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Chapter 5

Chapter 5. Microbial Biotechnology. Chapter Contents. 5.1 The Structure of Microbes 5.2 Microorganisms as Tools 5.3 Using Microbes for a Variety of Everyday Applications 5.4 Vaccines 5.5 Microbial Genomes 5.6 Microbial Diagnostics 5.7 Combating Bioterrorism.

joelle-daugherty
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Chapter 5

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  1. Chapter 5 Microbial Biotechnology

  2. Chapter Contents • 5.1 The Structure of Microbes • 5.2 Microorganisms as Tools • 5.3 Using Microbes for a Variety of Everyday Applications • 5.4 Vaccines • 5.5 Microbial Genomes • 5.6 Microbial Diagnostics • 5.7 Combating Bioterrorism

  3. 5.1 The Structure of Microbes • Microbes (microorganisms) are tiny organisms that are too small to be seen individually by the naked eye and must be viewed with the help of a microscope • Bacteria, cyanobacteria, fungi, algae, and protozoa

  4. A Brief History of Microbiology • Ancestors of bacteria were the first life on Earth. • In 1665, Robert Hooke using an early microscope, first reported that living things were composed of little boxes or cells. • In 1673-1723, Antoni van Leeuwenhoek described live microorganisms that he observed in teeth scrapings, rain water, and peppercorn infusions. • In 1858, Rudolf Virchow said cells arise from preexisting cells. • 1861: Louis Pasteur demonstrated that microorganisms are present in the air. 20 nm

  5. The Germ Theory of Disease • 1835: Agostino Bassi showed a silkworm disease was caused by a fungus. • 1840s: Ignaz Semmelwise advocated hand washing to prevent transmission of puerperal fever from one patient to another. • 1860s: Joseph Lister used a chemical disinfectant to prevent surgical wound infections after looking at Pasteur’s work showing microbes are in the air, can spoil food, and cause animal diseases. • 1876: Robert Koch provided proof that a bacterium causes anthrax and provided the experimental steps, Koch’s postulates, used to prove that a specific microbe causes a specific disease.

  6. Types of microorganisms: Yeast 1 m μ 10 μm TMV Tobacco Mosaic Virus (TMV) 20 nm 20 nm

  7. 5.1 The Structure of Microbes • Fungi: Yeasts and Moulds • Yeast – single-celled eukaryotic microbes • Sources of antibiotics and drugs that lower cholesterol • Mechanisms of gene expression resemble those in human cells

  8. 5.1 The Structure of Microbes • Yeast • Can grow in the presence of oxygen (aerobic) or in the absence of oxygen (anaerobic) • Pichia pastoris • Grows to a higher density in liquid culture than other yeast strains • Has a number of strong promoters that can be used for production of proteins • Can be used in batch processes to produce large number of cells

  9. 5.1 The Structure of Microbes • Structural Features of Bacteria • Small (1–5 µm) • No nucleus; DNA is contained in a single, circular chromosome • May contain plasmids • Cell wall that surrounds plasma membrane contains peptidoglycan; provides rigidity for protection • Some bacteria contain an outer layer of carbohydrates in a structure called a capsule

  10. 5.1 The Structure of Microbes • Bacteria are classified by the Gram stain • Gram + bacteria stain purple • Have simple cell walls rich in peptidoglycan • Gram – bacteria stain pink • Have complex cell wall structures with less peptidoglycan

  11. 5.1 The Structure of Microbes • Bacteria vary in size and shape • Most common shapes • Cocci – spherical cells • Bacilli – rod-shaped cells • Spiral – corkscrew-shaped cells

  12. Scientific names • Are italicized or underlined. • The genus is capitalized and the specific epithet is lower case. • Are “Latinized” and used worldwide. • May be descriptive or honor a scientist. • Staphylococcus aureus. • Describes the clustered arrangement of the cells (staphylo-) and the golden color of the colonies. • Escherichia coli - Honors the discoverer, Theodor Eshcerich, and describes the bacterium’s habitat, the large intestine or colon. • After the first use, scientific names may be abbreviated with the first letter of the genus and the specific epithet: • Staphylococcus aureus and Esherichia coli are found in the human body. S. aureus is on skin and E. coli, in the large intestine.

  13. 5.1 The Structure of Microbes Bacterial Genetics • Single, circular chromosome is relatively small • 2–4 million base pairs • Some bacteria contain plasmids as well • Plasmids often contain genes for antibiotic resistance and genes encoding proteins that form connecting tubes called pili • Plasmids are an essential tool for biotechnology

  14. 5.1 The Structure of Microbes • Bacteria grow and divide rapidly • Divide every 20 minutes or so • Millions of cells can be grown on small dishes of agar or in liquid culture media • Easy-to-make mutant strains to be used for molecular and genetic studies

  15. Normal Microbiota • Normal microbiota prevent growth of pathogens. • Normal microbiota produce growth factors such as folic acid and vitamin K. • Resistance is the ability of the body to ward off disease. • Resistance factors include skin, stomach acid, and antimicrobial chemicals.

  16. Modern Biotechnology and Genetic Engineering • MICROBIAL Biotechnology, the use of microbes to produce foods and chemicals, is centuries old. • Genetic engineering is a new technique for biotechnology. Through genetic engineering, bacteria and fungi can produce a variety of proteins including vaccines and enzymes. • Missing or defective genes in human cells can be replaced in gene therapy. • Genetically modified bacteria are used to protect crops from insects and freezing. IT IS IMPORTANT FOR BIOTECHNOLOGIST TO KNOW ABOUT MECHANISMS OF MICROBIAL GROWTH

  17. Microbial growth = increase in number of cells, not cell size The Requirements for Growth:1.Physical Requirements • Temperature range where organisms can grow: • Minimum growth temperature • Optimum growth temperature (best range) • Maximum growth temperature

  18. Temperature Requirements: Groups

  19. Physical Requirements: pH • Most bacteria grow between pH 6.5 and 7.5. • Molds and yeasts grow between pH 5 and 6. • Acidophiles grow in acidic environments (coal mines). • Osmotic Pressure • Hypertonic environments, increase salt or sugar, cause plasmolysis. It inhibits Cell Growth • This phenomenon is used to stop food spoilage. • Extreme or obligate halophiles require high osmotic pressure since they leave in high salt concentrations. • Facultative halophiles can tolerate high osmotic pressure, up to 15% salt. • Most microorganisms grow in a medium that is nearly all water.

  20. The Requirements for Growth:2.Chemical Requirements • Carbon: backbone of living matter • Structural organic molecules, energy source • Chemoheterotrophs use organic carbon sources energy from protein, carbohydrates and lipids • Autotrophs get it from CO2 • Nitrogen: also needed for synthesis of cellular material • In amino acids, proteins • Most bacteria decompose proteins • Some bacteria derive N from NH4+ or NO3 • A few bacteria use N2 in nitrogen fixation directly from atmosphere

  21. Chemical Requirements • Sulfur • used to synthesize sulfur-containing amino acids and vitamins (thiamine, biotin) • Phosphorus • used to synthesize nucleic acids and phospholipids • In DNA, RNA, ATP, and membranes • Phosphate ion, PO43is a source of phosphorus • Trace Elements • Iron, copper, molybdenum and zinc; (Inorganic elements required in small amounts) • Usually work as enzyme cofactors • Organic Growth Factors • Organic compounds that are only obtained fromthe environment • Vitamins, amino acids, purines, pyrimidines

  22. Culture Media • Culture Medium: Nutrients prepared for microbial growth • Sterility: No living microbes • Inoculum: Introduction of microbes into medium • Culture: Microbes growing in/on culture medium

  23. Agar • Complex polysaccharide • Used as solidifying agent for culture media in Petri plates, slants, and deeps • Generally not metabolized by microbes • Liquefies at 100°C • Solidifies ~40°C

  24. Culture Media • Chemically Defined Media: Exact chemical composition is known. The medium must contain organic growth factors that serve as a source of carbon and energy • Known for growth of “fastidious” and autotrophic organism • Used in tests to determine concentration of a vitamin in a substance • Complex Media: Extracts and digests of yeasts, meat, or plants • Nutrient broth: liquid • Nutrient agar: Solid

  25. Preserving Bacteria Cultures • Deep-freezing: -50°to -95°C (Glycerol is added to the culture the cells) • Lyophilization (freeze-drying): Frozen (-54° to -72°C) and dehydrated in a vacuum (sublimation)

  26. Culture Characteristics • A pure culture contains only one species or strain • A colony is a population of cells arising from a single cell or spore or from a group of attached cells • A colony is often called a colony-forming unit (CFU)

  27. Reproduction in Prokaryotes • Binary fission • Budding: initial outgrowth (bud) that enlarges until its size approaches that of a parent cell and then it separates • Conidiospores (actinomycetes): filamentous bacteria that reproduce by producing chains of conidiospores • Fragmentation of filaments:Few filamentous species simply fragment and the fragments initiate the growth of the new cells

  28. Binary Fission

  29. Generation Time: time required for a cell to divide and its population to double

  30. Phases of Growth • Lag Phase: period of little or no cell division since cells do not reproduce immediately a new medium (1 hour to several days) • Log Phase or Exponential Growth Phase: cells begin to divide, enter period of growth or logarithmic increase (straight line). Microorganisms are particularly sensitive to adverse conditions (radiation, antibiotics) • Stationary Phase: growth rate slows, number of microbial deaths balances number of new cells. This is due to exhaustion of nutrients, changes of pH. • Death Phase: logarithmic decline phase starts and continues until the population is diminished to a few cells or dies out completely

  31. http://www.youtube.com/watch?v=SuvGpMevLPU

  32. 1. Plate Count Method:Perform serial dilutions of a sample and plate each on an agar-medium plate After incubation, count colonies on plates that have 25-250 colonies (CFUs)

  33. Direct Measurements of Microbial Growth: 2. Filtration Bacteria are filtered out of a liquid and then transferred to a Petri dish

  34. Direct Measurements of Microbial Growth: 3. Direct Microscopic Count

  35. Estimating Bacterial Numbers by Indirect Methods Turbidityis not useful to measure contamination of liquids by relatively small numbers of bacteria

  36. Estimating Bacterial Numbersby Indirect methods • Metabolic activity: it assumes the amount of a certain metabolic product, such as acid or carbon dioxide, is in direct proportion to the number of bacteria present. • Determination of vitamin amount assay • Dry weight: used to measure the growth of filamentous organisms. • Fungus is removed from growth medium, filtered, and dried in a desiccator and then weighed. • HOMEWORK QUESTION: Which methods count for total cells and which ones for viable cells?

  37. The Control of Microbial Growth • Sepsis refers to microbial contamination. • Asepsis is the absence of significant contamination. • Aseptic surgery techniques prevent microbial contamination of wounds.

  38. 5.2 Microorganisms as Tools • Microbial Enzymes • Used in applications from food production to molecular biology research • Taq DNA polymerase • Isolated from a thermophile • Cellulase • Makes animal food more easily digestible • Stone-washed jeans • Subtilisin • Laundry detergents

  39. 5.2 Microorganisms as Tools • Hosts for cloning genes • Transformation – the ability of bacteria to take in DNA from their surrounding environment • Essential step in the recombinant DNA cloning process • Competent cells are cells that have been treated so they are ready to take up DNA easily • Treat cells with ice-cold solution of calcium chloride

  40. 5.2 Microorganisms as Tools • Transformation • Target DNA is introduced into a plasmid containing one or more antibiotic resistance genes (Ligation) • Plasmid vector is mixed in a tube with competent cells and placed on ice • Competent cells are heated briefly (heat shock) to allow DNA to enter cell • Growth in liquid media • Plate on agar plates containing antibiotics

  41. 5.2 Microorganisms as Tools

  42. 5.2 Microorganisms as Tools • Electroporation • An instrument called an electroporator produces a brief electrical shock that introduces DNA into the cells without killing them • Advantages • Rapid • Requires fewer cells • Can be used to introduce DNA into other cell types • More efficient process

  43. 5.2 Microorganisms as Tools • Bacteria can be used to mass-produce proteins • Plasmid vectors used are often called expression vectors • Incorporate prokaryotic promoter sequences • Purification of proteins after production: • Bacterial fusion proteins • Gene for protein of interest is inserted into a plasmid containing a gene for a well-known protein that serves as a “tag” • The tag protein allows for the isolation and purification of the recombinant protein as a fusion protein

  44. 5.2 Microorganisms as Tools Purification

  45. 5.2 Microorganisms as Tools • Microbial Proteins as Reporters • Bioluminescence – method of producing light used by marine organisms • Created by bacteria such a Vibrio fisheri that use marine organism as a host • Create light through action of lux genes

  46. 5.2 Microorganisms as Tools • Microbial Proteins as Reporters • Lux genes have been cloned and used to study gene expression • Clone lux genes into plasmid • If inserted into animal or plant cells, will produce luciferase and will fluoresce, providing a visual indicator of gene expression

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