Lecture 3

Question 1

What species carry out sporulation

Answer 1

Clostridium and Bacillus (e.g. Bacillus antrhaciis and Clostridium botulinum

Question 2

What was the structure of endospores

Answer 2

• 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

Question 3

What triggers sporulation

Answer 3

Starvation such as carbon and nitrogen

Question 4

Response to starvation

Answer 4

1. Aminoaceylated tRNA decreases because there aren’t enough amino acids to make proteins
2. There’s a 10-20 fold reduction in stable RNA
3. In response to amino acid starvation, ppGpp and pppGpp (signalling molecules) made in bacillus by RSH (ReIA SpoT homologue)
4. 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

Question 5

How does bacillus canabilse other cells

Answer 5

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

Question 6

When does cannibalism occur

Answer 6

Takes place during early sporulation

Question 7

Why is sporulation a last resort

Answer 7

It’s a long complicated process. It takes 7 hours in optimal conditions (37 degrees).

Question 8

What are the stages of sporulation

Answer 8

1. Assymetric cell division (helical repositioning of FtsZ to the pole. Ftsz sets where the division is happening
2. Engulfment - prespore is engulfed by mother cell. Forespore has two membranes of opposing polarity
3. Cortex biosynthesis- the forespore becomes dehydrated
4. Coat synthesis
   5/6. The mother cell lyses and the forespore becomes a mature spore

Question 9

What causes the initiation of sporulation

Answer 9

1. Cell density - only sporulate at high cell density
2. SpoA - transcriptional activator - controls 10% of all genes. Activated Spo0A triggers assymetric sporulation division and transcription of sigma factors E, A, K and G

Question 10

How is Spo0A controlled

Answer 10

It’s controlled at the level of transcription and phosphorylation. Sporulation is activated by increased levels of Spo0A phosphorylation

Question 11

SpoOA two component system

Answer 11

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

Question 12

How is KinA regulated

Answer 12

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)

Question 13

What’s KinAs role in the onset of spore fomation

Answer 13

It has 3 PAS domains - most of the amino terminal domains are important for spore formation and have been shown to bind to ATP.

Question 14

What do RAP proteins do

Answer 14

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

Question 15

How does Bacillus sense nutrient deprivation

Answer 15

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.

Question 16

What is AbRb

Answer 16

An unstable repressor of multiple stationary phase genes and it represses Spo0H

Question 17

What does SigH control

Answer 17

Some expression of Spo0A, Spo0F and KinA

Question 18

What are the sigma factors and what do they control

Answer 18

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

Question 19

What is sF controlled by

Answer 19

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.

Question 20

What is SpoIIE involved in

Answer 20

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

Question 21

What is sigma E made as and what’s it activated by

Answer 21

An inactive precursor and is membrane bound and is activated by proteolytic cleavage.

Question 22

When is sigma E activated

Answer 22

Once assymetric division has occured

Question 23

What does activation of Sg need

Answer 23

1. Engulfment (morphological cue)
2. Loss of interaction with SpoIIAB (anti-sigma factor)
3. SpoIIIA operon (transportor)
4. SpoIIIJ (membrane biogenesis)

Question 23

How do you get transcription of sigma k

Answer 23

Need to have expression of site specific recombinases (takes prophage out of the sigma gene)

Question 24

What is SpoIVFB and what is it regulated by

Answer 24

It’s a protease and is regulated by SpoIVFB and BofA

Question 25

What does Sigma G control

Answer 25

Controls the production of SpoIVB (which is a protease secreted into the intermembrane space).

Question 26

What does SpoIVB cleave

Answer 26

SpoIVFA and BofA (which controls the activity of SpoForeB). Once cleaved, SpoIVFB can activate sigma K

Question 27

How to revert endospores back to active

Answer 27

Need alert sensory mechanisms (ability to rehydrate).

1. 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
2. Release of ions with core hydrations, break down large macromolecular structures. Metabolism starts degrading sporulation proteins

Question 28

Examples of germinants

Answer 28

• 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).

Question 29

How do receptors sense germinants in the environment

Answer 29

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).

Question 30

What is GerA

Answer 30

Its a nutrient gated ion channel. Nutrients can bind to GerA, bringing about a confirmational change which causes release of ions

Question 31

What does PrkC do

Answer 31

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.

Question 32

What does activated EF-G do

Answer 32

1. modulates ribosome activirty (initiates translation)- enhances kick start of macromolecular synthesis
2. Blocks activity of protein Y (pY). pY prevents disassembly of ribosome