bacterial chemotaxis dr. habil. kőhidai lászló 2012
TRANSCRIPT
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
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
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
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
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