Dr. habil. Kőhidai László Bacterial chemotaxis Dr. habil. Kőhidai László 2016.

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

{ Bacterial flagellum - 12-30nm 5 monotrich 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

Gradient Gradient Length of linear path Number of tumblings

E. coli

E. coli

Without flagellum…

Myxococcus xanthus

! 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

sugars dipeptides 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

szignal transmitter domain Composition of Asp receptor ligand binding domain C O C O residues for methylation 8 db szignal transmitter domain in basal activity

Methylation of Asp chemotaxis receptor methyltransferase C O C O CH3 CH3 methylesterase

Repellent molecule CheW , CheB-P CheA CheA-P CheA-P + CheY 200 ms CheA CheA-P CheA-P + CheY CheY-P+ CheA Mg2+ CW rotation „tumbling” CheY-P + CheZ CheY + Pi 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 CheR CheB-P CheA CheW CheZ CheB CheA-P CheY MOTOR galactose ribose Ni2+, Asp Leu, Ser dipeptides Tap Trg Tsr Tar CheR CheB-P CheA CheW CheZ CheB CheA-P CheY MOTOR CheY-P

CheA CheW CheR CheB CheY CheZ Ser Asp Maltose Ribose D-Gal Dipeptide Tsr Tar Trg Tap Aer CheA CheW CheR CheB CheY CheZ MotA =MotB Ser -m Asp +P Maltose FliG FliM FliN MalE +P Ribose RbsB -P D-Gal MglB Dipeptide DppA +m Gases m = methylation P = phosphorylation

Repellent molecules Receptor Effector CH3 CheA CheY-P CheB-P Che A-P

L CheD H2O NH3 -CH3 CheR SAM Homocyst CheW CheV -P CheB H2O Methanol -P ATP ADP CheA CheY CheZ Pi Pi Sink P- CheY P- CheC CheX FliY Motor app

Regulatory role of FliM and CheY binding in CCW – CW switch

Time course of CCW-CW switch CheY-P binding – CCW  CW CheY-P release – CW  CCW

Structure of CheY

Structure of ChA - ChY complex

Significant flagellar proteins of bacteria 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-

Diversity of bacterial chemotactic signaling

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 which results 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) Che Y (kb. 120 AA) P1 P2 P3 P4 P5 N H C Phosphorylation RR-bdg. Dimer Catal. CheW rec bdg. Che Y (kb. 120 AA) N DD D T/S K C 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.

Characterization of Methyl-Accepting Chemotaxis proteines (MCP) MCP-k demethylation -CH3 Attractant MCP-CH3 CARRIER -CH3 rapid CARRIER -CH3 Methanol + CARRIER slow The non methylated intermedier results „tumbling”, then the ADAPTATION takes place.

Detection of MCP-fluorescence in diverse phenotype cells

Adaptation - Tumbling

Accumulation of cells in in the rings representing optimal concentrations - adaptation Ser ring Asp ring

Methylation – Effect of carbohydrate type ligands

Methylation – Time dependence

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 + ATTRACTANT DEMETHYLATION 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.

Chemotaxis - Evolution Bac.subst. CheY E.coli CheA Bac.subst. CheY-P E.coli CheZ CheY-P CheY 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

Che A (~ 650 AA) Che Y (~ 120 AA) P1 P2 P3 P4 P5 N H C Phosphoryl. RR-bdg. Dimer Catalyt. CheW rec bdg. Che Y (~ 120 AA) N DD D T/S K C Mg2+ bdg. Phosphoryl Catalyt.

Sigma factor Che ? Sigma28 Bas.body CheW 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

Chemoreceptor - ??? - Thermoreceptor

Low density Tsr – low methylation Thermophil response High density Tsr and Tar – high methylation Cryophil response

Chemotaxis-related receptors in bacteria

Pseudotaxis encounter agar matrix barrier strait swimming tumbling reorientation swim away

Measurement of bacterial chemotaxis in 3-channel system