Lecture 33

Question 1

What are the 2 regulators of light production by Vibrio fischeri?

Answer 1

1. Catabolite Repression => in response to starvation

2. Autoinducer Control => in response to high cell density (quorum sensing)

Question 2

What is an operon?

Answer 2

A set of genes hat are expressed on a single mRNA

Question 3

What do the left and righ operon encode?

Answer 3

Left operon => encodes LuxR

Right operon=> encodes luxICDABEF

Question 4

What is the baseline expression of V.fischeri lux operon?

Answer 4

• Left operon normally not expressed
• Right operon normally expressed at a very low level (constitutive expression)
• Cells always make a little bit of AI
  
  • Or expression not high enough for light production

Question 5

Explain catabolite repression

Answer 5

• The control of gene expression by [cAMP-CRP] that occurs in response to the availability of certain catabolic nutrients
• Model system for the study of catabolite repression is the LAC OPERON of E.Coli
• Found in many if not all chemoorganoheterotrophic bacteria, but note that different organisms utilize different catabolites
  - Catabolites are NUTRIENTS metabolized to generate energy
  - Repression is a reduction in gene expression

Question 6

How is the expression of lux gene controlled by cAMP-CRP?

Answer 6

• Lux genes can only expressed if V.fischeri cells are starving
  - Starvation signal conveyed via [cAMP-CRP]
• Starvation is an inevitable consequence of VERY HIGH CELL DENSITY in the squid light organ
• The constant supply of nutrients from the squid host is sufficient to allow growth of a V. Fischeri population to high density and maintain a high population density for a while, but insufficient to prevent starvation.
• Even though V.fischeri cells are “starving”, they receive enough nutrients to survice and to fuel light production

cAMP is a regulatory compound derived from ATP
- Increases in starving cells

CRP (catabolite repression protein )
- Gene regulatory protein activated by binding to cAMP

cAMP-CRP
- Can bind to gene promoters and thus turn expression of genes on or off when concentration high enough

Question 7

If nutrients are abundant….

Answer 7

• Cells are NOT starving
• LOW [cAMP-CRP]
• Lux genes are not expressed : NO LIGHT is emitted

Question 8

If nutrients are scarce…

Answer 8

• Cells are STARVING
• HIGH [cAMP-CRP]
• [cAMP-CRP] binds to DNA at the luxR gene promoter (OL)
  
  • Results in luxR gene expression and syntehsis of the LUXR protein
    - LUX R will then be available to bind to autoinducer (AI)
• High cell density is determind by high [AI]
  - Quorum sensing

Question 9

Explain the autoinducer control of light emission

Answer 9

Quorum sensing
- [AI] only reaches high enough level to activate sufficient LuxR for light production when cell density is 10^9-10^10 cells /mL

• A treshold [AI-LuxR] is reached at this high cell density
  - AI-LuxR binds to the luxI promoter DNA (Or)
  - Causes the luxICDABEF operon to be expressed
  - Light emission is a by-product of Luciferase reaction

Question 10

Why is it called autoinducer control?

Answer 10

• LuxI gene expressiosn is controlled by its gene product (AI)

Question 11

How does amplification of Lux expression occur?

Answer 11

Amplification occurs as [AI-LuxR] increases

- More and more light until sufficient for counterillumination

Question 12

When does repression of lux expression occur?

Answer 12

Repression of Lux expression occurs at VERY HIGH [AI-LuxR]
- Results in less LuxR, less AI-LuxR, less induction of O=> results in decreasing concentration of AI

• Prevents over-expression of system
• Allows efficient maintenance of a delicate balance
• Light organ will still be able to produce light until Vibrio are exprelled in the morning
  - Steady, sufficient light, energy efficiency