Ch. 16 Flashcards
Bacteria can be found in habitats that vary in pH from approximately pH 1 to 2 in acid springs to pH 11 in soda lakes and alkaline soils. What is bacterial pH usually maintained at, regardless of the external pH?
Within 1 to 2 units of neutrality (pH 7)
- Necessary to maintain viability
In general, what are the 2 main methods for adjusting internal pH?
- Proton pumps: extrude protons
- Antiporters: bring protons into the cell in exchange for sodium or potassium ions
Describe one method to demonstrate pH homeostasis.
Perturb the cytoplasmic pH by changing the external pH and then study the recovery process
Bacteria maintain a ΔpH across the cell membrane. Neutrophiles and acidophiles have a ____ ΔpH while alkaliphiles have a _____ ΔpH. (positive vs. negative)
Neutrophiles and acidophiles have a +ΔpH while alkaliphiles have a -ΔpH.
What is the major mechanism of pH homeostasis?
Controlling the flow of protons across the membrane
What occurs when the cytoplasm becomes too acidic? What about when the cytoplasm becomes too basic? (i.e. how does the cell respond?)
- Acidic: protons are pumped out
- Basic: protons are brought in via exchange with outgoing K+ or Na+
What condition is necessary for bulk proton flow to take place?
The pumping must be done electroneutrally
What implication arises from the exchange of protons with outgoing ions as a means of pH homeostasis?
Implies the existence of feedback mechanisms by which the intracellular pH can signal proton pumps and antiporters
Use the figure to describe the mechanisms of pH homeostasis.
In alkaliphiles, the Na+/H+ pump has what effect on the cytoplasm?
Acidifies the cytoplasm
What issue do alkaliphiles face in their basic environments? What is their solution to this issue?
- Main problem is keeping a cytoplasmic pH more acid than the external pH
- Solution: must always be bringing protons into the cell
In what way do acidophiles differ from other bacteria?
The external pH is several units lower than the cytoplasmic pH
- The maintenance of the large ΔpH requires an inverted ΔΨ at lower pHout
How is the membrane potential in acidophiles inverted?
Due to an inward flux of K+ greater than an outward flux of protons
- This might be due to the electrogenic influx of K+ catalyzed by an ATP-dependent K+ pump
How does E. coli respond to exposure to higher temperatures?
By transiently increasing the rate of synthesis of heat-shock proteins (Hsps) relative to other proteins
How are Hsps classified into families? What are the families?
Classified into families according to molecular weights
- Hsp70: MW of 70 kDa
- Hsp60
- Hsp40
- Hsp10
What happens to cells during increased temperatures? (i.e. Why are increased temperatures a problem?)
- Proteins denature or become misfolded
- Proteins aggregate
- DNA damage
- Cell membranes become fluid
What are the 4 main roles of Hsps?
- Folding of newly synthesized proteins at all temps
- Export of proteins at all temps
- Refolding of misfolded polypeptides
- Proteolysis of improperly folded/abnormal proteins
Following a temperature upshift, there is a transient increase in amount of sigma factor σ32 (aka RpoH), which is responsible for at least 30 Hsps the act in the cytoplasm. What factors contribute to the accumulation of σ32?
- σ32 is stabilized at higher temperatures (has a half-life of 1 minute at lower temperatures)
- Increased activity
- Increased rate of translation of σ32 mRNA
What is the function of sigma factor σ32?
Responsible for the synthesis of at least 30 Hsps that act in the cytoplasm
What does sigma factor σ32 recognize in a major heat-shock regulon?
Recognizes the promoters of genes in a major heat-shock regulon, known as the σ32 regulon
What happens to mutants that do not produce σ32?
They can’t grow at temperatures above 20ºC
What happens to the rpoH transcript under physiological temperature conditions vs. elevated temperatures?
- Physiological temps: rpoH transcript forms hairpin structure –> prevents ribosome binding
- Elevated temps: hairpin structure is melted –> ribosomes translate mRNA into σ32 (or oH)
How are the HSP genes recognized and transcribed at elevated temperatures?
They are recognized by RNA polymerase with σ32 and transcribed
What happens to the concentration of σH and Hsps after a sharp rise due to an increase in temperature?
The concentration decreases to a stable level due to feedback activity
Regulation of σ32: What happens when Hsps reaches a certain level?
- Separate σ32 from the RNA polymerase and degrade it
- Prevent overproduction of Hsps
- Hsps interact with σ32 mRNA, inhibiting its translation
At what levels does the regulation of σ32 occur?
- Stability of the protein
- Activity of the protein
- Translation of the mRNA
Several of the Hsps are chaperone proteins. What do chaperone proteins do?
- Folding of newly synthesized proteins
- Refolding of improperly folded proteins
- Export of proteins
List some Hsps that aid in the folding of newly synthesized proteins.
- DnaK, DnaJ, and GrpE
- GroEL and GroES
The Hsps DnaK, DnaJ, and GrpE appear to be ribosome associated. How does this help them do their job?
- Can bind to the protein as a team while the protein is still attached to the ribosome
- After protein has left the ribosome, they pass the protein into a cavity of the multimeric GroEL, where the final stages of folding take place
- Capped by GroES
What is SecB and what does it do?
- Major chaperone protein in E. coli (Hsp that aids in protein export)
- Prevents folding and brings unfolded presecretory proteins to SecA and the translocation machinery at the membrane
What is ClpB and what does it do?
- An ATPase in E. coli (Hsp that refold denatured proteins)
- Disentangles thermally aggregated proteins and transfers them to the DnaK-DnaJ-GrpE chaperone system
What happens if proteins are too damaged to be refolded?
Heat-shock proteases will destroy the damaged proteins
What is Lon and what does it do?
- Important in degrading damaged proteins (Hsps that are ATP-dependent proteases)
- Functions during both the heat-shock response and ordinary growth
- Degrades abnormal proteins
Define osmosis.
Diffusion of water into the more concentrated solution
Define osmotic pressure (Π).
The pressure that must be applied to stop the osmotic flow of water diffusion
Define osmotic potential (π).
Emphasizes that water flows from solutions of a high osmotic potential to solutions of a low osmotic potential
- Numerically equal to the osmotic pressure but negative
Explain why the cytoplasm has a more negative osmotic potential (a more positive osmotic pressure).
Because the cytoplasm of most bacterial cells is more concentrated than the medium
- Water flows into the cell and expands the cell membrane, exerting pressure against the cell wall (turgor pressure)
What is the turgor pressure in Gram-positive vs. Gram-negative bacteria?
- Gram-positive: ~15-20 atm
- Gram-negative: ~0.8-5 atm
Why is turgor pressure important?
It provides the forces that expands the cell wall
- Necessary for the growth of the wall and cell division
What provides the tensile strength of the bacterial cell walls that allows them to withstand turgor pressure?
Peptidoglycan
How do cells respond when placed in high osmolarity media?
They increase the intracellular concentration of certain solutes called osmolytes
- Ensures that the internal osmolarity is always higher than the external so turgor pressure is maintained
What are osmolytes used by bacteria called? Why?
Compatible solutes
- Relfects their relative nontoxicity
How do cells acquire compatible solutes? Name some.
Accumulate them intracellularly from the medium via transport
- K+
- Glutamine, glutamate, proline
- Betaine
- Trehalose
Describe the role of K+ in pH homeostasis.
- Bacteria pump protons across their membrane –> creates a membrane potential
- Membrane potential limits the number of protons that can be pumped across the membrane
- Cell needs to dissipate the extra membrane potential so it can continue to pump protons out to increase the intracellular pH
- Does so by pumping in cations or pumping out anions
- One of the cations it pumps in is K+
Explain osmotic homoeostasis in halobacteria.
- Cell keeps cytoplasm salty to prevent water from leaving the cell and maintain turgor pressure
- Take up K+, export Na+
- Ionic interactions are weakened in high salt concentration –> protein folding issues
- But halophiles are the opposite: they need high salt concentrations for protein stability and activity
Explain osmotic homeostasis in E. coli. (high osmolarity)
E. coli shifted to a high osmolarity medium
- Influx of K+ to respond to decreased turgor pressure
- Needs to preserve electrical neutrality in cytoplasm and prevent depolarization of cell membrane
- Pumping protons out prevents depolarization and maintains cytoplasmic neutrality
- Cytoplasm reacidifies, but glutamate provides counterions to K+
The total amounts of OmpF and OmpC are fairly constant, but the ratio changes with osmolarity and temperature of the medium. How do their levels change in high-osmolarity medium? What is the result of this change?
- Transcription of the ompF gene is repressed
- Transcription of the ompC gene is increased
- Result: switch to a smaller porin channel
What is one response to low-osmolarity media?
To decrease the concentration of cytoplasmic osmolytes
- Excretion of osmolytes
- Catabolism of osmolytes
- Involves mechanosensitive channels
Mechanosensitive channels are gated. What does this mean?
When water rushes into the cell from a dilute environment (increasing turgor pressure), the increased tension in the cell membrane causes a conformational change in the MS channel proteins to that the channels transiently open
What are the various methods of DNA damage repair?
- Nucleotide excision repair (NER)
- Recombination
- Base excision repair
Explain nucleotide excision repair.
- UvrABC (endonuclease) cuts the DNA near dimer
- UvrD (helicase) aids in removing the DNA segment and leaves a single stranded gap
- DNA Pol I fills gap
- DNA ligase seals gap
Explain how damaged DNA is repaired by recombination.
- DNA Pol III pauses when it arrives at a damaged base
- Nuclease will cut the opposite template strand
- Nuclease connects opposite template strand to fill gap (this is called “sister strand exchange”)
- DNA Pol I remakes the now missing template strand
- Crossover is cut, and the breaks are sealed by ligase
Explain base excision repair.
- Damaged base is removed by DNA glycosylase, leaving an AP site
- AP endonuclease cleaves the phosphodiester bond on the 5’ side
of the AP site - DNA Pol I extends the 3’ end while removing a portion of the damaged strand with its 5’-3’-exonuclease activity
- The nick is sealed by DNA ligase
Explain how toxic forms of oxygen are produced.
- Produced during the reduction of oxygen
- Oxygen is reduced by adding one electron to it at a time
- A small quantity of superoxide radical is released as oxygen is reduced
- Other toxic forms of oxygen are the hydroxyl radical and hydrogen peroxide, which are formed from the superoxide radical
When does the cell use the SOS response?
If a cell has prolonged exposure to a stressful environment or accumulates damage to many bases, replication will slow or even halt triggering the SOS response
Explain the role of RecA and LexA in the regulation of the SOS response.
- LexA regulon binds to LexA-binding sites in the SOS box to prevent the binding of RNA polymerase
- In an undamaged cell: prevents the transcription of SOS proteins (makes sense because those proteins are not needed)
- LexA represses the recA and lexA genes
- When DNA damage occur: RecA is activated to RecA*
- LexA binds to the RecA*-DNA complex and gets inactivated
- RecA* breaks the bond between glycine and alanine in LecA
The SOS response can induce translesion synthesis. What are the pros and cons of translesion synthesis?
- Pros
- Allows DNA replication to keeping going past the damaged part
- Increases the chances of the cell surviving - Cons
- Error-prone
- Increases the rate of mutation in the cell
Name the 2 transcriptoinal regulatory systems in E. coli that control the production of proteins in response to oxidative stress.
- SoxRS system
- OxyR system
Explain the SoxRS pathway.
- SoxR protein is activated in response to superoxide or nitric oxide
- Activated SoxR activates transcription of the soxS gene
- SoxS protein activates transcription of target genes whose products confer resistance to various toxic elements
- Activated genes: sodA (codes for superoxide dismutase) and nfo (codes for endonuclease IV)
Explain the OxyR pathway.
- OxyR is activated in vitro by O2
- Activates katG (codes for catalase)
- Also activates oxyS but function is unknown
