1. Bacterial chemotaxis Dr. habil. Kőhidai László 2012.

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

3. Bacterial flagellum - 12-30nm monotrich lopotrich peritrich Main composing protein: flagellin (53.000) pentahelical structure fast regeneration (3-6 min.)  5

4. stator rotor „hook” flagellum S L P M   22.5 nm 27 nm Structure of basal body of bacterial flagellum

5. CCW CW Correlation of swimming types and direction of flagellar rotation in bacteria tumbling

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

7. Gradient Length of linear path Number of tumblings Gradient Length of linear path Number of tumblings

9. E. coli

11. 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 !

12. periplasmaticbinding/transportmolecules sugars dipeptides amino acids chemotaxisreceptpors intracellullar signalling pathway

13. Detection of bacterial cheotaxis receptors receptor clusters division furrow/ring

14. Aspartate receptor ligand binding domain domain „coiled-coil” domain residues for methylation signal transmitter domain

15. COO COO ligand binding domain residues for methylation 8 db szignal transmitter domain in basal activity Composition of Asp receptor

16. COO COO C O O C O O COO COO CH 3 Methylation of Asp chemotaxis receptor methyltransferase methylesterase

17. CheA-P + CheY CheY-P+ CheA CheACheA-P CheY-P + CheZ CheY + P i CW rotation „tumbling” Mg 2+ CheW, CheB-P Repellent molecule 200 ms

18. CheA - activity CheY-P - amount direction of H + transport in the motor region of flagellum is reversed CCW rotation „swimming” Attractant molecule

19. T ap T rg T sr T ar dipeptides galactoseribose Leu, Ser Ni 2+, Asp CheR CheB-P CheB CheA CheA-P CheY CheY-P CheW CheZ MOTOR

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

21. CH 3 CheB-P CheB CheA CheA Che A-P CheY CheY-P Repellent molecules Receptor Effector

22. CheACheW CheV -P CheY CheC CheX FliY Motor app CheZ Pi P- Pi Sink P- CheB H2OH2O Methanol CheD H2OH2O NH 3 -P ATP ADP -CH 3 CheR SAM Homocyst L

23. Structure of CheY

24. Structure of ChA - ChY complex

27. Significant flagellar proteins of bacteria FlgK -„hook” region FlgK -„hook” region FlgD-determines the length FlgB, C, G - connecting „rod” FlgB, C, G - connecting „rod” FliF - M-ring FliF - M-ring Mot A - transmembrane proton-channel Mot A - transmembrane proton-channel Mot B -linker protein Mot B -linker protein Fli G -CheY-CheZ Fli G -CheY-CheZ Fli M-connections Fli N-

28. Flagellar proteins Determined by more than 30 genes organized into Determined by more than 30 genes organized into several operons several operons Their synthesis / expression is regulated by Their synthesis / expression is regulated by Sigma 28 factor Sigma 28 factor „Hook associated protein” (HAP) : „Hook associated protein” (HAP) : - nucleation point of flagellins - increases the mechanical stability Main classes: Fli, Flg, Flh Main classes: Fli, Flg, Flh

29. Characterization of bacterial chemotaxis proteins CheA - histidine autokinase 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 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 when cytopl. pH decreses below pH 7.6  ChAS (short) – possesses kinase activity, but the subunit does not autophosphorylating does not autophosphorylating - the aminoterminal 97 aa. long sequence - the aminoterminal 97 aa. long sequence is missing is missing

30. CheA hyper kinase – ponit mutation in Pro337 which results a faster phosphorylation CheA hyper kinase – ponit mutation in Pro337 which results a faster phosphorylation CheA - regulates phsphorylation of CheV CheA - regulates phsphorylation of CheV CheN - present in Bacillis substilisban and homologue to CheA of E. coli CheN - present in Bacillis substilisban and homologue to CheA of E. coli Characterization of bacterial chemotaxis proteins

31. CheY - Composed by 128 aa., its phosphorylation results a CheY - Composed 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, 85, 86, 87, 88, 94, 107, 109, 112, 113, 114, 121 85, 86, 87, 88, 94, 107, 109, 112, 113, 114, 121 Presence of Mg 2+ is essential for activation of CheY; Presence of Mg 2+ is essential for activation of CheY; Mg 2+ results the release of salt bond Lys109 - Asp 57 Mg 2+ results the release of salt bond Lys109 - Asp 57 which makes possible the phosphorylation Characterization of bacterial chemotaxis proteins

32. H P1 P2 P3 P4 P5 Phosphorylation RR-bdg. Dimer Catal. CheW rec bdg. Che A (kb. 650 AA) Che Y (kb. 120 AA) N C DD D T/S K DD D T/S K Mg 2+ bdg. Phosphorylation Catal. NC

33. Characterization of Methyl-Accepting Chemotaxis proteines (MCP) proteines (MCP) MCP1 - Tsr, MCP2 - Tar, MCP3 - Trg, MCP4 - Tap 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 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 DcrA - composed by 668 aa., oxygen sensor composed by hem and 2 hydrophobic sequences - hem and 2 hydrophobic sequences - induced by changes in redox-potential induced by changes in redox-potential (Desulfovibrio vulgaris) (Desulfovibrio vulgaris) Tlpc - 30% homology with E.coli MCP; Tlpc - 30% homology with E.coli MCP; its defect resulst the loss of pathological chemotaxis

34. Methylation is a food molecule dependent process (e.g. E.coli) Methylation is a food molecule dependent process (e.g. E.coli) Starvation results the methylation of a membrane associated Starvation results the methylation of a membrane associated 43kDprotein; - 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 essential common phylogenetical of chemotaxis points to the essential common phylogenetical background of chemotaxis receptor and the signalling background of chemotaxis receptor and the signalling process. process. Characterization of Methyl-Accepting Chemotaxis proteines (MCP) proteines (MCP)

35. MCP-k demethylation MCP-k demethylation Attractant MCP-CH 3 -CH 3 CARRIER CARRIER Methanol + CARRIER slow rapid The non methylated intermedier results „tumbling”, then the ADAPTATION takes place. then the ADAPTATION takes place. Characterization of Methyl-Accepting Chemotaxis proteines (MCP) proteines (MCP)

36. Detection of MCP-fluorescence in diverse phenotype cells phenotype cells

37. Adaptation - Tumbling

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

39. Methylation – Effect of carbohydrate type ligands

40. Methylation – Time dependence

41. Chemotaxis - Evolution Methyl-transferases CheR Homology: E.coli methyl-transferase methylates MCP of Bac. subst. Difference: Bac. subst. CheRB Adaptation to repellents E.coli CheREAdaptation to attractants

42. Methyl-esterases CheB Homology: Bac.subst. MCP E.coli CheB + ATTRACTANT DEMETHYLATION Bac.subst. CheB E.coli MCP + ATTRACTANT MCP determines the kinetics of reactions Chemotaxis - Evolution DEMETHYLATION

43. E. coli B. subst. C. gelida Dynamics of methanol-production and the ligand specificity

44. Chemotaxis - Evolution Bac.subst. CheY Bac.subst. CheY E.coli CheA E.coli CheA Bac.subst. CheY-P Bac.subst. CheY-P E.coli CheZ E.coli CheZ CheY-P CheY Bac.subst. positive chemotaxis - CheY-P Bac.subst. positive chemotaxis - CheY-P E.coli positive chemotaxis - Chey-P E.coli positive chemotaxis - Chey-P Bac.subst. and E. coli CheW 28.6% homology Bac.subst. and E. coli CheW 28.6% homology Bac. subst. CheB and E.coli CheY 36% homology Bac. subst. CheB and E.coli CheY 36% homology Bac. subst. and E. coli - M ring and rod Bac. subst. and E. coli - M ring and rod

45. Effect of Ca 2+ on the bacterial chemotaxis 38kD, Ca 2+ -binding protein is detectable 38kD, Ca 2+ -binding protein is detectable Ca 2+ channel blockers (e.g. verapamil, LaCl 3 ) Ca 2+ channel blockers (e.g. verapamil, LaCl 3 ) disturbs chemotaxis disturbs chemotaxis

46. Sigma factor Che ? Sigma28CheY CheB CheW Bas.body The Sigma28 factor coding gene is part of a 26 kb operon The 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 deficiency Deficiency: paralytic flagellum; MCP deficiency

47. Measurement of bacterial chemotaxis in 3-channel system