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Bacterial chemotaxis. Dr. habil. Kőhidai László 2011. Diverse swimming behaviours of chemotaxis and their interpretation regarding concentration gradients and cell size. Bacterial flagellum - 12-30nm. {. monotrich. 5. lopotrich. peritrich. Main composing protein: flagellin (53.000)
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Bacterial chemotaxis Dr. habil. Kőhidai László 2011.
Diverse swimming behaviours of chemotaxis and their interpretation regarding concentration gradients and cell size
Bacterial flagellum - 12-30nm { monotrich 5 lopotrich peritrich • Main composing protein: • flagellin(53.000) • pentahelical structure • fast regeneration (3-6 min.)
Structure of basal body of bacterial flagellum { flagellum 22.5 nm „hook” { L rotor P 27 nm S M stator
Correlation of swimming types and direction of flagellar rotation in bacteria CCW CW tumbling
R M Berry: Torque and switching in the bacterial flagellar motor. An electrostatic model. Biophys J. 1993 April; 64(4): 961–973
Length of linear path Number of tumblings Gradient Length of linear path Number of tumblings Gradient
Bacterial chemotaxis and adaptation Swimming of cells is influenced NOT ONLY by the changes of concentration of the ligand. ! Adaptation mechanisms refer to the presence of a ‘primitive’ memory of cells
dipeptides sugars amino acids periplasmatic binding/transport molecules chemotaxis receptpors intracellullar signalling pathway
Detection of bacterial cheotaxis receptors division furrow/ring receptor clusters
Aspartate receptor ligand binding domain „coiled-coil” domain residues for methylation signal transmitter domain
O O C C O O Composition of Asp receptor ligand binding domain residues for methylation 8 db szignal transmitter domain in basal activity
O O C C O O O O O O C C C C O O O O Methylation of Asp chemotaxis receptor methyltransferase CH3 CH3 methylesterase
CheA-P CheA CheA-P + CheY CheY-P+ CheA CheY + Pi CheY-P + CheZ Repellent molecule CheW , CheB-P 200 ms Mg2+ CW rotation „tumbling” Mg2+
Attractant molecule CheA - activity CheY-P - amount direction of H+ transport in the motor region of flagellum is reversed CCW rotation „swimming”
Trg Tsr Tar Tap galactose ribose Ni2+, Asp Leu, Ser dipeptides CheR CheA CheW CheB-P CheZ CheB CheA-P CheY MOTOR CheY-P
Tsr CheB Tar CheA CheY CheW Trg CheZ Tap CheR Aer MotA =MotB Ser -m Asp +P FliG FliM FliN Maltose MalE +P RbsB Ribose -P MglB D-Gal Dipeptide DppA +m Gases m = methylation P = phosphorylation
Repellent molecules CH3 CheY-P CheA CheB-P Che A-P CheY CheB CheA Receptor Effector
NH3 Homocyst CheD CheR -CH3 H2O SAM CheV -P Methanol ADP -P CheB ATP H2O Pi Pi Sink P- CheA CheW CheZ L CheY CheY P- CheC CheX FliY Motor app
FlgK - „hook” region FlgD- determines the length FlgB, C, G - connecting „rod” FliF - M-ring Mot A - transmembrane proton-channel Mot B - linker protein Fli G - CheY-CheZ Fli M- connections Fli N- Significant flagellar proteins of bacteria
Flagellar proteins • Determined by more than 30 genes organized into several operons • Their synthesis / expression is regulated by Sigma 28 factor • „Hook associated protein” (HAP) : • - nucleation point of flagellins • - increases the mechanical stability • Main classes: Fli, Flg, Flh
Characterization of bacterial chemotaxis proteins • CheA - histidine autokinase • P1 - 22 amino acids, non inhibited region • P2 - 25 amino acis, interacts with CheY • CheAL (long) - His48 autophosphorylation which is a component of the CheY and CheB activation • CheAL – its function is pH-dependent. Optimal pH 8.1 - 8.9 • - Tar és Trg receptors signalling is turned on • when cytopl. pH decreses below pH 7.6 • ChAS (short) – possesses kinase activity, but the subunit does not autophosphorylating • - the aminoterminal 97 aa. long sequence • is missing
Characterization of bacterial chemotaxis proteins • CheA hyper kinase – ponit mutation in Pro337 whichresults a faster phosphorylation • CheA - regulates phsphorylation of CheV • CheN - present in Bacillis substilisban and homologue to CheA of E. coli
Characterization of bacterial chemotaxis proteins • CheY - Composed by 128 aa., its phosphorylation results a conformational change in positions listed below: 17, 21, 23, 39, 60, 63, 64, 66, 67, 68, 69, • 85, 86, 87, 88, 94, 107, 109, 112, 113, 114, 121 • Presence of Mg2+ is essential for activation of CheY; • Mg2+ results the release of salt bond Lys109 - Asp 57 • which makes possible the phosphorylation
Che A (kb. 650 AA) P1 P2 P3 P4 P5 N H C Phosphorylation RR-bdg. Dimer Catal. CheW rec bdg. Che Y (kb. 120 AA) N C DD D T/S K Mg2+ bdg. Phosphorylation Catal.
Characterization of Methyl-Accepting Chemotaxis proteines (MCP) • MCP1 - Tsr, MCP2 - Tar, MCP3 - Trg, MCP4 - Tap • H1 - 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 hem and 2 hydrophobic sequences - • induced by changes in redox-potential • (Desulfovibrio vulgaris) • Tlpc - 30% homology with E.coli MCP; • its defect resulst the loss of pathological chemotaxis
Characterization of Methyl-Accepting Chemotaxis proteines (MCP) • Methylation is a food molecule dependent process (e.g. E.coli) • Starvation results the methylation of a membrane associated 43kD protein; • - in the presence of food the methylation is stopped • The link between the methylation system and activation • of chemotaxis points to the essential common phylogenetical • background of chemotaxis receptor and the signalling • process.
-CH3 CARRIER -CH3 CARRIER Characterization of Methyl-Accepting Chemotaxis proteines (MCP) • MCP-k demethylation -CH3 Attractant MCP-CH3 rapid Methanol + CARRIER slow The non methylated intermedier results „tumbling”, then the ADAPTATION takes place.
Detection of MCP-fluorescence in diverse phenotype cells
Accumulation of cells in in the rings representing optimal concentrations - adaptation Ser ring Asp ring
Chemotaxis - Evolution Methyl-transferases CheR Homology: E.coli methyl-transferase methylates MCP of Bac. subst. Difference: Bac. subst. CheRB Adaptation to repellents E.coli CheRE Adaptation to attractants
Chemotaxis - Evolution Methyl-esterases CheB Homology: Bac.subst. MCP E.coli CheB DEMETHYLATION + ATTRACTANT Bac.subst. CheB E.coli MCP DEMETHYLATION + ATTRACTANT MCP determines the kinetics of reactions
Dynamics of methanol-production and the ligand specificity C. gelida E. coli B. subst.
Bac.subst. CheY • E.coli CheA CheY-P • Bac.subst. CheY-P • E.coli CheZ CheY Chemotaxis - Evolution • Bac.subst. positive chemotaxis - CheY-P • E.coli positive chemotaxis - Chey-P • Bac.subst. and E. coli CheW 28.6% homology • Bac. subst. CheB and E.coli CheY 36% homology • Bac. subst. and E. coli - M ring and rod
Effect of Ca2+ on the bacterial chemotaxis • 38kD, Ca2+-binding protein is detectable • Ca2+ channel blockers (e.g. verapamil, LaCl3) • disturbs chemotaxis
Sigma factor Bas.body CheW Che ? Sigma28 CheY CheB • The Sigma28 factor coding gene is part of a 26 kb operon • Regulates synthesis of flagellin, „hook-assoc. protein” (HAP) and some motor proteins • Deficiency: paralytic flagellum; MCP deficiency
Measurement of bacterial chemotaxis in 3-channel system