Lecture 31 Flashcards
What are the typical relationships observed in luminescent bacteria?
They tend to have symbiotic relationships with their host - they often produce light for their host in return for a benefit.
Give an example of a symbiotic relationship involving luminescent bacteria.
Vibrio fischeri-squid
Explain how, in the vibrio fischeri - squid symobiosis, the light benefits the host animal.
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.
What is the typical habitat of the squid with which vibrio fischeri associate?
Shallow waters, and they come out at night.
Is the intensity of the light produced in the vibrio fischeri - squid interaction consistent? Explain why or why not.
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.
Explain the process by which the squid becomes associated with the vibrio fischeri.
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.
Where is the light organ inhabited by v. fischeri in the squid?
It is in the ink sac
Once the v. fischeri are associated with the squid, describe the conditions under which they live in the light organ.
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.
The squid host provides v. fischeri with […]
Organic and amino acids
Describe the turnover of v. fischeri in squid.
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.
Under what circumstances is light produced by v. fischeri?
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.
Explain what happens when the quorum is met in v. fischeri.
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).
What are milky seas? What is their cause?
It is a phenomenon that has been referenced historically - luminescent waters. They are likely due to vibrio spp. growing on decomposing algal blooms.
What is the light yielding reaction for v. fischeri?
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.
What is the role of luciferase in the light-yielding reaction of v. fischeri?
It consumes O2 + FMNH2 in the oxidation of R-CHO (tetradecanal). It produces light as a by-product.
Luciferase is a […] encoded by […] genes.
Mooxygenase, luxA and luxB
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.
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.
Describe the energy requirements of the light yielding v. fischeri reaction. What impact does this have on its mechanism?
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.
The primary mode of regulation in v. fischeri is […]
Quorum sensing
Explain how quorum sensing is done in v. fischeri, including how the relevant substances are produced.
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.
What is the role of quorum sensing in bacteria other than v. fischeri?
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.
The regulation of V. fischeri lux gene expression occurs by what two mechanisms?
- Catabolite repression (in response to starvation)
- Autoinducer control (in response to high cell density)
Describe the V. fischeri lux operon and describe its baseline functioning.
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.
What is the function of the catabolite repression regulation of v. fischeri gene expression? What signal does it use?
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.
