BIO 208 Class Notes
BIO 208 Class Notes BIO 208
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Lauren Van Atter
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Date Created: 08/16/16
The Microbial World and You 08/31/2015 ▯ Microbe Facts Most microbes do not cause disease o Chronic wasting disease o West nile virus o Mad Cow disease o E. Coli/E. Colie 08757 o Meningitis All thought to be caused by microbes, but they were actually prions Microbes first appeared on the earth about 3.8 billion years ago. o They are critically important in sustaining life on our planet Microbes make up most living matter and display tremendous diversity o Less than 1% have been cultured and studied Microbes generate at least 50% of the oxygen we breath Microbes offer unusual capabilities reflecting the diversity of their environmental niches o These may be organisms of value in addressing global change, biotechnology, and energy production What do they look like? o Macroscopic Mushrooms Molds Algal blooms Biofilms (slimes) on rocks Plaque o Microscopic All kinds of shapes Cocci--sphere Streptococci—chains Diplococcic—pairs Staphylococci—clumps Rods Curved Straight Spiral Other Squares Club Filamentous Branching No distinct shape ▯ ▯ The Beginning of Microbiology ▯ I. Discovery of Microbes a. Ancient Chinese i. Chinese medical text 1766 B.C. ii. Describstsymptoms of polio and smallpox b. Romans (1 Century B.C.) i. Hypothesized that invisible animals caused disease ii. Malaria led to the fall of the roman empire c. Antoni van Leeuwenhoek (1673) i. First to see microbes~”animalcules” ▯ ▯ The Transition Period ▯ I. Battle over Spontaneous Generation a. Redi (1668) i. Maggots do not arise from rotting meat 1. Jar with meat and no top a. Over time, flies on jar and maggots on meat 2. Jar with meat and top a. No flies or maggots 3. Jar with meat and cheesecloth a. Maggots on cheesecloth, but not on meat i. *No support for spontaneous generation b. Needham (1745) i. Boiled broth in a beaker, poured it into a flask, and corked it. 1. Teemed with growth a. *Supports spontaneous generation c. Spallanzani (1765) i. 4 treatments with broth in flask Growth? 1. boiled 5 minutes, cooled, corked. + 2. Boiled 60 minutes, cooled, corked. + 3. Boiled 5 minutes, corked immediately + 4. Boiled 60 minutes, corked immediately - a. *No support for spontaneous generation d. Pasteur (1861) i. Swan-necked flask 1. Non-sterile liquid poured into flask neck of flask drawn out in flame liquid sterilized by heating liquid cooled slowly liquid remains sterile indefinitely 2. Flask tipped so microorganism-laden dust contacts sterile liquid microorganisms grow in liquid *Spontaneous generation disproved in full ▯ ▯ The Golden Age of Microbiology ▯ I. Germ Theory of Disease—diseases are caused by microbes a. Basi (1835) i. Silkworm disease caused by fungus b. Berkeley (1845) i. Potato blight caused by fungus 1. i.e. Irish potato famine c. Semmelweis (1840s) i. Child-bed fever was contagious 1. Women died during childbirth from fever they would get. 2. ~13% death rate in hospital ii. Instituted washing hands between surgical/childbirth patients 1. ~2% death rate d. Lister (1867) i. Phenol—known to kill microorganisms 1. Now found in Listerine ii. Aseptic surgery 1. Used phenol during/prior to surgery to control infection —antiseptic spray e. Koch i. One specific microbial agent causes one specific disease 1. 1876—Bacillus anthracis a. “Anthrax is a disease of animals” 2. 1882—Mycobacterium tuberculosis 3. 1883—Vibrio cholera ii. Koch’s Postulates 1. Associate—specific microbe with a specific disease 2. Isolate—in pure culture 3. Inoculate—causes same disease 4. Re-isolate—same organism f. Microbial effects on matter i. Schwann (1837) 1. Yeast: sugaralcohol = wine ii. Pasteur (1857-1860) 1. All fermentations are due to the activities of specific yeasts and bacteria iii. Winogradsky (1892) 1. Microbes are involved in the cycling of nutrients in soil/aquatic environments 2. ▯ I. How do we classify microbes? a. What is a species? i. Interbreeding organisms b. What is a microbial species? i. Lack obvious morphological differences ii. Traditional versus genetic approaches 1. Traditional a. Staining—shape, morphology, etc. b. Metabolic differences c. Biochemical tests 2. Genetic a. Based on DNA similarity c. Three domain system i. How is the tree of life produced? 1. DNA sequences determined and aligned 2. Differences in bases are identified 3. Computer program generates tree illustrating similarities 4. Distance b/w organisms directly due to their relatives II. How do we visualize microbes? a. Problems to overcome i. They’re small ii. They’re fuzzy iii. They look distorted b. Units of Measure i. 1 μm (Micrometer) = 0.000001 or 10^-6m 1. 1m = 1,000,000μ ii. 1 nm (nanometer) = 0.000000001 or 10^-9m 1. 1m = 1,000,000,000 nm c. Properties of light i. Wavelength (λ) of light (or electron beam) used ii. Resolution—ability to see 2 things that are close together as discrete objects 1. Shorter λ = better resolution 2. Visible light: λ = 550 nm a. Resolution max = 200 nm (0.2μ) 3. Ultraviolet light: λ = 100-400 nm a. Resolution max = 100 nm 4. Electrons: λ = 0.005 nm a. Resolution max = 0.2 nm iii. Refraction—bending of light as it passes from one medium to another of different density d. Microscopy types i. Light microscopy—uses visible light 1. Magnification up to 1,000x 2. Resolving power = 0.2μm a. Bright field—light goes direction through specimen i. Dark specimen, bright background ii. Used to view killed/stained microbes b. Dark field—light doesn’t go through specimen, but is refracted off i. Light specimen, dark background ii. Used to view living/unstained microbes c. Phase contrast—light is moved out of phase i. Enhances small differences ii. Used to view internal structures in living microbes, esp. eukaryotes ii. Fluorescence 1. UV light is used, shorter λ (100-400nm) a. Invisible 2. Molecules are excited to release light with a longer λ a. Visible 3. Bright, fluorescent specimen, dark background 4. Natural or a fluorochrome—stain/dye a. Can be used to view a specimen against a complex background b. R.P. 0.1μm iii. Electron microscopy 1. Uses electrons, even shorter λ (0.005nm) a. Transmission Electron Microscopy (TEM) i. Can view internal structures—have to slice cells into thin layers ii. R.P. 0.2nm (light = 0.2μm) iii. Magnification = 10,000 – 100,000x 1. (Light = 1,000x) b. Scanning Electron Microscopy (SEM) i. Surface view of cells—fix cells ii. R.P. = 20nm iii. Magnification = 1,000 -10,000x iv. ▯ The Prokaryotic Cell I. Composition a. H20—70% b. Macromolecules—26% i. Proteins ii. Group of lipids iii. carbs c. Small molecules and ions—4% i. Lipids ii. Amino acids iii. Glucose II. Size a. “average”—E. coli i. 1μm x 2-6 μm b. smallest i. 0.2μm ii. mycoplasmas c. Oscillatoria i. 8μm x 50 μm d. Thiomargarita namibiensis i. 0.3mm (300μm) III. Shapes a. Coccus (cocci) Ο i. Pair—diplococcic ΟΟ ii. Chains—streptococci ΟΟΟ iii. Clusters—staphylococci b. Bacillus (bacilli) i. Straight rods and in chains c. Spiral i. Vibrio—curved ii. Spirochete—spiral IV. Prokaryote Structure a. Internal Structures—found in ALL i. Cytoplasm 1. 80% water 2. 20% dissolved/suspended stuff ii. ribosomes 1. protein + ribonucleic acid (RNA) 2. site of protein synthesis 3. 70S (vs 80S for eukaryotes) 4. important target for antibiotics iii. nucleoid (nuclear region) 1. mostly DNA (double stranded) 2. circular chromosome b. Internal Structures—found in SOME i. Plasmids 1. Extrachromosomal DNA 2. Circular 3. Contain extra genes (non-essential) a. Antibiotic resistance, toxin production ii. Spores 1. Endospores—resting structures a. Ex. Bacillus anthracis; Clostridium botulinum b. Function—allows microbes to survive long term under adverse conditions c. Step 1—sporulation—in response to some unfavorable environmental cue i. Forms spore d. Step 2—germinations—in response to some favorable environmental cue i. Spore vegetative cell c. Plasma Membrane i. Cytoplasmic membrane ii. Composition 1. Phospholipids a. Polar end and non polar end (hydrophilic and hydrophobic end) 2. Proteins a. Peripheral and integral 3. Bacteria a. Have a bilayer b. Some archaea have a monolayer iii. Function 1. Retains the cytoplasm 2. Site of crucial metabolic processes a. Respiration b. Photosynthesis 3. Contain receptor molecules a. “nose” b. “eyes” 4. selectively permeable barrier a. simple diffusion—small molecules move from high concentration to low concentration i. Ex. CO2, O2 ii. Not very efficient iii. Not used much b. Osmosis—solvents move from high concentration to low concentration i. Ex. H20 c. Facilitated diffusion—uses transporter proteins to move molecules across membrane i. Ex. Amino acids ii. Important in euks, not proks 1. *None of the above require energy— can not be used against concentration gradient d. Active transport—transport molecules against a concentration gradient i. Uses carrier proteins ii. Organism can concentrate substances e. Group translocation—substance is chemically altered during movement i. Ex. Glucose 1. *Both the above require energy d. Cell wall i. Most proks have, with few exceptions (mycoplasmas) ii. Functions 1. Shape—elastic, rigid 2. Pathogenicity 3. Protects from changes in water pressure—strong iii. Bacteria—divided into 2 groups with different thickness of peptidoglycan (murein) 1. Gram + cells a. Thick layers of peptidoglycan b. Techoic acids—toxicity c. Single plasma membrane 2. Gram – cells a. Thin layer of peptidoglycan b. Lipopolysaccharide (LPS)—toxicity c. Two plasma membranes e. Structures external to the cell wall i. Glycocalyx—like a spider web; sticky 1. Polysaccharide (sugar) or polypeptide (amino acids) 2. If tight—capsule (remember anthrax? 3. If loose—slime layer 4. Functions: a. Attachment—other cells, surfaces (teeth) b. Prevents phagocytosis—helps them survive and not be engulfed by other cells c. Protects from drying out ii. Flagella—like a tail/propeller 1. Made of protein flagellin 2. Function—movement = taxis a. Chemotaxis = chemical b. Phototaxis = light i. Cell may want to move toward light but ends up zigzagging its way there instead of moving in a straight line, because flagella cause it to run and tumble. 3. Used for vaccines and identification a. Bad E. coli = 0157:H7 4. Arrangement—in lab manual iii. Axial filament—like a corkscrew 1. Function—movement in spirochetes (spiral shaped) iv. Fimbriae—like short bristles 1. Functions— a. Attachment b. Prevents phagocytosis i. Ex. Neisseria gonorrhoeae v. Pili—like long hairs, hollow 1. Function—DNA exchange (plasmid) a. Antibiotic resistance ▯ ▯ Eukaryotes I. Differences between prokaryotes and eukaryotes a. Prokaryotes are the size of an organelle, therefore smaller than eukaryotes b. Major differences: i. Membrane bound nucleus 1. Prokaryote: absent 2. Eukaryote: present ii. Organelles—allow separation of biochemical and physiological functions 1. Prokaryote: absent 2. Eukaryote: present c. Similarities: i. Genetic code—DNA: GCAT ii. Biochemical pathways—Krebs; glycolysis II. Origins of the Eukaryotic Cell a. Theory of Serial Endosymbiosis i. Credited to Lynn Margulis ii. Eukaryotic cells are the result of prokaryotic mergers iii. Symbiosis—living together iv. Endosymbiont—living inside v. How did they get in? 1. Engulfed by other bacteria b. Chloroplasts and mitochondria were bacteria i. Evidence: 1. Same size as bacteria 2. Surrounded by a plasma membrane 3. Contain 70s ribosomes 4. Have their own DNA 5. Grow and divide on their own schedule 6. Same pigments (cyanos) ii. Some organisms don’t have mitochondria, instead they have endosymbiotic bacteria 1. Mixotricha paradoxa a. Protist—has a nucleus b. Mitochondria = free-living cocci c. 3 types of cilia i. 250,000 spirochetes ii. 250,000 rods iii. 200 large spirochetes ▯ Microbial Growth 09/28/2015 Good vs. Bad Microbial Growth Good: fermented food, antibiotics, water treatment Bad: food spoilage, corrosion, biofilms, clogging, corrosion, growth on medical devices I. Growth of Bacterial Cultures a. Bacterial division—binary fission i. Growth is exponential b. Generation time—time required for a bacterial population to double i. 12 or 100,000200,00 takes the same amount of time ii. 24 iii. vary by organism 1. Not all organisms double at the same rate; some are fasner than others iv. Cell # = 2 1. n = # of doublings 2. ex. 2 = 4 3. initial # cells x 2enerati= cells ▯ ▯ II. Effects of nutrients on microbial growth a. Cell growth requirements i. Source of monomers—surroundings and/or synthesized by cell 1. i.e. glucose or amino acids ii. Source of information—DNA iii. Source of energy—biochemical reactions 1. i.e. ATP b. nutritional categories i. energy source (E source) 1. organic molecules—organotroph 2. inorganic molecules—lithotroph a. *only prokaryotic cells 3. light—phototroph c. macronutrients (CHNOPS) i. Needs in large amounts ii. Carbon source 1. CO —2utotroph 2. Organic molecules—heterotroph a. i.e. polysaccharides, fats, other organisms iii. Nitrogen source 1. Proteins, nucleic acids 2. Amino groups (-NH ) 2 3. Ammonia (NH ) 3 - 4. Nitrate (NO ) 3 5. Atmospheric nitrogen (N ) 2 iv. Phosphorous source 3- 1. Phosphate (PO ) 4 2. Organic molecules v. Sulfur source 2- 1. Sulfate (SO )4 2. Sulfide d. Micronutrients (K, Mg, Fe) i. Needed in small amounts ii. Act as cofactors for many enzymes iii. Are important in cell structures 1. Ca in diatoms e. Trace elements i. Required by a small number of enzymes ii. Co, Zn, Cu III. Growth Curve a. Lag phase i. New medium ii. Cell numbers do not increase iii. Cell size does increase iv. Synthesis (DNA, RNA, protein) v. Length varies b. Log phase i. Growth at max. rate ii. Population doubling iii. Metabolically active—sensitive to antibiotics iv. Industrial fermentation c. Stationary phase i. Death = growth ii. Live cells constant iii. Cells resistant to stress iv. Antibiotic production v. Why does this occur? 1. Depleted nutrients 2. Depleted O 2 3. Waste build-up vi. Death phase 1. Death > growth 2. Sporulation IV. Measurement of Growth a. Cell numbers i. Direct counts 1. Advantages—quick, cheap, cell size, cell morphology 2. Disadvantages—need larger number or cells, can’t tell living from dead 3. Methods—microscope, Coulter Counter ii. Plate counts—indirect 1. Dilute sample dispersed over solid agar surface a. 1 cell 1 colony 2. cells in sample can then be determined 3. a countable plate has between 30-300 colonies 4. advantages—simple, sensitive 5. disadvantages—have to know how to culture the target organism, time b. cell mass i. dry weight—filter cells, dry, weigh ii. light scatter—spectrophotometer 1. determine how much light passes through a turbid tube, gives an idea as to how many cells are in the tube blocking the light. iii. disadvantages—need LOTS of cells; dead cells? 1. i.e. even 10 cells would not be turbid 2. dead cells would be counted because they are still there. V. What affects microbial growth? a. Nutrient availability i. Copiotrophs—require high nutrient levels 1. copious ii. Oligotrophs—require low nutrient levels 1. Few iii. Physical environment 1. Temperature a. Important points: i. Cell temperature is the same as the environment ii. Enzyme catalyzed reactions are temperature sensitive iii. Higher temps speed up chemical reactions b. cardinal temps i. minimum—membranes gel, slow transport across membrane ii. optimum—enzyme reactions occur at max rate iii. maximum—proteins and nucleic acids denature, cytoplasmic membrane collapses c. temp classification i. psychrophile—cold loving 1. optimum—15° C 2. Ex. Chlamydomonas nivalis (-36°, 0°, 4°) ii. Psychrotroph—cold tolerant 1. Optimum—20°-30°C iii. Mesophile—middle temps 1. Optimum—25°-40°C 2. Ex. Most pathogens iv. Thermophile—heat loving 1. Optimum—50°-60°C 2. Ex. Thermoplasma v. Hyperthermophile—extreme heat 1. Optimum— >80°C 2. Ex. Pyrolobus fumarii (90°, 106°, 113°) 2. pH a. important points i. internal pH of cell is neutral ii. each species has a range and optimum iii. protozoa and most bacteria—neural iv. fungi and algae—slightly acidic b. effects of wrong pH i. disrupts membranes ii. inhibits transport across membrane iii. inactivates enzymes c. pH classification i. neutrophils—5.5-8 1. most organisms ii. acidophiles—0-5.5 1. some molds and bacteria 2. habitats—ore mines, bogs, stomach 3. Ex. Helicobacter pylori –ulcers iii. Alkaliphiles—8.5-11.5 1. Habitats—soda lakes 2. Uses—laundry detergent 3. Water a. Important points i. Water is essential to all life b. Effects of water imbalance i. Isotonic—solute concentration is balanced ii. Hypotonic—outside cell concentration is low relative to inside iii. Hypertonic—outside cell solute concentration is high relative to inside c. Adaptations to hypertonic conditions i. Halotolerant—tolerate high salt 1. Habitat—skin 2. Ex. Staphylococcus aureus ii. Halophiles—require salt (1-15%) 1. Habitat—seawater iii. Extreme halophiles—15-30% salt 1. Habitat—dead sea, Great Salt Lake 2. Ex. Halobacterium (Archaea) iv. Osmophiles—high sugar 1. Habitat—maple syrup, ham, honey v. Xerophiles—extremely dry 1. Habitat—dried foods, deserts, cereal 4. Oxygen a. Evolution of earth’s atmosphere i. 4.8 bya no atmosphere ii. 2.25 bya oxygenic photosynthesis iii. 2 bya 1% O2 iv. today 21% O 2 b. why is O 2ad? i. Oxygen strongly accepts electrons c. What protects cells from damage? i. Superoxide disumtase (SOD) ii. Catalase d. Oxygen tolerance classification i. Obligate aeroe—requires O f2r growth 1. B. subtilis ii. Facultative anaerobe—can grow with or w/o O 2 1. E. coli iii. Obligate anaerobe—does not require O fo2 growth, can’t survive exposure 1. Clostridium iv. Microaerophile—needs low amounts of O 2 v. Aerotolerant—does not require O2 I. Terminology a. Sterilization—destroys all viable cells, spores, viruses b. Disinfection—kills pathogens on inanimate objects i. Non-living c. Antiseptic—kills pathogens on living tissue d. Sanitize—lowers number of pathogens to an “acceptable” level II. How do we kill microbes? a. Nonspecific methods i. Physical methods—heat ii. Chemical methods 1. Phenols—denature proteins, disrupt membranes a. Ex. Lysol 2. Alcohols—denature proteins, dissolve membranes a. Ex. Ethanol, isopropanol 3. Halogens— b. Specific methods—antibiotics i. Definition—natural substances produced by one organism that inhibits growth of another ii. How were they discovered? 1. Fleming (1928)—Penicillin notatum iii. How do they work? 1. Bacteriocidal—kill 2. Bacteriostatic—inhibit 3. Selective toxicity—“no” harm to host iv. Cellular target sites 1. Cell wall a. Ex. Penicillin—antibacterial prevent synthesis of peptidoglycan works only on log phase cells 2. Plasma membrane a. Ex. Nystatin—antifungal alters permeability 3. Nucleic acids a. Ex. Rifampin—penetrates tissues interferes with mRNA synthesis 4. Proteins a. Ex. Tetracycline—wide spectrum inhibits protein synthesis targets 70s ribosomes c. Antibiotic resistance i. Timeline 1. 1928—penicillin discovered 2. 1930s & on—rapid discovery of many new antibiotics 3. 1940s—penicillin use begins 4. 1960s—many infectious diseases declining; government cuts research funding 5. 1980s—less research to discover new antibiotics (penicillin = magic bullet) 6. 1990s—rapid increase in resistance ii. increase in antibiotic resistance 1. post-surgical prophylaxis 2. needless prescriptions 3. not regulated in all foreign countries 4. half of the antibiotics produced in the US are used in animal feed I. Metabolism a. Metabolism is the sum of all chemical reactions in a cell a. Catabolism i. Breakdown of complex organic molecules into simpler ones ii. Degradation reactions 1. Release energy 2. Protein amino acids b. Anabolism i. Building complex organic molecules from smaller ones ii. Synthesis reactions 1. Use energy 2. Amino acids proteins 3. Sugars carbohydrates c. ATP i. Energy currency that links catabolism and anabolism II. Enzymes a. Catalytic proteins b. Properties i. Speed up reactions 1. Ex. DNA polymerase; lactate dehydrogenase ii. Powerful 1. Ex. N2+ 3H 2 2NH 3 2. In the lab—450° C, 400 atm 3. Bacteria—20° C, 1 atm. –nitrogenase iii. Specific iv. Structure 1. Large 2. Globular 3. Cofactors (inorganic: metals—Zn, Cu, etc.) 4. Coenzymers (organic: vitamins—riboflavin) c. Factors that influence activity i. Temperature 1. Heat denatures ii. pH 1. hi and low denature iii. substrate concentration 1. increase concentration, increase enzyme activity iv. inhibitors 1. competitive—compete for active site 2. non-competitive—binds somewhere else III.Energy Production a. Oxidation i. Reduction reactions (redox) ii. Terms - 1. Oxidation—loss of e 2. Reduction—gain of e - 3. “OIL RIG” iii. electron carriers—temporarily accept e- and release them to other molecules 1. cycle between reduced and oxidized + 2. ex. NAD --coenzyme b. Catabolism i. Breakdown to release energy ii. Overview—carbohydrate catabolism 1. 2 ways for getting energy from carbs a. fermentation - i. e-donor is the substrate (organic) ii. e acceptor is an intermediate formed from initial substrate iii. *does not use O 2 iv. Ex. Saccharomyces cerevisae; E. coli b. Respiration i. E donor is the substrate ii. Electron acceptos is a molecule other than substrate or its itermediates iii. Aerobic—e acceptor is O 2 1. Ex. Pseudomonas aueruginosa; Escherichia coli iv. Anaerobic—e acceptor is not O 2 1. Unique to bacteria and archaea 2. Ex. Desulfovibrio 2. Glycolysis—breakdown of glucose - a. Oxidation of glucose (loses e ) b. Glucose (6C) 2 intermediates (3C) 2 pyruvate (3C) + ATP 3. Fermentation—use organic molecule derived from substrate as e acceptor a. Re-oxidize NAD+ b. Dump waste product from cell c. Types—defined by end products i. Ex. Lactic acid, alcohol, mixed acid d. In nature i. Primarily bacteria ii. Wherever organic matter accumulates and no O 2 iii. Also some protozoa and fungi 4. Respiration—series of redox reactions that generate ATP - a. Involves membrane bound e transport chain b. Transfers e to terminal e acceptor c. Re-oxidizes NAD+ - d. Krebs Cycle—e carriers are reduced i. NAD NADH e. Electron Transport chain—ATP synthesis; LOTS! i. Ferm—2ATP ii. Resp—up to 38 ATP f. Aerobic respiration—O is 2he terminal e acceptor g. Anaerobic respiration—O is not 2he terminal e - acceptor i. Less energy generated ii. Exclusive to prokaryotes - iii. Ex. Denitrifiers—NO bro3h, gas bubble 1. Sulfate reducers 2. methanogens 3. ▯ I. Way of Life a. C to make organic molecules comes from CO 2 b. Energy source ATP CO gluc2se II. Types of Autotrophs a. Chemolithoautotrophs i. Energy source = inorganic chemicals ii. PROKARYOTES ONLY iii. Requires O 2 iv. Subtypes 1. Hydrogen-oxidizing bacteria - a. Hydrogen is the e donor 2. Sulfur-oxidizing bacteria - a. Sulfur is the e donor 3. Nitrifying bacteria (N oxidizers) a. Nitrogen is the e donor b. Photoautotrophs i. Energy source = sunlight ii. Oxygenic phototrophs - 1. E donor is H O 2 2. Ex. Cyanobacteria, algae, plants 3. H O 2e + 2H + ½ O+ 2 2 iii. Anoxygenic phototrophs 1. Proks only - 2. E donor is a reduced substance from the environment (something other than water) 3. Ex. Chromatium - + 4. H S2 2e + 2H + S Microbial Genetics 10/26/2015 ▯ I. Background a. Terms i. Nucleotides—monomers that make up nucleic acids 1. Nitrogen base (purine or pyrimidine) 2. 5 C sugar (ribose or deoxyribose) 3. PO4group ii. nucleic acids—macromolecules made up of nucleotides (DNA and RNA) 1. DNA—deoxyribonucleic acid a. Storage molecule for genetic information b. 5 C sugar is deoxyribose c. double stranded (ds) polymer d. sequence of nucleotides has mean4ng (ATCG) i. 4 bases can be arranged in 4 (256) different combinations ii. So, if there are 100 nucleotides in a chain of DNA = 4 10different combinations (1.61x10 ) 1. E. coli = >4x10 bases 2. RNA—ribonucleic acid a. Primarily involved in protein synthesis b. 5 C sugar is ribose c. single strand (ss) polymer d. 3 types i. mRNA—messenger RNA—scaffold ii. tRNA—transfer RNA—transfers a.a. to messenger RNA iii. rRNA—ribosomal RNA—makes up ribosome 3. genes—specific sequences of nucleotides in DNA that code for a functional product = protein a. Avery—a non protein was responsible for heritable lethal traits in bacteria; worked with Streptococcus pneumonia Protein Protein Protein RNA RNA RNA DNA DNA DNA + + b. Helped Avery purify the “transforming” factor c. DNA was isolated, a compound found in all living organisms d. Not sure how it worked though II. Structure of DNA a. Franklin—DNA image b. Crick & Watson—structure c. Double helix structure i. Polymer of nucleotides—A, T, C, G 1. Base Pairing a. Bases are complementary b. Strands are antiparallel c. New bases are always added to the 3’ end (5’3’) i. A—T ii. C—G ii. Backbone is deoxyribose—phosphate iii. Notice number of H bonds between bases 1. A-T = 2 bonds 2. C-G = 3 bonds III.How is DNA copied? a. DNA replication—DNADNA b. Semiconservative—resulting new ds DNA after replication is made up of one old strand and one new strand c. Prokaryotic DNA replication i. Typically one circular chromosome ii. One origin of replication (euks have many) iii. Replication Fork—origin of replication (2) iv. Leading strand—continuous v. Lagging Strand—discontinuous vi. RNA primer— vii. RNA polymerase—adds RNA primer viii. DNA polymerase—adds DNA bases to primer ix. DNA ligase—connects all of the fragments x. Okazaki fragments—pieces of DNA that haven’t yet been stitched together IV. DNA to Protein a. Transcription i. Copy; DNA mRNA ii. Nucleotides Nucleotides (DNA RNA) iii. Promoter—tells RNA polymerase where to bind (genes begin) iv. RNA polymerase—unwinds DNA and adds RNA nucleotides to the DNA template v. Terminator—where the gene ends vi. DNA Strand: 5’ G C T A G T 3’ Read right to left; start at 3’ vii. mRNA strand: 3’ C G A U C A 5’ b. Translation i. mRNA protein ii. nucleotides amino acids (RNA protein) 1. codon—3 nucleotide bases in a row that code for amino acids a. 4 nucleotides (ACGU) i. 3 nucleotides make a codon ii. 4 different codons possible = 64 2. however, only 20 amino acids 3. degeneracy—multiple codons can form an amino acid a. Ex. Leucine has 6 4. Sense codons—code for amino acids (61) 5. Nonsense codons—not translated into amino acids (Stop; 3) iii. Ribosomes—where protein synthesis occurs 1. Made up of rRNA and protein 2. 70s ribosomes in prokaryotes; 80s in eukaryotes iv. tRNA—brings (transfers) the amino acids to the mRNA v. anticodon—region on tRNA that is complementary to the codon V. Regulation of Gene expression a. DNA RNA protein Transcription translation b. Constitutive vs. Inducible i. Constitutive—genes are always expressed 1. Ex. Breakdown of glucose (glycolysis) ii. Inducible—genes are regulated on/off to conserve energy 1. Ex. Breadown of lactose 2. Repression—the ability to turn genes off a. Components: i. RNA polymerase—binds to promoter ii. Structural genes—encode enzymes iii. Promoter—RNA polymerase binding site iv. Operator—where the repressor binds v. I gene—codes for a repressor protein 3. Induction—the ability to turn genes on a. Ex. The lactose (lac) operon b. Operon—a group of coordinately regulated genes with related functions c. Structure of the lac operon i. Consists of promoter, operator sites, structural genes which code for the protein, and regulated by the product of regulatory gene 1. Regulatory gene—encodes the repressor, is always transcribed and translated d. When do bacteria control the expression of these genes? i. Absence of lactose = of ii. When lactose is present and glucose is not = on iii. When both lactose and glucose are present – of iv. Ex. E. coli grow better on glucose than lactose; when given both, the lac operon is repressed until glucose is depleted— catabolite repression. E. coli cells have the genes to metabolize lactose, but do not always express them VI. Mutations a. Change in the sequence of nucleotides in DNA; can be good, bad, and neutral b. Types of mutations i. Point mutation—base substitution; a single base is replaced with another 1. Missense—results in a different amino acid 2. Nonsense—results in a stop codon, produces a shortened protein 3. Silent—no change in amino acid ii. Frame shift mutation—causes greater damage than point mutation 1. Insertion—adding a nucleotide 2. Deletion—subtracting a nucleotide c. Spontaneous mutations—happens in the absence of a mutagen d. Induced mutations—produced by agents (mutagens) i. Can be physical or chemical ii. Ex. Ethidium bromide (chemical); ultraviolet light (physical) e. Sickle cell anemia—mutation in Beta-globin protein; hemoglobin is misshaped and doesn’t stick; point shift mutation resulting in missense VII. Exchange of genetic information a. Types of genetic transfer i. Transformation—DNA released by one bacterium (naked DNA) is taken up by another 1. Can by DNA fragments or plasmids 2. Some organisms—ex. Bacillus—are naturally competent 3. Works best when donor and recipient are closely related 4. Griffith’s experiment a. Worked with mice and Streptococcus pneumonia b. Smooth, capsule colony Live strain mouse mouse dies c. Smooth, capsule colony Strain heat fixed mouse mouse alive d. Rough colony live strain mouse mouse lives e. Rough live cells + smooth, dead cells mouse dead mouse ii. Conjugation—mediated by a plasmid 1. Plasmid—circular, self replicating extrachromosomal DNA that carries genes not essential for cell survival or growth under normal conditions. 2. Requires direct cell-cell contact pilus 3. Cells must be opposite mating types a. F+ x F- F+ & F+ 4. Plasmid examples a. Pseudomonas—degrade contaminants b. Clostridium tetani—neurotoxin c. Bacillus anthracis—toxin, capsule d. Antibiotic resistance iii. Transduction—bacterial DNA is transferred from donor to recipient inside a virus 1. Bacteriophage—viruses specific for bacteria ▯ I. General Characteristics of Viruses a. Distinctive features i. Nucleic acid –DNA or RNA, not both ii. Protein coat—surrounds and protects nucleic acid 1. May also have an envelope (lipid, carbohydrate, and/or protein) iii. Obligate intracellular parasites—multiply inside living cells using machinery of host iv. Cause the synthesis of specialized structures that can transfer viral nucleic acids to other cells b. Host range—determined by attachment i. Animal cells-receptor for virus is on plasma membrane of host cell ii. Bacteria—receptor on cell wall, fimbriae or flagella c. Sizes i. Very small sizes, some extremely large ii. Measured in nm range II. Structure a. Viewed with Electron Microscope b. Virion—complete, fully developed viral particle (infectious) c. Nucleic acid—DNA or RNA; ss or ds d. Capsids and Envelopes i. Capsid—the protein coat ii. Capsomere—protein subunit that makes up capsid iii. Envelope—lipids + carbs + proteins 1. Naked vs. enveloped iv. Spikes—carbo-protein structures e. Morphology—used in classification i. Helical—long rod 1. Capsid and nucleic acid are helical ii. Polyhedral—many sided 1. Most viruses iii. Complex 1. Ex. Bacteriophages III.Multiplication a. Bacteriophages i. Lytic cycle—lysis and death of host cell 1. Attachment—phage attaches to host cell 2. Penetration—phage penetrates host cell and injects its DNA 3. Biosynthesis—phage DNA directs synthesis of viral components by the host cell. 4. Maturation—viral components are assembled into virions. 5. Release—host cell lyses and new virions are released ii. Lysogenic cycle—host remains alive 1. capable of lytic cycle, can also have it’s own cycle. a. First 2 steps of lytic cycle b. Phage DNA integrates within the bacterial chromosome by recombination, becoming a prophage c. Lysogenic bacterium reproduces normally d. Occasionally, the prophage may excise from the bacterial chromosome by another recombination event, initiating a lytic cycle. iii. Viral Growth Curve 1. Eclipse—period of decline, very few detectable; penetration through maturation b. Animal viruses i. Attachment—viruses attaches to cell membrane ii. Penetration—by endocytosis (whole virus) or fusion (nucleic acid only) iii. Uncoating—by viral or host enzymes iv. Biosynthesis—production of nucleic acid and proteins v. Maturation—nucleic acid and capsid proteins assemble vi. Release—by budding (enveloped viruses) or rupture IV.Viruses and Viral Infections a. DNA viruses i. Replication 1. Transcription and translation ii. Examples 1. Papovaviridae— a. Ds b. Naked c. Includes viruses that cause warts 2. Herpesviridae— a. Ds b. Enveloped (budding) c. Nearly 100 known—includes viruses that cause cold sores, genital herpes, chickenpox, infectious mono 3. Hepadnaviridae— a. Ds b. Enveloped c. Hepatitis B virus—transmitted by blood, needles, saliva, sexual contact b. RNA viruses i. Replication 1. Some make host cell produce RNA-dependent RNA polymerase 2. Can have sense (+) or antisense (-) RNA a. + acts as mRNA b. – acts as mRNA template c. RNA sense strand: i. RNA goes through translation to form protein ii. Can also use RNA-dependent RNA polymerase to become –RNA, using RNA-dependent polymerase again to become +RNA d. RNA Antisense Strand i. –RNA RNA-dependent polymerase +RNA protein ii. –RNA polymerase +RNA polymerase -RNA e. RNA (retrovirus) rt = reverse transcriptase i. Retroviruse carries rt inside premade +RNA DNA ii. Contain reverse transcriptase iii. Makes DNA from viral RNA ii. Examples 1. Picornaviridae— a. Ss b. + strand c. naked d. Rhinovirus—one of many viruses that cause the common cold e. Norovirus—Norwalk agent, causes gastroenteritis, cruise ships 2. Orthomyxoviridae a. Ss b. – strand c. Influenzavirus—the flu V. Prions—Proteinaceous Infections Particles a. Background i. Defective proteins that have a weird life of their own ii. Self replicating proteins 1. Can catalyze their own proteins iii. Cause problems with brain tissue 1. Spongiform—white holes in brain tissue iv. Can be passed on in a number of different ways 1. Inherited, transmitted, consumed v. Some have been around for some time 1. Kuru from cannibalism vi. Cannot be cooked or even sterilized b. Examples in mammals i. Scrapie—sheep ii. Kuru—humans iii. Creutzfeld-Jacob—humans iv. Bovine spongiform encephalopathy (BSE)—mad cow disease v. Chronic wasting disease (CWD) 1. First deer discovered in MI in 2008 in Kent County 2. Precautionary measure—no baiting ▯
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