1. 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)
  2. 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
  3. 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
  4. 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
  5. 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
  6. 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
  7. 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
  8. 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
  9. 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
  10. 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
  11. 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
  12. 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
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