Lecture 14 - Sporulation

Question 1

What is a spore?

Answer 1

Spores could be thought of as dead as they are not metabolically active

A spore is an offspring from an organism’s life cycle

78 genera of bacteria produce spores, particularly: Clostridia and Bacillus

Question 2

Properties of endospores

Answer 2

Protection from the environment:

• Heat
• UV
• Dessication
• Nutrient starvation

Resistant to antibacterial processes

• Boiling/cooking
• Disinfectant
• AntiB’s
• Alcohol
• Acid/alkali

Autoclaving and formaldehyde fumigation only ways to destroy

Question 3

Structure of an endospore

Answer 3

DNA is packaged tightly within an impermeable, multi-layered structure

6 separate layers to ensure the DNA is protected

DNA is Protected by small acid soluble spore proteins SASP’s (they condense DNA and make it inactive - possible antimicrobial therapy?)

Layers:
1. Exosporium
 Function unknown but may play a role in pathogenesis
 Composition:
 - predominantly protein (43–52% of dry weight)
 - lipids (15–18% of dry weight)
 - carbohydrates (20–22% of dry weight)
 - calcium and magnesium(around 4%)

Not always present/ hard to retain during purification
The protein BclA of B. anthracis is a major component of the exosporium and interacts with a specific macrophage receptor, CD14

BclA mutants of B. anthracis show a reduced adherence to certain surfaces

2. Spore cortex
   Composed of peptidoglycan chains of alternating N-acetlyglucosamine (NAG) and N-acetylmuramic acid (NAM) and muramyl lactam (this is ‘cortex peptidoglycan’ - normal peptidoglycan does not have this)
   Structure breakdown is an important step in germination and outgrowth
   Selectively degraded during germination

Germ cell wall
Becomes the cell wall of the germinating cell, remains intact during germination

3. Inner membrane
   Very low permeability, even to water
   Membrane structure is highly compressed i.e. low fluidity = low permeability
   Most important membrane for conferring resistance to DNA damaging molecules
   Contains germinant receptors (GR’s)
4. Core
   Contains genomic DNA, RNA, ribosomes and enzymes
   Has low water content (25-50% wet weight)
   Has high Ca-DPA, (calcium chelated dipicolinic acid)
   Contains DNA repair proteins that repair damaged DNA upon germination
   DNA in the core is saturated with α/β small acid soluble proteins, exclusive to spores

Question 4

Spore formation

Answer 4

1. DNA is replicated
2. DNA aligns along cell’s long axis
3. Cytoplasmic membrane invagintes to form forespore
4. Cytopplasmic membrane grows and engulfs forespore within a second membrane. Vegetative cell’s DNA disintegrates
5. A coretex of calcium and diplicolinic acid is deposited between the membranes
6. Spore coat forms around endospore
7. Maturation of endospore; completion of spore coat and increase in heat and chemical resistance by unknown processes
8. Endospore released from original cell.

Question 5

Genetic regulation of sporulation.

Answer 5

A regulon is a bunch of genes regulated by one sigma factor

Sigma factors couple with RNA pol to transcribe specific genes

In a normal vegetative cell RNA polymerase is associated with the sigma factors sA and sH

The sporulation cascade is initiated when environmental conditions trigger a “phosphorylation relay,” in which a phosphate group is passed along a series of Spo proteins by histidine kinases until it reaches Spo0A

Spo0A is the master regulator of sporulation. Once phosphorylated Spo0A is a transcriptional activator positively regulating genes that are required for sporulation and negatively regulating those that are not.

One of these positively regulated genes is SpoIIE which activates SpoIIAA by removal of a phosphate group. Removal of a phosphate from SpoIIAA causes a sigma factor F to be released from SpoIIAB

The whole process of sporulation takes about 8 hours in Bacillus

What does sF do?
In the forespore, sF binds to RNA polymerase and directs transcription of the sF regulon. One such gene encodes a protease that is mobilised from the developing endospore to the mother cell to activate pro- sE.
Another gene that sF bound RNA polymerase, along with signals from the mother cell, initiates the transcription of is sigG (sG) which remains in the forespore, replacing sF.

Sporulation is temporally and spatially ordered!

Question 6

How spores germinate

Answer 6

In the cell inner membrane there are Germination Receptors

The peptidoglycan of the germ cell wall forms the new cell wall of the new vegetative cell

Stage 1

1. cation release
2. ca2+ dap release
3. partial core hydration
4. partial loss of resistance

Stage 2

1. cortex hydrolysis
2. further core hydration
3. core expansion
4. more loss of resistance
5. loss of dormancy

Stage 3

1. metabolism
2. SASP degradation
3. macromolecular synthesis
4. escape from spore coats

But what causes spore activation?

Spores can detect specific germinants:
Can confer niche specificity
- C. difficile, intestinal pathogen, bile acid sodium taurocholate
- C. perfringens Type A, food poisoning pathogen, KCl, NaPi, common food preservatives

Common germinants include:
L-amino acids
D-sugars
Purine nucleosides

What is the molecular basis for this?

nGR’s – nutrient germination receptors are buried through the exosporium to ensure environment is favourable
nGR proteins are expressed within the forespore under regulation by the sG transcription factor
Located in the spore inner membrane
Redundancy in nGR’s
Receptors are made from three genes: Tripartite operons gerAA, gerAB, gerAC. Have intracellular signalling domains

Germinant–GR interaction results in transduction of a signal that leads to the release of DPA and associated cations, likely via a channel composed at least in part of SpoVA proteins
DPA release then triggers degradation of the spore’s peptidoglycan cortex by cortex-lytic enzyme, eventually leading to resumption of and vegetative growth
Other proteins such as the GerD lipoprotein also required.

Question 7

Are nutrients the only signal for germination?

Answer 7

Proposed that a peptidoglycan binding kinase may be involved in germination
A PASTA containing eukaryotic like kinase is present in Bacillus - PrkC

PASTA (penicillin and serine/threonine kinase associated) repeat - membrane protein with ser/thr domains (signalling proteins, kinases, cause a cascade). Had an extracellular domain that resembled a penicillin receptor

Spores from Bacillus that have a mutation in PrkC fail to germinate in the presence of cell free supernatants from late log phase cultures

What is the molecular recognition that leads to germination via PrkC?
All dependent on the third amino acid in the stem of peptidoglycan - you have to have peptidoglycan and it has to be m-Dpm (only G+) and NOT L-Lys (G-)
This is so the spore can sense if there are other bacteria present and what they are - it is HIGHLY SPECIFIC and works with the tripartite Ger operon to regulate germination