VL6: Two component systems

Question 1

What are the take home messages?

Answer 1

1. TCS are very widely used regulators
2. They are used to regulate behaviour and/or gene expression
3. They detect the environment and transmit a signal onwards

Question 2

What are TCS?

Answer 2

1. Sensor (Kinase), senses the environment
   phosphorylation of second component when sensor is stimulated
2. Response Regulator mediates a response
   phophrylated form of second componend is the active state of the regulator

Question 3

What components/domains are TCS made up of?

Answer 3

Histidine Kinase:

• Sensing domain (N-terminal)
• Dimerization domain (carries phosphate, that gets passed on)
• ATP binding kinase domain (Transmitter=autokinase)

Response regulator:

• Regulatory domain (gets phosporylated)
• Effector domain

Question 4

What 2 groups of kinases exist?

Answer 4

1. Membrane associated proteins sense envrionmental signal
2. Cytoplasmic proteins are usually part of a pathway that is more complex than a simploe TC mechanism and they respond to intracellular signals

Question 5

What is chemotaxis and how does it work?

Answer 5

Straight run followed by tumble (to randomize direction), random walk up or down a concentration gradient
attractant or repellents cause a biased random walk

they swim and taste environment and decide if it getting better or worse

Question 6

How does chemotaxis work on a molecular level?

Answer 6

Attractants are caught by transducer (MCP= methyl accepting chemotacis proteins)
if bound, CheA (=sensor kinase) autophosphorylates
CheY (=response regulator) controls flagellar rotation
CheZ-dephosphorylates CheY-P
CheB also response regulator, regulates methylation level of MCP

Question 7

What are flagella?

Answer 7

Flagella are long whiplike filaments composed of protein that originate in the cell membrane
Flagella rotate and impart swimming movement on the cells

Question 8

Name/Explain 4 types of flagella

Answer 8

1. Monotrichous: a single flagellum usually at one pole (vibrio cholerae)
2. Amphitrichous: a single flagellum at both ends of the organism
3. Lophotrichous: two or more flagella at one or both poles
4. Peritrichous: flagella over entire surface

Question 9

What is the opportunity cost of flagella?

Answer 9

flagella synthesis is expensive and requires up to 30 genes and about 10 more genes encoding proteins for hook and basal body

Question 10

How do flagella move?

Answer 10

rotation of the filament is driven by diffusion of protons into the cell through the basal apparatus after the protons have been actively transported by proton pumps in the plasma membrane

filament is rotated by a protein motor in the cell membrane
motor is powered by proton motive force (pmf)

Question 11

What parts do flagella consist of?

Answer 11

• basal body (series of rings that drive the flagella)
• hook (short curved segment that links flagellin to the basal body
• Flagellin (hollow rigid cylinder constructed of a single protein (Filament)

Question 12

How do chemoreceptors influence flagella movement?

Answer 12

The methyl-accepting chemotaxis protein (MCPs) form clusters associated with the CheA and CheW proteins

lots of different receptors for different signals (serine, aspartate, dipeptides sugars ect)

Question 13

How is chemotaxis influenced by signal transduction?

Answer 13

-small molecules diffuse through pores of OM, bind to protein receptors in cell membrane, binding of atttractant reduces the rate of phosphorylation and thus prolongsthe runs (less tumbling)

methylatioon resets the cells sensitivity to the attractant, sot it requires a higher concentraion of attractant next time to reduce phosphorylation and prolong the run thus the bacteria can sense a concentration gradient by measuring a difference between what it was before and what it is now

–> attractant binding reduces tumbling frequency and drives swimming

Question 14

Signal transduction step by step

Answer 14

1. if no attractant is bound to MCP
2. CheW stimulates autophosphorylation (cheA -> CheA-P)
3. CheA-P transfers P to CheY (making CheY-P)
4. CheY-P interacts with flagella motor protein ->tumbling
5. CheZ de-phosphorylates CheY-P (making Che-Y)
6. CheZ activity ensures bacteria can go from trumble ->Run
7. CheA-P also transfers P to CheB (making CheB-P)
   8: CheB-P de-methylates MCP receptor protein
8. Unmethylated MCP is more sensitive (binds ligand)
9. MCP with bound ligand slows the CheA autophosphorylation
10. Low rate of CheY phosphorylation
11. Low rate of interaction with flagella motor
12. Flagella mortor moves CCW (bacteria runs)
13. If the concentration of ligand is high the run is long
14. CheR continuously methylates the MCP
15. Methylated MCP is less sensitive to ligand
16. Methylated MCP stimulates CheA->CheA-P autophosphorylation
17. Methylated MCP stimulates tumbling
18. CheR and CHeB activity ensure system can adeapt to new concentrations
19. CheZ ensures the system does not get stuch in on mode

Question 15

what is RpoS?

Answer 15

=Sigma S (sigmayeichen)S

• sigma factor induced in response to stress like lack of nutrients
• re-directs RNA polymerase transcription activity
• regulated via TCS

Question 16

How can an increaded level of RpoS be achieved?

Answer 16

• Stimulating rpoS transcription (reduced growth rate)
• Stimulating rpoS mRNA translation (high cell density, low temperature, high osmolarity, low pH)
• Inhibiting RpoS proteolysis (high osmolarity, low pH, carbon starvation, high temperature)

combination is most efficient

Question 17

What have TCS to do with RpoS?

Answer 17

TCS regulate RpoS at all levels

example ArcB=histidine sensor kinase/ArcA=rpoS repressor

Question 18

What is the PhoP/PhoQ system?

Answer 18

master regulatior of salmonella virulence

inactivation of PhoP/PhoQ attenuatates virulence, no survival in macrophages, susceptible to killing by antimicrobial peptides

PhoQ (sensor kinase) senses periplasmic concentration of Mg2+ and Ca2+ ions,
Ionic concentration is low if it is in phagosome -> regulates expression of lots of genes to survive in phagocytic vacuole interacts with other TCS

Question 19

How do TCS regulate porins?

Answer 19

Influence which porin (OmpF > OmpC) with different channel sizes based on osmolarity (when low osmolaritz -> larger channel radius OmpF -> faster diffusion rate)