chapter 5 - Chapter 5: Chapter 5: Microbial Nutrition...

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Unformatted text preview: Chapter 5: Chapter 5: Microbial Nutrition Nutrient Acquisition Nutrient Acquisition Nutrients must pass from the environment through the cytoplasmic membrane In the environment, some nutrients are abundant, others are scarce Diffusion is based on the tendency for molecules to be in constant motion Move from areas of higher to lower concentration Membrane blocks diffusion of some molecules Nutrient Transport Mechanisms Nutrient Transport Mechanisms 1. Passive diffusion Molecules move by thermal agitation Inefficient Movement from higher to lower concentrations Works for small molecules like O2 and CO2 2. Facilitated diffusion Carrier protein helps movement from higher to lower concentrations Saturable: the rate of transport is dependent on concentration of substance vs. carriers available Nutrient Transport Mechanisms Nutrient Transport Mechanisms 3. Active transport Energy is used to transport against a concentration gradient (lower to higher) Uses a carrier protein Saturable Molecule in transport is chemically altered before it passes through the cell membrane Chemical change ‘tricks’ gradient situation 4. Group translocation Facilitated Diffusion Facilitated Diffusion Carrier proteins (permeases) Spans cell membrane High specificity­ binds only to specific (large) molecules Binding may change conformational shape of the permease Open and close the ‘gate’ Facilitated Diffusion Facilitated Diffusion Facilitated Diffusion Facilitated Diffusion Process is saturable Faster rates when transportable substance concentration is low Plateaus when transportable substance concentration = permease concentration Gradient must be maintained Cells maintain concentration gradient by transforming transported nutrient Facilitated Diffusion Facilitated Diffusion Reversible: cellular waste products exported from the cell Not the prominent transport mechanism of nutrients into prokaryotic cells [Nutrient] is usually lower outside cell than inside the cytoplasm Example: glycerol transport Active Transport Active Transport Transport which occurs against concentration gradients Resembles facilitated diffusion in some ways Permeases in membrane are used Specificity or recognition of molecules to transport Saturable Requires energy ATP or H+ (that would be used in respiration to produce more ATP) Active Transport Types Active Transport Types Uniport­ one molecule at a time, one way Symport­ two molecules at a time, one way and done simultaneously Antiport­ two molecules at a time, one in and one out, and done simultaneously Export system for many antibiotics/toxic compounds ABC Transporters­ one molecule at a time, uses special peripheral binding proteins that cleave ATP Active Transport Types Active Transport Types Coupled Symport & Antiport Coupled Symport & Antiport Proton (H+) and Na+ Pumps H+ pumped outside membrane during electron transport Proton­motive force (high H+ concentration external to cell) drives expulsion of sodium from the cell as H+ enters (see antiport box next slide) Externally, sodium binds to carrier protein complex and allows solute to enter protein channel Solute binding produces conformational change in carrier, releasing both sodium and solute inside the cytoplasmic membrane (see symport box next slide) Coupled Symport & Antiport Coupled Symport & Antiport Antiport Symport ATP­Binding Cassette (ABC) ATP­Binding Cassette (ABC) Transporters Solute­binding protein External to cytoplasmic membrane In Gram –’s: found in periplasmic space In Gram +’s: found as tethered lipoproteins on the external phospholipid bilayer Binds to molecule to be transported Complex interacts with transport domain of permease to open the channel ATP­Binding Cassette (ABC) ATP­Binding Cassette (ABC) Transporters Permease has two domains One spans phopsholipid bilayer = Transport domain Acts as channel for transport of solutes One within the cytoplasm = Nucleotide­binding domain Binds to ATP, cleaves it to ADP, and opens the channel so solute can pass into cytoplasm ATP­Binding Cassette (ABC) ATP­Binding Cassette (ABC) Transporters Group Translocation Group Translocation Molecule is transported into cell while being chemically altered i.e. ­PO4 moiety is added Metabolic energy used in the process to alter transported substance i.e. the moiety is cleaved from an existing compound, not newly synthesized Group Translocation Group Translocation Example: phosphoenolpyruvate (PEP)­ dependent sugar phosphotransferase system, or PTS transport PEP used as a phosphate donor to phosphorylate sugars during transport PEP becomes pyruvate (substrate for glycolysis) Mechanism widely distributed in prokaryotes May also play a role in chemotaxis Group Translocation Group Translocation Nutritional Requirements Nutritional Requirements 1. Carbon 2. Energy/H+ source 3. Terminal electron acceptors 4. Major elements 5. Trace elements 6. Growth factors Nutritional Requirements Nutritional Requirements 1. Carbon: Autotrophs use inorganic carbon CO2 Heterotrophs use organic carbon Simple sugars Amino acids Proteins Complex carbohydrates Lipids Nutritional Requirements Nutritional Requirements 2. Energy Source Phototrophs capture energy from sunlight Cyanobacteria, purple & green sulfur bacteria Chemotrophs oxidize organic or inorganic molecules for energy Organic: glucose Inorganic: H2: Hydrogenomonas H2S; FeS2 : Thiobacillus NO2­: Nitrobacter Nutritional Requirements Nutritional Requirements 2. Energy source: cont. Hydrogen/electron source (H+) Organotrophs: reduced organic molecules Glucose Lithotrophs: reduced inorganic molecules HS 2 Nutritional Requirements Nutritional Requirements Groupings based on energy/H+ diversity Photolithoautotrophs Cyanobacteria, purple & green sulfur bacteria Purple & green non­sulfur bacteria Sulfur oxidizers, H2 oxidizers, methanogens Some sulfur oxidizers Photoorganoheterotrophs Chemolithoautotrophs Chemolithoheterotroph Chemoorganoheterotroph Bacterial pathogens, fungi, archaea Nutritional Requirements Nutritional Requirements 3. Terminal electron acceptors: Aerobic respiration: O2 Anaerobic respiration: inorganic molecules Fermentation: organic molecules Nutritional Requirements Nutritional Requirements 4. Major elements: N, P, S, O: Along with C and H are basis for proteins, carbohydrates, nucleic acids, and lipids Nutritional Requirements Nutritional Requirements 5. Trace elements: Co, Mo, Mn, Zn, Cu: Necessary for enzyme cofactors, vitamins, etc. Very minute quantities Nutritional Requirements Nutritional Requirements 6. Growth factors Components that cannot be synthesized by an organism from the essential elements Purines & pyrimidines Vitamins Certain amino acids Varies with species, even strains Microbes requiring many growth factors are considered fastidious Cultivating Bacteria in the Lab Cultivating Bacteria in the Lab Agar Complex polysaccharide Extracted from marine algae (seaweed) Melts at about 95°C Solidifies at about 45°C Inert to most bacteria Some marine bacteria produce agarase Cultivating Bacteria in the Lab Cultivating Bacteria in the Lab Culture media: provides appropriate nutritional conditions for growth Complex media Contains things like meat extracts, protein digests like peptone, yeast extract Exact chemical formula is unknown Nutrient broth, nutrient agar and blood agar are examples Chemically defined Contain pure chemicals like NaCl, glucose, MgSO 4 Buffered to maintain a neutral pH Cultivating Bacteria in the Lab Cultivating Bacteria in the Lab Specialty Medias: Selective Allows only the growth a few kinds of organisms while inhibiting the growth of others that inhibit all but Neisseria sp. Thayer­Martin agar contains antibiotics Selective Media: Thayer­Martin Agar Selective Media: Thayer­Martin Agar Cultivating Bacteria in the Lab Cultivating Bacteria in the Lab Specialty Medias: Differential Does not inhibit any bacteria from growing Growth is somehow differentiated: i.e. biochemical Blood agar shows hemolysis patterns Alpha­ partial breakdown (red to green) Beta­ complete breakdown (red to clear) Gamma­ no breakdown Differential Medium: Blood Agar Differential Medium: Blood Agar Cultivating Bacteria in the Lab Cultivating Bacteria in the Lab Specialty Medias: Selective and Differential MacConkey agar Selects against Gram positive organisms Differentiates lactose fermenters (pink/red colonies) from other bacteria incapable of using lactose (cream/colorless) Selective/Differential Medium: Selective/Differential Medium: MacConkey Agar Isolation of Pure Cultures Isolation of Pure Cultures A colony of bacteria arises from a single original parental cell Multiplies by binary fission All progeny are identical 1 colony = 1 million cells Isolation of Pure Cultures Isolation of Pure Cultures 1. Streak plate (Quadrant streak): Loopful of bacteria is sequentially diluted over the surface of an agar plate Flame sterilization of loop used to remove excess cells from loop in between quadrants Not quantitative Streak Plate Isolation Streak Plate Isolation Isolation of Pure Cultures Isolation of Pure Cultures 2. Spread plate: Bacteria are serially diluted (usually 10­fold) in a known volume of diluent A specific volume is placed on the surface of the agar and spread over it to evenly distribute cells Cells grow up on surface of agar Quantitative Isolation of Pure Cultures Isolation of Pure Cultures 2. Spread plate: Isolation of Pure Cultures Isolation of Pure Cultures 3. Pour plate: Similar to spread plate isolation practice Serial dilutions Quantitative The difference is that inoculum is mixed with warm agar then poured into empty sterile Petri dish­ colonies form within agar as well as on surface Pour Plate Isolation Pour Plate Isolation ...
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This note was uploaded on 03/24/2009 for the course MIBO 3500 taught by Professor Dustman during the Fall '09 term at University of Georgia Athens.

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