Module 6: Reg. of Gene Expression (Quorum Sensing) Flashcards
Quorum Sensing
A chemical signaling system that allows for detection of the PRESENCE and NUMBER of microbes in the environment
Quorum sensing leads to the regulation of what kind of genes?
Density dependent genes!
“Quorum” refers to…
“The number of members of a group that needs to be present in order to conduct business”
In bacteria, what does it mean to “achieve quorum”?
Means that enough cells are present to accomplish a task that individual cells cannot do alone
Quorum sensing involves the release of…
Release of autoinducers into the environment by bacterial cells
Autoinducers
Specific signaling molecules used in quorum sensing that increase in concentration as density of a population increases
What is the relationship between population density and autoinducer concentration?
As population density increases, autoinducer concentration increases
(and same for decreases)
–> They change WITH each other!
How can bacterial cells assess their population density?
WHY?
By detecting autoinducer concentration in their surrounding environment
–> Works because of the relationship between autoinducer concentration and population density!
As detected population density changes, what is likely to occur in the detecting cell?
Change in gene expression!
Quorum sensing specificity
–> What does it depend on?
Communication via quorum sensing CAN BE specific!
–> Different populations use different autoinducers for signaling so molecules that can be detected by one population may not be detectable by another == specificity
What is a prototypical quorum signaling example?
The LUX system within Aliivibrio fischeri
What is special about Aliivibrio fischeri?
This bacterium has a lux system
The lux system was one of the…
FIRST quorum sensing systems to be characterized!
A. fischeri can exist in one of two states:
1) Free-living in the marine environment
2) As a symbiont in the light organ of the Hawaiian Bobtail Squid
In what state can A. fischeri produce light?
In its symbiont state
–> Living within the light organ of the bobtail squid
Why does A. fischeri produce light in its symbiont state but not when it’s free living?
Possibly because when in the light organ, high cell density is achieved (which is needed for lux)
Whereas in free living, the cells are dispersed within the marine environment and don’t have a high enough cell density to produce light
How can A. fischeri be MADE to produce light outside of the bobtail squid light organ?
One of two ways by culture:
1) By plating on agar in HIGH cell densities (mimics the densities found in the light organ)
2) By plating on agar with LOW cell densities and ADDING sufficient amount of AHL
The regulation of light production depends on the production of what?
What type of system regulates this?
Light production depends on the production of LUCIFERASE
–> Quorum sensing system regulates this production
What does AHL stand for?
N-acyl-homoserine lactone
What is AHL?
An autoinducer with the ability to induce luminescence
–> the autoinducer of the lux system in A. fischeri
Other than A. fischeri, where is AHL used and for what?
Used by a # of other members of the PROTEOBACTERIUM phylum for intercellular signalling
What is the structure of AHL?
A lactone (ester in a 5 member ring) substituent of the nitro group on an AMIDE
What differentiates AHL molecules in different organisms and pathways?
Why is this important?
The 1) Length and 2) Chemical modifications of the amide R side chain differentiates between AHL molecules
–> Important for signaling specificity
What type of bacteria do not use AHLs?
Gram (+) Bacteria
What autoinducers do Gram (+) bacteria use?
How do they work in quorum sensing?
Small peptides
–> Get transported out of the cell upon production to bind to cell surface receptors and trigger cell signaling pathways that alter gene expression
What makes AHL?
LuxI enzyme
LuxI is AKA?
Autoinducer Synthase
LuxI
An enzyme that catalyzes the production of AHL
What is the expression of the luxI gene?
What does this mean?
Always expressed to some degree (part of the lux operon LEAK)
==LuxI is always produced at least at a low level, meaning AHL is always produced at least at a low level
In the lux system, the basic production of what molecule is NOT dependent on cell density?
The production of AHL!
–> Is produced at least at low levels ALL the time in ALL cells!
LuxR
A transcriptional activator protein that detects/binds to AHL
What is the issue with LuxR by itself?
It cannot bind to the activator binding site of DNA on its own!
–> Requires a co-activator to form an active activator complex!
(This co-activator is AHL)
When LuxR-AHL complex forms, what happens?
The activator complex (LuxR-AHL) binds to the LUX BOX
What is the Lux Box?
(Where is it found?)
A lux regulatory site (activator binding site) on DNA situated upstream of the lux operon
Binding of LuxR-AHL to the lux box causes…
RNA polymerase to bind to the lux operon operator and trigger the transcription of the lux operon
== Activates the lux operon and ultimately leads to light production!
What structural genes encode for the production of luciferase?
luxA and luxB
–> Each encodes for one of the subunits needed to form luciferase
luxA and luxB do what?
What do their products form?
luxA = Encodes for alpha subunit of luciferase
luxB = Encodes for beta subunit of luciferase
–> alpha and beta subunits come together to form an active luciferase enzyme!
What is the order of genes in the lux operon?
(LuxR)
(Lux Box = ABS)
(Promoter)
(Operator)
1) luxI
2) luxC
3) luxD
4) luxA
5) luxB
6) luxE
7) luxG
What is the function of EACH gene in the lux operon? (6)
luxI= Encodes for LuxI protein which produces AHL
luxC, luxD, luxE = Encode for polypeptides needed for substrate of luciferase
luxA, luxB = Encode for the alpha and beta subunits of luciferase
luxG = No function in lux production
luxC, luxD, luxE
Genes in the lux operon that encode for polypeptides needed to produce the substrate used by luciferase to produce light
What is the density dependent component of the lux system?
The accumulation of AHL within the cell!
(Allowing for detection by LuxR activator)
In LOW cell density, what happens within the lux system of a cell?
1) luxI gene expressed at low level to produce some LuxI enzyme
2) LuxI produces low level of AHL
3) AHL IMMEDIATELY diffuses OUT of and away from the cell
== Low concentration of AHL in the cell
4) LuxR present from constitutive expression remains unbound/inactive
== No activation of the lux operon = no light
What is the expression of the luxR gene?
Constitutively expressed! == Always producing LuxR
–> Not a part of the lux operon so not regulated by its activation!
In HIGH cell density, what happens within the lux system of a cell?
1) luxI gene expressed at low level to produce some LuxI enzyme
2) LuxI produces low level of AHL
2) AHL diffuses OUT and INTO cell! (due to high concentration of AHL in the surrounding ECF from the production of other cells)
3) AHL accumulates in the cell to the high enough critical threshold concentration
4) LuxR produced via constitutive expression binds to AHL forming the active LuxR-AHL complex
5) LuxR-AHL binds to the lux box
6) RNA polymerase is able to bind to the operator
7) Transcription of the lux operon is activated
8) lux operon activation increases expression of luxI which triggers (+) feedback loop
9) luxA and luxB are transcribed leading to alpha and beta subunit production
10) Alpha and beta subunits combine to form luciferase
== LIGHT produced by luciferase!
Upon activation of the lux operon, what kind of feedback occurs?
What causes this?
POSITIVE FEEDBACK LOOP
–> Once the lux operon is activated, luxI gene is transcribed more!
== More production of LuxI == more production of AHL == more activation of LuxR leading to further activation of the lux operon!
What survival benefits does light production give to the bobtail squid?
Provides camoflauge from predators that cannot detect the illuminated squid against the background of filtered light from the surface above
== Increased survival and therefore increased reproductive success
What are the benefits of A. fischeri symbiosis?
Symbiosis provides them with a safe home + nutrient access
BUT: The benefits to A. fischeri are not fully understood
–> Some suggest that the symbiosis allows for the generation of a lot of A. fischeri cells (high density) which can then disperse in large numbers back into the water = increased survival
What are the cellular processes that quorum signaling has been found to control?
1) Motility
2) Conjugation
3) Biofilm formation
4) Secondary metabolite formation
5) Pathogenesis
Many events triggered by quorum sensing in various microbes involves…
The colonization or infection of multicellular organisms
(generating the high cell density)
Example of quorum sensing in pathogenesis: Organism
Vibrio cholerae
Vibrio cholerae:
What can they NOT do alone?
A single cell cannot produce enough toxin to have much effect on host organism cells
Since V. cholerae cannot produce enough toxin on their own, how does this alter their toxin production?
Will not produce ANY toxin unless triggered by high cell density via quorum sensing!
== Allows for conservation of energy and resources; why make the toxin when its effect won’t even be felt?
Vibrio cholerae:
In high cell density, what occurs?
Why does this help V. cholerae?
Greater toxin production results in a summated effect that is enough to cause an effect in host organism
–> This toxin production allows for evasion of the host organism immune system
Vibrio cholerae:
The production of toxin is controlled by…
Quorum sensing!
High cell density = toxin produced
Low cell density = toxin NOT produced
Autoinducers can also have a potential negative impact by playing a role in….
How do they do this?
Competition!
–> By acting as inhibitors or interrupting a control pathway in another organism (not the producer of the autoinducer) in the environment
