Lecture 31

Question 1

What are the typical relationships observed in luminescent bacteria?

Answer 1

They tend to have symbiotic relationships with their host - they often produce light for their host in return for a benefit.

Question 2

Give an example of a symbiotic relationship involving luminescent bacteria.

Answer 2

Vibrio fischeri-squid

Question 3

Explain how, in the vibrio fischeri - squid symobiosis, the light benefits the host animal.

Answer 3

The light provides the squid with counter-illumination. This protects the squid from predators below, as it will allow them not to cast a shadow. Squid without bioluminescent bacteria will cast a shadow and will thus be easy prey.

Question 4

What is the typical habitat of the squid with which vibrio fischeri associate?

Answer 4

Shallow waters, and they come out at night.

Question 5

Is the intensity of the light produced in the vibrio fischeri - squid interaction consistent? Explain why or why not.

Answer 5

No - the amount of light produced is proportional to the amount of downwelling light. This way, they are not too bright compared to their surroundings.

Question 6

Explain the process by which the squid becomes associated with the vibrio fischeri.

Answer 6

When squid eggs hatch, they become infected by only the vibrio fischeri and no other species, forming a pure culture. They enter through a pore that is full of mucus. This mucus will kill other species of bacteria trying to get in.

Question 7

Where is the light organ inhabited by v. fischeri in the squid?

Answer 7

It is in the ink sac

Question 8

Once the v. fischeri are associated with the squid, describe the conditions under which they live in the light organ.

Answer 8

They live in ‘crypts’ as a pure culture in close association with host cells and blood supply. The microvilli of the crypt allow for the transmission of amino acids and organic acids to the bacteria (allowing for high surface area).

There is also the formation of a bacterial biofilm in these crypts - this is critical in making them a permanent part of that light organ.

Question 9

The squid host provides v. fischeri with […]

Answer 9

Organic and amino acids

Question 10

Describe the turnover of v. fischeri in squid.

Answer 10

There is a fresh culture grown every day. Every morning, 90% of symbionts are expelled from the squid. These expelled bacteria become VBNC but remain able to infect baby squids later.

The culture in the light organ will then grow back to full density within 12 hours, by nightfall.

Question 11

Under what circumstances is light produced by v. fischeri?

Answer 11

Light is only produced when the population collective sense a high cell density (quorum) of 10^9 to 10^10 cells/ml. These high densities only really occur in the squid. This also will only happen when the cells are starving.

Question 12

Explain what happens when the quorum is met in v. fischeri.

Answer 12

The lux genes, which encode the luciferase enzyme, are only expressed when the cells are starving AND at a high cell density (which tend to occur together because large populations deplete nutrients more rapidly).

Question 13

What are milky seas? What is their cause?

Answer 13

It is a phenomenon that has been referenced historically - luminescent waters. They are likely due to vibrio spp. growing on decomposing algal blooms.

Question 14

What is the light yielding reaction for v. fischeri?

Answer 14

R-CHO + O2 + FMNH2 + ATP — (luciferase) –> R-COOH + hv/light + H2O + FMN + AMP + PPi

The aldehyde gets oxidized to a fatty acid. This organic compound gets recycled continuously.

This is an energy-intensive reaction (hence ATP consumption). Despite this and the fact that it’s largely a futile cycle, it persists because the light is so evolutionarily important.

Question 15

What is the role of luciferase in the light-yielding reaction of v. fischeri?

Answer 15

It consumes O2 + FMNH2 in the oxidation of R-CHO (tetradecanal). It produces light as a by-product.

Question 16

Luciferase is a […] encoded by […] genes.

Answer 16

Mooxygenase, luxA and luxB

Question 17

What is the fate of the byproducts of the v. fischeri light-yielding reaction? Explain the enzymes involved and name the genes that encode them.

Answer 17

The R-COOH gets recycled by a fatty acid reductase, encoded by the luxCDE genes. This will regenerate the tetradecanal (R-CHOO) and consume ATP and NADPH.

The FMN has electrons transferred to it from NADH by an oxidoreductase, which is encoded by luxG. This will renegerate FMN2.

Question 18

Describe the energy requirements of the light yielding v. fischeri reaction. What impact does this have on its mechanism?

Answer 18

it is extremely energy-intensive. The fatty acid reductase consumes NADH to recycle the fatty acid back to tetradecanal. The oxidoreductase also consumes an NADH to transfer electrons onto FMN to produce FMN2. Plus, the reaction requires an input of ATP.

Each photon essentially costs 2 NADH + ATP, which is a huge energy cost. Because of this high cost, it is very tightly regulated.

Question 19

The primary mode of regulation in v. fischeri is […]

Answer 19

Quorum sensing

Question 20

Explain how quorum sensing is done in v. fischeri, including how the relevant substances are produced.

Answer 20

This is done through a quorum sensing signal called the autoinducer AI. It’s a homoserine lactone. It is encoded by the enzyme AI synthease, which is from the gene LuxI.

AI concentration is proportional to the cell density/quorum, and its concentration is the same outside and inside the cell because it can cross the membrane.

AI gets detected by being bound to the LuxR protein. AI-LuxR binds to the luxI gene promoter, causing luxICDABEG to be expressed.

This is why light production only occurs at high cell density - binding of AI-LuxR to the promoter requires a high AI concentration.

Question 21

What is the role of quorum sensing in bacteria other than v. fischeri?

Answer 21

It is perhaps present in all bacterial species. N-acyl-homoserine lactone AI are the most common type in gram - bacteria. The precise N-acyl-homoserine lactone will vary depending on the organism.

In many abcteria, quorum sensing is required for induction of virulence genes.

In all these cases, the AI only works when the population reaches the threshold density.

Question 22

The regulation of V. fischeri lux gene expression occurs by what two mechanisms?

Answer 22

1. Catabolite repression (in response to starvation)
2. Autoinducer control (in response to high cell density)

Question 23

Describe the V. fischeri lux operon and describe its baseline functioning.

Answer 23

The left operon (OL) encodes luxR, and the right operon (OR) encodes luxICDABEG.

Baseline (not at particularly high cell densities):
The left operon is normally not expressed, and the right operon is normally expressed at very low levels. Cells always make a little bit of AI (because luxI is slightly active in OR), but not enough for light production.

Question 24

What is the function of the catabolite repression regulation of v. fischeri gene expression? What signal does it use?

Answer 24

It signals when the community is starving, which allows for the expression of the lux genes. The starvation signal is conveyed by a high concentration of cAMP-CRP.

Question 25

Give an example of catabolite repression in an organism aside from v. fischeri.

Answer 25

The lac operon of E. Coli

Question 26

Explain the circumstances under which V. fischeri starve in the squid

Answer 26

The constant supply of nutrients from the squid host is sufficient to allow growth of a V. fischeri population high density and maintain it for a while, but once the population gets really high, starvation is inevitable.

Question 27

Explain the role of cAMP, CRP, and cAMP-CRP in the catabolite repression mechanism.

Answer 27

cAMP increases in starving cells and is effective at low concentrations.

CRP is the catabolite repression protein. It is activated by binding to cAMP.

cAMP-CRP can bind to gene promoters and thus turn on or off gene expression when their concentration is high enough. Its concentration is proportional to [cAMP].

Question 28

Describe the sequence of events that take place in the catabolite repression and autoinducer mechanism when nutrients are abundant.

Answer 28

If nutrients are abundant, cells are not starving. There will therefore be a low concentration of cAMP-CRP. lux genes are therefore not expressed and no light is emitted.

Under these circumstances, the cells are making a bit of AI (as a baseline) but there’s not enough OR expression for light production.

Question 29

Describe the sequences of events that take place in the catabolite repression and autoinducer mechanism when nutrients are scarce.

Answer 29

Here, cells are starving. There is therefore a high concentration of cAMP-CRP. cAMP-CRP will bind to the the luxR promoter (OL), resulting in luxR gene expression and the synthesis of the luxR protein. LuxR is now available to bind to AI.

The LuxR-AI can now bind to the LuxI promoter (OR) and bolster the expression of luxICDABEG. This allows light to be produced if there’s enough AI to activate sufficient luxR.

Question 30

Describe how autoinducer control works.

Answer 30

It works via quorum sensing. [AI] only reaches high enough levels to activate sufficient LuxR for light production when cell density is 10^9-10^10 cells per ml.

Once this threshold is reached, AI-LuxR binds to the LuxI promoter. This will create much more AI and lead to a positive feedback. Light will be produced once there’s enough

Question 31

Describe the role of autoinduction in v. fischeri gene expression.

Answer 31

luxI gene expression is controlled by its gene production (AI).

Question 32

Describe the role of amplification in v. fischeri gene expression.

Answer 32

Amplification of lux expression occurs as [AI-LuxR] increases. These causes more and more light to be produced until it’s sufficient for counterillumination.