fungi, bacteria. 86 Check Your Understanding Almost all medically important microbes belong to which of the four groups as mentioned above? 5-23 87  QUIZ TIME!!! Microbial Metabolism 88  What’s next? 1. Review notes and readings for LO4. 2. Prepare readings for LO5.