Microbiology Review PDF

Micro Test 2 Review Central Metabolism Part 2 Glycolysis Pathway ★ Glycolysis is a glucose catabolism pathway that does not require oxygen ★ Some parts of the pathway occur in all living cells ★ During glycolysis a net of 2 ATP is produced via substrate level phosphorylation ★ Glucose...

Micro Test 2 Review Central Metabolism Part 2 Glycolysis Pathway ★ Glycolysis is a glucose catabolism pathway that does not require oxygen ★ Some parts of the pathway occur in all living cells ★ During glycolysis a net of 2 ATP is produced via substrate level phosphorylation ★ Glucose (6C) + 2 ATP + 2 NAD+ —> 2 pyruvate (3C) + 4 ATP + 2 NADH ○ NADH needs to be recycled and used in more REDOX reactions ★ Glycolysis also produces precursor molecules for biosynthetic pathways Recycling reduced Coenzymes: Fermentation ★ Uses 2 pyruvate produced from glycolysis and allows for recycling of reduced coenzymes ★ Fermentation does not require the electron transport chain and produces Ethanol (Alcohol) and Lactate- (yeast) ★ If an organism does not have a good terminal acceptor like oxygen, fermentation occurs ★ Fermentation is done under anaerobic conditions (does not need oxygen!!) Recycling reduced Coenzymes: Respiration; Citric Acid/Krebs/TCA cycle ★ Catabolism of 1 glucose with aerobic conditions (with oxygen) yields 36-38 ATP ★ 2 Pyruvate (3C) + [cofactors] —> 6 CO2 + 8 NADH + 2 FADH + 2GTP (produced via substrate level phosphorylation ) ★ CoA is recycled!!! Electron Transport Chain (ETC) ★ Oxidizes electron carriers (NADH and FADH2) by going down the redox tower ★ Protons are pumped out generating a proton motor force (PTM) ★ Coenzymes ○ NAD+, NADP+, FAD+, Quinones ★ Prosthetic groups ○ FMN(Flavin mononucleotide), Heme, Iron sulfur complexes (Fe-S) ★ Oxygen is the terminal electron acceptor to make water  ATP Synthase ★ Movement of protons down the gradient provides energy for ATP synthesis ★ The enzyme for ATP synthesis is ATP Synthase ★ The reaction is reversible, cells that do not require oxygen run the reaction in reverse to generate a proton motor force!! ★ Fo channels protons to F1 and F1 generates ATP!!  Anabolic Metabolism ★ Biosynthesis of sugars and polysaccharides ○ Not all organisms grow on sugar sources ○ Gluconeogenesis If a cell does not have glucose, it makes glucose ★ Biosynthesis of amino acids and proteins ○ Transaminases move amino groups between molecules ○ Adds NH2 near the end of biosynthesis ★ Biosynthesis of nucleotides and nucleic acids ○ Carbon and nitrogen atoms from amino acids ○ Single carbons from CO2 and folic acid ★ Biosynthesis of fatty acids and lipids ○ ACP = acyl carrier protein is what fatty acids are built on ○ Reaction is repeated lengthening the chain by 2 C in each step ○ The 16C chain is transferred to glycerol to produce the lipid ○ Make important components of the cell membrane Microbial Division and Growth Microbial Growth ➔ Culture Media ◆ Defined medium ◆ Complex medium Defined Medium (Minimal Medium) ➔ Minimum nutrients necessary for growth ➔ Made with purified chemicals ➔ The exact composition is known ◆ Ex: glucose, amino acids, salts, buffers, vitamins, etc ➔ Works well for species with high biosynthetic capacity ◆ They have the ability to make all of the amino acids, nucleotides, etc from basic C and N sources ➔ Good for studying physiology and metabolism ➔ We can add or remove specific metabolites ➔ Bad for organisms with unknown growth requirements Complex Medium (Undefined Medium) ➔ Made with animal digests or plant products ◆ Ex: yeast extract, casein (milk protein), soy beans, beef extract, etc ➔ Required for species with multiple nutritional requirements ◆ Ex: many animal pathogens don’t grow outside the animal so they don’t usually need to make many of the biosynthetic building blocks ➔ Good for broad ranges of organisms Growth Media ➔ Media used for isolating/identifying organisms within a mixed population ➔ Common in clinical labs for identifying pathogens ➔ Selective media ➔ Differential media ➔ Solid media ➔ Liquid media Selective Media ➔ Contains compounds that inhibit the growth of certain organisms while allowing others to grow Differential Media ➔ Contains compounds that cause different colony appearance for different organisms ➔ Dyes can signal the presence of specific biochemical reactions Liquid Medium ➔ Allows for rapid and constant mixing so all cells experience the same conditions ➔ Simple recovery which can concentrate by centrifugation or filtration Solid Medium ➔ Allows for isolation of individual (pure) colonies ➔ Can look at colony morphology ➔ Can count the colony forming units (CFU) Bacterial Cell Division ➔ Binary fission results in symmetric cell division Binary Fission Phase-contrast microscopy Fluorescent DNA stain Fluorescent divisome stain Steps of Cell Division ➔ 1. DNA replication and segregation of DNA ◆ This happens well before the start of division ➔ 2. Cytokinesis = division of the cytoplasm into 2 ◆ Septum formation Choose the center of the cell Divisome (a protein complex associated with cell division) forms Invagination of the membrane ➔ 3. Synthesizing the new cell wall ◆ polymerization of peptidoglycan strands ◆ Cell shape is determined Measuring Population Growth ➔ 1. Direct Count/total count with Petroff-Hausser counting chamber ➔ Pros ◆ Rapid ◆ Inexpensive ◆ Can differentiate between single and clumped cells ➔ Cons ◆ Cannot differentiate between alive and dead cells ◆ Need to immobilize motile cells ◆ Need a high concentration of cells Measuring Population Growth Monitoring Population Growth ➔ 2. Viable Count using serial dilutions and either spread plate or pour plate ➔ Pros ◆ Can count with dilute concentrations (or low concentrations) ➔ Cons ◆ Cannot differentiate between single and clumped cells Monitoring Population Growth ➔ Spread Plate Monitoring Population Growth ➔ Pour Plate Serial Dilutions ➔ Used to obtain a cells suspension that can be accurately counted ➔ Statistically significant counts are found between 30-300 colonies/plate ◆ Over 300 colonies/plate → TMTC (too many too count) ◆ Under 30 colonies/plate → TFTC (too few to count) ➔ To calculate:  Serial Dilutions Example: Find the concentration of bacteria in the sample ➔ Find colony number (between 30-300): 159 colonies ➔ How much is plated: 0.1 mL ➔ ➔ What is the total dilution? 10-3 ➔ 1/total dilution= 1/10-3 = 103 ➔ 1.59 X 103 CFU/mL * 1/10-3 = 0.1 mL 1.59 X 103 CFU/mL * 103 = 1.59 X 106 CFU/mL Monitoring Population Growth ➔ 3. Measure turbidity or optical density ◆ Spectrophotometer which measures absorbance ➔ Pros: ◆ Fast ◆ Simple ◆ Low cost ◆ Accurate ◆ Reproducible ➔ Cons ◆ Can’t differentiate between live and dead cells ◆ Inaccurate at high cell densities  Monitoring Population Growth Cell size changes the optical density (OD) At high densities, numbers can be underestimated Growth curve  Growth of Bacterial Populations ➔ Exponential = logarithmic ➔ g = generation time = the time for cells to divide ➔ n = # of generations ➔ t = time ➔ 2n = # of cells after “n” generations ➔  Population Growth ➔ We plot growth on semi-log scales (log of cells/mL versus time) ➔ Standard measure of growth rate is doubling time = generation time ➔ The whole number intervals = when the cell number is doubled Population Growth Example ➔ What would the generation time be if ◆ 10 cells @ 0 minutes ◆ 80 cells @ 60 minutes ◆ ◆ t = 60 minutes ◆ (doubling) 10 → 20 → 40 → 80 (3 arrows → n=3) ◆ g = 60 minutes/3 = 20 min Calculating Population Growth ➔ If we know the generation tim and the initial number of cells (N0), we can calculate the number of cells (N) at a later time (t) ➔ Ex: How many cells after 60 minutes if N0=2 and g = 20 minutes N=? N0 = 2 t=60min g=20min n=60min/20min = 3  Calculating Population Growth ➔ Ex: How many cells after 60 minutes if N0= 2 cells and g = 20 minutes ◆ Using less math → N0 = 2 cells Doubles every 20 minutes for 60 minutes 2 cells x2→ 4 cells in 20 total minutes 4 cells x2→ 8 cells in 40 total minutes 8 cells x2→ 16 cells in 60 total minutes Growth Phases in Batch Culture Same medium in a fixed volume ➔ Lag: adapting to new conditions ➔ Exponential/logarith mic (log): maximal growth, abundance of nutrients ➔ Stationary: growth slows due to nutrient limitations and accumulation of waste ➔ Death: insufficient energy for cell maintenance or repair We will see no lag phase if transferring to new (identical) growth conditions → The cells will not need time to adapt Growth in a Continuous Culture ➔ Pros ◆ grows cultures for long time ◆ Can mimic a low nutrient environment found in nature ◆ Can enrich for specific organisms with specific conditions ➔ Too little fresh media; bacteria is in stationary/death phase ➔ Low bacterial viability ➔ Steady state: bacterial growth and loss is balanced ➔ Lots of nutrients, exponential growth ➔ Wash out: cells leave faster than growing Growth in a Continuous Culture ➔ Chemostat Factors Affecting Microbial Growth ➔ Medium: complex vs defined ➔ Medium: liquid vs solid ➔ Temperature ➔ pH ➔ Osmotic strength (NaCl + other salts) (Halophiles) ➔ Oxygen levels Factors Affecting Microbial Growth: Temperature ➔ Cardinal temperatures: minimal, optimal, maximum Factors Affecting Microbial Growth: Temperature - Class of Organisms ➔ Psychrophile: growth in cold temperatures (0°C - 20°C) ➔ Mesophile: growth between 15°C - 45°C ➔ Thermophile: growth between 45°C - 80°C ➔ Hyperthermophiles: growth over 80°C ◆ Only archaea above 100°C and at high pressure to avoid boiling  Factors Affecting Microbial Growth - pH ➔ Acidophiles: below pH 8 ➔ Neutrophiles: between pH 6-8 ➔ Alkalophiles: above pH 8 ➔ Cells still maintain an internal pH between pH 6-8 ➔ We often use buffers in artificial medium to maintain a constant pH ➔ For food preservation: acidic/basic conditions inhibit most microbes  Factors Affecting Microbial Growth - Osmotic Strength ➔ External salt conditions ➔ Nonhalophile: growth in less than 1% salt ➔ Halotolerant: growth in less than 10% salt ➔ Halophile: growth in 2.5% - 10% salt ➔ Extreme halophile: growth in over 15% salt  Factors Affecting Microbial Growth - Oxygen Levels ➔ A. Aerobes: must have oxygen (21% O2) ➔ B. Obligate/strict anaerobes: cannot have oxygen ➔ C. Facultative (aerobic or anaerobic): can grow with or without O2 (usually grow faster with oxygen) ➔ D. Microaerophile: grows in minimal O2 (between 0%-21%) ➔ E. Aerotolerant anaerobes: doesn’t use oxygen, can grow in oxygen Growth of Anaerobes ➔ Strict anaerobes frequently in anaerobic (anoxic) chamber ➔ Aerotolerant anaerobes can be be anoxic jars (chemically removes oxygen)  Oxygen levels ➔ Toxic forms of oxygen ➔ Produced during metabolism in the presence of oxygen ➔ The intermediates (highlighted in the figure) are very reactive and are damaging to cell components  Oxygen levels ➔ Cellular enzymes for removal of toxic forms of oxygen ➔ Microaerophiles can do respiration but they are not able to survive high oxygen levels since they do not have 1 or more of these enzymes Cell Structure and Function Part 1  Morphology (shape) of Prokaryotes Coccus Rod Spirillum - One to several turns Spirochete Budding and appendaged Filamentous bacteria bacteria - Tens to hundreds of - Stalk & hypha turns Vibrio - Curved rod  Cocci Diplococci Streptococci Tetrads - remain in pairs; divide - forms chains; divide in - form groups of 4; in one plane one plane divide in two planes Sarcinae Staphylococci or Micrococci - forms cubes; divide at - forms irregular right angles in three “bunches”; divides planes irregularly in many planes Factors affecting evolution of cell shape and size ➔ Surface Area and Volume ◆ Surface area to volume ratio (S/V) can affect: 1) SHAPE - Rate of nutrient uptake and thus rate of growth ○ Faster nutrient uptake → outcompete neighbors ○ High S/V ratio → faster uptake 2) SIZE - Number of cells produced per unit of nutrients available ○ Smaller cells means less material per cell and more generations per nutrient ○ Faster growth & more generations → faster evolution ➔ Sphere has lowest S/V ration → appendages can increase S/V Factors affecting evolution of cell shape and size ➔ 1) Motility ◆ Rods have a greater capability for movement in specific direction ◆ Spirilli and spirochetes seem to have the greatest capability of moving through highly viscous media ➔ 2) Attachment to surfaces ◆ Stalked bacteria tend to adhere to surfaces via their stalks ◆ Stalk provide a higher surface to volume ratio for nutrient exchange Structure of cytoplasmic membranes ➔ Phospholipid bilayer ➔ Amphipathic Structure of Cytoplasmic Membranes ➔ Membrane fluidity ◆ Lipids and proteins can move within the membrane ➔ Composition ◆ 50/50 lipid/protein ➔ Integral proteins ◆ Significantly buried in membrane Example: Transmembrane protein; exposed on both faces of membrane ➔ Peripheral associated ◆ Stuck to membrane but not embedded Example: Lipoprotein; binds to lipids ➔ Asymmetric organization ◆ Inside vs outside faces Distribution of lipids in rafts Structure of Cytoplasmic Membranes Variation in cytoplasmic membranes ➔ Bacteria Eukarya = Ester VS ➔ Archaea = Ether ◆ Utilize isoprene groups ** Lipids are different **  Archael cytoplasmic membranes Bilayer (diether with 20-carbon phytanyl) Monolayer (tetraether with 40-carbon biphytanyl)  Archael cytoplasmic membranes Bilayer (diether with 20-carbon phytanyl) Monolayer (tetraether with 40-carbon biphytanyl) - Monolayer is very stable at high temperatures Functions of Cytoplasmic membranes The Need for Transport Proteins ➔ Dissolved gasses permeate well ◆ Examples: O2, CO2, N2H2 ➔ Larger & more charged molecules have bad permeability ➔ Smaller & more hydrophobic, uncharged molecules have good permeability ➔ Protein channels can improve transport of molecules (includes water) → necessary to maximize growth Transport across cytoplasmic membranes ➔ Passive vs Active Transport ◆ Passive = moves DOWN concentration graduation (high → low) NO energy required ◆ Active = moves UP concentration gradient YES requires energy Passive Mechanisms ➔ Simple diffusion vs Facilitated diffusion ◆ Simple diffusion Small non-polar & uncharged polar molecules ○ Examples: glycerol, H2O, O2, and CO2 ◆ Facilitated diffusion Requires channel proteins or carrier proteins Selective for specific chemicals Can be regulated by cell, turned on and off (to accelerate transport if needed) Active Transport ➔ Requires an energy source - 3 classes  Simple Transport ➔ Depends on ion concentration gradients Really just facilitated diffusion Ion gradient in SAME Ion gradient in OPPOSITE direction of substrate Direction of substrate Group Translocation ➔ Example: Phosphotransferase system (PTS) - imports sugars like glucose for glycolysis ➔ Phosphate from phosphoenolpyruvate (PEP) → through 5 enzymes → phosphorylates imported glucose ➔ Energy from PEP hydrolysis (late in glycolysis) is used to import and phosphorylate glucose (early in glycolysis) ABC (ATP-binding cassette) system ➔ 3 proteins ➔ Periplasmic binding protein binds a substrate, and brings it to the transporter ➔ ATP hydrolysis by ATP-hydrolyzing protein ◆ Induced conformational change in transporter ➔ Substrate is imported Protein secretion is a transport system! ➔ Cells must place many proteins: on the outer surface of the membrane in the cell wall into the medium ➔ Getting a large protein across the membrane is more complex than transporting a small ion, sugar, or amino acid ◆ But this is still essentially an active transport system ➔ Sec system – at least 7 protein components ➔ Energy from ATP and PMF IMPORTANT NOTE! ➔ Structure of bacterial cell envelopes ➔ The textbook uses a terminology that is different and less precise than that used by most microbiologists. Dr. Hsu will use the following terminology in describing the cell envelope and wall: ◆ The CELL ENVELOPE refers to all layers surrounding the cell, including any membranes and wall material ◆ The CELL WALL refers only to the peptidoglycan layer Structure of bacterial cell envelopes: Gram Positive (G+) ➔ One membrane (“Cytoplasmic”) ➔ Thick peptidoglycan (PG) cell wall Structure of bacterial cell envelopes: Gram Negative (G-) ➔ Two membranes ◆ Outer ◆ Cytoplasmic (“inner”) ➔ Thin PG cell wall Reminder: - Cell wall in G+ and G- - Keeps cell from bursting due to osmosis - Determines cell shape Gram Positive (G+) & Gram Negative (G-) Gram Positive (G+) & Gram Negative (G-) Cell wall = Peptidoglycan = Murein Cell Structure and Function Part 2 Bacterial Cell Wall Structures ➔ Interconnected glycan chains make a net ➔ Gram negative: ◆ Direct x-link between peptides of the peptidoglycan ➔ Gram positive: ◆ X-link by glycan interbridge Adding New Cell Wall Material ➔ Peptidoglycan synthesis - four steps ◆ Transglycosylase: forms glycan chain Energy from phosphate bond ◆ Transpeptidase: X-links peptide sidechains ◆ Autolysins: cuts glycan for cell wall expansion ◆ Bactoprenol: lipid carrier for cell wall precursors ➔ PBPs: Penicillin-Binding Proteins catalyze BOTH the glycosyl transferase and transpeptidase activities. Transpeptidation ➔ D-ala cleaved ◆ Energy release ➔ Forms peptide bond between di-amino acid and other chain ◆ Pentapeptide → tetrapeptide Gram-Positive Cell Walls ➔ Teichoic acid: embedded in cell wall ➔ Lipoteichoic acid: embedded in cell wall and cell membrane ➔ Negative cell surface Degradation of Bacterial Cell Walls ➔ To degrade the bacterial cell walls, lysosomes cleave the beta(1,4) glycosidic bond ◆ Low solute solutions ◆ Isotonic solute solutions ➔ Antibiotics prevent cross-linking of peptide chains ◆ Only a few prokaryotes can live without a cell wall Gram-Negative Cells ➔ Cell envelope: thin PG cell wall, outer membrane present, and cytoplasmic membrane present ◆ Lipid A in outer leaflet, porins for PT, lipoproteins connecting OM to PG, periplasm with PG & PMF ➔ Outer membrane: lipopolysaccharide (LPS) that contains lipid A (endotoxin), core polysaccharide, and O-specific polysaccharide (variable) Archaeal Cell Walls ➔ Called the S-Layer ➔ Pseudomurein in some ➔ N-acetyltalosaminuronic acid ➔ Beta(1,3) glycosidic bonds ➔ Not sensitive to penicillin  Cell Comparison Gram-Negative Gram-Positive Archaea Cytoplasmic Present (ester Present (ester Present (ether Membrane linked) linked) linked) Cell Wall Thin PG Thick PG Can be pseudomurein (not PG) Outer Membrane Present (LPS Absent Absent containing Lipid A) Cell Surface Structures/Appendages ➔ Capsule: surface attachment, biofilm formation, phagocytosis protection. ➔ Fimbriae: protein structures that are highly absorbent, short, and promote adherence ➔ Pili: protein structures that are few per cell, long, and retractile ◆ Adherence, motility, and DNA exchange ◆ Type IV pili attach to a surface and retract to move ➔ Flagella: polar on one pole (lophotrichous), both poles (amphitrichous), or around the cell (peritrichous) ◆ Swimming motility; PMF drives the flagellum (1,200 protons per rotation) Chemotaxis ➔ “Chemical” movements ◆ Movement based on a concentration gradient in a cell ◆ “Biased random walk” ➔ Other forms of “taxes” ◆ Phototaxis ◆ Aerotaxis ◆ osmotaxis Studying Taxes ➔ Capillary assay ➔ Have a control, attractant, repellent ➔ Count cells per tube on a spread plate and compare Questions? Good Luck!!!