Lecture 3 - Regulation + Functions of VFs Flashcards
(T/F) In bacteria, genes present in the genome are expressed at all times.
False!
Bacteria respond to changes in the environment by sensing the surrounding conditions and then altering gene expression accordingly.
This is demonstrated in part by quorum sensing + two-component systems, but there are other ways to regulate gene expression (of VFs).
How did microbiologists see bacterial populations before compared to now?
For decades, microbiologists thought of bacterial populations as collections of individual cells growing and behaving independently.
We now know that bacteria can COMMUNICATE WITH EACH OTHER USING CHEMICAL SIGNALS that allow them to act as COMMUNITIES, able to accomplish tasks that they could not perform as single entities. This is important for pathogenesis!
Studies on which organisms helped microbiologists learn about the cell-cell communication between bacteria?
Studies on gram-negative bioluminescent bacterium aka V. fischeri.
This bacteria is found in salt water and exists in a unique SYMBIOTIC relationship with the squid EUPRYMNA SCOLOPES (hawaiian bobtail squid).
Describe the symbiotic relationship of V. fischeri and Hawaiian bobtail squid,.
Vibrio fischeri colonize the LIGHT ORGAN of the squid. Living here is beneficial fo bacteria because the organ is rich in nutrients.
When feeding, the squid uses its light organ (bioluminescence from V. fischeri) to provide camouflage by projecting light downwards, helping to limit detection by predators.
E. scolopes is _____ and emerges at ____ to hunt. During this period, the light organ is ____ of V. fishceri.
At dawn, the host expels ____ of the light-organ bacteria into the surrounding environment.
The squid then _____ beneath the sand and during the day the remaining ____ of bacteria that remain in the light organ _____.
By mid-afternoon, the light organ is ____ of bacteria and the cycle repeats itself.
nocturnal; dusk; full
95%
buries; 5%; grow
full
Briefly answer the questions regarding how V. fisheri gave us insights into bacterial cell-cell communication.
1) How does V. fischeri control its ability to glow?
2) What is now known about autoinducers and bacteria?
1) V. fischeri controls its ability to glow by producing a small, diffusible hormone-like substance called an AUTOINDUCER. This was identifies as ACYL-HOMOSERINE LACTONE (AHL).
2) It is now known that many bacteria (not just pathogenic) produce similar autoinducers (vary in chemical composition), each of which has DIFFERENT FUNCTIONS in the regulation of cellular processes (i.e. virulence, antibiotic resistance, plasmid transfer etc).
Gram-negative bacteria use ___________ as cell-cell communication molecules.
Gram-positive bacteria use ______ called ___________ as cell-cell communication molecules.
Bacterial cell communication is ______-dependent.
acyl-homoserine lactones (AHLs)
peptides; auto-inducing peptides (AIPs)
density
1) What happens to autoinducers at low cell densities?
2) What happens to autoinducers at high cell densities?
1) At low cell densities, the autoinducer diffuses OUT of the cell. The functional outcome is not displayed (i.e. no bioluminescence).
2) At high cell densities, there is an INCREASE in autoinducer in the external environment. When the population reaches a SPECIFIC DENSITY, the DIFFUSION GRADIENT REVERSES whereby the autoinducer enters the cell. The influx of autoinducer promotes signalling events that ALTER GENE EXPRESSION to regulate specific functions (i.e. production of bioluminescence).
Because the influx of autoinducers is density-dependent, it allows single cells to assess the _______ ________.
This communication ensures cells perform specific beneficial functions when conducted in ______ with other cells, rather than as _______ cells.
This is the general concept of ______ ________!
population density
synchrony; individual
quorum sensing
What is Quorum Sensing?
Quorum sensing is a process of CELL-CELL COMMUNICATION that involves the production, detection and response to AUTOINDUCERS, ultimately allowing bacteria to share information about cell density and to then ADJUST GENE EXPRESSION IN UNISON.
Bacteria can therefore synchronize particular behaviours on a POPULATION-WIDE SCALE.
*a quorum usually refers to the minimum number of members in an organization needed to execute a role.
*not all processes are regulated by quorum sensing.
1) What is the luciferase operon?
2) What is LuxR?
3) What is LuxI?
4) What happens when the autoinducer concentration reaches a critical threshold concentration?
1) V. Fischeri contains an operon known as the luciferase operon, denoted as luxICDABE (or lux operon). Expression of the lux operon is required for light production.
2) LuxR is the autoinducer receptor and DNA-binding transcription activator.
3) LuxI is an autoinducer synthase, which produces the AHL autoinducer.
4) When the autoinducer concentration reaches a critical threshold concentration, it is bound by LuxR and this complex activates transcription of the operon encoding luciferase which, in turn, produces bioluminescence.
Briefly describe why light is not produced at low cell densities of V. fischeri.
At low cell densities; there is a LOW LEVEL OF TRANSCRIPTION from the lux operon.
The RNA polymerase binds weakly to the promoter in the lux box, allowing for low expression of luxI, which produces low levels of AHL.
AHL produced diffuses out of the cell; there is no AHL to bind and form a complex with LuxR. This complex is needed for full activity of the lux operon (luciferase + its substrates) to produce light.
No complex = no LIGHT!
Describe the functions of LuxA, LuxB, LuxC, LuxD, and LuxE of the lux operon.
LuxA and LuxB: encode for luciferase subunits.
LuxC, LuxD, and LuxE: encode polypeptides needed for substrate of luciferase.
*luciferase uses its substrates to produce light.
Briefly describe how light is produced at high cell densities of V. fischeri.
At high population density, there is a high level of transcription from the lux operon.
There is a high concentration of AHL in the cell (diffusion gradient changes; AHL from the external environment goes into bacterial cell).
AHL and luxR interact and luxR binds lux box for activation (increases the processivity of RNA pol), allowing full activity of lux operon (not just luxI).
This produces luciferase and substrates, generating light!
Give examples of processes controlled by quorum sensing besides the expression of bioluminescence.
1) Biofilm formation
2) Regulation of motility
3) Expression of VFs
4) Antibiotic production
S. aureus is a gram _____ bacterium that uses a ____-component quorum sensing system encoded by the ____locus, which encodes for:
positive; two; agr; AgrA, AgrB, AgrC, AgrD.
Briefly describe the functions of the different proteins of the agr locus (AgrA, AgrB, AgrC, AgrD) and how they aid in virulence using quorum sensing.
AgrD encodes for a pre-AIP that becomes a mature autoinducer once secreted.
AgrB is a membrane transporter that secretes pre-AIP in the external environment.
AgrC is a sensor kinase that is embedded in the bacterial membrane that binds to AIP at a certain population density, causing the kinase to be activated.
Activated AgrC phosphorylates AgrA which now serves as a transcription factor that promotes expression of promoter 2 and promoter 3.
In S. aureus, phosphorylated AgrA promotes the expression of promoter 2 and promoter 3.
What do these promoters regulate? How does this help achieve virulence?
Promoter 2 encodes for the agr locus. By stimulating P2, AgrA can promote the expression of the Agr locus, producing more AIP.
Promoter 3 regulates the expression of RNAIII. RNAIII upregulates secreted virulence factors (toxins, proteases, lipases etc) specific to S. aureus and represses ROT (repressor of toxins).
ROT inhibits the expression of virulence factors when they are not needed.
Quorum sensing is one way bacteria can sense and adapt to their environments and regulate the expression of their virulence factors.
What is another way?
Another way bacteria can sense and adapt to a range of environments (and regulate expression of their virulence factors) is by using the TWO-COMPONENT SIGNALLING SYSTEM (TCSS).
(T/F) Production of virulence factors in S.aureus is an example of TCSS where the environment sensing molecule is an autoinducer (density-dependent process).
True!
Thus, it is said that this bacterium uses a ‘two-component’ quorum sensing system!
