Unit 2 Exam for Microbiology 3050
Unit 2 Exam for Microbiology 3050 MICRO 3050
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UNIT 2 STUDY GUIDE Fall 2015 MICR 3050 OBJECTIVES: Chapter 3.1 – 3.5 1. Describe the structure and functions of lipopolysaccharide (LPS). • Lipopolysaccharides act as anchors that are connected to the outer membrane to peptidoglycan in gram negative bacteria (unique feature) • LPS are seen as replacing most phospholipids in this outer membrane • 3 parts to LPS: a) Lipid A: Embedded in the outer membrane (like phosphor lipids) b) Core polysaccharide: Core because it’s always the same structure c) O side chain (O-antigen) à antigens are markers on cells that the immune system attacks since they are foreigners • Core polysaccharide and the O side chain extend out form the cell like thin sugary hair • LPS contribute to the negative charge on cell surface (core polysaccharide) • Helps stabilize outer membrane structure (lipid A = structural component) • May contribute to attachment to surfaces (sugars sticking out because will help with places that will more likely have food) and biofilm formation (bacteria stick together à strength in numbers; middle of the biofilm = protected) • Creates a permeability barrier: Toxins and anti-microbials are sometimes able to be kept out and not get through via the LPS binding to the substance • May mutate to protect from host defenses (O antigen) à are able to mutate easily as cells reproduce and can be an advantage for bacteria (Even if there is only a slight mutation, one has to wait for the immune system to build of those particular antibodies) • Can act as an endotoxin à lipid A = a poison: a toxin part of the cell (vs. exotoxin where bacteria release the toxin) - Endotoxin= synonymous with lipid A of LPS - Ex: Salmonella dies and is reabsorbed in the body with cell turnover à gas, bloating, diarrhea, vomiting and these are all due to the endotoxin 2. Explain how bacteria may survive without a cell wall. • Survival in isotonic environments where there is no net cincrease - Protoplasts: Gram positive - Spheroplasts: Gram negative à includes outer membrane - Top 2 are given in terms of when the cell walls of both types have been removed from the bacteria for testing • Mycoplasma: - Has no cell wall - Plasma membrane is more resistant to osmotic pressure than those with cell walls because they have hopanoids and sterols in their cell membrane - Concern because some of these cause disease and can’t use typical antibiotics with them because they usually go after cell walls, which Mycoplasmas do not have - Atypical pneumonia, pelvic inflammatory disease, arthritis, postpartum fever 3. Describe capsules and slime layers and discuss their functions. • Capsules: - Usually composed of polysaccharides - Well organized (uniform shape) and not easily removed from the cell - Have protective advantages (these are not necessary for survival though): a) Resistant to phagocytosis: Phagocytes are the first responders in an immune response; bacteria with capsules are usually seen as pathogens while those without capsules are not seen as pathogens b) Protect from desiccation: The bacteria can drink/eat the capsule to survive longer and if put back into good environment, the capsule can regrow again c) Exclude viruses and detergents (1/3 mortality of bacteria is because of viruses) d) Polysaccharides are sticky, so help with sticking to surfaces e) Absorb nutrients and store nutrients f) Act as waste dump for cell also • Slime Layers: - Similar to capsules except they diffuse, are unorganized, and are easily removed à slime layers are secreted and are not in a uniform shape - May aid in motility (ex: Snail secretions; bacteria with slime layers use gliding motility on solid surface) Chapter 3.6 – 3.9 4. Describe the following bacterial structures and their functions: cytoskeletal proteins, cell inclusions, flagella, and endospores. • Cytoskeletal proteins: Cytoplasm is made out of the cytoskeletal proteins a) FtsZ Protein: - Bacteria divide and multiply via binary fission à process of elongating the cell, making copies, and cloning themselves - FtsZ proteins line up in the plane of division all the way around the entire cell before the cell divides (takes ~10,000 proteins) and facilitate the cell division à know where the middle is because of MinD proteins b) MinD Protein: - Ensures that the FtsZ proteins do not polymerize at the poles of the cell and directs them to the middle where the plane of division is c) MreB/MbI Protein: - If present, determines the bacilli (rod shape) for the new cell; if not present, then bacteria would be cocci, which is the default shape - Scaffolding of peptidoglycan • Cell inclusions: - Located in the cytoplasm (for all bacterial cell types) - Aggregates of organic or inorganic material such as granules, crystals, or globules à depends on the cell and what is being stored; act as micro compartments that have functions - Some inclusions are enclosed by a single-layered membrane or invaginations of the plasma membrane (but they are still not considered an organelle) - Considered to be the “refrigerators” of the cell: Where extra food is stored and feast/famine phases deplete the inclusions - Come out in the area between the folds and store things there à ex: Cyanobacteria: Store chlorophyll that is continuous with the cell membrane (invagination of the plasma membrane) - Storage inclusions: o Involved with storage of nutrients, metabolic end products, energy, building blocks: a) Carbon à glycogen and poly-B-hydroxybutyrate (PHB) = ways to store carbon b) Phosphate à stored in polyphosphate granules c) Sulfur globules à take sulfur component and use it as a source of electrons in metabolism d) Nitrogen à stored via cyanophysicn granules (these store different amino acids for N source) - Other inclusions: o Gas vacuoles: Provide buoyancy – “life jackets” Photosynthetic bacteria that have a collection of gas vesicles with simple membrane proteins that are impermeable to water and some gases; able to inflate for optimization of oxygen/nutrients in the water column and are able to deflate o Magnetosomes: Magnetic particles for orientation in the Earth’s field Rare collection of magnetized particles in inclusions à also live in water and the magnets help them be at the bottom because there is more nutrients available due to sediments for food; these bacteria don’t need a high level of oxygen • Flagella: External structure because extends beyond the cell envelope - Threadlike appendages that extend outward from the plasma membrane and the cell wall (flagella is attached to both parts) - Can be up to 20 nm in diameter - Functions: a) Motility and swarming behavior (sometimes bacterial species with flagella can have coordinated movement so they all move at the same time) b) Attachment to surfaces: In terms of helping to swim and burrow (ex: A particular bacteria has 6 flagella that helps it burrow into the lining of the stomach to get away from the acid) c) May be virulence factors: Make the cell more virulent (deadly) à makes a better pathogen; usually referring to more proteins when talking about virulence d) Antigens on the flagella: Help with identifying the pathogen - Patterns of Flagella Distribution: Trichous = “hair” a) Monotrichous: One flagellum b) Polar flagellum: Flagellum at the end of a cell (Can see combo of polar monotrichous for ex.) c) Amphitrichous: One flagellum at each end of the cell d) Lophotrichous: Cluster of flagella at one or both ends of the cell; “tuft of hair” e) Peritrichous: Spread over the entire surface of a cell - Spirals in general have flagella, half of bacilli have flagella, and very few cocci have flagella • Endospores: - Complex, dormant structures that are formed by some bacteria à clone of the cell that makes it that occurs when the original bacteria cannot grow and divide anymore - Various locations within the cell for where the endospore is created (species specific) - Resistant to numerous environmental conditions: Heat, radiation, chemicals (such as disinfectants), desiccation (can withstand dry conditions) - Overall gives selective advantage - Ex: Bacillus species, Clostridium species (require anoxic conditions to grow à botulism in cans- 1 tsp can kill 100,000 people), soil bacteria - Oldest endospore= 25,000,000 years old (found in frozen gut of bee) 5. Describe flagellar structure and movement. • Bacterial flagella = rigid (unlike with eukaryotic cells) • Flagellar structure contains 3 parts: a) Filament: Tail made out of the flagellin molecule—makes hollow tub à filament cap protein organizes the flagellin molecule into the filament; the filament is built from the tip (in contact with the cap protein) and not from the base b) Hook: Attaches the filament to the basal body moter c) Basal body motor: Includes the P, MS, and C rings (in gram negative bacteria) • Gram negative bacterial flagella: Has 4 rings a) L Ring: LPS layer; stationary b) P Ring: Peptidoglycan; stationary c) MS Ring: Supra membrane (looks like 2 membranes but really 1 together); spins d) C Ring: Cytoplasm; spins - Fli proteins: Responsible for being the motor switch that turns the flagellar movement off and determines which direction (some bacteria with flagella are bi directional and important because it has to do with what they’re doing) • Gram positive bacterial flagella: Only has 2 rings - One ring is attached to the cell membrane while the other one is attached to the peptidoglycan - Not studied as much as the gram negative bacteria with flagella • Mechanism of Flagellar Movement: - The flagellum is a two part motor that produces torque àhave stator = stationary (Motor A and Motor B proteins on the side in the cytoplasm) that interact with the rotor = mobile (C ring and MS ring) - Uses proton motive force to spin the flagellum à electron transport chain in bacteria is located in the cell membrane which shuttles H + outside the cell to create a negative charge inside to create potential energy = concentration gradient + - Energy is used to do work when the H are shuttled back inside the cell down their concentration gradient and go through something that is like a revolving door that causes the C and MS rings to spin à ** need 1000 H to spin the flagellum 360 degrees • Flagellar Movement: - Flagellum rotate like a propeller - Up to 1000 rev/sec (vibrio cholera) - Counterclockwise (CCW) rotation to move forward and in a straight path = run - Clockwise (CW) rotation to disrupt the run and cause the cell to stop and tumble (random) à may be because have to generate more H + motive force 6. Define chemotaxis and describe how bacteria move toward an attractant (or away from a repellent). • Chemotaxis is movement toward a chemical attractant or away from a chemical repellent • Concentrations of chemoattracts and chemorepellants are detected by chemoreceptors on the surface of cells in the cell membranes; some are even located in the periplasm of gram negative bacterial cells • Complex but rapid movements à responses occur in less than 20 ms and the bacteria is able to run 2 – 60 cell length/s sec (vibrio cholera = 60 cell lengths/min) • Positive/negative chemotaxis: - When an organism runs toward something, it’s only for so long until it tumbles - Toward: Caused by lowering the frequency of tumbles (flagellum moving clockwise) - Runs in the direction of the attractant are longer - Biased random “walk” à away from the repellent and involves similar but more frequent tumble responses so it’s ending up moving in random directions until it finally gets in a direction away from the repellent • Others: - Aerotaxis: When a bacteria moves towards oxygen - Thermotaxis 7. Describe other types of motility (spirochete, twitching, and gliding). a) Spirochete Motility: Ex: Syphilis (gram negative, flexible) - Multiple flagella form axial fibril, which winds around the cell inside the periplasm - Flagella remain in periplasmic space inside the outer sheath - Corkscrew shape exhibits flexing and spinning movements à advantageous for syphilis for example because it helps it burrow into the mucosal layers b) Twitching: - May involve type IV pili and/or slime - Pili at the ends of the cell - Short, intermittent, jerky motions - Cells are in contact with each other and surface c) Gliding: - Mary involve type IV pili and/or slime - Smooth movements against solid surface and is usually in connection with others **Secrete slime: Pores at the end of the cell and others say that they are adhesion complexes that are like feet and secrete slime (with gliding movements?) 8. Understand the structure and functions of bacterial endospores, the basics of sporulation and germination, and endospore resistance. • Endospore structure includes: a) Exosporium: Thin layer of protein on the outside b) Spore coat: Consists of layers of protein (more than 50 is possible); the proteins are impermeable to toxins/environment c) Outer membrane: Like another lipid bilayer and is also decently protected d) Cortex: Is made out of peptidoglycan but less cross-linked than that of a normal cell e) Germ cell wall f) Inner membrane g) Core: Consists of everything inside a normal cell: cytoplasm, ribosomes, etc. ** Noted parts of the endospore structure = what the cell will lose when it germinates (should be left with a gram positive bacterial cell) • Endospore resistance: a) Core: - Has low water content à not metabolically active and no enzymes are working because of the low water content - Calcium dipicolinate (Ca-DPA): Goes in between the bases of DNA to stabilize it and it protects it as a result - SASPs: Small, acid-soluble, DNA-binding proteins à not found in vegetative cells and are saturated to protect DNA from desiccation, radiation, chemicals, etc. - Slightly lower pH also causes the enzymes to not work properly (they normally want a pH of about 7) - Exosporium, spore coat (mainly impermeable), and cortex • Bad conditions (such as depleted nutrients) trigger vegetative cells no longer being able to divide and reproduce which therefore causes them to make endospores = sporulation à original cells are sacrificed and the sporangia dies under the conditions are good for germination activation • Sporulation overall process: - Cell division occurs that is not binary fission - A protoplast develops (gram positive with no cell wall) and then is engulfed by the cell membrane which becomes its outer membrane - Other layers develop on top of it with the cortex developing next with the less cross-linked peptidoglycan and then the others - Ends with lysing of original cell and freeing the now dormant endospore • Germination overall process: - Activation: Prepares the spore for germination and often results form treatments like heating and nutrient presence - Germination: Occurs when environmental nutrients are detected at high enough levels via receptor; spore swelling and rupture of the spore coat (water starts to rush in) which leads to loss of resistances; increased metabolic activity - Outgrowth: Emergence of the vegetative cell (gram positive) Chapters 11.1, 10.1 – 10.4 9. Know the requirements for microbial survival and growth and their sources. • Source of energy: - Used for cellular work such as repair, maintenance, growing in length before division, spinning of flagella, etc. • Source of electrons: - Play a role in in energy production à ex: ATP made with the ETC eventually - Reduce CO t2 form organic molecules (contain C and H) • Nutrients: - Carbon, hydrogen, oxygen (CHNOPS) needed to synthesize organic building blocks needed for cell maintenance and growth • Energy and electrons: - Sources: Inorganic or organic chemical compounds à energy is obtained via oxidizing (removing electrons from) a compound - Sunlight: Provides energy only; not all organisms are able to do this o Involves getting photons of light (NOT electrons- they need to come from a different source) - Energy is usually conserved in cells as ATP (adenosine triphosphate) and is not used directly when it is made; ATP = most common form • Chemoorganootrophs (Source of energy/electrons from organic compounds + chemotrophy) and chemolithotrophs (Source of energy/electrons from inorganic compounds + chemotrophy) • Phototrophs (Source of energy only from sunlight + phototrophy) 10. Define and recognize the major nutritional types of microorganisms based on their energy source, electron source, and carbon source. • Based on energy source: - Phototrophs: Use light - Chemotrophs: Obtain energy from oxidation of chemical compounds (organic or inorganic) • Based on electron source (reducing power) - Lithotrophs: Use reduced inorganic substances; “litho” = “rock eating” - Organotrophs: Obtain electrons from organic compounds • Based on carbon source: - Heterotrophs: Use organic molecules as carbon sources (pre-formed organic molecules) – which often serve as an energy an electron source as well - Autotrophs (self-producing): Use carbon dioxide as their sole/principle carbon source and must obtain energy from other sources (ex: sunlight) because carbon dioxide= most oxidized carbon on earth; known as primary producers • 5 Major Nutrition Classifications: First half of the name= energy source and second half = electron source Nutritional Type Energy Source Electron Source Carbon Source Photolithoautotroph Light Inorganic CO 2 compound Photoorganoheterotroph Light Organic compound Organic compound Chemolithoautotroph Inorganic Inorganic CO 2 compound compound Chemolithoheterotroph Inorganic Inorganic Organic compound compound compound Chemoorganoheterotroph Organic Organic compound Organic compound compound 11. Define metabolism, catabolism, and anabolism. a) Metabolism: Total of all chemical reactions occurring in the cell - Metabolites: Products of metabolism (Ex: Vitamins made by bacteria in the colon) b) Catabolism: Breaking down food/energy sources to make energy (spontaneous) - Fueling reactions - Energy-conserving - Provide ready source or reducing power (electrons) - Generate precursors for biosynthesis c) Anabolism: - The synthesis of complex organic molecules from simpler ones - Requires energy and building blocks form fueling reactions 12. Underotand the concepts of free energy (G) and standard free energy change (Δ G ʹ′). • Energy: The capacity to do work or to cause particular changes • G = free energy (the amount of energy that is available to do useful work - Energy leftover after a reaction has occurred that can be used to do work • ΔG = the change in energy that can occur in a chemical reaction à calculate for a chemical reaction which tells you how much energy is being released • Types of work carried out by microorganisms with G: - Chemical: Synthesis of new cellular material - Transport: Take up of nutrients, repair and replace, elimination of wastes, maintenance of ion balances - Mechanical: Motility of cells, chemotaxis o Ex: Movement in a non-motile bacteria with moving two chromosomes apart with cell division • 1 kCal = 4.18 KJ • Energy (G) is measured in KJ/mol • Δ G ʹ′ = Standard free energy change - At pH 7 - Temperature at 25 ºC - 1 atm - Reactants and products at 1 M concentration ** Standard conditions but different environments will cause various values to change slightly 13. Distinguish between exergonic and endergonic chemical reactions and their o relationship to Δ G ʹ′. • Exergonic: Catabolism - Release energy (that can be used for work) - A + B à C + D + energy - Δ G ʹ′ = negative, which means the reaction is spontaneous à no energy has to be put in - Some energy is lost as heat in the reaction but the rest is used for the formation of ATP (high P transfer potential- easy to break these bonds) - Breaking bonds = no energy requirement • Endergonic: Anabolism - Requires energy - A + B + energy à C + D o - Δ G ʹ′ = positive, which means that energy has to be put in to make the reaction go 14. Explain the importance of ATP. • ATP = ~ 31 kJ/mol = ~ 7.3 kCal/mol of energy is released when ATP is hydrolyzed to ADP + Pi • About 46 kJ/mol of energy is released when ATP is hydrolyzed to AMP + Pi ** Can’t double the amount of energy released for AMP in terms of ADP because it depends on how close the second bond is, for example, to the overall molecule that have other interactions affecting it • Breaking of the first bond is easier than the second bond because it is less tightly held to the molecule (ATP à ADP is easier to break than ADP à AMP) • Role of ATP in metabolism: - Endergonic reaction alone will not go - Add ATP, P bond breaks to ADP + Pi and the reaction will go 15. Be aware of other high-energy compounds, and know the change in standard free energy requirement for cells to use them. • Overall, cells want to use energy-rich compounds that give off energy greater than 30 kJ/mol when breaking a bond Δ G ʹ′ > 30 kJ o • Phosphoenolpyruvate (PEP): -61.9 Δ G ʹ′ kJ/mol - In glycolysis, PEP à pyruvate here P bond broken to give ATP • 1,3 Bisphosphosphoglycerate: Δ G ʹ′ -49.3 kJ/mol o • Acetyl phosphate: Δ Goʹ′ -44.8 kJ/mol • ATP (à ADP): Δ G ʹ′ -30.5 kJ/mol • ATP (à AMP): Δ G ʹ′ -45.6 kJ/mol Δ G ʹ′ < 30 kJ o • AMP: Δ G ʹ′ -14.2 • Glucose-6-phosphate: Δ G ʹ′ -13.8 16. Understand redox reactions including the standard reduction potential (E ʹ′) 0 o of half reactions, the electron tower, and their relationship to Δ G ʹ′. • Oxidation-Reduction Reactions (REDOX): - Many metabolic processes involve redox reactions (electron transfers) - Electron carriers are often used to transfer electrons form an electron donor to an electron acceptor - Can result in energy release, which can be conserved as ATP or another energy-rich compound • Oxidation: The removal of an electron(s) from a substance + - Ex: NAD = oxidized form - ½ O + 2 e à O 2- 2 (oxid.) (red.) à Electron-accepting half of the reaction • Reduction: Addition of an electron (s) to a substance - Ex: NADH = reduced form - + - 1 H 2 2 e + 2 H (red.) (oxid.) à Electron donating half of the reaction • Formation of water: 2 H + O à H O 2 • Net reaction: H +2½ O à 2 O 2 - Where H = electron donor (à oxid.) and O = electron acceptor (à red.) • Oxidations and reductions frequently involve the transfer of electrons and also the electron (e ) plus a proton (H )+ • Reduction and oxidation occur at the same time (an oxidized molecule is immediately reduced) • Standard Reduction Potential: E ʹ′ 0 - Measurement of the tendency for something to give up/lose electrons (in volts) - Equilibrium constant for an oxidation-reduction reaction - The more negative E ʹ′ = the better electron donor 0 - The more positive E ʹ′0= the better electron acceptor - Written as oxidation on the left and reduction on the right à electrons will always flow from the reduced to oxidized form (right to left) • Electron tower: - The greater the difference between E ʹ′ 0f the donor and the E ʹ′ o0 the acceptor, the more negative Δ G ʹ′ o - AKA, the further electrons fall down the tower, the more energy is released 17. Describe the location, organization, and functions of the Electron Transport Chains in bacteria. • Occurs in both anaerobic and aerobic respiration • Electron carriers are organized into the ETC with the first electron having the most negative E ʹ′0 - The potential energy stored in the first redox couple is released and used to form ATP - The first carrier is reduced and electrons move on to the next carrier and so on • The net energy of the complete reaction sequence is determined by the difference in reduction potentials between the primary donor and the final acceptor à Δ G is calculated to see how much energy is released to do work • Electron carriers come to the ETC in the mitochondria in the cristae in eukaryotic cells while they come to the ETC in the cellular membrane in bacterial cells (except for in the thylakoid membrane in some) • NADH dehydrogenase is the first +lectron acceptor as it becomes reduced and oxidizes NADH à NAD • Nutrition/bacterial species depends on the height of the redox tower à the shorter the tower, the less energy is to be released when electrons are being transferred down the tower to the next more positive electron acceptor 18. Define the two classes of electron carriers. a) Coenzymes: - Freely diffusible, meaning they can easily move in and out of the cell - Can transfer electrons from one place to another in the cell - Ex: NAD b) Prosthetic Groups: - Firmly attached (fixed) to enzymes in the plasma membrane - Function in membrane-associated electron transport reactions - Ex: Cytochromes + + 19. Describe how NAD /NADH and NADP /NADPH carry electrons and their roles in metabolism. • NAD : Nicotamide adenine dinucleotide (oxidative form usually used in anabolism) + • NADP : Nicotinamde adenine dinucleotide phosphate (oxidative form usually used in anabolism) • NADH and NADPH are good electron donors (Reduction potential of redox +ouple is -0.32 V) • NAD /NADH: - Coenzyme—freely diffusible - Carries 2 e + 1 H (1 H is released) - Involved in catabolism - Can be reused and NADH dehydrogenase in the ETC is what oxidizes NADH to NAD so it can be recycled back into glycolysis for example - This is good for the cell because there isn’t a lot of NAD and not a lot is +eeded since it’s being recycled anyway • NADP /NADPH: - Works the same way except it is involved in anabolism Chapter 11.2 – 11.8 20. Compare and contrast aerobic respiration, anaerobic respiration, and fermentation in bacteria. • Electrons released during the oxidation of chemical energy sources must be accepted by an electron acceptor • Aerobic Respiration: - Organic electron donor - Exogenous (meaning the electron acceptor cam from outside of the cell and had to be brought in) electron acceptor: O (2inal electron acceptor) - Most amount of energy released since O is the most positive on the 2 tower • Anaerobic Respiration: - Organic electron donor - 2- - Exogenous electron acceptors: NO , SO3, CO 4 fumar2te (all of these are about O 2n the tower so less energy is released from anaerobic respiration) • Fermentation: - Organic electron donor - Endogenous (meaning the acceptor originated inside of the cell) organic electron acceptor: Ex: pyruvate or derivative of pyruvate - Least amount of energy released via pyruvate since high up on tower 21. Compare and contrast substrate-level phosphorylation and oxidative phosphorylation. • Substrate-Level Phosphorylation: - Used in fermentation and other pathways (respiration also) - ATP is synthesized during steps in catabolism of an organic compound - Where the substrate is phosphorylated and a P is taken off and added to ADP to give ATP • Oxidative Phosphorylation: - Used in aerobic and anaerobic respiration - Where electron carriers take electrons to the ETC - ATP is produced by proton motive force which is established with electron transport from the electron carriers in the ETC + - - H are shuttled to the outside of the cell which are attracted to the OH inside the cell which creates the concentration gradient (potential energy) - The potential energy is used to make ATP and is also the source for the spinning flagellum - When H go back to the cell, this gives the energy needed to give P to + ADP à ATP then the H motive force has to be regenerated - Proton Motive Force occurs right after the ETC and where most of the ATP produced is gotten • Photophosphorylation: - Used by phototrophic organisms - Light drives the redox reactions that generate the proton motive force 22. Describe aerobic catabolism (overview). • Bacteria are unicellular so nutrients such as protein and starch are too big to be brought directly into the cell so it’s necessary to have them broken down outside of the cell first via exoenzymes that are inducible— regulatory à dependent on detection in the environment (Stage 1) • Stage 2: Occurs inside the cell; substrate phosphorylation (glycolysis) - Endoenzymes: constitutive à means these are always on/being made because they are so similar in function and don’t have to wait to detect substrate in the environment - Includes the CAC (?) = intermediate that can be used for biosynthesis (anabolism) and ex: AA à means that glucose is not always completely metabolized • Stage 3: ETC with oxidative phosphorylation • Stages 2 and 3: Amphibolic pathways meaning that the enzymes can be used in both catabolic and anabolic pathways (getting building blocks for anabolism) • Process that can completely catabolize an organic energy source to CO 2 using: a) Glycolytic Pathway: Takes glucose and breaks it down for ex. (glycolysis); PPP, fermentation, Entner-Doudoroff Pathway (aka various pathways to get to pyruvate) b) TCA Cycle: Can be different depending on the organism c) ETC with O as 2he final electron acceptor (varies on the species) • Produces ATP and recycles electron carriers à cannot have a pathway without recycling the electron carriers (including with fermentation) because they are how we get the ETC and make ATP through oxidative + + phosphorylation à cells don’t have many NAD or FAD because they are recycled 23. Describe the organization and functions of the electron transport chain in aerobic respiration including its role in ATP production. • Series of electron carriers that operate together • Transfer electrons from NADH and FADH (which2are not a part of the ETC itself) to a terminal electron acceptor • Electrons flow from carriers with more negative E ʹ0 • As electrons are transferred, energy is released to make ATP by oxidative phosphorylation • Important to note that electrons can’t be delivered to something higher in E 0′ than its own E0ʹ′ on the electron tower • NADH dehydrogenase is what NADH delivers to in order to be oxidized + and have its electrons/H removed • Carriers are arranged in the ETC with the most negative/better donors at the top and the most positive/better acceptors at the bottom + • See H accumulation on the outside of the cell membrane à proton motive force generated • NADH delivers electrons to the ETC and then it is recycled to NAD ; will give off more energy/ATP because it is higher on the electron tower • ETC for P. denitrifican: - Difference in E 0′ between NADH and O à 2arge amount of energy is released + - P/O (ATP/oxygen molecule = H consumed) - P/O = 2.5 for NADH - P/O = 1.5 for FADH 2 - The previously listed P/O are best case scenario for mitochondrial ETC 24. Understand the Chemiosmotic Hypothesis. • The movement of protons establishes the proton motive force (H are+ going outside of the cell into the periplasm) • ATP Synthase uses proton flow down gradient from the periplasm to make ATP à when Pi is added to ADP à ATP into the cell; this is the ending enzyme of the ETC that makes ATP - Smallest known biological motor 25. Explain the function of ATP synthase. • F 1 headpiece • F = motor part o • C proteins start spinning à energy to epsilon (e) and gamma and cant spin so it’s used to change the active site on the B subunit • B subunit binds ADP and Pi à ATP + • When H go through the motor, this causes ATP to be released when the B subunit goes back to its original conformation (Empty active site) 26. Know the functions of proton motive force and how it is established. • Established via transfer of electrons from electron carriers like NADH and FADH 2 • H pumped out to the outside of the cellular membrane to create concentration gradient • In organisms that use respiration: have the ETC + proton motive force in this form • For bacteria that only use fermentation: Have ATP synthase which has been discovered as a reversible enzyme (breaks ATP to make the proton motive force) that is used to make energy in another form for the spinning of the flagellum (doesn’t use ATP) • Some types of transport will also use proton motive force • Proton motive force may also be used to reduce electron carriers 27. For aerobic respiration, explain where in the pathway ATP is produced (glycolysis, TCA cycle, and ETC), the methods of ATP production used for each ATP generated, the electron carriers used, and the number of ATPs produced (during the process and the final net yield). • Total of 32 ATP is produced in Aerobic Respiration - Best case scenario since the total amount that’s actually produced depends on how tall the electron tower is, species, and nutrition etc. • Glycolysis: 4 ATP molecules produced - Net of 2 ATP since before G3P, input of 2 ATP molecules was required and then 4 ATP were produced after G3P • Oxidative Phosphorylation (ETC): 28 ATP molecules produced - When NADH and FADH are oxidized in the ETC 2 • Overall ATP produced with glycolysis (SLP), Bridge between glycolysis and Krebs Cycle, and the Krebs Cycle to oxidative phosphorylation with ETC: a) Between G3P and Pyruvate: 2 NADH * 2.5 = 5 ATP à oxidative phosphorylation 4 ATP (produced) -2 ATP = 2 ATP 7 total ATP from glycolysis b) Bridge between Pyruvate and Acetyl-CoA à Krebs Cycle: 2 NADH * 2.5 = 5 ATP à oxidative phosphorylation c) Krebs Cycle: 6 NADH * 2.5 = 15 ATP à oxidative phosphorylation 2 FADH *21.5 = 3 ATP à oxidative phosphorylation SLP: 2 ATP (2 GTP) 20 total ATP from Krebs cycle **Electron carriers go to the ETC from these pathways and are how ATP is indirectly obtained How many ATP are made via the Krebs Cycle through oxidative phosphorylation? 18 ATP How many ATP are made via SLP to 1 pyruvate (glycolysis)? 1 ATP 28. Summarize the major features of the Entner-Doudoroff pathway. • Used by some soil bacteria (ex: Pseudomonis) • Alternative way to get to pyruvate • Yields pyruvate and glyceraldehyde-3-phosphate • Key intermediate: 2-keto-3-deoxy-6-phosphogluconate (KDPG) • Glucose à glucose-6-phosphate à gluconate-6-phosphate (via glucose-6- dehydrogenase = enzyme from PPP and this is where one NADPH is produced) à KDPG (via 6-phosphogluconate dehydrogenase) à G3P à pyruvate OR pyruvate (via 2-keto-3-deoxygluconate-6-phosphate aldolase) • 6-phosphogluconate dehydrogenase and 2-keto-3-deoxygluconate-6- phosphate aldolase = indicate that the Entner-Doudoroff Pathway is being used • Net yield: 1 ATP (via substrate-level phosphorylation), 1 NADH, and 1 NADPH (goes to PPP) when coupled with the second half of Embden- Meyerhof (Glycolysis) 29. Describe the process of fermentation, its functions, and its products. • Uses an endogenous electron acceptor (Such as pyruvate or a derivative of pyruvate where pyruvate is reduced) – takes place in the absence of an exogenous electron acceptor where oxygen is not needed • Does not involve the use of an ETC or proton motive force • ATP synthesized only by substrate-level phosphorylation • Electrons go to pyruvate which produces ATP and fermentation products can include lactic acid, ethanol etc. and other products that can be used in biosynthesis • Main importance: Continues the recycling of electron carriers (NADH from glycolysis is oxidized to NAD ) + 30. Know why bacteria produce fermentation products and how these products are useful to humans. • Produces products such as lactate, propionate, isopropanol etc. • To the bacteria, fermentation products are seen as waste because they are only used as an electron dump to recycle NAD + • Fermentation Classes: a) Ethanol: Breads, wine, beer via yeast b) Lactic Acid: Homolactic and heterolactic c) Mixed Acid d) 2,3-Butanediol e) Propionic Acid: Lowers the pH and is used in making Swiss cheese • Acids: Lower the pH when they are produced via fermentation; way to preserve food and also gives food a distinct flavor and consistency 31. Distinguish between homolactic and heterolactic acid fermentation. • Homolactic: Glycolysis - Cheeses, sour cream, yogurt - Makes only lactic acid • Heterolactic: PPP - Sauerkraut, pickles, buttermilk, and involved in food spoilage (increases risk) - Makes lactic acid, ethanol and CO (2an see some bubbling) 32. Distinguish between mixed acid and butanediol fermentation. • Mixed Acid Fermentation: - NOT lactic acid bacteria - Gram-positive bacteria such as E. Coli, Salmonella etc. - Use the Methyl Red test which is an acid indicator à acidic fermentation products lower the pH and at pH of 5 or below, the indicator will turn pink - If Methyl Red positive, usually will not be VP positive too • Butanediol Fermentation: - Vogues-Proskauer Test à detects intermediate acetoin - If positive, will not be MR positive too; uses Barrit’s A/B to detect the acetoin which is the compound right before 2,3-butanediol - pH does not usually get as a low with these more neutral products than with those in the methyl red test 33. Explain the purpose of the MR-VP test and know how it works. • Used to detect the presence of acid • MR Test: - Acid indicator - Build up of acidic fermentation products lowers the pH and when a pH of less than or equal to 5 is reached, the indicator will turn red = positive result • VP Test: - Detects the acetoin intermediate of 2,3-butanediol - pH does not get as low - Addition of Barrit’s A/B indicates positive via red color Chapter 7.1, 7.3 – 7.4, 7.6 – 7.7 1. Describe the growth of bacterial cells (binary fission). • Growth: Refers to the increase in the number of cells (population rather than individual cell growth is studied) • Binary Fission: - Two cells arise from one; occurs because bacteria are haploid and can’t use meiosis/mitosis (conjugation = closest to sexual reproduction) o Resulting daughter cell may not be completely identical because plasmids are not necessarily evenly divided due to possibilities of there being an odd number and because plasmids do not replicate along with the chromosome (they are under their own control of their own replication) - Cell elongation: Cellular constituents increase proportionally o More cell membrane and wall have to be created o Penicillin works by first putting holes in the peptidoglycan during cell growth activity - Genome is replicated and separated - Cell division: o Septum formed at the mid cell à pinching in of the cellular membrane and wall o FtsZ proteins line up in the center of the cell (cytoskeletal proteins) and facilitate the cell division - Increase in cell number - One cell à two cells = one generation (prokaryotes and bacteria have small generation times) - The two cells may or may not completely separate from each other, it depends on the species 2. Describe in detail the four phases of bacterial growth observed in a batch culture. • Batch Culture: Culture incubated in a closed vessel with a single batch of medium • Growth curve is usually plotted as a logarithm of cell number vs. time - Done so experimenters can get the healthiest cells possible - Done because you want the inoculum to be the same phase of growth - May want to see how fast bacteria can grow/reproduce via calculating the generation time under those certain conditions • 4 phases: a) Lag Phase: - Cell synthesizes new components to replenish spent materials (such as enzymes for ex.) and to adapt to the new medium or other conditions - Varies in length: In some cases it can be very short or even absent if cells are already very healthy b) Log (Exponential) Phase: - Rate of growth is constant - Rate of growth is maximal (for those conditions; why slope is the steepest) - Population is most uniform in terms of chemical and physical properties during this phase - Where have the healthiest bacterial cells c) Stationary Phase: - Total number of viable (means having the ability to reproduce) cells remains constant – metabolically active cells stop reproducing but are not dead OR reproductive rate is balanced by the death rate where there is no increase in cell number - Possible reason for this phase: o Nutrient limitation because not adding any nutrients since it’s a batch culture à starvation responses resulting depending on the species o Limited O ava2lability (even though the batch culture is on the shaker) o Toxic waste accumulation: Many fermentation products are toxic to the bacteria o Critical population density reached so there is no more room for anymore cells (10 cells/mL = max) d) Death/Decline Phase: - Total number of viable cells is decreasing due to removal of critical nutrient (s) below a threshold level and metabolic end product toxic level - Death: Irreversible los of the ability to reproduce (ex: may think the cell is dead but it may be persistent and revive itself and reproduce so this does not mean it is dead) and lysis may occur ** When measuring cell number, important to not include the dead cell bodies and can be hard to do this because they may not be obvious through not being lysed • Starvation Responses: - Can occur at any phase but more like to see them at stationary phase - May be caused by morphological changes such as endospore formation (sporulation triggered by any bad conditions) or decrease in size (chromosomes may have condensed and also protoplast formation could occur do to shrinking from the cell wall) - May be indicated by production of starvation proteins (enzymes) that make metabolism more efficient à increase cross-linking in cell wall to make the cells stronger, Dps protein protects the DN, and chaperone protein prevent protein damage (protect other enzymes from denaturing and can fix some - May be indicated by persister cells which are for long-term survival and increased virulence à concern with pathogens since often times when one thinks they’ve been killed they are still there); can get sticky which makes them more virulent - On a growth curve, there may be a bit of increase of cell numbers due to these starvation responses 3. Be able to label a growth curve. 4. Define generation time, and be able to calculate it. • Generation (doubling) time: - The time required for the population to double in size (looking at the rate of how fast the bacteria are doubling) - Varies depending on species and environmental conditions - Exponential growth: Cell number doubles within a fixed time period à this is the fastest period for bacterial cells being able to divide for those particular conditions o Choose points from the middle so points won’t be chosen that are too close to the lag/stationary phases • Calculating Generation Time: - n = 3.3 (logN t log N ) 0 gives the number of generations where N = population number (choose two points for N and N0on the t exponential part of the growth curve) - Generation Time: g = t/n - K = growth rate constant (reciprocal of g = n/t) 5. Explain the methods of measuring the growth (number) of microbes (microscopic count, plating methods, turbidity measurements). • Direct: - Total cell counts—count the number of cells that are observed in the medium o Use of a counting chamber: ex: hemocytometer o Electronic counter: Where bacteria are forced through a small tube that has an electrical current and every time one passes through and interrupts the current = a count § Photocytometer= similar but with light interruption instead - Viable cell counts—Count the number of CFU’s that are culturable from the medium o Plating techniques o Membrane filter: Used for dilute samples (ex: looking at drinking water à put the water under the filter and catch the bacteria and count them) • Indirect: - Dry weight: Draw water out of the bacterial sample and weigh it (error prone) - Turbidity: Absorbance of light is equivalent to the number of bacteria but don’t actually get the physical number **Each colony represents one bacterium • Types for measuring cell numbers: a) Petroff-Hausser Counting Chamber (Hemocytometer) - Total cell count, direct count - Want units to be in bacteria/mL so have to do a conversion - Likely to get an overestimate of the living cells due to presence of dead bodies that have not lysed yet b) Plating Methods: - Viable cell count, direct count - Plate dilutions of population on suitable solid medium à count number of colonies à calculate number of cells in original population - Population size is expressed as Colony Forming Units (CFU’s) - Likely to get an underestimate of the real numbers à 2 colonies may be stuck together which may lead to counting errors - Concern of whether or not the medium is the best choice to grow the bacteria in and what if the agar is too hot etc. - Types of plating methods: a) Spread Plate Method: b) Pour Plate Method: o Agar has to be warm when it is poured on the plate with the sample and the heat may kill some of the bacteria o Have to be careful to include the sesame colonies that may be embedded into the agar c) Turbidity Measurements: - Indirect count - Involves using a spectrophotometer - The more cells there are, the more light absorbed/scattered which means the less transmitted/unscattered light is detected - Resulting transmittance number is the indirect measurement that is converted to absorbance to tell the concentration of bacteria in the medium - Even after this is done, it is necessary to obtain a direct count via a plating method for example and then a standard curve can be created to be able to be used later in counting 6. Describe how water activity, pH, temperature, and oxygen affect microbial growth. • Microbes must be able to respond to changing environmental conditions • Environmental factors that affect growth: water availability, pH, temperature, oxygen, pressure, radiation • Extremophiles: Microbes that grow under harsh or extreme environmental conditions (includes some archeans in addition to bacteria) • Water activity (and solutes): - Water activity = a w - Amount of water available to organisms - a values range from 0 to 1 with 1 = pure water (completely available w to the organism) - Water associated with solutes is unavailable to microorganisms - The higher [solute] à lower a w - Ex: Hypertonic environment: Low a so twe bacterial cell experiences plasmolysis - Adaptations: a) Hypotonic Solutions: o Use mechanosensitive (MS) channels in the membrane to allow solutes to leave the cell so less water comes into the cell o Works when too much water is coming into the cell, it swells the channels and they open for solutes to leave the cell which in turn causes the water to not come in as much (gets away from too steep of a concentration gradient) b) Hypertonic Solutions: o Increase the internal solute concentrations with stock piled compatible solutes (solutes that the cell doesn’t really need to use, ex. Some AA) which causes more water to come into the cell - See jams in the refrigerator with fungi for example because they can sand a lower water activity environment - Lower water contents = why some foods can’t spoil as fast • Effects of NaCl on microbial growth: - Non-halophile: o Does not require NaCl o Can grow if <1% NaCl - Halotolerant: Can withstand presence of NaCl but don’t need it o Can grow on our skin - Halophile: o Requires NaCl for growth o Grow optimally at >0.2 M (1-15% NaCl) o Would be in the ocean: 3% salt for example – found in some fish and squid - Extreme halophiles: o Requires 2-6.2 M (15-30% NaCl) o Great salt lake or archeans in the dead sea (one bacteria has a pink pigment so makes the dead sea pink) • pH: - Measure of the acidity of a solution + - Negative logarithm of the H concentration - Affects enzyme function, solubility and even can disrupt the cellular membrane - pH scale: up/down unit = 10-fold - Optimum pH: Can cover only about 2-3 units of pH (range) - Response to pH: o Most microbes maintain an internal pH in the cytoplasm near neutrality (including with acidophiles and alkaliphiles) o Maintenance of neutrality achieved by buffers such as an ion exchange of K for H for example o Acid Shock Proteins: Similar to the chaperone proteins found in starvation responses; they protect the cell/denaturing of enzymes from acid to a certain point o Many microbes change the pH of their habitat by producing acidic or basic waste products - Acidophiles: o Growth optimum between pH 0 and pH 5.5 - Neutrophiles: o Growth optimum between pH 5.5 and pH 8 - Alkaliphiles (Alkalophiles) o Growth optimum between pH 8.5 and pH 11.5 o The ocean has a pH of about 8.3 so alkaliphiles live here and may have some neutrophils in other parts of the ocean that are less basic • Temperature: - Microbes cannot regulate internal temperature - Exhibit distinct cardinal growth temperatures (3): a) Minimum: Cold o Membrane gelling occurs (ex. of adaptation: change in saturated FA to unsaturated FA to prevent this) o Transport processes so slow that growth cannot occur b) Maximum: o Protein denaturation occurs o Collapse of the cytoplasmic membrane o Thermal lysis o Ex. of adaptation: Membranes with more saturated FA can withstand higher temperatures so the membrane does not fall apart c) Optimum: o Where are the enzymatic reactions occur at the fastest possible rate o Happens to be closer to the maximum temperature than the minimum temperature - Temperature classes of microbes: a) Psychrophiles: o <0-20 °C (optimum < 15 °C) o Found in Antarctica b) Psychotrophs: o 0-35 °C (optimum 20-30 °C) o Overlap part of the mesophilic range and also contains the refrigerator temperature) c) Mesophiles: o 15-45 °C (optimum 20-45 °C) o What most pathogens fit into d) Thermophiles: o 45-85 °C (optimum 55-65 °C) o Similar to hot water heater o > 65 °C: Where archeans also live e) Hyperthermophiles: o 65-113 °C (optimum 85-113 °C) o Consists of mainly archeans with lipid monolayers that don’t fall apart at this high of temperatures - 4 °C: Refrigerators are set at this temperature and where psychrophiles and psychrotrophs (no
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