Motility and Chemotaxis

Question 1

What are the two types of movement used by bacteria?

Answer 1

By using flagella:
Pertrichous (E. coli, Salmonella) – several, decorated.
Lophotrichous (Rhodospirillum) – several, one/both end(s).
Monotrichous (Pseudomonas) – one, one end.

By gliding:
‘rolling’ in slime across a solid surface (phototrophic cyanobacteria).

Many prokaryotes are able to move in an ordered way. Not all bacteria are motile, but of those that are most are thought to be able to react by taxis (i.e. swim towrds something, or swim away from something bad).

Bacteria are fast swimmers:
up to 200 microm / sec (40-50 body lengths / sec).
Flagella rotate at 60-300 Hz.

Question 2

What is the “random walk”?

Answer 2

‘run’ – in a straight line for about 1 second.

‘tumble’ – briefly, about 0.1 second.

Change course by ~ 60°.

Question 3

How does the bacterium run and tumble?

Answer 3

Run = counter-clockwise (CCW) rotation of flagella
(viewed from the end of the filament towards the cell).

Tumble = clockwise (CW) rotation of flagella.

Question 4

What is the structure of the flagellum?

Answer 4

How big is a flagellum, what is it made of, how is it powered. Need to do this first as chemotaxis part involve sinteractions with the base of the flagellum.

1. 26 x FliF proteins = ‘MS’ ring of the rotor.
2. 23-36 x FliG-FliM-FliN complexes = C ring of the rotor.
3. 11 x MotA4-MotB2 complexes = ‘stator’ (energy transduction).
4. FlgI complex = P-ring.
5. FliE protein = MS ring / rod junction protein.
6. FlgB-FlgC-FlgF complex = the proximal rod (drive shaft).
7. FlgH complex = L Ring.
8. FlgG = distal rod (drive shaft).
9. FlgE = hook (universal joint).
10. FlgK = joint = hook-associated protein 1.
11. FlgL = joint = hook-associated protein 3.
12. FliC = flagellin = filament (propeller).
13. FliD = cap = hook-associated protein 2.

There are thought to be 11-17 MotAB motor/stator complexes.

Question 5

How is rotation of the flagellum powered?

Answer 5

Rotation is powered by the proton (or sodium) motive force.

Energetics is next – together with ‘torque’ and mechanism (MotA-FliG). Note that CCW is default setting.

Through MotA- H+ from periplasm (1200 per rotation), into cytoplasm.

Inner membrane- Δp = Δ – 59ΔpH.

Torque is generated by direct interaction between MotA and FliG.

2700 pN / nm at 10 Hz.

The flagellum is hollow – a 2 nm / 20 Å channel runs throughout .

Question 6

How does the flagellum machinery overcome wear and tear?

Answer 6

Wear and tear on the stator is relentless.

MotAB is continuously synthesised and replaced.

Only ~22 MotBs associated with flagella at any one moment in time.

~200 ‘free’ copies of MotB in the E. coli membrane.

Question 7

What is “biased random walk”?

Answer 7

Seminal experiment in 19th Century by Pfeffer, Engelmann & Stahl.

“schrekreaktion” or phobotaxis (avoiding reaction).

No steering, but merely avoidance of the wrong direction.

1960’s – chemotaxis revisited (Julius Adler, Madison, Wisconsin).

Noticed ‘run’ was prolonged in favourable direction.

Noticed bacteria seem to ‘remember’ a previous condition and compare that to the present one.

Bacteria possess a temporal sensing mechanism – or memory.

Mix a non-stimulated solution (mostly tumbling bacteria) with a solution containing attractant (no gradient) – the tumbling stops immediately and the bacteria run for up to 5 mins in a straight line!

Question 8

What signal tranduction pathways are involved in chemotaxis?

Answer 8

Multiple signal transduction pathways are involved:
Methyl-accepting chemotaxis proteins.

The phosphotransferase system.

Respiratory electron transport chains.

Question 9

What is the methyl-accepting chemotaxis protein system?

Answer 9

4 MCP receptor proteins located at opposite pole of the cell from the flagellal bundle.

Integral membrane proteins of 550 amino acids.

Respond to signals outside cytoplasm (no transport involved).

Tsr – binds serine (attractant), leucine (repellant).

Tar – binds aspartate, maltose binding-protein (attractants), nickel and cobalt ions (repellants).

Trg – binds galactose and ribose binding-proteins (attractants).

Tap – binds the dipepetide binding-protein (attractant).

Question 10

Wat does attractant and repellant binding do?

Answer 10

Attractant binding induces a counter clockwise signalling (CCWS) state.

Repellant binding induces a clockwise signalling (CWS) state.

Question 11

What are the parts of the two component system involved in MCP function?

Answer 11

CheA – a soluble, cytolasmic histidine kinase ‘transmitter’ domain of a two-component system.

CheY – is the ‘response regulator’, but does not bind DNA.

Question 12

How does the MCP two component system work?

Answer 12

MCP in the membrane detects a positive extracellular signal and interacts with the ‘transmitter’ CheA.

CheA is an auto-kinase but is INACTIVATED by a positive stimulus to MCP.

Nothing else happens – the flagellum freely rotates CCW.

‘run’.

Question 13

What does CheY~P do?

Answer 13

CheY~P interacts with FliM and reverses rotation of the flagellum.

Question 14

What does CheZ do?

Answer 14

CheZ dephosphorylates CheY~P and rotation returns to CCW.

Question 15

What is needed for adaptation to stimuli?

Answer 15

Adaptation to stimuli requires methylation / demethylation and a bacterial “memory”.

MCPs are continuously methylated by CheR.

As methylation increases, sensitivity to stimuli reduces.

Question 16

What happens in the absence of stimuli with CheY?

Answer 16

No stimulus, or repellant binding.

CheA forms a tight complex with CheW on the MCP.

Autokinase activity is induced and CheA is phosphorylated on His-48.

CheY is phosphorylated on Asp-57.

CW rotation.

Question 17

What is CheB~P?

Answer 17

CheB~P is a highly active protein-glutamate methylesterase.

Question 18

What happens in the absence of stimuli with CheB?

Answer 18

No stimulus, or repellant binding.

CheA forms a tight complex with CheW on the MCP.

Autokinase activity is induced and CheA is phosphorylated on His-48.

CheB is phosphorylated on Asp-56.

Demethylation and sensitisation of MCPs.

Question 19

How does CheB affect methylation in the absence and presence of stimuli?

Answer 19

Absence of stimuli; CheB~P active; MCP demethylated.

When completely demethylated, the MCP is at its most sensitive and can detect tiny amounts of stimulants.

Presence of stimuli; CheB inactive; MCP methylated and gradually de-sensitised.

The ‘dynamic range’ of the receptors is increased.

As sensitivity of MCP decreases upon methylation, the relative signal from the MCP decreases with increasing stimulation, and so activity of CheA changes.

Question 20

What happens when there is some stimulus, but MCP is desensitised?

Answer 20

Some stimulus, but MCP desensitised.

CheA forms a tight complex with CheW on the MCP.

Autokinase activity is induced and CheA is phosphorylated on His-48.

CheB and CheY are phosphorylated.

Demethylation / sensitisation of MCPs / CW rotation ‘tumbling’.

Methylation reduces MCP sensitivity; extra stimulus required to turn off CheA autokinase.

Question 21

What happens when an extra stimulus overcomes the desensitisation threshold?

Answer 21

Extra stimulus overcomes desensitisation threshold.

CheA/CheW/MCP complex falls apart.

CheA autokinase activity is lost.

CheB and CheY are inactive.

Methylation of MCPs resumes /CCW rotation ‘run’/sensitivity is lost.

Question 22

Where does “memory” come from?

Answer 22

“memory” comes from fast response to stimuli, but slower adaptation to that stimuli.

It takes several seconds to adapt to each new concentration of stimulant.

In the meantime, signalling from CheA is very high.

Gives the impression the cell has ‘remembered’ the previous condition – and it has, in a primitive way.

Question 23

The hook is always 55 nm long – how / why?

Answer 23

The ‘Type III’ export apparatus contains eight proteins: FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ, FliR.

FlhB is an ‘export switch’.

FliH, FliI- share homology with F1FO ion-driven ATPases. FliI hexamer when full formed- monomers exist in cytoplasm.

Question 24

Biosynthesis of the flagellum?

Answer 24

FlhB sits under the channel.

1. The hook cap (FlgD) is exported first.
2. FliK is unstable, but its N-terminus binds to the hook cap.
3. The FliK C-terminus binds to FlhB and stops growth of the hook.
4. FliH interacts with FliN.
5. FliJ interacts with FliM.
6. The FliH-FliI-FliC complex interacts with the FlhA-FlhB platform.
7. FliI oligomerises and activates. FliI-dependent ATP hydrolysis drives export of FliC.

Question 25

How are flagellins exported?

Answer 25

Flagellins are exported by a chaperone-based system.

FliC is maintained unfolded by the FliJ chaperone.

FliH and FliI then bind the FliJ-FliC complex.