Lecture 7 - Cell Division and Divisome

Question 1

(i) What occurs During Division Septum Formation?
(ii) Is the Mechanism Conserved across Bacterial Species?

Answer 1

(i) Division Septum Formation involves the coordinated remodelling of different layers that make up the bacterial envelope

(ii) Overall Mechanism - highly conserved in B. Subtilis and E. coli, with some differences due to cell wall anatomy (Gram +ve vs Gram -ve)

Question 2

During B. Subtilis Division, how are the two daughter cells separated?

(2 Points)

Answer 2

1. Synthesis of complete cross wall of peptidoglycan, thats sandwiched by lipid bilayers of two daughter cells
2. Invagination develops at division site due to degradation of inner part of septal peptidoglycan wall

Question 3

Compare cell division in E. coli and B. Subtilis

(3 Points)

Answer 3

(i) E. Coli - synthesis of division septum is concomitant with constriction (i.e. always produces separated cells)

(ii) B. Subtilis - due to the thick cell wall taking longer to degrade, there is a delay between cell division and separation
* In Fast Growing Cultures, this results in cells being associated in long chains

Question 4

Define Filamentous Temperature Sensitive Mutant Z (FtsZ)

Answer 4

FtsZ is a tubulin homologue which polymerises into dynamic filaments via a GTP-dependent process to form a cytokinetic ring structure (Z-ring) essential for cell division

Question 5

Why is FtsZ known as the Bacterial Master Division Protein?

(3 Points)

Answer 5

1. Its inactivation produces block in division earlier than any other division protein
2. Widely Conserved, with all known division proteins depending on FtsZ for correct localisation to division site
3. Cellular FtsZ Level is primary determinant of division frequency

Question 6

Describe the process of FtsZ filament assembly

(4 Points)

Answer 6

1. GTP binds to nucleotide-binding pocket of FtsZ N-terminal domain, inducing conformational change that allows polymerisation
2. C-domain of one monomer interacts with N-terminal domain of another monomer, leading to polymerisation
3. Filament assembly leads to activation of GTPase activity of FtsZ monomers, resulting in hydrolysis of GTP
4. GDP-bound FtsZ generates unstable filaments and subsequent disassembly, hence FtsZ polymers are highly dynamic (assemble/disassemble regularly during cell cycle)

Question 7

How does the cell control location of Septum formation?

Answer 7

1. MinC and MinD - proteins localised to poles, which inhibit formation of division septum
2. Noc - prevent Z-ring formation in vicinity of nucleoids

Question 8

Define the three components of the Min System, explaining their Function

(2 Points)

Answer 8

1. MinC and MinD - interact to form membrane-associated complex that is capable of inhibiting Z-ring formation in site-specific manner
2. DivIVA - essential for site-selectivity of Min Inhibitor, with localisation of MinCD being random in its absence

Question 9

What are the two suggested molecular mechanisms for pole localisation of DivIVA?

(4 Points)

Answer 9

1. Interaction with Mature divisome:
   * DivIVA-MinCD complex has no inhibitory effect on mature divisome, only on early stages
   * DivIVA-MinCD would be retained at newly formed poles
2. May be attracted to the poles via a physical property e.g., Curvature

Question 10

How does the MinCD complex differ in E.coli?

Answer 10

Unlike the Static Localisation of MinCD in B. subtilis, in E. coli the complex displays dynamic oscillation, rapidly moving from pole-to-pole to prevent formation of polar septum

Question 11

What is the Nucleoid Occlusion (NO) effect?

Answer 11

Nucleoids can exert “VETO effect” on septum formation in their vicinity, which occurs at level of Z-ring formation

Question 12

Give Two pieces of Evidence that support the theory of Nucleoid Occlusion

Answer 12

1. Once Nucleoid undergoes partial replication and segregation by assuming a bilobed shape, NO is relieved at midcell
2. Mutations that reduce organisation/condensation of nucleoids greatly reduce NO efficacy

Question 13

Define Sporulation in terms of:
(i) What are spores?
(ii) Why do they form?

Answer 13

(i) Spores are highly specialised cells, capable of resisting many environmental stresses and remaining dormant for many years

(ii) During Nutrient Deprivation/Environmental Stress, with spores germinating into vegetative cells when conditions improve

Question 14

How are Spores produced from vegetative cells?

(4 Points)

Answer 14

1. Asymmetric division of a progenitor cell into two unequal cellular compartments:
   * Smaller Cell (Forespore) - becomes resistant spore
   * Larger cell - known as mother cell

2, Establishment of Morphological asymmetry by polar septation is critical to triggering distinct programs of gene expression in each compartment (promotes spore differentiation

Question 15

Describe the Mechanism of Z-ring repositioning during sporulation

Answer 15

• Repositioning of Z-ring from midcell to cell poles involves a transient FtsZ spiral intermediate, that will eventually produce a cell with two polar Z-rings

Question 16

(i) How is Asymmetric Division during Sporulation Regulated?

(ii) How do these proteins regulate Asymmetric Division

Answer 16

(i) Regulated by two proteins:
1. Response Regulator Spo0A
2. Alternative Sigma Factor H

(ii) These proteins regulate transcription of approx. 100 genes, only two are required for Z-ring switch:
1. FtsZ - displays 2 fold increase during sporulation
2. SpoIIE - encodes multi-functional protein expressed exclusively during sporulation

Question 17

What are the two suggested mechanisms by which the MinCD inhibitory effect is overriden in sporulating cells?

Answer 17

Possibility 1 - SpoIIE Interaction with FtsZ makes polymer formation refractory to inhibition

Possibility 2 - MinCD may be outcompeted for binding to DivIVA by RacA (involved in Axial Filament formation), hence not present at poles

Question 18

What is the Role of NO in determing
Z-ring polarity?

Answer 18

• Formation of Axial Filament connecting the two poles prevents Z-ring formation at the midcell
• Special property of edges of nucleoid (e.g., Local DNA concentration) reduces effectiveness of NO at nucleoid ends only