1. What is diauxic growth?
    • Preferential growth on one carbon source BEFORE growing on a second
    • ex- E. coli consuming all glucose before using lactose
  2. 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
  3. 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)
  4. 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+
  5. 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
  6. 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
  7. 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
  8. 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
  9. 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
  10. 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)
  11. 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
  12. 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
  13. 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
  14. 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
  15. 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
  16. 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
  17. 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
  18. 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
  19. 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
  20. 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
  21. 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)
    • 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
  22. 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
  23. 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)
  24. 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
  25. 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
  26. 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.
  27. 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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