Lecture 3 Flashcards
What species carry out sporulation
Clostridium and Bacillus (e.g. Bacillus antrhaciis and Clostridium botulinum
What was the structure of endospores
- Highly cross linked proteaceous coat (resistant to solvents and enzymes so can survive chemical attack)
- Electron transparent cortex which maintains dormancy and the core in a dehydrated state
-The core contains DNA proteins and others (as it’s dehydrated due to diploclinic acid which chelates metal ions that reduce osmotic potential- makes spores heat resistant. Small acid soluble proteins binds to chromosomes to protect it from UV and oxidative stress
What triggers sporulation
Starvation such as carbon and nitrogen
Response to starvation
- Aminoaceylated tRNA decreases because there aren’t enough amino acids to make proteins
- There’s a 10-20 fold reduction in stable RNA
- In response to amino acid starvation, ppGpp and pppGpp (signalling molecules) made in bacillus by RSH (ReIA SpoT homologue)
- Drop in the GTP pool.
-Depression of biosynthetic pathways (Trp)
-Induction of transport systems for alternative nutrient sources
-motility towards nutrients (can be controlled by sigma factor D)
-macromolecule degrading systems
-competence (take DNA from environment), allows it to recombine with chromosome
-Production of antibiotics (allows them to compete and kill off other organisms)
-Cannibalism
How does bacillus canabilse other cells
They secrete extracellular killing factors such as SkfA (bacteriocin-like) and Sdpc (C-terminal 63 amino acids).
The targeted cells do not express immunity proteins such as SkfEF, sdpl
When does cannibalism occur
Takes place during early sporulation
Why is sporulation a last resort
It’s a long complicated process. It takes 7 hours in optimal conditions (37 degrees).
What are the stages of sporulation
- Assymetric cell division (helical repositioning of FtsZ to the pole. Ftsz sets where the division is happening
- Engulfment - prespore is engulfed by mother cell. Forespore has two membranes of opposing polarity
- Cortex biosynthesis- the forespore becomes dehydrated
- Coat synthesis
5/6. The mother cell lyses and the forespore becomes a mature spore
What causes the initiation of sporulation
- Cell density - only sporulate at high cell density
- SpoA - transcriptional activator - controls 10% of all genes. Activated Spo0A triggers assymetric sporulation division and transcription of sigma factors E, A, K and G
How is Spo0A controlled
It’s controlled at the level of transcription and phosphorylation. Sporulation is activated by increased levels of Spo0A phosphorylation
SpoOA two component system
Spo0B- receiver
So0F- receiver
KinA-E (transmitter - histidine protein kinase)
The signalling cascade allows further levels of control. Mutated Spo0 genes don’t sporulate at all
How is KinA regulated
By inhibition of SDA (binds directly to form a hetero tetramer) and activation of PAS domains. Expression of SDA is impaired by DNA replication (prevents sporulation)
What’s KinAs role in the onset of spore fomation
It has 3 PAS domains - most of the amino terminal domains are important for spore formation and have been shown to bind to ATP.
What do RAP proteins do
They cause dephosphorylation of SpoF0-P.
Rap-C inactivates target protein compA-B by binding to the protein.
Rap A, B and E can cause dephosphorylation of Spo0F by binding and stimulating autophosphoratase activity
How does Bacillus sense nutrient deprivation
CodY (natural dappener on the environment). phrA, C, E and kinB are repressed by CodY. CodY sense guanine nucleotide levels. There’s a drop in the GTP pool. CodY is inactivated.
What is AbRb
An unstable repressor of multiple stationary phase genes and it represses Spo0H
What does SigH control
Some expression of Spo0A, Spo0F and KinA
What are the sigma factors and what do they control
sH- assymetric septation
sF- early forespore associated gene expression
sE- early mother cell gene expression
sG- active in the late forespore
sK- late mother cell
What is sF controlled by
SpoIIAA (an anti-anti sigma factor) and SpoIIAB (an anti sigma factor - kinase)
sF is inactivated by binding to SpoIIAB. SpoIIAA causes it to dissociate from AB. Once dissociated sigma factor F can become active.
What is SpoIIE involved in
Its a DNA translocase involved in getting chromosome through the developing asymmetric septum to the forespore.
It stalls with 30% of 1 chromosome in forespore.
SpoIIE dephosphorylates SpoIIAA which inactivates AB. AB is degraded allowing SF to become active
What is sigma E made as and what’s it activated by
An inactive precursor and is membrane bound and is activated by proteolytic cleavage.
When is sigma E activated
Once assymetric division has occured
What does activation of Sg need
- Engulfment (morphological cue)
- Loss of interaction with SpoIIAB (anti-sigma factor)
- SpoIIIA operon (transportor)
- SpoIIIJ (membrane biogenesis)
How do you get transcription of sigma k
Need to have expression of site specific recombinases (takes prophage out of the sigma gene)
What is SpoIVFB and what is it regulated by
It’s a protease and is regulated by SpoIVFB and BofA
What does Sigma G control
Controls the production of SpoIVB (which is a protease secreted into the intermembrane space).
What does SpoIVB cleave
SpoIVFA and BofA (which controls the activity of SpoForeB). Once cleaved, SpoIVFB can activate sigma K
How to revert endospores back to active
Need alert sensory mechanisms (ability to rehydrate).
- Loss of heat resistance (immobilisation of ions that are inside the cytoplasm). Uptake of water > onset of metabolism (cell can swell to become dehydrated > outgrowth
- Release of ions with core hydrations, break down large macromolecular structures. Metabolism starts degrading sporulation proteins
Examples of germinants
- Nutrients (amino acids, L-ala). Specific receptors for specific germinants. L-ala is a marker for nutrients in the environment
- Peptidoglycan - ubiquitous in bacterial environments (when they grow and divide, they shed peptidoglycan so is a marker for growing cells and growth permissive conditions).
How do receptors sense germinants in the environment
GerA and GerB are membrane proteins that respond to germinants (brings about a confirmational change). This leads to a cascade of events, leading to cortex hydrolysis.
Cortex hydrolysis removes the cortex allowing expansion (activation of cortex lytic enzymes).
What is GerA
Its a nutrient gated ion channel. Nutrients can bind to GerA, bringing about a confirmational change which causes release of ions
What does PrkC do
It’s associated with the membrane and responds to PG muropeptides. It phosphorylates itself and transduces a signal to bring about an outcome to optimise germination.
What does activated EF-G do
- modulates ribosome activirty (initiates translation)- enhances kick start of macromolecular synthesis
- Blocks activity of protein Y (pY). pY prevents disassembly of ribosome
