Lecture 3. Communities, Quorum Sensing, Biofilms

Question 1

Because microbes live in a community in their natural state, what does this mean to the bacteria and how they live?

Answer 1

Exponential growth means before long there are many cells
20 generations = 5 x 10⁵ cells

Question 2

In a mixed culture, which bacterias cells will dominate in the environment?

Answer 2

The one with the faster doubling time
After 7 hours, a bacteria with a doubling time of 20 minutes will have a million cells vs 30 minutes having 8,000

Question 3

What influences the abundance of different organisms in the community?

Answer 3

The environment they find themselves in

Question 4

In microbial communities, what does a positive interaction mean?

Answer 4

Toxic product of one organism may be substrate for another

Question 5

What is the main example of a positive interaction?

Answer 5

Cyanobacteria, fix carbon in water and double and grow based on how much they fix and how much light they capture in order to fix the carbon

Question 6

What happens when cyanobacteria are kept in a pure culture?

Answer 6

Die off after ~8 weeks because they run out of energy and produce toxic products that end up killing them

Question 7

What happens when a heterotrophic bacteria is present in a culture of cyanobacteria?

Answer 7

Both bacteria can survive for months
Cyanobacteria tend to produce waste products that are toxic to themselves. Heterotroph bacteria use and recycle these waste products to produce products that the cyanobacteria use to survive (as long as light is present or any other limitations)

Question 8

What are Windogradsky columns?

Answer 8

Made by mixing soil/sediment with water and adding some carbon (cellulose) and nutrients
Creates a whole ecosystem within a tube
Microbes fix carbon and recycle all nutrients so allowing these columns to grow for many years
Carbon fixation by cyanobacteria
Can have layers of aerobes and anaerobes

Question 9

In microbial communities, what are examples of negative interactions?

Answer 9

Antibiotic production by fungi, targets other microbes (always resistant to own antibiotics) - Penicillium strain produces an antibiotic inhibiting bacteria
Competition - sulphate reducing bacteria outcompete methanogens for substrates
Add acetate, little methane produced, inhibit sulphate reduction, lots of methane produced - These organisms are competing for substartes

Question 10

How do microbial communities affect each other in molecular terms?

Answer 10

They “sense” each other using chemical signals
How many are we? - Quorum sensing (Staphylococcus aureus, talking to itself, cells interacting from the same source)
What are we doing? - Biofilm formation (80-90% of all microbes on the planet are found in biofilms)

Question 11

How does quorums sensing work?

Answer 11

Detection of an autoinducer (gene that induces own production/a compound that will induce the infection of itself) - signal molecules that allows cell to sense population size (“quorum”)
Some processes require a certain density of cells to be activated (switch from attaching to invading form) such as production of a toxin
Once autoinducer reaches a threshold then cells respond - Changes expression and function

Question 12

What are most autoinducers?

Answer 12

Acyl-homoserine lactones
Autoinducing peptides with a thiolactone ring

Question 13

How does quorum sensing function in Staphylococcus aureus?

Answer 13

Staphylococcus aureus is a major opportunist pathogen
Also forms biofilms in wounds and on implanted medical devices (Staphylococcus always ends up on wounds, but normally harmless)
Switch from a biofilm-forming to an invasive phenotype is driven by quorum sensing
One quorum sensing mechanism is the Agr (accessory gene regulator) regulatory system

Question 14

What is Agr (accessory gene regulator)?

Answer 14

A two component regulatory system based around an operon with 2 promoters

Question 15

What does Agr constitutively express?

Answer 15

AgrD – Autoinducing peptide (AIP) - produces quorum sensing molecule
AgrB – Transmembrane protein, secretes mature AIP
AgrC – AIP receptor, binds AIP then phosphorylates AgrA
AgrA – Response protein, activates P3 after being phosphorylated by AgrC producing RNAIII

Question 16

What happens when the S. aureus cell density concentration is low?

Answer 16

Concentration of AIP is low
AgrC inactive so no phosphorylation of AgrA to activate P3 and produce RNAIII
Cell expresses many surface adhesins, which enhances biofilm formation

Question 17

What happens when the AIP (autoinducing peptide) reaches its threshold value due to biofilm maturation increasing the cell density?

Answer 17

At a threshold AIP activates enough ArgC which phosphorylates ArgA
ArgA-P induces expression of RNAIII (regulatory RNA)
RNAIII acts as a regulatory RNA controlling many genes

Question 18

What is the role of RNAIII in S. aureus?

Answer 18

Represses adhesion (represses cell wall compartments) - adhesisns
Induces elements that drive invasion (cytotoxins etc)
Also the mRNA holder of hld (δ-hemolysin) - damages red blood cells, also helps the invading staphylococcus seek out iron

Question 19

What does the activation of the agr system switch S. aureus from and in to?

Answer 19

Activation of of agr system switches S. aureus from an adhesive colonising commensal to an invasive aggressive pathogen

Question 19

What does RNAIII regulate?

Answer 19

Regulates some genes directly: Many of these are other regulatory genes which are the starting point for a major regulatory network
Also directly regulates α-hemolysin expression
Forms a complex secondary and tertiary structure: 14 hairpins bends and a globular shape brings 3’ and 5’ ends close together
This binds to hlaA mRNA at the shine-delgarno region allowing its translation
A rare example of RNA activating translation of a mRNA molecule

Question 20

What are biofilms?

Answer 20

Structured clusters of cells, enclosed in a self-produced polymer matrix and attached to a surface
Cells and tissues solid surfaces
Estimated that 80% all microbial biomass is in biofilms - most microbes attach to surfaces or other cells to allow for the cell to survive
Enable pathogens survival in the environment and in hosts

Question 21

What can a biofilm on contact lenses cause?

Answer 21

Keratitis - can damage eye sight
Particularly caused by Ps. aeruginosa and Staph. epidermis

Question 22

What can a biofilm on urinary catheters cause?

Answer 22

Bacteriuria - can cause major complications
Particularly caused by E. coli , Ps. aeruginosa and Proteus mirabilis

Question 23

What can a biofilm on joint replacements cause?

Answer 23

Septicemia or device failure
Particularly caused by Staph. epidermis and Staph. aureus

Question 24

What are the common characteristics of biofilms?

Answer 24

Cells enclosed in polymer matrix of exopolysaccharides, proteins and nucleic acid
Formation initiated by extracellular signals present in the environment
Biofilm protects bacteria against the host immune response, desiccation and biocides (e.g. antibiotics or disinfectants)

Question 25

How do biofilms form?

Answer 25

1. Initial attachment – bacteria attaches to surface using flagella and type I pili
2. Irreversible attachment – usually after doubling once or twice, pumps lipopolysaccharide outside of the cell, functions like glue, type I pili change expression to type IV pili
3. Maturation I – mature into mirocolonies, produce aliginate (also sticky), repress flagella, starting to change function
4. Maturation II – Quorum sensing, biofilm becomes large and complex, covers itself with goo to protect cells
5. Dispersion – changes some cells to planktonic cells and releases them

Question 26

What are mutlispecies biofilms?

Answer 26

Form from multiple species
Interactions of many different cells allow the biofilm to function like an ecosystem
Often lack oxygen so many anaerobic processes
Share waste products to function

Question 27

What is found in multispecies biofilms?

Answer 27

Lots of intracellular signalling organising the system and metabolic interactions

Question 28

What processes happen the further inside the multispecies biofilm you go?

Answer 28

Fermentation processes that produces organic acids
These are degraded producing H₂
H₂ used by other anaerobes removing end products
Constantly removing waste products by turning things back into CO₂, methane and water
Whole system keeps on functioning

Question 29

What is essential for Pseudomonas aeroginosa maturation into microcolonies?

Answer 29

Twitching motility of type IV pili - seek each other out on surfaces
Once they come together, they induce alginate production (exopolysaccharide stiky layer) to live in and repress flagella

Question 30

What are the steps in P. aeruginosa maturation linked to?

Answer 30

Specific sigma factor, σ²²
σ²² is encoded by algT, in the aliginate synthesis operon

Question 31

What is essential for Vibrio parahaemolyticus maturation?

Answer 31

A switch between 2 flagella systems is important in maturation
Interference with flagella rotation by a surface induces a secondary flagella system linked to swarming motility
Swarming is movement along a surface
Example of an environmental signal altering expression

Question 32

What is an example of changing expression using a different sigma factor in microbial communities?

Answer 32

σ²² in Pseudomonas aeruginosa (change the suite of genes you are using in your cells)

Question 33

What are examples of sensing environment to induce change in microbial communities?

Answer 33

Signal molecules inducing changes in gene expression
Quorum sensing in Staphylococcus aureus
Flagella sensing a surface in Vibrio parahaemolyticus

Question 34

What is an example of RNA acting as a regulatory element in microbial communities?

Answer 34

RNAIII in S. aureus
Role as direct and indirect regulator