Bacterial chemotaxisBacterial chemotaxis

Dr. habil. Kőhidai LászlóDr. habil. Kőhidai László2012.2012.

Diverse swimming Diverse swimming behaviours of behaviours of chemotaxis and chemotaxis and their interpretation their interpretation regarding regarding concentration concentration gradients and cell gradients and cell sizesize

Bacterial flagellum - 12-30nm Bacterial flagellum - 12-30nm

monotrich

lopotrich

peritrich

Main composing protein: flagellin (53.000) pentahelical structure fast regeneration (3-6 min.)

55

statorstator

rotorrotor

„„hook”hook”

flagellumflagellum

SS

LL

PP

MM

22.5 nm22.5 nm

27 nm27 nm

Structure of basal body of bacterial Structure of basal body of bacterial flagellumflagellum

CCWCCW

CWCW

Correlation of swimming types Correlation of swimming types and direction of flagellar and direction of flagellar rotation in bacteriarotation in bacteria

tumblingtumbling

R M BerryR M Berry::Torque and switching in the bacterial flagellar motor. An Torque and switching in the bacterial flagellar motor. An electrostatic model. electrostatic model. Biophys J. 1993 April; 64(4): 961–973Biophys J. 1993 April; 64(4): 961–973

Gradient Gradient Length of linear path Length of linear path Number of tumblingsNumber of tumblings

Gradient Gradient Length of linear pathLength of linear pathNumber of tumblingsNumber of tumblings

E. coliE. coli

E. coliE. coli

Bacterial chemotaxis and adaptationBacterial chemotaxis and adaptation

Swimming of cells is influenced Swimming of cells is influenced NOT ONLYNOT ONLY by the by the changes of concentration of the ligand.changes of concentration of the ligand.

Adaptation mechanismsAdaptation mechanisms refer to the presence of refer to the presence of a a ‘primitive’ memory‘primitive’ memory of cells of cells!!

periplasmaticperiplasmaticbinding/transportbinding/transportmoleculesmolecules

sugarssugars dipeptidesdipeptides amino acidsamino acids

chemotaxischemotaxisreceptporsreceptpors

intracellullar signalling pathwayintracellullar signalling pathway

Detection of bacterial cheotaxis receptorsDetection of bacterial cheotaxis receptors

receptor clustersreceptor clusters

division furrow/ringdivision furrow/ring

Aspartate receptorAspartate receptor

ligand binding ligand binding domaindomain

„„coiled-coil” domaincoiled-coil” domain

residues for residues for methylationmethylation

signal transmittersignal transmitterdomaindomain

CCOO

OOCC

OO

OO

ligand binding domainligand binding domain

residues forresidues formethylationmethylation

8 db8 db

szignal transmitter domainszignal transmitter domainin in basal activitybasal activity

Composition of Asp receptorComposition of Asp receptor

CCOO

OOCC

OO

OO

CCOO

OOCC

OO

OOCC

OO

OOCC

OO

OOCHCH33CHCH33

Methylation of Asp chemotaxis receptorMethylation of Asp chemotaxis receptor

methyltransferasemethyltransferase

methylesterasemethylesterase

CheA-P + CheYCheA-P + CheY CheY-P+ CheACheY-P+ CheA

CheACheA CheA-PCheA-P

CheY-P + CheZ CheY-P + CheZ CheY + PCheY + Pii

CW rotationCW rotation„„tumbling”tumbling”

MgMg2+2+

MgMg2+2+

CheW , CheB-PCheW , CheB-P

Repellent moleculeRepellent molecule

200 ms200 ms

CheA - activityCheA - activity

CheY-P - amountCheY-P - amount

direction of Hdirection of H++ transport in the transport in themotor region of flagellum is reversedmotor region of flagellum is reversed

CCW rotationCCW rotation„„swimming”swimming”

Attractant moleculeAttractant molecule

TTapapTTrgrg TTsrsr TTarar

dipeptidesdipeptidesgalactosegalactose

riboseriboseLeu, SerLeu, Ser NiNi2+2+, Asp, Asp

CheRCheR

CheB-PCheB-P

CheBCheB

CheACheA

CheA-PCheA-P CheYCheY

CheY-PCheY-P

CheWCheW

CheZCheZ

MOTORMOTOR

MalEMalE

RbsBRbsB

MglBMglB

DppADppA

SerSer

AspAsp

MaltoseMaltose

RiboseRibose

D-GalD-Gal

DipeptideDipeptide

GasesGases

TsrTsr

TarTar

TrgTrg

TapTap

AerAer

CheACheACheWCheW

CheRCheR

CheBCheB

CheYCheY

CheZCheZ

FliGFliGFliMFliMFliNFliN

MotA =MotBMotA =MotB

+m+m

-m-m

+P+P

+P+P

-P-P

m = methylationm = methylationP = phosphorylationP = phosphorylation

CHCH33

CheB-PCheB-P

CheBCheB

CheACheA

CheACheA

Che A-PChe A-P CheYCheY

CheY-PCheY-P

Repellent moleculesRepellent molecules

ReceptorReceptor EffectorEffector

CheA

CheW CheV -P

CheY

CheYCheCCheXFliYMotor app

CheZ

Pi

P-

PiSink

P-

CheB

H2O

Methanol

CheD

H2O

NH3

-P

ATP

ADP

-CH3CheR

SAM

Homocyst

L

Structure of Structure of CheYCheY

Structure of Structure of ChA - ChY ChA - ChY ccomplexomplex

Significant flagellar proteinsSignificant flagellar proteins of bacteria of bacteria

FlgK -FlgK - „hook” region„hook” region

FlgD-FlgD-determines the lengthdetermines the length FlgB, C, G - FlgB, C, G - connecting „rod”connecting „rod” FliF - FliF - M-ringM-ring Mot A - Mot A - transmembrane transmembrane

proton-channelproton-channel Mot B -Mot B - linker proteinlinker protein Fli G -Fli G - CheY-CheZCheY-CheZ

Fli M-Fli M- connectionsconnections

Fli N-Fli N-

Flagellar proteinsFlagellar proteins Determined by more than 30 genes organized intoDetermined by more than 30 genes organized into several operonsseveral operons

Their synthesis / expression is regulated by Their synthesis / expression is regulated by Sigma 28Sigma 28 factor factor

„ „Hook associated protein” (Hook associated protein” (HAPHAP) : ) : - nucleation point of flagellins- nucleation point of flagellins- increases the mechanical stability - increases the mechanical stability

Main classes: Main classes: Fli, Flg, FlhFli, Flg, Flh

Characterization of bacterial chemotaxis proteinsCharacterization of bacterial chemotaxis proteins

CheA - histidine autokinase CheA - histidine autokinase P1P1 - 22 amino acids, non inhibited region - 22 amino acids, non inhibited regionP2P2 - 25 amino acis, interacts with CheY - 25 amino acis, interacts with CheY

CheAL (long) -CheAL (long) - His48 His48 autophosphorylation which is a autophosphorylation which is a component of the CheY and CheB activationcomponent of the CheY and CheB activation CheAL – its function is pH-dependent. Optimal CheAL – its function is pH-dependent. Optimal pH 8.1 - 8.9pH 8.1 - 8.9

- - Tar és TrgTar és Trg receptors signalling is turned on receptors signalling is turned on when cytopl. pH decreses below when cytopl. pH decreses below pH 7.6pH 7.6

ChAS (short) – possesses ChAS (short) – possesses kinase activitykinase activity, but the subunit , but the subunit does not autophosphorylatingdoes not autophosphorylating - the aminoterminal 97 aa. long sequence- the aminoterminal 97 aa. long sequence is is missingmissing

CheA hyper kinase – ponit mutation in CheA hyper kinase – ponit mutation in Pro337 Pro337 whichwhich results a results a fasterfaster phosphorylation phosphorylation

CheA - regulates phsphorylation of CheA - regulates phsphorylation of CheVCheV

CheN -CheN - present in present in Bacillis substilisBacillis substilisban and ban and homologue to CheA of homologue to CheA of E. coliE. coli

Characterization of bacterial chemotaxis proteinsCharacterization of bacterial chemotaxis proteins

CheY - CheY - Composed by 128 aa., its phosphorylation results aComposed by 128 aa., its phosphorylation results a conformational change in positions listed below:conformational change in positions listed below:

17, 21, 23, 39, 60, 63, 64, 66, 67, 68, 69,17, 21, 23, 39, 60, 63, 64, 66, 67, 68, 69, 85, 86, 87, 88, 94, 107, 109, 112, 113, 114, 12185, 86, 87, 88, 94, 107, 109, 112, 113, 114, 121

Presence of MgPresence of Mg2+2+ is essential for activation of CheY; is essential for activation of CheY; MgMg2+2+ results the release of salt bond results the release of salt bond Lys109 - Asp 57Lys109 - Asp 57

which makes possible the which makes possible the phosphorylationphosphorylation

Characterization of bacterial chemotaxis proteinsCharacterization of bacterial chemotaxis proteins

HH

P1 P2 P3 P4 P5P1 P2 P3 P4 P5

Phosphorylation RR-bdg. Dimer Catal. CheW rec bdg.

Che A (kb. 650 AA)Che A (kb. 650 AA)

Che Y (kb. 120 AA)Che Y (kb. 120 AA)

NN CC

DD D T/S KDD D T/S K

Mg2+ bdg. Phosphorylation Catal.

NN CC

Characterization of Methyl-Accepting ChemotaxisCharacterization of Methyl-Accepting Chemotaxis proteines (MCP)proteines (MCP)

MCP1 - Tsr, MCP2 - Tar, MCP3 - Trg, MCP4 - TapMCP1 - Tsr, MCP2 - Tar, MCP3 - Trg, MCP4 - Tap H1 - 97 kD pI 5.1; H2 - 86 kD pI 5.1; H3 - 76 kD pI 5.3H1 - 97 kD pI 5.1; H2 - 86 kD pI 5.1; H3 - 76 kD pI 5.3

DcrA - composed by 668 aa., oxygen sensor composed by DcrA - composed by 668 aa., oxygen sensor composed by hem and 2 hydrophobic sequences - hem and 2 hydrophobic sequences - induced by changes in redox-potentialinduced by changes in redox-potential ((Desulfovibrio vulgarisDesulfovibrio vulgaris))

Tlpc - 30% homology with E.coli MCP;Tlpc - 30% homology with E.coli MCP;its defect resulst the loss of pathological chemotaxisits defect resulst the loss of pathological chemotaxis

Methylation is aMethylation is a food molecule dependent food molecule dependent process (e.g. E.coli)process (e.g. E.coli) StarvationStarvation results the methylation of a membrane associated results the methylation of a membrane associated

43kD43kD protein;protein;- in the presence of food the methylation is stopped - in the presence of food the methylation is stopped

The link between the methylation system and activation The link between the methylation system and activation of chemotaxis points to the of chemotaxis points to the essential common phylogeneticalessential common phylogenetical backgroundbackground of chemotaxis receptor and the signalling of chemotaxis receptor and the signalling process.process.

Characterization of Methyl-Accepting ChemotaxisCharacterization of Methyl-Accepting Chemotaxis proteines (MCP)proteines (MCP)

MCP-k demethylationMCP-k demethylation

AttractantAttractant MCPMCP--CHCH33

--CHCH33

CARRIERCARRIER--CHCH33

CARRIERCARRIER--CHCH33 Methanol Methanol + CARRIER+ CARRIERslowslow

rapidrapid

The non methylated intermedier results The non methylated intermedier results „tumbling”„tumbling”,, then the then the ADAPTATION ADAPTATION takes place.takes place.

Characterization of Methyl-Accepting ChemotaxisCharacterization of Methyl-Accepting Chemotaxis proteines (MCP)proteines (MCP)

Detection of MCP-fluorescence in diverseDetection of MCP-fluorescence in diverse phenotype cellsphenotype cells

Adaptation - TumblingAdaptation - Tumbling

Accumulation of cells in in the rings representingAccumulation of cells in in the rings representingoptimal concentrations - adaptationoptimal concentrations - adaptation

Ser ringSer ring

Asp ringAsp ring

Methylation – Effect of carbohydrate type ligandsMethylation – Effect of carbohydrate type ligands

Methylation – Time dependenceMethylation – Time dependence

Chemotaxis - EvolutionChemotaxis - Evolution

Methyl-transferases CheRMethyl-transferases CheR

Homology:Homology:E.coli methyl-transferase methylates MCP of Bac. subst.E.coli methyl-transferase methylates MCP of Bac. subst.

Difference:Difference:Bac. subst. CheRB Bac. subst. CheRB Adaptation to repellentsAdaptation to repellentsE.coli CheREE.coli CheRE Adaptation to attractantsAdaptation to attractants

Methyl-esterases CheBMethyl-esterases CheBHomology: Homology:

Bac.subst. MCPBac.subst. MCPE.coli CheBE.coli CheB + + ATTRACTANTATTRACTANT DEMETHYLATIONDEMETHYLATION

Bac.subst. CheBBac.subst. CheBE.coli MCPE.coli MCP + + ATTRACTANTATTRACTANT

MCP determines the kinetics of reactionsMCP determines the kinetics of reactions

Chemotaxis - EvolutionChemotaxis - Evolution

DEMETHYLATIONDEMETHYLATION

E. coliE. coli

B. subst. B. subst.

C. gelidaC. gelida

Dynamics ofDynamics ofmethanol-productionmethanol-production

and the ligand and the ligand specificity specificity

Chemotaxis - EvolutionChemotaxis - Evolution

Bac.subst. CheYBac.subst. CheY E.coli CheAE.coli CheA

Bac.subst. CheY-PBac.subst. CheY-P E.coli CheZE.coli CheZ

CheY-PCheY-P

CheYCheY

Bac.subst. positive chemotaxis - CheY-PBac.subst. positive chemotaxis - CheY-P

E.coli positive chemotaxis - Chey-PE.coli positive chemotaxis - Chey-P

Bac.subst. and E. coli CheW 28.6% homologyBac.subst. and E. coli CheW 28.6% homologyBac. subst. CheB and E.coli CheY 36% homologyBac. subst. CheB and E.coli CheY 36% homologyBac. subst. and E. coli - M ring and rodBac. subst. and E. coli - M ring and rod

Effect of CaEffect of Ca2+2+ on the bacterial chemotaxis on the bacterial chemotaxis

38kD, Ca38kD, Ca2+2+-binding protein is detectable-binding protein is detectable

CaCa2+2+ channel blockers (e.g. verapamil, LaCl channel blockers (e.g. verapamil, LaCl33) )

disturbs chemotaxisdisturbs chemotaxis

Sigma factorSigma factor

Che ?Che ? Sigma28Sigma28 CheYCheY CheBCheBCheWCheWBas.bodyBas.body

The Sigma28 factor coding gene is part of a 26 kb operonThe Sigma28 factor coding gene is part of a 26 kb operon Regulates synthesis of flagellin, „hook-assoc. protein” (HAP) Regulates synthesis of flagellin, „hook-assoc. protein” (HAP) and some motor proteins and some motor proteins Deficiency: paralytic flagellum; MCP deficiencyDeficiency: paralytic flagellum; MCP deficiency

Measurement of bacterial chemotaxis Measurement of bacterial chemotaxis in 3-channel systemin 3-channel system