MGY377 – Midterm 2 Study Note All.docx

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Molecular Genetics and Microbiology
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MGY377H1
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William Navarre

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MGY377 – Midterm 2 Study Note! Page 1 Oct 3 Bacterial Cell Cycle and Growth FtsZ: Key protein in this group *scaffold that start Slonczewski, Chapter 4 process (with membrane, around divisome proteins) Highly conserved and distributed among Bacterial growth prokaryotes • An increase in the number of cells Structural homology to tubulin • Every cell has a lifespan, species must replenish found in eukaryotic cells (tubulin makes itself by growth microtubules, essential for cell division) • Important to understand microbial growth a GTPase, forms polymers Food (pasteurization) Forms a membrane-associated ring Biotechnology (hormone) structure at bacterial midcell during division Fighting disease (FtsZ-ring) • During growth cycle, all cellular constituents increase in number Fts Proteins and Cell Division 1. FTsZ form at middle of the • Partitioning of replicated DNA depends on its association with membranes cell into Z ring • Time required for growth cycle varies greatly 2. Recruitment of other cell Different species division proteins needed for cytokinesis and construction Nutritional conditions Stress and environment of Z ring e.g. E. coli replicates every 20 min in rich 3. Daughter cells formed at zing medium in vitro, only about once every 24 h in the ring gut (have competition) Binary Fission  ZipA: FtsZ membrane Cell divides into two anchor cells  FtsA: ATPase, provides 1. Increase # of energy for divisome macromolecule, assembly DNA replicated,  FtsI: involved in PG both copy anchored transpeptidation to cell membrane  FtsK: assists with 2. First, elongate to chromosome approx. twice the separation length of the smallest cell Paradigm shift: Daughter cells are not created equal 3. Partition forms in Bacteria divide into two equal daughter cells…but….. the middle, called a septum, as a result of inward ―Aging and death in an organism that reproduces by growth of cytoplasmic membrane and cell wall morphologically symmetric division‖ Explored the 4. Two daughter cells are pinched off concept of whether daughter cells are really equal Tracked the growth characteristics of individual cells Dividing bacteria In agar and ALL their progeny using video microscopy Salmonella dividing in our cells The concept of inequality Get into human cell, can replicate - When divided, new material rapidly in human cell cytosol. - During cell division, each daughter cell gets one NEW cell pole, and one OLD cell pole Bacterial Cell Division - Subsequent division gives daughter cells that • Many proteins involved in cell arise either from the new pole end (i.e. ‗new pole‘ division cell), or the old pole end (I.e. ‗old pole‘ cell) • Fts =filamentous temperature sensitive - Old pole grows slower, have impact of growing Describes mutants lacking these - Found that old pole progeny cells grow genes (elongate) on average 2.2% slower than new pole These proteins interact to form a progeny cells! division apparatus called the Divisome - Old pole cells also divides less and less FtsZ, FtsA, FtsW, FtsI, FtsQ, FtsK…. frequently than new pole cells, thus less offspring biomass. MGY377 – Midterm 2 Study Note! Page 2 - Concept of aging chew and inactivate C 5. CD prevent ring and the ring is right at the Control of cell shape in bacteria midpoint PG synthesis and Growth • New cell wall biosynthesis must take place for growth • Must be added to existing cell wall • Beginning at FtsZ ring, small openings in cell wall are created by autolysins Present in divisome protein complex Related to lysozyme in animals MreB  rob shape • New cell wall material is then added to fill in these areas  Junction of new and old cell wall Crescentin  used to get crescentin shape, helps leaves a ‗scar‘, called wall bands bend bacteria Localization of new cell wall How do cells know to divide in the middle? synthesis during cell division. In cocci, new cell wall synthesis is localized at only one Proteins of the min family help determine this : point. MinC, MinD, and MinE  exclusion to make ring in the middle PG synthesis and Growth • MinC: • Controlled cutting of existing PG simultaneous An inhibitor of FtsZ ring formation with new PG insertion Interacts with MinD • MinD: • Carrier molecule, Bactoprenol, plays major role in this An ATPase C55 alcohol, very hydrophobic Interacts with MinC, which can Bonds to N-acetylglucosamine/N- recruit MinC to the inner cell membrane • MinE: acetylmuramic acid/pentapeptide PG precursors and transports across the Oscillates like MinC/D in live cells, cytoplasmic membrane. stimulates MinC/D removal from membrane by stimulating ATP hydrolysis by MinD 1. Bind PG precursors in cytoplasm Dynamic Min proteins 2. Carries them across • Localization of MinC and D is not static. • MinC and D oscillates from one pole to another, cytoplasmic membrane 3. Interacts with enzymes that remaining at one pole for only 10-30 seconds insert precursors into before moving to the other pole. It is really at the growing cell wall middle point, exclude it so it forms in the middle Transpeptidation • Final step in 1. FtsZ want to form a ring cell wall (randomly) synthesis • Involves 2. Min CD, C formation of inhibiting FtsZ peptide cross- 3. Min E links between muramic acid forming a residues in adjacent glycan chains ring inhibiting Gram(-)ve: typically between diaminopimelic acid (DAP) and D-Ala and moving Gram(+): typically from L-Lys of one peptide to D- the other poll Ala on the other using a glycine interbridge 4. Min E (ring) go to end and • Inhibited by penicillin MGY377 – Midterm 2 Study Note! Page 3 Population growth 2. Exponential (Log) phase • Growth rate = change in # (or mass)/unit time • Rapid cell growth (exponential growth) • Generation = interval for formation of two cells • Also known as logarithmic growth • Generation time = time for one generation • Straight line on semi-log graph (log cell # vs Also known as doubling time time) Can vary widely, depending on • Population doubles every generation species and conditions • Healthiest population, usually chosen for study • e.g. E. coli (20 minutes) vs. Mycobacterium • May also be ideal for virulence factor tuberculosis (~24 hours) expression (e.g., invasion is optimal for Salmonella in late Log phase) Exponential growth • Occurs when bacteria keep doubling, no 3. Stationary phase restrictions present (enriched broth) • Death rate = rate of reproduction • e.g.: E. coli - generation time of 20 min. • Cells begin to encounter environmental stress • 20 generations (about 7 hrs.) • lack of nutrients • 1 million cells • lack of water • 30 generations (about 10 hrs.) • not enough space • 1 billion cells • metabolic wastes • 72 generations (about 24 hrs.) • oxygen, pH • 1 x 1021 • Bacteria adapt:: e.g. E. coli gets smaller • For convenience, bacterial #‘s plotted on log or • In some species, spores will form now semi-log plot 4. Death phase The Growth Cycle • Death rate > rate of reproduction • Can be exponential death rate at times • Due to limiting factors in the environment • Bacteria can actually lyse (burst open) • May be a programmed cell death response, similar to that seen in eukaryotic cells Population growth • Remember, what you see and measure are AVERAGES of the population • Averages good for handling populations, but always remember that individual bacteria Bacteria not always growing in exponential phase 4 Phases of growth in a batch culture can have a big impact since they have high Lag phase  Exponential phase (rapid growth)  potential for growth Stationary phase (growth stops)  Death phase • Individual bacteria may behave differently from bulk of population Optical density is method of getting # readout (turbidity) • Possible implications for bacterial pathogens • Evidence that subset of bacteria may express 1. Lag phase different genes in otherwise identical culture • Bacteria are first introduced into an • Subset of Salmonella are highly invasive environment or media (20-25%) • Bacteria are sensing their surroundings • Evidence that a subset of Salmonella grow rapidly in host cells while others grow • Need to alter gene expression for rapid growth or to accommodate new nutrient slowly, or are killed. This is observed even sources late after infection of host cells initiated • Lag also occurs if cells need to undergo (chronic vs. acute colonization) repair due to damage sustained in previous environment (e.g. use HO, lag) • 1 hour to several days depending on conditions • Complex medium (nutrient rich) to complex - short lag • Complex medium to minimal defined medium - long lag MGY377 – Midterm 2 Study Note! Page 4 Counting bacteria Direct methods (microscopy): Counting bacteria: Turbidity • Quick and easy - Cells in suspension will scatter light. More cells, • Doesn‘t always differentiate live vs dead more scatter, more turbidity. (depends on method) - Spectrophotomer measures unscattered light • Not that precise after passage through a liquid culture. • Doesn‘t work at low concentrations - OD 600often used for measuring bacterial #‘s Viable counts: o generate standard curve vs CFU • Measure only live bacteria (Colony forming o accurate except at high turbidity (scattered units, CFU) light may be RE-scattered back to the • Assume each viable cell gives one colony (but not spectrophotometer, giving false readings) always true) Some bacteria aggregate Counting bacteria • Measurement of total nitrogen, or protein Some don‘t survive plating conditions content • Determine total cell yield from very dense Counting bacteria cultures Drop of solution, count the area in grid, only with • Only practical in research laboratory high [] bacteria • Measurement of Biochemical activity • E.g. oxygen uptake, CO pr2duction, ATP LIVE/DEAD BacLight TM stain production, light production, etc. Viability stain (from Molecular Probes) • Measurement of dry weight of cells, or volume of • Allows quantitation of total bacteria cells, after centrifugation of culture • Also allows determination of viability • Flow cytometry • Two nucleic acid stains: • Detector measures number of particles (in 1. SYTO 9: this case bacteria) that pass through Green fluorescent dye chamber in solution Membrane permeable • Fluorescence (e.g. GFP expression) can be Labels live bacteria used to measure specific bacteria in a 2. Propridium iodide: complex biological mixture (e.g. tissue Red fluorescent dye homogenate) Highly charged, does not penetrate intact membranes Continuous cultures Dead bacteria cannot exclude this • studies, desire constant conditions for long dye, become strongly labeled periods of time (like exponential phase only) • This is done with use of continuous cultures TM LIVE/DEAD BacLight stain • Flow system of constant volume to which Caveats: fresh medium is constantly added, and waste - Damaged bacteria can recover is constantly expelled - Some bacteria with seemingly intact membranes • Steady state: system in equilibrium, cell number may be unculturable and nutrient status is constant Counting bacteria: Plate counts Continuous cultures  Like the gut (constant input of bacteria and nutrient) - Bacteria need to survive the plating process - Dilution series made MGY377 – Midterm 2 Study Note! Page 5 The Chemostat • Growth rate and growth yield (cell density) are controlled separately Two important factors 1 Steady-state relationships in the chemostat. The • Concentration of Nutrients (controls cell dilution rate is determined from the flow rate and the volume of the culture vessel. Thus, with a vessel density) At a given flow rate, higher nutrient of 1000 ml and a flow rate through the vessel of 500 concentration leads to a higher density… ml/h, the dilution rate would be 0.5 h-1. Note that at same as in batch culture high dilution rates, growth cannot balance dilution, and the population washes out. Note also that Batch Culture although the population density remains constant - Increase nutrient, growth density should during steady state, the growth rate (doubling time) increase constantly, as N increase, growth rate can vary over a wide range. Thus, the experimenter can obtain populations with widely varying growth increase till plateau, density is affect, not the growth rate rates without affecting population density. - Nutrients effects o Growth rate increase and plateu at high Advantages of the Chemostat density, in flow is rich, higher density • Growth rate and yield controlled independently • Any growth rate can be obtained The Chemostat • Maintain bacterial cultures in exponential growth phase for long periods Two important factors 2 o Concentration of Nutrients (controls cell • Can study mixed populations of bacteria density) • Competitiveness? o Dilution rate (controls growth rate) • Enrichment and isolation of specific bacteria  Faster dilution rate means faster clearance of waste and faster influx of fresh media… therefore faster growth rate  However, can actually have washout of bacteria when the dilution rate is too fast for the bacteria to keep up! Higher flow rate (high rate of dilution): faster nutrients in  faster waste out  faster growth rate  (lower doubling time) MGY377 – Midterm 2 Study Note! Page 6 Oct. 10 Environmental effects on bacterial Temperature classes o growth Typical temperature range of 30 C for any organiso Slonczewski Chapter 5, - Psychrophile: Optimal temp for growth is 15 C or less Environmental effects on bacterial growth - Psychrotolerant: Grow at 0 C, but optimal at 20- o - Normal growth conditions based on human 40 C frame of reference: - Mesophile: Mid-range temp optima, e.g. 39 C for o Sea level E. coli. All human pathogens are mesophiles o 20-40 Celcius o - Thermophile: Optimal temp for growth is 45 C o Neutral pH or higher (mysteria) o Salt concentration 0.9% (like our blood) - Hyperthermophile: Optimal temp for growth is o Ample nutrients 80 C or higher - Any ecological niche outside this range considered ―Exteme‖ Temperature classes o R. MacElroy first used term ―Extremophile‖ to describe prokaryotes able to grow in extreme environments - Conditions on earth when life began likely ―extreme‖. Therefore earliest life likely extremophiles Four Key Factors: - Temperature - pH - Water availability Growth range in many case do not overlap, grow in - Oxygen specific temperature (Adapted to specific environment) Temperature (Probably most important factor) ‗Cardinal‘ temperatures characteristic of each Psychrophiles organism - Most of the earth is cold A) Minimum temperature o Oceans are 5 C on average, colder in deep B) Optimal temperature ocean C) Maximum temperature (that sustain life) o Arctic, Antarctic permanently frozen Note: these can be modified by other environmental - Psychrophiles live in these environments factors  e.g. pH affect temp allowing growth - Survive in small pockets of water in ice, glaciers, glacial lakes, snowfield Cardinal Temperatures Adaptations of Psychrophiles - Protein structural alterations: o Allows enzymes to function in cold o More alpha-helices (flexible) o Less beta-sheets (rigid) o More polar, less hydrophobic amino acids - Membrane alterations: o Transport activities not affected by cold o Higher content of unsaturated fatty acids o Polyunsaturated fatty acids (butter) o Helps maintain semifluid state of membrane at low temperatures Snow bacteria - Isolated from snow at the South Pole - Using fluorescent DNA dye to enumerate bacteria, estimated ~5000 cells/ml of melted Effect of temperature on growth rate and the molecular consequences for the cell. The three snow!!! - Metabolically active at -17 C (DNA and protein cardinal temperatures vary by organism. synthesis) MGY377 – Midterm 2 Study Note! Page 7 Freezing Acidophiles - Freezing prevents microbial growth, but doesn‘t - Fungi tend to be acidophilic necessarily kill them o E.g. contaminated vinegar - Medium affects impact of freezing: - Other examples: Thiobacillus, some Archaea o Cryoprotectants such as glycerol and DMSO (Sulfolobus). prevent damage due to ice crystal formation o Picrophilus oshimae, grows in extremely and dehydration acidic volcanic soils, pH optima 0.7, lyses o Preserve lab strains this way and store at -80 above pH 4 C for years - Most critical factor is stability of cytoplasmic membrane Thermophiles and Hyperthermophiles - Internal pH normally must stay near neutral (6- - Grow in hot places: Soils w/ full sunlight (50 C) 8) but can be as low as pH4.6 in some Metabolism generate heat acidophiles o - Fermenting compost piles (70 C) - Man-made machinery, pipes, effluent Alkaliphiles - Hot springs and steam vents, hydrothermal - Usually found in soda lakes, high carbonate o vents (100-350 C)!!! soils, iron ore waste sludge (approaching pH 13!) - Yellowstone hot springs (102 C) - Bioenergetic problem: How to establish proton motive force across cytoplasmic membrane in Adaptations of Thermophiles basic environment? - Protein alterations: o External surface is alkaline, will neutralize o Little sequence divergence from mesophiles H gradient o But, critical amino acid substitutions - Alkaliphiles can use Na gradient to drive o Increased ionic bonds, densely packed transport, ATP synthesis and motility hydrophobic cores, contribute to resistance to unfolding Alkaliphiles - High solute production in cytoplasm, helps stabilize proteins: Di-inositol phosphate, diglycerol phosphate etc. - Membrane alterations: Rich in saturated fatty acids, form stronger hydrophobic bonds to stabilize membrane at high temp. Importance of Thermophiles - DNA polymerases from Thermus aquaticus (Taq polymerase), Pyrococcus furiosus (Pfu polymerase) o Highly heat stability, robust, high activity - Used to amplify DNA transcripts in Polymerase Chain Reaction (PCR) - PCR Invented by Kary Mullis, Nobel prize recipient Decarboxylase couple Na++ transport, cytochromes (ETC – Sodium gradient), sodium driven anti-porter pH ..etc. - pH is a measure of the H concentration Osmotic Effects - Most organisms show a growth pH range of 2-3 units - All life requires water - pH classes for organisms: - Water availability depends on: o Acidophiles, grow best at low pH o water content of environment o concentration of solutes o Neutrophiles, grow best near pH 7 o Alkaliphiles, grow best at high pH - Water activity (aw): Physical measure of water o Acidophiles availability o Ratio of vapor pressure of the air in - The pH scale. Note that although some microorganisms can live at very low or very high equilibrium with a substance or solution to pH, the cell‘s internal pH remains near the vapor pressure of pure water neutrality. Different environment with different pH as well MGY377 – Midterm 2 Study Note! Page 8 Water activity Compatible Solutes - Pure water is 1 - Amino aicd types solutes - Very few microbes grow at a 2 O · + NADP + H + Enzymes that destroy toxic oxygen species. (a) Catalases with 2 2 hydrogen oxide and (b) peroxidases are porphyrin-containing The superoxide produced is subsequently converted proteins, although some flavoproteins may consume toxic oxygen to hydrogen peroxide (H O ), h2po2hlorous acid species as well. (c) Superoxide dismutases are metal-containing proteins, the metals being copper and zinc, manganese, or iron. (HOCl) and other microbicidal products. (d) Combined reaction of superoxide dismutase and catalase. (e) - Superoxide reductase catalyzes the one electron reduction2of O to H2O2using reduced cytochrome c as the electron donor. MGY377 – Midterm 2 Study Note! Page 10 NADPH oxidase in phagocytes Oct 12. Bacterial cell death, injury and spores Slonczewski Chapter 5 The need for microbial growth control - Decline in Salmonella typhi (typhoid fever) deaths in US from 1 in 1000 (1900) to 1 in 50 million (1970) - Attributed to many control measures: o Water chlorination o Milk pasteurization o Sewage treatment o Vaccinations o Antibiotics o Isolation of patients 1. Phagocytosis (uptake of bacteria into membrane S. typhi still endemic in developing world, causes bound compartment, the phagosome) hundreds of thousands of deaths each year 2. NADPH oxidase assembly on phagosome membrane Typhoid Mary: An extreme case of transmission 3. Bacterial killing with ROS control (Healthy chronic carrier of S. Typhi) Functions of the NADPH oxidase Microbial Growth Control Cross Presentation, Chemotaxis, signaling, immune Physical Methods modulation, antimicrobial activity, autophagy Chemical Methods (antimicrobial peptides) Antimicrobial chemotherapy Salmonella can prevent recruitment of NADPH oxidase Heat Sterilizaton • Most widespread method • Works for all microbes at right temp. • Denaturation: loss of molecular function (e.g. unfolding of proteins) • Death from heat is exponential (survival fraction – log vs. time  drop = time for 1 logdrop in bacterial #‘s | 1D = 10% survive  5D 0.001% Factors affecting Heat Sterilizaton • Moist heat better than dry heat, more penetrating • Matrix/medium effects Salmonella in meat harder to kill than free in solution pH effects, solute effects Human, makes static electricity vs. one in phagosome (kills) • Higher Temperature, faster killing - Salmonella can invade and live inside eukaryotic Increased severity of challenge (temp) = faster killing (D) cells Mesophile and [Thermophile  need more]( - Intracellular bacteria reside in a vacuole (Salmonella-containing vacuole; SCV) optimal temp, different D to kill) - Can secrete bacterial proteins into the host cell The autoclave (typically 15 min for sterilization time) cytosol using needle-like type III secretion - Sealed device, heats samples with steam and system - Wild-type SCVs avoid NADPH oxidase pressure - Salmonella deficient in intracellular type III - heat kills, not pressure - pressure is used to achieve very high steam heat secretion do NOT avoid NADPH oxidase - Use of the autoclave for sterilization. A typical - Other intracellular pathogens can also inhibit the NADPH oxidase during infection; Important autoclave cycle. Sterilization of a fairly bulky for their virulence object. The temperature of the object rises more slowly than the temperature of the autoclave. MGY377 – Midterm 2 Study Note! Page 11 Pasteurization Microbial Growth Control - Reduces (but does not eliminate) microbial numbers in milk, juices or other heat-sensitive foods o Named for Louis Pasteur o Kills pathogens and improves shelf life - Typical methods: o 63-66 C for 30 min (usually in large vats) o 71 C for 15 sec (flash pasteurization; more efficient) Radiation Ionizing: High energy, e.g. X and  rays Generates ions or reactive metabolites from molecules struck by radiation particles, e.g.: OH . Action of antimicrobial agents total counts determined by direct measurement Damages DNA and proteins Used to sterilize medical devices Used to sterilize food Non-ionizing: Typically UV radiation, 200-300 nm Induces DNA damage Cannot penetrate solids or liquids Three types of action of antimicrobial agents. At the time indicated by the arrow, a growth-inhibitory Disinfect exposed surfaces (biosafety cabinet) concentration was added to the exponentially Decimal reduction dose, D 10 growing culture. Note the relationships between • D = Amount of energy to viable and total cell counts. 10 reduce bacterial #‘s by 1 Measuring antimicrobial activity log • Higher dose, faster killing Minimum inhibitory concentration (MIC) - Smallest amount needed to inhibit growth • Varies dramatically - Different between organisms between different species Salmonella, 200 Gy - Affected by many factors: inoculum size, temp, Vegetative C. botulinum, pH, aeration etc. Various methods 3300 Gy Tube dilution: A series of increasing concentrations C. botulinum spores, 39,600 Gy • Standard killing dose is 12 D 10 of antibiotic is prepared in the culture medium. Each tube is inoculated, and incubation is allowed to proceed. Growth (turbidity) occurs in those tubes Filtration with antibiotic concentrations below the MIC. MIC is Physically remove bacteria from solutions The structure of (a) a depth filter. Depth filters are where there is no turbidity. Agar diffusion method: Agar diffusion method for used as prefilters and for the filtration of liquids with assaying antibiotic activity a high amount of suspended particles (prefilter step like Sari) (b) Membrane fiter (Polymer) (b) Nucleopore (polycarbonate film with few small pore) Antimicrobial peptides Major component of innate immunity - Constitutively present on all surfaces of the body Chemical methods of growth control - Antimicrobial agent: Natural or synthetic - Other peptides expressed during infection Found in all living species (Including plants and chemical that kills or inhibits growth of bacteria!) microorganisms Small peptides (12-100 aa) that kill bacteria - Antimicrobial agents vary with regard to their selective toxicity| Ability to kill microbes vs man - Four main structural classes - Net positive charge of +2 to +7 - 50% or more of aa are hydrophobic - Amphipathic: hydrophobic and hydrophilic faces; Allows solubility and insertion in membranes MGY377 – Midterm 2 Study Note! Page 12 Antimicrobial peptides Antimicrobial peptides Over 500 known antibacterial peptides - Cows have 38 different antimicrobial peptides - Can act synergistically - Found from frog, permeabililize bacterial membrane  swimming around - AA K and R Antimicrobial peptides Mixed, beta sheet, loop, helical, extended Synthetic peptides being developed for clinical use: - The next antibiotics? o Potent o Selective (eukaryotic cell membranes very different) o Resistance harder to develop in bacteria - Enhancers of antibiotic function? o New treatment for endotoxinemia/shock? o Binding to LPS, preventing its activation of immune cells - Other uses? o Fight cancer? - Pick Hole ,interact with hydrophilic face o Vaccine enhancement? Dr. Monisha Scott Other AMP targets are intracellular mRMA Synthesis, Protein Synthesis, Protein folding, - Studying the use of host defense peptide variants aminoglycoside-modifying enzyme, Cell wall in stimulating host immune response synthesis, DNA replication - These peptides prepare the host to better deal with infections AMPs in action - Designed peptide IDR-1 (extended conformation), net charge - Cell turnover rate at intestinal mucosa is high. of +3 - IDR-1 lacks direct antimicrobial activity - Continuous production of new cells from stem - Injected mice with mice with IDR-1 48hrs before infection cells at base of microvilli (crypt) with S. aureus, S. Typhimurium, MRSA, VRE - Paneth cells secrete high levels of AMPs (a- - Significantly decreased mortality rates and bacterial counts defensins) to protect stem cells from intestinal in mice were observed microbes Mechanism of IDR-1 action - Shown to act on macrophages and monocytes Antimicrobial peptides| Other immune effects - Stimulate prostaglandin release (inflammatory - Induced upregulation of key signaling pathways - Increased production of important modulator, vasodilation) immunomodulatory chemokines - Promote phagocyte recruitment (chemoattractant: recruits more - Enhance phagocytosis - Promote angiogenesis and wound repair monocytes/macrophages) - Also decreased production of proinflammatory - Coordinate innate and adaptive immune cytokines like IL-6, TNF  (i.e. prevent excessive responses o T cell recruitment and activation inflammation) o DC development - Recently shown to bind a host protein (p62/SQSTM1) which regulates innate immune - AMPs often referred to as ―Host defence gene expression peptides‖ o alpha Defensins: antimicrobial at high - Since IDR-1 acts on HOST immune system, it is concentrations, an immunomodulator at low more difficult to achieve antimicrobial resistance concentrations o Antiviral peptides may work by interfering with viral attachment to host cells or by disrupting viral envelopes. MGY377 – Midterm 2 Study Note! Page 13 Cell Injury and Stress Responses - Sporulation is not a reproductive process, that is - Injury is very common for bacteria  Field of 1 vegetative cell = 1 spore. When environmental stress response in microbiology and cell biology conditions return to to normal, one spore will - Bacteria that are injured (but not killed) often transform into one vegetative cell which then will recover undergoes cell division - A second injury during this time will be lethal  E.g. plating bacteria on selective media after A survival strategy electroporation, or pasteurization - Spores are metabolically dormant and highly - Bacteria that recover induce a stress response resistant to a variety of environmental insults, o Many gene regulons (in general, called heat including UV and gamma radiation, free shock response) radicals, high and low temperature, acid and - Recovered bacteria more capable of surviving a alkali conditions, hydrolytic enzymes, and organic solvents. For example, spores can second injury o o Same stress (heat tolerant after heat shock) survive at 120 C,which kills all bacterial cells o Different stresses (tolerance to metals after - Spores can remain dormant for long periods of heat shock) time (perhaps indefinitely) but will germinate when they encounter a nutrient-rich Cell Injury and Stress Responses environment Treated with OH, 2D gel separating protein, # protein induced to damage (23 H2O2 stress protein) Features of Spores - Spores can be seen with a Cell Injury and Stress Responses light microscope due to the - Many genes induced with stress: high protein levels causing a high refractivity - Regulatory genes: o e.g. Sigma 32 in E. coli, unique to heat shock - Spore represents almost the o An ―alternative sigma factor‖ specific for heat entire dry mass of the cell shock genes although it occupies only 10% - Chaperones: of the volume of the vegetative cell o e.g. GroEL (Hsp60) in E. coli, helps prevent protein denaturation Spore Structure| Spore coat: The outmost structure common to spores of all - Proteases: o e.g. DegS in E. coli, degrades misfolded species proteins in periplasmic space Consists of a series of one or more morphologically distinct layers that protect spore Sporulation (endospore formation) in Gram-positive from a variety of toxic molecules and from bacteria mechanical damage Typical feature of some Gram+ve Is largely protein, e.g., ~20 proteins in B. subtilis bacteria such as Bacillus, Clostridium and Sporosarcina, including the pathogenic bacteria Bacillus anthracis Spore coat| Spore cortex: (cause of anthrax), Clostridium - Cortex surrounds the spore core and is botulinum (botulism), Clostridium tetani composed predominantly of peptidoglycan (PG) (tetanus), Clostridium perfringerns (gas with a different structure from that of vegetative gangrene and food poisoning), and Clostridium difficile (antibiotic- cell wall associated and pseudomembranous - ~50% of N-acetyl muramic acid (NAM) residues colitis) - Not found in Gram-ve in the cortex are present as muramic acid - lactam (MAL) bacteria - ~25% of NAM residues carry only a single L- alanine, therefore there are only ~25% as many Sporulation: A survival strategy DAP residues available to participate cross-link - Sporulation usually occurs as a response to formation in spore contex adverse environmental conditions. In the - Total cross-linking in cortex PG is ~3%, 10- to laboratory, sporulation can be induced by 12- fold below that is vegetative cell wall PG starvation for a carbon, nitrogen, and/or (~35%) phosphorus source. In these conditions, - Cortex is important for maintenance of spore sporulation begins immediately following core dehydration, which increases spore wet- exponential growth heat resistance MGY377 – Midterm 2 Study Note! Page 14 Core wall Spore formation - Immediately underlying the cortex is a second - One of the most complex differentiation layer of PG with a different structure from that processes in bacteria of cortex. This second PG layer makes up only a - >200 genes are involved small amount of total spore PG and is the spore‘s - Spore formation is a highly controlled process, nascent, or germ cell wall (core wall). many ‗checkpoint‘ mechanisms exist to ensure - Core wall PG is similar to that of vegetative cell that sporulation initiates only under conditions wall in which cells are capable of faithfully replicating - Cortex is targeted for degradation early in spore and segregating their chromosomes germination, the germ cell wall is not degraded and provides the template for new cell wall synthesis during spore outgrowth - Unlike the walls of growing cells, in which teichoic acids are a major component, teichoic acids are not present in spores Spore core - Contains cytoplasmic membrane, cytoplasm and nucleoid The core cytoplasm: - Is highly dehydrated- the water content is ~1/3 of growing cells - Contains high concentration of dipicolinic acid (DPA) and calcium, which forms calcium dipicolinic acid. The presence of DPC is Initiation of sporulation - Initiation of spore formation requires multiple associated with low water contents and heat conditions or signals, including nutrient resistance deprivation, high cell density, the absence of - pH is ~1 unit lower (6.3-6.5) than that in growing cells (7.5-8.2) DNA damage - Multiple environmental and physiological - Spore nucleoid is highly condensed and bound signals control the activation of Spo0A, which is by SASPs (small acid-soluble spore proteins), required for the initiation of sporulation which protect DNA from potential damage from UV radiation, desiccation and dry heat Spo0A~P - Activates genes required for later events in Differences between endospores and vegetative cells sporulation - Represses genes involved in stationary-phase growth - A phosphorelay pathway produces Spo0A~P, which initiates sporulation MGY377 – Midterm 2 Study Note! Page 15 Oct. 15 Microbial Genomics Sanger Method The method is based Chapter 7-9 on the DNA polymerase- Genomics dependent synthesis The branch of science/technology which specializes of a complementary in the systematic study of genomes, (including their DNA strand in the molecular characterization) and the production of presence of natural their gene products (proteins), their role in health 2′-deoxynucleotides (dNTPs) and 2′,3′- and disease, and the effects of manipulation of these dideoxynucleotides systems by agents such as pharmaceuticals and (ddNTPs) that serve radiation. as nonreversible synthesis terminators. The Sequencing DNA synthesis Procedures for determining of exact order of reaction is randomly terminated whenever nucleotides in the DNA fragment. a ddNTP is added to the growing Sequencing virus genomes oligonucleotide In 1976 the complete RNA sequence of Bacteriophage MS2 was chain, resultingin determined, compared to the DNA sequence of the Phi-X174 truncated products of phage, which was determined by Fred Sanger and his team in varying lengths with 1977. These two genomes were the first to be determined in an appropriate ddNTP at their 3′ terminus. scientific history. - DNA primer and a DNA used for sequencing - Need primer compliment to the sequence First cellular genome (limitation)  or clone into plasmid  primer In 1995, Haemophilus influenzae was the first free-living organism to have its entire chromosome sequenced (~2 million  or adaptor, now sequence and go from there bp), sneaking in just ahead of Escherichia coli in that race, mainly Need DNA pol and a mixture of nt, running in 4 because its genome is smaller in size than E. coli's. For a relativelyseparate reaction, where in which reaction is obscure bacterium, there was already a good understanding of its genetic processes, especially transforation. dope to terminate the sequence of DNA (dideoxynucleotide)  primer extend, need to incorporate and stop, hit, keep going, Steps towards DNA Sequencing terminating C, stop, million polymerase and • The Plus and Minus Method (1975) was one of fragements, sager fragements spaced acrossed the first methods used to sequence DNA, and required a comparison of both the ―plus‖ and ―minus‖ sequences to determine the actual sequence. It could be used only for sequencing Modern automated DNA sequencing ssDNA • The Maxim and Gilbert Method (1977) was published just before the inhibitor method, however it required a series of complex modification of DNA, and was therefore a much more difficult method of sequencing. Like the inhibitor method, it can be applied to dsDNA. - Originally, four different reactions were required • The Sanger et al (1977) method of DNA per template, each reaction containing a Sequencing was a method that incorporated the different ddNTP terminator, ddATP, ddCTP, idea that inhibitors can terminate elongation of ddTTP, or ddGTP. DNA at specific points. THIS TECHNIQUE - Advances in fluorescence detection have allowed REMAINS THE STANDARD TODAY. for combining the four terminators into one reaction by having them labeled with fluorescent dyes of different colors. - Overall throughput has been increased by the advent of capillary arrays whereby many samples could be analyzed in parallel - - ~100 bp read before reaction is out MGY377 – Midterm 2 Study Note! Page 16
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