Bacterial Responses to Environmental Stress Flashcards
What are some environmental stresses?
Nutrient shortage (phosphate, nitrogen, carbon, trace elements).
pH changes.
Temperature (cold shock, heat shock).
Osmolarity.
Oxygen.
Light quality, UV damage.
Toxins, phage attack, reactive oxygen species.
General stress (“envelope stress”, pressure of exams, etc.)
What is osmolarity and turgor?
Osmolarity is a measure of the solute concentration per litre of water (or other solvent). (units = Osmols).
Turgor describes the pressure (units = Pascals) exerted against a cell wall from inside a cell.
Gram -ve pressure = 3-5 bar = 0.3-0.5 MPa.
Gram +ve pressure = 20 bar = 2 MPa.
Cells need to maintain a positive pressure to maintain cell shape.
Easy to lyse cells due to inside positive pressure.
Pack inside of cell with compounds that are always +300mmol.
How do bacteria maintain an internal positive pressure?
Pack cytoplasm with ‘compatible solutes’: amino acids (glutamate (E), proline (P)).
amino acid derivatives (ectoine, proline betaine).
small peptides.
sulphate esthers (choline-o-sulphate).
polyols (glycerol, glycosylglycerol).
sugars (trehalose, sucrose).
methylamines (glycine betaines, carnitine),
and their sulphonium analogues (e.g. DMSP).
Compatible- don’t affect biological processes.
Why is it important to sense osmolarity?
Hyperosmotic shock: entering a very salty or dry environment- dehydration, plasmolysis.
Hypoosmotic shock: entering a freshwater environment- bacteria control turgor by actively modulating the pool of osmotically active solutes in the cytoplasm.
How does water cross the cytoplasmic membrane?
Through aquaporins (water channels) in the inner membrane.
Cross by osmosis.
Passes very slowly through lipids.
Proteins that assist water movement.
Aquaporins- 5nm across, span membrane. Only allow water molecules through, across biological membranes.
How to bacteria protect against hypoosmotic shock?
Hypoosmotic shock – protection against extreme turgor-
mechanosensitive channels.
very rapid release of solutes: K+, glutamate, glycine betaine, trehalose-
> 1 M glycine betaine in 200 ms.
Rain, flooding, wash-out into fresh water.
Cell swells up.
Very quickly releases all its compatible solutes in response- responds at millisecond level.
Relaxes back to normal size.
Too fast for a component system.
How to mechanosensitive channels work?
Quicker way.
Rapid response to turgor pressure.
Sensed by mechanosensitive channels.
Sits in membrane- spans membrane.
Normal conditions- channel closed, nothing happens. Membrane thickness is 5nm.
When cell fills up with water, membrane stretches. Thickness of bilayer slightly reduced- to about 4.5nm. Pulls apart channel- it opens.
Pore big enough to let out solutes.
Relaxes and goes back to normal shape.
Describe MscS.
Small 3 helix protein compiled into channel that then opens to show a massive hole that even small proteins can travel through.
Well characterised.
One protein- 3 transmembrane hydrophobic alpha helices.
7 form ring. Pore in middle.
Closed- small pore. Open- bigger pore.
Emergency response.
What doe the EnvZ / OmpR two component system regulate?
Slower response- 2 component system.
Regulate relative abundance of 2 transmembrane proteins. OmpC: abundant at high osmolarity. OmpF: abundant at low osmolarity.
Synthesis of 2 channels regulated by 2 component system.
EnvZ is histidine kinase. OmpR is response regulator.
Describe the EnvZ / OmpR two component system in high osmolarity.
Senses something in high osmolarity.
Hydrolyses ATP.
Phosphorylated through EnvZ autokinase. Pi on His 243 passes to OmpR (Asp 55). OmpR phosphate switches on genes for ompC, and switches off genes for ompF.
Describe the EnvZ / OmpR two component system in low osmolarity.
Mechanosensitive channel activated.
Stretch in membrane may switch off kinase.
Everything goes backward- Pi on OmpR passed back to EnvZ. Dephosphorylates OmpR. Switches off genes for ompC. Switches on genes for ompF.
What does heat shock do?
Heat shock denatures proteins. Normal folded protein- water soluble polar globule. Unfolded polypeptide- exposed hydrophobic core.
Proteins are fragile.
Conc. of protein in cytoplasm- 300mg/ml. If protein denatured, cores can aggregate with other cores, proteins can’t refold. Bad.
For E. coli, a temperature shift from 37o to 42oC is enough.
What are the thermodynamics of heat shock?
Proteins are very delicate.
Globular proteins are only marginally stable.
Slight changes in pH/temperature can convert a protein from the native to the denatured state.
So, the free energy difference (DeltaG) between these states is small, about 5-15 kcal/mol.
What is the Cyrus Levinthal paradox?
Consider a protein of 100 amino acids.
Assume each amino acid can sample 10 different conformations.
Total number of possible conformations is 10^100 for the whole protein.
Each conformation to be tested in the shortest possible time, 10-13 sec (0.1 picosec), for a single bond vibration.
Total time required to sample all conformations is 10^77 years!!
In reality a protein of 100 amino acids at 37 ˚C folds within 5 sec.
So, protein folding is not a ‘trial-and-error’ process, even in vitro.
Something intrinsic in protein- knows folding structure.
Protein folding in the bacterial cell?
Even under ‘normal’ conditions, protein folding will start to occur as soon as polypeptide emerges from the ribosome (sometimes before!).
Cells contain many proteins (about 300 mg/ml in concentration), so aggregation can occur easily, especially when proteins are still to be folded (hydrophobic residues exposed). This is much worse under heat shock conditions.
Aggregation can be prevented by molecular chaperones.
Chaperones recognize and interact with partially folded or improperly folded proteins, and some classes even provide micro-environments in which folding can occur.
