5. Bacterial growth and the cell division cycle

Question 1

The typical bacterial growth cycle

Answer 1

1. Lag phase
2. Exponential phase
3. Stationary phase
4. Death phase

Question 2

Lag phase

Answer 2

Cells adjust to new conditions and synthesise required metabolic enzymes and metabolites

Question 3

Exponential phase

Answer 3

Optimal growth with regular doubling in cell numbers

Question 4

Stationary phase

Answer 4

Growth limited by nutrient depletion or accumulation of toxic metabolites. Rate of new cell production equals cell death

Question 5

Death phase

Answer 5

Gradual loss of viability with some cell turnover

Question 6

Measuring bacterial growth: Plating methods - Method

Answer 6

Culture plate onto solid nutrient medium after serial dilution.
Each colony assumed to represent progeny of single viable cell.
Colony forming units extrapolated giving cell numbers in original culture.

Question 7

Measuring bacterial growth: Plating methods - Positives

Answer 7

Highly sensitive.

Growth conditions customizable to growth of just one species.

Question 8

Measuring bacterial growth: Plating methods - Measurements

Answer 8

Only measures viable cells.
Underestimates cells in chains or clusters.
Number of colonies dependent on growth conditions
Inaccurate

Question 9

Measuring bacterial growth: Plating methods - Uses

Answer 9

Food
Medicine
Aquatic microbiology

Question 10

Measuring bacterial growth: Turbidity - Method

Answer 10

Measures light scattering by cells

Question 11

Measuring bacterial growth: Turbidity - Positives

Answer 11

Simple and convenient
Non destructive
Can be done continuously
Measures all cells (including dead cells)

Question 12

Measuring bacterial growth: Turbidity - Negatives

Answer 12

Low sensitivity

Culture turbidity has to be within a certain range for accuracy

Question 13

Measuring bacterial growth: Direct counting - Method

Answer 13

Simplest method

Microscopic count of known volume

Question 14

Measuring bacterial growth: Direct counting - Positives

Answer 14

Can accommodate clumping/chaining.

Doesn’t discriminate against live or dead cells - Via staining method

Question 15

Measuring bacterial growth: Direct counting - Negatives

Answer 15

Laborious but can be automated

Question 16

Measuring bacterial growth: Cytometry and FACs - Method

Answer 16

Flow cytometry: Measures particles in microfluidic flow

Question 17

Measuring bacterial growth: Cytometry and FACs - Positives

Answer 17

Highly automated
Can measure fluorescence at multiple wavelengths
Call sorting possible via FACs (FLuorescent activated cell sorting)

Question 18

Measuring bacterial growth: Cytometry and FACs - Negatives

Answer 18

Required right equipment, reagents and expetise

Question 19

Cell division cycle

Answer 19

1. New born cell
2. Cell elongates and increases in size
3. Cell structure duplicates
4. Chromosomes duplicate
5. Daughter chromosomes segregate to different ends of the cell
6. Septum forms at mid cell as z-ring contracts
7. New pole of cell synthesised as z-ring constricts
8. Cell division at mid cell

Question 20

Chromosome replication - Type of replication

Answer 20

Bidirectional replication.

Question 21

Chromosome replication - Proteins used

Answer 21

Regulated by 2 proteins - dnaA initiates replication and seqA blocks replication.

Question 22

Chromosome replication - Steps

Answer 22

1. OriC recruits replisome
2. 2 replisomes bind to give replication forks
3. Replication forks move in opposite direction (bidirectional replication)
4. Forks meet at TerC
5. Chromosomes separate

Question 23

Chromosome replication - Time taken for E.coli

Answer 23

40 minutes

Question 24

Chromosome replication choreography

Answer 24

Important that each cell gets copy of chromosome.
OriC located at centre of cell and left/right arms of chromosome positioned.
During chromosome replication, daughter OriC’s localised to quarter cell.
After division, cells have one cope of chromosome.

Question 25

Septum formation

Answer 25

Governed by divisome.
Assembles to form ring structure (z-ring) - Ftsz protein key role in formation.
Recruits 10 other essential proteins - Membrane and cell wall invagination, construct of new cell pole.

Question 26

Z-ring contraction

Answer 26

Causes septum formation, cell pole synthesis and cell division

Question 27

Cell division spatial cues

Answer 27

2 negative regulators govern division.

1. Min system: inhibits division at cell pole
2. Nucleoid occlusion system: Inhibits division in vicinity of nucleoid.