1. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Chapter 7 Outline Microbial Physiology –Introduction –Microbial Nutritional Requirements –Categorizing Microorganisms According to Their Energy and Carbon Sources Metabolic Enzymes –Biologic Catalysts –Factors That Affect the Efficiency of Enzymes Metabolism –Catabolism –Anabolism Bacterial Genetics –Mutations –Ways in Which Bacteria Acquire New Genetic Information Genetic Engineering Gene Therapy

2. 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)

3. 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.

4. 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. –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. organic

5. 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 (CO 2 ) as their sole source of carbon. –Heterotrophs use organic compounds other than CO 2 as carbon sources. Terms that combine both energy and carbon source: –Photoautotrophs use light as a carbon source and CO 2 as an energy source. –Chemoautotrophs use chemicals as a carbon source and CO 2 as an energy source. –Chemoheterotrophs use chemicals as a carbon source and organic compounds other than CO 2 as an energy source.

6. 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.

7. 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.

8. 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!)

9. 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

10. 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-Qjdj88http://youtu.be/v0OM-Qjdj88

11. 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.

12. 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).

13. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Interrelationships among ATP, ADP, and AMP molecules.

14. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Metabolism, cont. Energy is required for metabolic pathways, growth, reproduction, sporulation, and movement of the organism, and active transport of substances across membranes. a Marine dinoflagellates use energy for bioluminescence.

15. 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 http://youtu.be/pnKih-4SRAE

16. 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.

17. 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 http://youtu.be/6JGXayUyNV w –The Krebs cycle –The electron transport chain The 1 st 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.

18. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Aerobic Respiration of Glucose: First Step = Glycolysis.

19. 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 +, FADH 2 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.

20. 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!

21. 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.

22. 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.

23. 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.

24. 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).

25. 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.

26. 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.

27. 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.

28. 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

29. 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.

30. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

31. 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.

32. 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.

33. 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.”

34. Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins Conjugation in Escherichia coli. Sex pilus Copyright © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins

35. 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.

36. 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.)