BMB - bacterial chemotaxis

chemostimuli

most sugars are attractants

serine and aspartate are attractants

leucine is a repellent

secondary metabolites e.g. nickel are repellents

phenol is attractant in E.coli and repellent in salmonella

cell tethering experiment - rotation of flagellum

take slide and coat with antibody against flagellin

add blended E.coli with sheared flagella

some E.coli attach by single flagellum

can see movement of single flagellum under microscope

basis of tumbles and runs

repellents increase CW rotation = tumble

attractants suppress clockwise rotation = runs

2 isoforms of flagella

normal = long wavelength

curly = short wavelength

one flagellar filament

11 protofilaments

domains of protofilaments

D0 = filamentous, involved in monomer interactions

D1-3

normal vs curly protofilaments

repeat distances are different in normal and curly

less than 1A difference in distance - additive

CW rotation = RH screw configuration = curly isoform

CCW rotation = LH screw configuration = normal isoform

transition between normal and curly isoforms

reversal in torque of LH screw configuration leads to knot

formation

transitions between normal and curly leads to kink in flagellum and therefore bundle falls apart = tumble

evidence of molecular mechanism of MCPs

transposon mutagenesis of e.coli = non motile, Tsr mutant, Tar mutant

swarm plates on semi solid agar = prevents convection but allows swimming

circles develop as bacteria swim away from point of inoculation corresponding to two different nutrients

non motile = no circles

Tsr mutant = only inner circle

Tar mutant = only outer circle

identified as chemotaxis receptors

MCP receptor conformational change

binding of ligand can cause twist, piston or crank motion

small conformational change in periplasmic domain when attractant binds

repellent binding leads to less dynamic receptor

negative cooperativity - half a binding site in the monomer, binding of ligand decreases ability of another ligand to bind

cell envelope experiment investigating rotation direction

empty E.coli re sealed with purified proteins

no protein = CCW only

CheY added = CW only

role of CheY

response regulator

phosphorylated by CheA - sensor histidine kinase

CheY-P is clockwise signal

chemotaxis towards attractants

1. unstimulated state, CheR and B levels are at steady state

2. attractant binds, CheA autophosphorylation is inhibited = less CheY-P = less CW rotation

3. less CheB demethylation, receptor methylation increases due to CheR

4. increased methylation activates CheA autophosphorylation and CheY-P levels increase = more CW rotation

5. CheB activated by CheA = receptor demethylation

6. receptor is back in steady state

chemotaxis away from repellents

1. unstimulated state, CheR and B are in steady state

2. repellent binds, CheA autophosphorylation activity increases = more CheY-P = more CW rotation

3. CheB activity increases = receptor demethylation

4. CheA activity decreases = less CheY-P = less CW

5. CheB activity decreases = receptor methylated

6. steady state

arrangement of receptors

clusters at pole of cell

trimers of dimers

mixed composition trimers can be linked by single CheA and W pairs = higher activity

higher order arrangements may explain dynamic range of response

how methylation affects receptor activity

high CheB = low methylation = ultrasensitive receptor due to low occupancy, high kinase inhibition

high CheR = high methylation = insensitive receptor due to high occupancy, low kinase inhibition

phosphorelay

CheA phosphorylated on histidine

phosphoamidate bond is highly reactive, so fast transfer to aspartate of CheY

phosphoanhydride is very reactive, only stable for seconds = CheY-P falls off

CheY mediates phosphotransfer

CheY-P dephosphorylated spontaneously by CheZ which forms oligomers in presence of CheY-P

CheY binding partners

CheA

CheZ

FliM

interaction of CheY with FliM

CheY-P binds FliM

conformational change in FliM causes conformational change in FliG

ultimately leads to switch to CW rotation

cooperative motor response to CheY-P

n = 10

not due to FliM binding CheY-P, FRET studies showed this is hyperbolic

motor is driven by pmf

components of motor

rotor and stator

stator components

MotA = proton pore

MotB = anchor, secures inner membrane components to PG, can diffuse in and out of membrane

arrangement of MotA and B

form ring structures around central motor component

found by freeze fracture experiments and confirmed by Cryo ET

structure of stator complex

MotA pentamer and MotB dimer = force generating units

avoiding uncoupling when not bound motor

MotB doesn’t bind PG until bound motor, instead blocks MotA proton pore

proton flux only when bound motor

experiment proving MotA is proton pore

make vesicles with high potassium conc inside

with expressed MotA or knockout

suspend in non-buffer solution and add pH meter

add potassium ionophore valinomycin

protons then flow down ECG into the vesicle as potassium flows out

detect alkalisation with pH meter of solution

experiment showing diffusion of force generating units

tethered cells containing MotA under controlled promoter

speed of tethered cell increases stepwise corresponding to addition of force generating unit when MotA expression is induced

motor can accommodate up to 13 force generating units

rotor components

FliM/N/G make up C ring

role of FliG

switching role from CCW to CW

associates with stator

conformational change in FliG = different contact of MotA and B, leading to CW or CCW rotation

C ring contacting different points of stator means rotation forced in different directions

motor function - power stroke model

conformational change pushes ring to rotate

more proton flow = more pushing of c ring

T3SS relationship to flagella

T3SS penetrate host membrane and injects virulence factors - contact dependent

similar structures

no stator components

flagellar assembly

FlgJ makes holes in membrane - also used in phage needles

T3SS moves proteins through membrane