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Chapter 7 Outline • Microbial Physiology • Metabolism – Introduction – Catabolism – Microbial Nutritional Requirements – Anabolism – Categorizing Microorganisms According to Their Energy and Carbon Sources • Metabolic Enzymes – Biologic Catalysts – Factors That Affect the Efficiency of Enzymes • Bacterial Genetics – Mutations – Ways in Which Bacteria Acquire New Genetic Information • Genetic Engineering • Gene Therapy Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Microbial Physiology Introduction • Physiology is the study of the vital life processes of organisms. – Microbial physiology is very much chemical reactions (metabolism) Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Microbial Physiology Nutritional Requirements • All living protoplasm contains 6 major chemical elements: carbon, hydrogen, oxygen, nitrogen, phosphorus, and sulfur. – Combinations of these and other elements make up vital macromolecules of life, including carbohydrates, lipids, proteins, and nucleic acids, vitamins, etc. • Essential Nutrients: • materials that organisms are unable to synthesize, but are required for building macromolecules and sustaining life, • e.g., certain essential amino acids and essential fatty acids. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Categorizing Microorganisms by Energy and Carbon Sources • Terms relating to an organism’s energy source. – – Phototrophs use light as an energy source. organic Chemotrophs use either inorganic or organic chemicals as an energy source. • Chemolithotrophs use inorganic chemicals as an energy source. • Chemoorganotrop hs use organic chemicals as an energy source. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Microbial Physiology Categorizing Microorganisms According to Their Energy and Carbon Sources, cont. • Terms relating to an organism’s carbon source: – Autotrophs use carbon dioxide (CO2) as their sole source of carbon. – Heterotrophs use organic compounds other than CO2 as carbon sources. • Terms that combine both energy and carbon source: – Photoautotrophs use light as a carbon source and CO2 as an energy source. – Chemoautotrophs use chemicals as a carbon source and CO2 as an energy source. – Chemoheterotrophs use chemicals as a carbon source and organic compounds other than CO2 as an energy source. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Categorizing Microorganisms According to Their Energy and Carbon Sources, cont. • Ecology is the study of the interactions between living organisms and the world around them. • Ecosystem refers to the interactions between living organisms and their nonliving environment. • Interrelationships among the different nutritional types are important in the functioning of the ecosystem. – Example: Phototrophs, such as algae and plants, are the producers of food and oxygen for chemoheterotrophs, such as animals. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Metabolic Enzymes • Metabolism refers to all the chemical reactions that occur in a cell. The chemical reactions are referred to as metabolic reactions. – Metabolic reactions are carried out by enzymes. • Biologic Catalysts – Enzymes are biologic catalysts; they are proteins that cause a particular chemical reaction to occur or accelerate it. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Metabolic Enzymes Biologic Catalysts, cont. • Enzymes are specific, they only catalyze one particular chemical reaction. • An enzyme only affects one particular substance, known as the substrate for that enzyme. • The unique 3-dimensional shape of an enzyme enables it to fit the substrate like a key fits into a lock. • http://youtu.be/PILzvT3spCQ • An enzyme does not become altered during the chemical reaction it catalyzes. (They don’t last forever!) Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Factors That Affect the Efficiency of Enzymes – pH - extreme acidity for example – Temperature - heat can denature enzymes by breaking bonds – Concentration of enzyme and/or substrate – may be too high or too low – Inhibitors, for example heavy metals like lead, zinc, mercury and arsenic Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Metabolism • Metabolism refers to all of the chemical reactions within a cell • A metabolite is any molecule that is a nutrient, an intermediary product, or an end product in a metabolic reaction. • Metabolic reactions fall into 2 categories: catabolism and anabolism. – Catabolism refers to all catabolic reactions in a cell. – Anabolism refers to all anabolic reactions in a cell. – http://youtu.be/v0OM-Qjdj88 Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Metabolism, cont. • Catabolic reactions involve the breaking down of larger molecules into smaller ones. – Energy is released. Catabolic reactions are a cell’s major source of energy. • Anabolic reactions involve the assembly of smaller molecules into larger molecules, requiring the formation of bonds. The bonds are stored energy. • Much of the energy released during catabolic reactions is used to build molecules in anabolic reactions. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Metabolism, cont. • Energy is temporarily stored in bonds in adenosine triphosphate (ATP). • When ATP is used as an energy source, it is hydrolyzed (split) to adenosine diphosphate (ADP). • ADP can be used as an energy source by hydrolysis to adenosine monophosphate (AMP). Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Interrelationships among ATP, ADP, and AMP molecules. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Metabolism, cont. a Marine dinoflagellates use energy for bioluminescence. Energy is required for metabolic pathways, growth, reproduction, sporulation, and movement of the organism, and active transport of substances across membranes. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Metabolism Catabolism • Catabolic reactions release energy (by breaking bonds) and are a cell’s major source of energy. – Some energy is lost as heat in catabolic reactions. • Biochemical pathways are a series of linked biochemical reactions, with a starting chemical and an end product (chemical). • Think of nutrients as energy sources for organisms and think of chemical bonds as stored energy. • Glucose, for example, can be catabolized by either aerobic respiration or fermentation. • Glycolysis is shared by both: http://youtu.be/pnKih-4SRAE Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins A biochemical pathway with 4 steps. Compound A is ultimately converted to compound E. Four enzymes are required in this biochemical pathway. Compound A is the substrate for Enzyme 1, Compound B for Enzyme 2, etc. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Metabolism Catabolism, cont. • Catabolism of glucose by aerobic respiration occurs in 3 phases (each is a biochemical pathway): – Glycolysis http://youtu.be/6JGXayUyNV w – The Krebs cycle – The electron transport chain • The 1st phase (glycolysis) is anaerobic, but the other 2 phases are aerobic. So, the whole process is considered aerobic. • Glycolysis is a 9-step biochemical pathway. Each step requires a specific enzyme. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Aerobic Respiration of Glucose: First Step = Glycolysis. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Catabolism Aerobic Respiration of Glucose, cont. • The Krebs Cycle, aka citric acid cycle and TCA cycle: – A biochemical pathway consisting of 8 separate reactions, each controlled by a different enzyme. – Only 2 ATP molecules are produced, but NADH, H+, FADH2 are formed, which enter the electron transport chain. • In eucaryotes, the Krebs/TCA cycle and the electron transport chain occur in mitochondria. • In procaryotes, both occur at the inner surface of the cell membrane. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Catabolism Aerobic Respiration of Glucose, cont. • The electron transport chain: – A series of oxidation-reduction reactions, where energy is released as electrons which are transferred from one compound to another. – Many enzymes are involved in the electron transport chain, including cytochrome oxidase, which transfers electrons to oxygen (the electron final acceptor). – A large number of ATP molecules are produced by oxidative phosphorylation in the electron transport chain. http://youtu.be/DNReloT3QYU • Aerobic respiration is very efficient! Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Catabolism Fermentation of Glucose • Fermentation reactions do not involve oxygen. They take place in anaerobic (no oxygen) environments. – First step is glycolysis (anaerobic). – The next step is conversion of pyruvic acid into an end product. – The end product varies from one organism to another. Example: yeasts are used to make wine and beer; the end product is ethanol. – Fermentation reactions produce very little energy, ~ 2 ATP molecules. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Catabolism Oxidation-Reducton (Redox) Reactions • Oxidation-reduction reactions are paired reactions in which electrons are transferred from one compound to another. • Oxidation occurs whenever an atom, ion, or molecule loses one or more electrons in a reaction; in which case, the molecule is said to be oxidized. • The gain of one or more electrons by a molecule is called reduction and the molecule is said to be reduced. • Within a cell, an oxidation reaction is always paired with a reduction reaction; hence the term, oxidation-reduction reaction. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Catabolism Oxidation-Reduction (Redox) Reactions, cont. • In a redox reaction, the electron donor (compound A) is the reducing agent, and the electron acceptor (compound B) is the oxidizing agent. • Many biologic oxidations are referred to as dehydrogenation reactions because hydrogen ions, as well as electrons, are removed. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Anabolism • Anabolic reactions require energy because chemical bonds are being formed. The energy that is used comes from catabolic reactions, which are occurring simultaneously. • Biosynthesis of organic compounds requires energy. The energy may be obtained through photosynthesis (from light) or chemosynthesis (from chemicals). – Photosynthetic reactions trap the radiant energy of light and convert it into chemical bond energy in ATP and carbohydrates (e.g., glucose). Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Bacterial Genetics • Genetics = the study of heredity. • An organism’s genotype is its complete collection of genes. • An organism’s phenotype refers to its physical traits (e.g., includes hair and eye color in humans). • An organism’s phenotype is the manifestation of that organism’s genotype because genes control all functions of the cell. • Gene: a particular segment of the chromosome. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Bacterial Genetics Mutations • A change in a DNA molecule (genetic alteration) that is transmissible to offspring is called a mutation. – 3 categories of mutations: • Beneficial mutations • Harmful mutations (some are lethal mutations) • Silent mutations • Mutation rate (the rate at which mutations occur) can be increased by exposing cells to physical or chemical agents called mutagens. • The organism containing the mutation is called a mutant. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Bacterial Genetics Ways in Which Bacteria Acquire New Genetic Information • Ways in which bacteria acquire new genetic information (i.e., acquire new genes): – Lysogenic Conversion – Transduction – Transformation – Conjugation • An extrachromosomal DNA molecule is called a plasmid. An organism that acquires a plasmid acquires new genes. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins (A) A disrupted E. coli cell, in which the DNA has spilled out. A plasmid can be seen slightly to the left of top center (arrow). (B) Enlargement of plasmid. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Ways in Which Bacteria Acquire New Genetic Information, cont. • Lysogenic Conversion – Temperate phages (or lysogenic phages) inject their DNA into a bacterial cell. – The phage DNA integrates into the bacterial chromosome, but does not cause the lytic cycle to occur – this is known as lysogeny. This is the opposite of a lytic cycle, that causes the lytic cycle TO occur, resulting in the lysis (rupturing) of the host cell. – A phage is called a prophage (early or first phage/virus) when all that remains of it is its DNA. – The bacterial cell containing the prophage is referred to as a lysogenic cell. – The bacterial cell exhibits new properties, directed by the viral genes – this is referred to as lysogenic conversion. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins how Bacteria Acquire New Genetic Information, cont. • Transduction (“to carry across”): – Also involves bacteriophages. – In transduction, bacterial genetic material is “carried across” from one bacterial cell to another by a bacterial virus; thus, in transduction, bacteria acquire new bacterial genes. – Note how this differs from lysogenic conversion, wherein bacteria acquire new genetic information in the form of viral genes. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins How Bacteria Acquire New Genetic Information, cont. • Transformation – A bacterial cell becomes genetically transformed following the uptake of DNA fragments (“naked DNA”) from its environment. – The ability to absorb naked DNA into the cell is called competence and bacteria capable of absorbing naked DNA are said to be competent bacteria. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins How Bacteria Acquire New Genetic Information, cont. • Conjugation – Involves a specialized type of pilus called a sex pilus. – A bacterial cell with a sex pilus (called the donor cell) attaches by means of the sex pilus to another bacterial cell (called the recipient cell). – Some genetic material (usually a plasmid) is transferred through the hollow sex pilus from the donor cell to the recipient cell. – A plasmid that contains multiple genes for antibiotic resistance is known as a resistance factor or R-factor. A bacterial cell that receives a R-factor becomes a “superbug.” Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Conjugation in Escherichia coli. Sex pilus Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Genetic Engineering • Genetic engineering or recombinant DNA technology involves techniques to transfer eucaryotic genes (particularly human genes) into easily cultured cells to manufacture important gene products (mostly proteins). • Plasmids are frequently used as vehicles for inserting genes into cells. • There are many industrial and medical benefits from genetic engineering. – Examples: synthesis of antibodies, antibiotics, drugs and vaccines; also, for synthesis of important enzymes and hormones for treatment of diseases. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Gene Therapy • Gene therapy of human diseases involves the insertion of a normal gene into cells to correct a specific genetic disorder caused by a defective gene. • Viral delivery is the most common method for inserting genes into cells; specific viruses are selected to target the DNA of specific cells. • Genes may someday be regularly prescribed as “drugs” in the treatment of diseases (e.g., autoimmune diseases, sickle cell anemia, cancer, cystic fibrosis, heart disease, etc.) Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins