Lecture 7 Bacterial Motility and Chemotaxis

Question 1

An introduction to motility & flagella

Answer 1

Many prokaryotes show swimming motility, mediated by a structure called the flagellum (plural, flagella)

Bacterial flagella are long, thin appendages attached to the bacterial cell (one end attached, the other end free)
Typically only 15-20 nm thick
The flagellum functions by rotation, pushing or pulling the bacterial cell through the liquid medium

Question 2

Cellular localization of flagella

Answer 2

Flagella can be attached to bacterial cells in different places

Organisms are normally polarly flagellated, but can form peritrichous flagella under certain growth conditions

Question 3

Flagellar structure - overview

Answer 3

Flagella are helical structures, the filament of which is composed of many copies of a protein called flagellin

Flagellin is highly conserved in species of Bacteria, suggesting that flagellar-mediated motility evolved early within this domain

The base of the flagellum is structurally distinct from the filament

Wider region at the base called the hook, which connects the filament to the motor portion in the base
This motor is anchored in the cytoplasmic membrane & cell wall, and consists of a central rod passing through a series of rings

Question 4

Flagellar structure – in detail

Answer 4

Gram-negative flagellar structure:

L-ring embedded in LPS
P-ring in peptidoglycan layer within periplasm
MS ring embedded in cytoplasmic membrane
C-ring within the cytoplasm

In Gram-positive bacteria, only the inner pair of rings is present

Question 5

Flagellar assembly

Answer 5

20-30 genes are involved in flagellar assembly, including those encoding:

• Flagellin
• 10+ hook & basal body proteins
• Proteins involved in control of flagellar construction & function

Flagella filaments grow at the tip of the structure – not from the base

Rod & filament structures contain a narrow channel through which flagellin molecules diffuse to reach the site of flagellar assembly

Approx. 20,000 flagellin molecules per filament

When flagellin reaches the tip, it spontaneously aggregates under the direction of a filament cap

Example of self assembly (i.e. without the aid of enzymes)

Question 6

Flagellar movement

Answer 6

“Proton turbine model” is proposed as explaining flagellum rotation

Mot proteins form a proton channel and function as the flagellar motor

The Fli proteins function as the flagellar switch, reversing direction of rotation in response to signals

Question 7

Proton-mediated flagellar movement

Answer 7

Energy required for flagellar rotation comes from the proton motive force

Protons move across the cytoplasmic membrane through the Mot complex, driving rotation of the flagellum

In the “Proton turbine model”, protons flowing through Mot proteins exert electrostatic forces on helically arranged charges on the rotor proteins

Attractions between positive & negative charges then cause the basal body to rotate

Proton motive force is a source of energy that results from the separation of protons from hydroxyl ions across the cytoplasmic membrane, generating a “membrane potential”. Causes the membrane to be energized, much like a battery.

Question 8

Archaeal flagella

Answer 8

Flagellar motility is also widespread among Archaea species

Archaeal flagella also confer movement by rotation, however:

• the structural component of their flagella is unrelated to flagellin
• In documented cases, it appears rotation is powered directly by ATP rather than by proton motive force

Suggests that flagellar motility in Bacteria and Archaea evolved after the two prokaryotic domains had diverged (> 3 billion years ago)

Question 9

Bacterial taxis

Answer 9

“Taxis”, a movement towards or away from a stimulus

Most commonly studied in the context of chemotaxis – the directed movement towards chemical attractants (e.g. nutrients) and away from repellents

Chemotaxis and motility are not synonymous; cells can be motile, but not chemotactic

Other forms of taxis include thermotaxis (temperature), phototaxis (light), aerotaxis (oxygen) and osmotaxis (osmolarity)

Question 10

Bacterial chemotaxis: a biased random walk

Answer 10

Because of their small size, bacteria can not perform spatial sensing of gradients.

Instead they use temporal sensing:

Cells swim randomly, alternating between periods of smooth swimming and brief direction changes

Smooth swimming is favoured when cells are moving up a concentration gradient of attractant:

If conditions are improving then the cells are swimming in the right direction so they keep swimming that way (“forward run”)
If conditions are worsening then the cells are swimming in the wrong direction and so need to change direction (“tumble”)

Memory is essential for temporal sensing. Bacteria remember their previous environment and compare it with the current conditions

Question 11

Flagellar rotation, tumbling & running

Answer 11

Forward run: Counter clockwise

Tumble: Clockwise

Question 12

Chemotaxis is regulated by a two-component system

Answer 12

Two-component systems discussed previously are EnvZ-OmpR, PhoP-PhoQ and PmrA-PmrB

In each of these cases:
The sensor kinase is a transmembrane protein
The response regulator is a transcriptional regulator

Two-component systems involved in chemotaxis differ:
The sensor kinase (CheA) is cytoplasmic
The response regulator (CheY) interacts directly with the motor proteins of the flagellar basal body

Question 13

Proteins of the chemotaxis signalling pathway

Answer 13

The cytoplasmic CheA sensor kinase; interacts with chemoreceptors

When activated, CheA phosphorylates CheY (response regulator) and CheB (a methyl esterase)

Phosphorylated CheY interacts with the FliM protein of the flagellar motor

Question 14

The chemotaxis signalling pathway

Answer 14

Numerous chemoreceptors have been identified; one of the most widely studied classes being methyl-accepting chemotaxis proteins (MCPs)

MCPs span the membrane:
Periplasmic domain has binding sites for attractant molecule(s)
Cytoplasmic domain interacts with CheA & CheW

CheW binds to the MCP and helps attach the CheA protein

The default mode for flagellar rotation is counter clockwise (CCW)

Active CheA > phosphorylated CheY

CheY-P diffuses through the cytoplasm and interacts with FliM

Direction of rotation is switched from CCW to clockwise (CW)

Question 15

The chemotaxis signalling pathway

Answer 15

Moving up a concentration gradient of attractant (i.e. towards attractant)
Attractant is bound to MCP, which inhibits CheA autophosphorylation
> CheY is inactive > flagellar rotation remains CCW (forward run)

Moving down a concentration gradient (i.e. away from attractant)
If attractant levels fall, MCP-bound attractant decreases, activating CheA autophosphorylation
> Activation of CheY (CheY-P) > Interacts with FliM
> CW rotation (tumbling)
CheZ dephosphorylates CheY-P to reset the system (CCW rotation)

Question 16

Role of MCP methylation in chemotaxis

Answer 16

How does the bacterial cell know if it is moving towards the attractant?
Bacteria measure the concentration of chemoattractant every few seconds and determine if it is increasing or decreasing over time
(i.e. temporal sensing rather than spatial sensing)

Bacteria can ‘remember’ the previous concentration of attractant
Achieved through methylation of the MCPs, governed by:
CheR (methyl transferase)
CheB (methyl esterase)

Methylation is favoured when attractant is bound

Bacteria monitor concentration gradients by comparing the overall methylation status of MCPs with the amount of chemoattractant bound

Moving up concentration gradient
Concentration of attractant keeps increasing > number of MCPs bound to attractant remain high > methylation of MCPs is favoured
Balance between attractant bound and high methylation state

Moving down concentration gradient
Concentration of attractant decreases > number of MCPs bound to attractant drops > MCP methylation exceeds attractant bound
This disparity triggers CheA autophosphorylation > CheY-P > tumbling
Active CheA also phosphorylates CheB (methyl esterase) > reduces the methylation status to reset the system

Question 17

Flagella, motility & chemotaxis in virulence

Answer 17

Various mutants can be employed to tease apart role of different systems, targeting genes encoding:
Structural components of the flagella (e.g. flagellin)
Motor proteins that facilitate flagellar rotation
Proteins that govern chemotaxis (CheAY, MCPs etc.)

Flagella, motility &/or chemotaxis often implicated in virulence, but can be species-specific and/or infection model-specific

Flagellar-mediated motility frequently implicated in cell invasion
Sometimes virulence attenuation of flagella-deficient mutants can be due to flagella promoting adherence, rather than motility

Flagella are also highly immunogenic, with flagellin being recognised by Toll-like receptor 5 (TLR5)
TLR5 activation results in significant proinflammatory response
During chronic infection, it is not uncommon for flagella to be lost from bacteria through mutation

Question 18

Conclusions – motility & chemotaxis

Answer 18

Flagellar-mediated motility is widespread in bacteria
Frequently implicated in virulence
Essential for taxis towards attractants and away from repellents

Direction of movement governed by direction of rotation of the helical flagella (CCW favours forward run; CW favours tumbling)

Two-component system CheA-CheY regulates the chemotactic response through direct interaction with flagellar motor proteins, influencing direction of rotation