Kelly 18 - Differentiation

Question 1

Give an example of a bacteria that does not undergo standard binary fission - what type of division is present here?

Answer 1

Caulobacter crescentus

polar growth and budding from one pole (asymmetrical division)

Question 2

Define morphogenesis

Answer 2

morphological changes that occur during the cell cycle

Question 3

Define the cell cycle

Answer 3

sum of biochemical, genetic and morphological changes that occur during the cell cycle

Question 4

Define differentiation

Answer 4

production of functionally distinct organisms

can be REVERSIBLE eg endospore formation in Bacillus or IRREVERSIBLE eg heterocyst formation in cyanobacteria

Question 5

Define polymorphic

Answer 5

the production of 3 or more morphologically distinct cell - types - can occur by more than one cell cycle & can be influenced by the environment

Question 6

Give the 2 methods (& describe them further) to measure and observe differentiation

Answer 6

• microscopy from batch cultures - light microscope (fluorescence) or electron microscopy. however not good for biochemical analysis
• use a synchronised culture in which a population behaves like 1 cell type. all progress through same stages of cell cycle together

Question 7

Give the 2 methods, & give examples of each, of how to obtain a synchronous population

Answer 7

INDUCTION -> starvation of culture and reinoculate w/ enough substrates for growth etc
-> stop DNA synthesis by using substrate analogues & then remove

SELECTION -> use a centrifuge & a gradient of an inert substance (eg sucrose) to identify small (newborn) cells
-> baby factory method

Question 8

Give one problem w/ using the induction method to obtain a synchronous population

Answer 8

metabolic processes will be affected - may affect analysis

Question 9

Describe the baby factory method

Answer 9

use a unit with a filter w/ small pores in it
inoculate the culture on the medium and pass warm media through it to stimulate growth
any small newborn cells will pass through pores and into the synchronous culture below

Question 10

Describe the 2 types of cells that result from the asymmetric division of Caulobacter crescentus

Answer 10

• swarmer cells

- stalk cells

Question 11

Describe the features of swarmer & stalk cells

Answer 11

swarmer - flagella, no RNA/DNA/protein synthesis therefore no growth UNTIL nutrients are reached, chemotactic and motile
stalk - non motile, asymmetrical division to produce stalk & swarmer cells, has a stalk w/ holdfast on end of it

Question 12

Stalk and swarmer cells have _____ cell times

Answer 12

unequal

Question 13

Give the cell cycle sequence (eg S,G1,G2 phases) and describe what happens in each phase

Answer 13

G1 - S - G2
G1 = swarmer cell w/ flagella
S - swarmer has found nutrients & begins to grow -> stalk cell. DNA synthesis and cell growth. flagella starts to grow @ pole
G2 = before division and after DNA replication

mini cycle of stalk cells also exists when stalk cell divides to give another stalk cell & a swarmer cell

Question 14

What is pulse labelling used for in terms of flagella production?

Answer 14

biochemical analysis of the cell cycle

shows when genes for the biosynthesis of the flagellum are expressed

Question 15

Describe the process of pulse - labelling

Answer 15

add 35S - Met (radio labelled) to medium
incubate for 10 mins
make cell extract
add antibodies to detect for proteins FlaA/B and Hook protein
centrifuge and collect precipitate
separate the proteins out on poly acrylamide gel and make autoradiograph

Question 16

What is the purpose of Rifampicin

Answer 16

inhibitor of RNAP

Question 17

Describe the protein composition in stalk, swarm and pre - div state cells

Answer 17

stalk - no proteins
swarmer - FlaA
pre - div - FlaA , FlaB and Hook proteins

Question 18

What is the significance of FlaA proteins being present only is swarmer cells when there is Rifampicin present?

Answer 18

FlaA mRNA is preserved from the pre-div cells
as FlaA is at top of flagella and is used for swimming it will be damaged etc therefore new FlaA will allow continual synthesis of new FlaA protein

Question 19

Describe 2 genetic tools that can be used for genetic manipulation of Caulobacter

Answer 19

• Tn5 mutagenesis

- whole genome sequencing; showing transcriptomics and proteomics, can also identify genes for differentiation

Question 20

Give the advantages of using Tn5 insertions

Answer 20

• allows null mutants to be made and therefore mutations in non - essential genes can be identified (eg in pill, stalk, flagella), chemotaxis
• polar effects in downstream genes can help define operons
• insertions are normally stable and not reversible
• knR allows genes to mapped and cloned

Question 21

Differential _____ ______ is coupled to the different stages of the swarmer cell cycle

Answer 21

gene expression

Question 22

What is the name of the master regulator that controls different phases of cell differentiation

Answer 22

CtrA - controls different levels of gene expression during different phases of cell cycle

Question 23

Give the 3 proteins involved in the Two Component System and state how they were defined

Answer 23

CtrA
CckA
ChpT
- ts mutants identified that could grow @ 27C but not at 38C and had defects in regulating flagellum biosynthesis

Question 24

How is CtrA levels controlled in the cell?

Answer 24

phosphorylation
proteolysis
transcription

Question 25

When phosphorylated what are the 2 effects of CtrA response regulator?

Answer 25

when P , CtrA binds to ori of chromosome and inhibits initiation of replication in SWARMER cells
also causes differentiation genes to be switched on

Question 26

Draw a diagram explaining how CtrA is P/unP at the poles of swarmer and stalk cells

Answer 26

kelly 3 notes

Question 27

The activity of CtrA depends on the _____ of the Div ___ and ___ proteins and their ability to transfer CckA from a kinase to a ____

Answer 27

localisation
J and K
phosphatase