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What is diauxic growth?
- Preferential growth on one carbon source BEFORE growing on a second
- ex- E. coli consuming all glucose before using lactose
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What is catabolite repression by glucose?
- Glucose inhibits synthesis of enzymes used in catabolism of non-primary carbon sources
- Very common in bacteria
- Exceptions in Rhizobium and P. aeruginosa which prefer organic acids over glucose
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Adaptive responses of bacteria to nutrient limitation
- Changes in cell size: become smaller
- reductive division (division with no growth)
- Dwarfing - continuous cell size reduction after reductive division
- Self-digesting process
- Morphological changes: rod-shape to coccoid
- Changes in surface properties: surface becomes hydrophobic and adhesive
- Allows cells to aggregate and adhere to adsorbed nutrients on surfaces
- Changes in metabolic activities: rate slows
- Turnover of proteins and RNA to serve as energy sources
- Changes in protein composition: specific genes turned on (survival proteins)
- Changes in resistance to environmental stress: more resistant to high temp, osmotic stress, harsh chemicals
- Control of rRNA synthesis: starvation slows rRNA synthesis (lack of AA)
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Changes that can occur for the final electron carrier?
- Bacteria have many final electron carriers (and pathways) which can be substituted to be the most efficient under a given circumstance.
- E. coli has 2 quinol oxidases (cyt o oxidase and cyt d oxidase) based on O2 levels available
- E. coli can synthesize reductases under anaerobic conditions (fumerate, nitrate, etc)
- E. coli can ferment if there is NO final electron acceptor available to regen NAD+
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Pathways can be anabolic OR catabolic based on need....
- AA <-> proteins
- dNTPs/NTRs <-> nucleic acids
- fatty acid/glycerol <-> lipids/cell membranes
- NAM/NAG/AA <-> cell wall (peptidoglycan)
- All 3 pathways (EMP, PPP, ED) convert glucose to PGALD in the different way, but have same pathway for PGALD -> pyruvate
- These pathways share intermediates
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Fate of pyruvate based on O2 availability
- aerobic respiration: pyruvate -> Acetyl Coa which feeds into the citric acid cycle
- anaerobic respiration: part of TCA cycle functional, uses ETC w/ diff final e- acceptor
- Fermentation: doesn't produce ATP, meant to regen NAD+ for continuation of glycolysis
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Compare/contrast citric acid cycle with and without O2
- With O2: Acetyl-Coa oxidized to CO2
- Enzymes no made without O2, they inactivate
- Operates as a cycle
- Feedback inhibition and irreversible reactions regulate
- Without O2: provides precursors to 10/20 AA and intermediates for other pathways
- NOT a cycle
- Citrate synthase allosterically inhibited by NADH and alpha ketoglutarate
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Determine oxygen toxicity in strict anaerobes, aerobes, and aerotolerants
- Strict anaerobes: killed by toxic forms of oxygen
- Superoxide radicals (O2-*) - forms because e- transferred to oxygen one at a time
- Hydrogen peroxide (H2O2) - form when superoxide radicals are reduced
- Hydroxyl radicals (OH-*) - forms when H2O2 in presence of transition metals
- Aerobes and aerotolerants posses enzymes that remove these forms of oxygen.
- Superoxide dismutases: 2 O2-* +2H -> H2O2 + O2
- Found in aerobes and aerotolerants
- Catalases: 2 H2O2 -> 2H2O + O2
- Found in aerobes
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Why is fermentation a less energy efficient process than aerobic or anaerobic processes?
- Most reactions take place in cytoplasm
- ATP is made by substrate-level phosphorylation
- No ETC = no proton gradient = very small amount of ATP production
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How are bacteria able to maintain homeostasis re: pH (general)
- Regardless of external pH, internal pH is maintain 6-8
- When exposed to change in external pH internal initially changes WITH external followed by recovery back to initial value
- Factors influencing pH inside cell...
- 1. Buffering capacity of cytoplasm
- 2. Metabolic reactions producing acids/bases
- 3. Flow of protons across membrane (most important)
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How are bacteria able to maintain homeostasis re: pH (specific mechanisms)
- Proton pumping: raises internal pH and membrane potential
- Requires membrane potential
- If no protons are pumped, they equilibrate across membrane
- Sometime membrane potential must be dissipated to continue proton flow (influx of cation/efflux of anions)
- Uptake of K+ by neutrophillic bacteria: dissipates membrane potential
- Allows extrusion of protons
- Increases pH
- Na+/H+ antiport or K+/H+ antiport: K or Na are exported while H+ is imported
- Decreases pH
- Important in alkiliphiles
- Na+ uptake: completes Na+ circuit
- Allows other mechanisms to continue
- Acidophiles: influx of K+ required to depolarize membrane
- Protons rapidly consumed during metabolism rather than pumping
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How are bacteria able to maintain homeostasis re: Osmolarity (general)
- Osmotic pressure: pressure needed to STOP the flow of water across a membrane (measured in Osm)
- Osmotic potential: water flows from low osmotic potential to high osmotic potential
- Higher [solute] = higher osmotic potential
- Turgor pressure required for bacterial growth
- Higher T. Pressure in gram + due to increased amt peptidoglycan
- Increase to osmolytes can come from solutes synthesized or transported into cell
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How are bacteria able to maintain homeostasis re: high Osmolarity media (mechanisms)
- In halobacteria: cytoplasm is kept satly with K+ to prevent water exiting cell
- High ionic strength normally causes proteins to denature, but but halophilic proteins REQUIRE high ionic strength
- In E. coli: shift to .5 M NaCl
- 1. Influx of K+ in response to decreased turgor pressure
- 2. Accumulation of other solutes
- Increased synthesis, decreased utilization
- Transport from media
- Effect on transcription and enzymes...
- Synthesis of new enzymes and transporters compatible with solutes
- Different sigma factors used to transcribe genes under environmental stress
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How are bacteria able to maintain homeostasis re: Low Osmolarity media (mechanisms)
- Water enters cytoplasm, potentially increasing turgor pressure too much
- Adapts by excretion of solutes (K+) via mechanosensative channels embedded in membrane
- MS channels: allow rapid exit of internal solutes, stimulated to open by water flow into cell and increased turgor pressure
- Homeostasis in the periplasm: synthesis of membrane-derived oligosaccharides to increase osmolarity of periplasm
- IMPORTANT TO GRAM NEG
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How are bacteria able to maintain homeostasis re: Temperature
- Membrane fluidity: dependent on degree of saturation in fatty acids (more saturation = better stacking = higher mp) and chain length (longer chain = higher mp)
- Principle temperature environment determines what mixture of fatty acids a given bacteria will have in its membrane
- Growth rate: increase in temperature increases growth rate until enzymes denature
- Protein patterns: increase/decrease based on temperature shifts
- Different protein sets made by same bacteria
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Describe the heat shock response
- Induced by ALL stress environments when translation is interfered with or there is denaturation of proteins
- Increase in heat increases synthesis of heat shock proteins (HSPs)
- HSPs repair/eliminate proteins damaged by heat and ensure proper folding/protein export occurs at all temperatures
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What are the various sigma factors?
- δ32 (RpoH) regulon: made constantly at low concentrations, increased rate at higher temperature (42deg)
- 1. More stable, can be utilized more frequently before proteolysis
- 2. Activity AND amount is increased
- 3. Increases to rate of translation for the regulon
- δ32 stimulated by amount of denatured proteins, temperature, ethanol, starvation, and oxidative stress
- δ24 (δE) regulon: Activated by high temperatures (50deg) and envelope stress
- Protects against damage to extracytoplasmic proteins (folding/refolding/degredation of misfolded proteins)
- Low activity under nonstress conditions
- Envelope stress releases sigma factor which binds to RNAP to initiate transcription
- δS (RpoS) regulon: "Master regulator" activated by starvation, stationary phase, hyperosmolarity (.3M), and low pH (5)
- Kept at low concentration in log phase
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Types of DNA repair
- Mismatched base pairing during DNA replcication
- Breaks or gaps in DNA
- 1. SS - sealed by DNA ligase, nonlethal
- 2. DS - often lethal, can't be resealed by ligase
- Base modifications: oxidative damage, usually lethal
- Barrier to replication
- Thymine dimers: photodimerization caused by UV radiation
- Stalls DNAP, blocking replication
- Leads to "snakes" - long cells that can't divide
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Describe repairing UV damaged DNA
- Thymine diamers repaired by removing cyclobutane rings
- Photoreactivation: photolyase absorbs blue light energe and cleaves dimer
- Photolyase not found in placental mammals
- Nucleotide excision repair: recognizes distortion in DNA helix
- cuts on either side of the dimer
- DNAP 1 fills in complementary bases
- DNA ligase seals them together
- Recombination: Daughter-strand gap repair uses ReCA and good sister template strand to fix
- Base Excision repair: repairs single bases damaged by deamination
- *cytosine --deamination-> uracil
- Involves DNA glycosylases
- 1. Remove base of nucleotide -> AP site
- 2. AP endonuclease cleaves phosphodiester bond of 5' site
- 3. DNAP extends the 3' end while removing portion of bases ahead (5' exonuclease activity)
- 4. DNA ligase fills in the gap
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Describe the GO system
- Involves 3 proteins...
- MutM: removes 8-oxoguanine (GO)
- Cuts damaged DNA
- DNAP and DNA ligase repair
- MutY: functions when adenine is paird with GO, before GO is removed
- removes adenine -> AP site
- Repair by MutM
- MutT: Phosphatase that converts 8-oxo-GTP to 8-oxoGMAP preventing its incorpration
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SOS Response (detail)
- Stimulated when RecA protein beins to ssDNA at a stalled replication fork
- Induction of SOS regulon due to INACTIVATION of LexA (a repressor) -> synthesis of repair protein
- RecA is activated by DNA damage -> inactivation of LexA
- Activated RecA complexes with ssDNA
- LexA binds to RecA-ssDNA complex and undergoes cleavage (inactivation)
- ERROR-PRONE REPAIR
- DNA Pol V is produced, and does not have stringent prooreading capability.
- Incorporates bases randomly opposite T dimers and abasic sites
- Replication proceeds, but mutations abound
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Describe anaerobiosis (general)
- Anaerobiosis: shift from aerobic to anaerobic environment
- 1. Metabolic changes
- Citric acid cycle is replaced by reductive TCA (noncyclic)
- NADH no longer produced (no need to regen NAD+)
- 2. Induction of anaerobic genes
- Electron acceptor synthesis regulated based on availability
- +O2 represses synthesis of anaerobic reductase
- +NO3- represses synthesis of other reductases
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Describe the ARC system in detail
- anaerobic respiratory control OR anoxic redox control system
- Arc B: transmembrane sensor kinase
- Activated by anoxia (increase in reduced electron carriers in cell [NADH], anaerobic metabolite buildup [pyruvate, lactate]
- Arc A: response regulator
- Represses genes for aerobic growth
- Induction of genes for cytochrome d oxidase (high O2 affinity at low O2 levels)
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Describe the FNR system in detail
- Fumerate Nitrate Reductase system
- Global regulator protein activated during anaerobic growth
- + transcription of anaerobic growth genes
- - transcription of aerobic growth genes
- may regulate the same genes as ARC system
- Has DNA binding ability
- Iron-Sulfur center (reduced/oxidized based on O2 levels)
- Oxidized: inactivated
- Reduced: activated
- binds to DNA to inhibit aerobic genes
- binds to DNA to induce anaerobic genes
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Describe the PHO regulon in detail
- Regulon: set of nontcontiguous operons controlled by a common regulator
- PHO regulon
- contains genes for regulation of phosphate assimilation when phosphate supply is limited
- Under low phosphate conditions stimulates at least 38 genes for phosphate uptake
- PtsS: periplasmic binding protein
- Forms repressor complex if P is bound
- PhoR: histidine kinase/phosphotase
- Detects P, can activate or deactivate PhoB
- PhoB: response regulator
- positively regulates PHO regulon when active
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How do bacterial cells protect themselves from osmotic stress?
- Bacteria need to adjust the amount of solutes within the cell to match that outside the cell
- Regulate outer membrane proteins (porins)
- OmpC: small pore
- OmpF: large pore
- Hypteronic/Hot: increased OmpC (retards inflow of solutes)
- Hypotonic/Cold: increased OmpF (increase inflow of dilute nutrients)
- Lakes/streams
- Enz/OmpR two component system is regulator.
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Describe EnvZ/OmpR system in detail
- EnvZ: inner membrane histidine kinase
- osmotic sensor
- transmembrane; periplasm and cytoplasm
- OmpR: Response Regulator
- Cytoplasmic protein
- Hypertonic: OmpR phosphorylated
- stimulates transcription of OmpC (stimulates kinase activity of EnvZ)
- represses ompF byinducing micF (antisense RNA that prevents OmpF mRNA expression)
- Hypotonic: OmpR unphosphorylated
- stimulates transcription of OmpF
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