Communication in unicellular organisms LECTURE 3

Two key levels of communication identified in unicellular organisms

1. Communication with other members of cell population

2. Sensing and response to environmental stimuli

Life cycle of slime mould

How is aggregation initiated

1. founder cell begins to emit a pulse of cAMP

2. Attracts neighbouring cells

3. Neighbouring cells emit own pulse of cyclic AMP→ acts as relay signal

4. Result: waves on cAMP radiating from aggregation centre every few mins→ 2cm/hour

5. Cells respond by chemotaxis towards founder cell

How is signal realy and cehmotaxis initiated?

• cyclic AMP receptor

  → has 7 helical transmembrane segments

• one of the family of G-protein activating receptors

What does cyclic AMP receptor do?

Activate a number of signalling pathways

1. adenylate cyclase dependent cyclic AMP pathway

   → relay system

2. phospholipase C dependent phosphoinositide pathway

   → Triggers local cytoskeletal rearrangements →chemotaxis

Three properties of signal relay and chemotaxis

1. Positive feedback by cyclic AMP→ ensures propagation of signal wave occurs

2. Cyclic AMP signal is degraded by phosphodiesterase

3. In presence of cAMP, cyclic AMP receptor is reversibly inactivated by phosphorylation

   • makes cell transiently insensitive to signals produced by neighbours

   • ensures: cAMP signal is pulsed and directional

cAMP chemotaxis in slime mould 1

1. cAMP from outside bind to receptor

2. comformational change component 3→ G protein→ adeylate cylcase ATP→ cAMP

3. More cAMP packed into vesciles→ released elsewhere

OVERALL: sends to other cells→ positive feedback

cAMP chemotaxis in slime mould 2 Creating oscillating dynamic

1. High cAMP conc in the cell

   → causes desensitissation of the cAMP recetepor

BUT ALSO

2. cAMP→ degraded into AMP

• Resensitise of receptor

→ Causes oscillations of desensitisation and resensitisation

These sensitiation oscillations happen due to phosphylation of receptor adatpatation

• Phosphylation→ can bind cAMP

• Desphosphylation→ cannot bind cAMP

cAMP chemotaxis in slime mould 3 Localaised extension

1. Only are points in the cell with highest cAMP still there

2. activates Gq recetpros→ PLC→ PIP2→ IP3 +DAG

3. IP3→ Ca2+ cytoskeleton

4. Chemotaxis

5. elongation of the pseodpods

This process has been tested with mathematical models

How do bacteria determine population size

• Quorum sensing

Why do bacterium Vibrio fisheri use quorum sensing

• emission of light is growth phase dependent

• only induced when bacterial population raeches a critical cell density

  → dependent on expression of luxA and luxB enoding enzyme luciferase

→ Quorum sensing regulated by Luxland LuxR

OVERALL: only activated when it actually become beneficial

• if not enough of them→ light to dim to have any value

How is the quorum sensing signal produced?

1. OHHL synthesised by Luxl operon

2. freely diffusible across the cell membrane into external growth medium

3. Luxl constitutively expressed during exponential growth

4. concentration of OHHL is directly dependent on the bacterial cell density

How is OHHL conentration regulated?

1. Sensed by transcriptional regulator LuxR

2. OHHL leaves and moves back into the cells continuously

3. When OHHL reaches critical concentration for luxR binding (must mean there are enough cells around

What does LucR induce

LuxR induces 2 sets of genes:

1. luxl → more OHHL synthesis (positive feedback loop)

• ensures all cells are induced at the same time

2. second cell density dependent target genes induced→ response

   • lucuciferase

What is the response in this case?

• bioluminescence due to

• expression of luxA and luxB

Other examples of function regulated by this process?

• antibiotic production

• extracellular enzyme secretion

  → by pathogenetic bacteria

What does chemotaxis help bacteria do?

• Respond to chemical gradients

What do bacteria use to do this?

Two component sensor regulator systems:

• highly conserved protein pairs

What do these comprise?

1. Environmental sensor

   • gets phosphylayed at a conserved histidine residue when a signal is received by the input domain

   • Auto-phosphylation

2. Response regulator

   • Senosr P is psated to receiver (trans-phosphylation)

   • phosphorylated at conserved asparatate residue by cognate sensor→ activate output domain

   • phosphate physically transferred from sensor protein to response regulator→ ouput

   • This then act at either:

     • DNA level → alter transcription

     • Protein level→ regulate protein function

How does chemotaxis move E.coli

10-50 umsec^-1

• Towards attractants

• Away from repellents

How does the bacterium move?

• Rotation of 6-10 flagella

• helical protein filametns made up of flagellin

Flagellar motor facts

• located in cell membrane

• driven by proton motive force

• Reversible

• Rotates at 10 Hz, requiring 1200 protons per turn

What are the sensor components in this case?

Methyl-accepting chemotaxis proteins (MSP)

• theres are at the other end of the flagella

• THEREFORE: must be some kind of signalling pathway to get to flagella movement

Flagella rotate counter and clockwise

How does chemotaxis work?

Biased random walk

1. cell swim in relatively straight lines → runs

2. rotating flagella in counter-clockwise direction for 1 sec

3. Then reverse flagellar motar for 0.1 sec (tumble)

4. New run in random direction

5. Length of run is biased→ runs are longer in favourable and shorter in unfavourable directions

How are changes in concentration of attractant/repellents detected?

• Temporally

NOT a spatial sensing mechanism

• Biased random walk when there is a chemattractant→ longer runs, shorter tumbles

4 sets of proteins which help carry chemotaxis out

1. Membrane bound signal transducer→ E.Coli has 5 receptor protein classes (MCPs) proteins (methyl accepting chemotaxis proteins)

   • mediate response to sepcific chemoattractants

   • e.g Tar→ aspartate and maltose

   - > SENSOR

2. Cytoplasmic signal transduction

   • CheA (histidine kinase), CheY (response regulator), CheW (adapter protein) and CheZ (an accelerator of CheY dephosphylation)

   → NB. ALSO THE SENSOR BUT A SEPARATE PROTEIN (unlike before)

3. Flagellar switch

   • FliG, FliM and FliN

   • determine direction of flagellar rotation

4. Adaptation

   • CheR (methyl transferase)

   • CheB (response regulator which acts as a methylesterase)

     → controls level of methylation of the chemotaxis receptor proteins

   • These help with the temporal memory of the concentration gradient thing

How flagella proteins organised

• Slightly different type of 2 component sensor

Chemotaxis transduction pathway A→ Attrctant absent/repellent present

1. CheW couple the receptor CheA

2. Due to lack of attractant, CheA = auto-phosphylated at His48

3. CheA-P transfers the P to CheY at Asp57 (transphosphylation of response regulator)

4. CheY-P interacts with the switch protein FliM at flagellar rotor

5. Flagella rotate clockwise and induce a tumble

Chemotaxis transduction pathway B→ Attractant present

1. Attractants (AT) bind to chemotaxis receptor protein

2. Binding = low rate CheA autophosphylation

   → CheA is unphosphylated

3. Unphosphylated CheA cannot transfer the P to CheY

4. CheY also dephosphylated by CheZ

5. When CheY is dephosphylated, it cannot interact with FliM

   → flagella rotates counter-clockwise→ with long smooth runs

E.coli are too small to sense concentration differences at either end (coz like treacle). How do they detect concentration gradients?

• use temporal memory

• Achieved through a negative feedback mechanism→ based on methylation of MCP proteins

• Uses the different timings of methylation and phosphorylation:

  • Methylation is slower than phosphorylation

  • Methylation is slower than de-methylation

Negative feedback mechanism→ based on methylation of MCP proteins

Chemotaxis transduction pathway C→ Adaptation

1. methylation of chemotaxis receptor protien

2. Acts as negative feedback→ returns system to an unstimulated state

3. CheR continuously methylates receptor whose response is influenced by degree of methylation state

4. CheB demethylating enzyme is determined by the phosphylation of CheA

5. Methylation of receptor CheR causes CheA stimulation and phosphylation

   → Methylation of receptor restores CheA activity→ following dephosphylation, caused by attractant binding to the receptor

6. Stimulation of CheA and phosphylation→ phosphylation and activity of CheB→ demethylation of the receptor

7. Causes→ restoration of CheA to dephosphylated low activity state

CheR→ constituively active

CheB→ regulated by CheA

These two types of protein modifications (de/phosphylation and de/methylation)

• Occur at different timescales

  → methylation provides memory for system

→ essential for biased random walk

Temporal adaptation no attractant

1. NO attractant→ auto phophylation high→ CheA-P→ CheY-P= tumble

but

2. High CheA acitivty= CheB increased activity

3. CheB demthylated CheA

4. CheA now reduces activity

5. CheY dephosphylated by CheZ

   → swim

Temporal adaptation with attractant

1. Attractant= low autophophylation= Swimming

but

2. Consistent gradual methylation by CheR (and CheB inactive)

3. CheA methylated= high activity

4. Swimming favoured over tumbling

Self assessment questions

1. Why are there oscillations in the signal relay of aggregating slime molds?

2. What is quorum sensing?

3. What is a two component signaling system?

4. How do temporal delays contribute to chemotaxis in E. coli?