Bacterial chemotaxis

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

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

5. Correlation of swimming types and direction of flagellar rotation in bacteria CCW CW 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. Length of linear path Number of tumblings Gradient Length of linear path Number of tumblings Gradient

9. E. coli

10. 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. dipeptides sugars amino acids periplasmatic binding/transport molecules chemotaxis receptpors intracellullar signalling pathway

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

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

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

16. 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

17. 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+

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

19. Trg Tsr Tar Tap galactose ribose Ni2+, Asp Leu, Ser dipeptides CheR CheA CheW CheB-P CheZ CheB CheA-P CheY MOTOR CheY-P

20. 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

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

22. 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

23. Structure of CheY

24. Structure of ChA - ChY complex

27. 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

28. 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

29. 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

30. 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

31. 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

32. 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.

33. 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

34. 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.

35. -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.

36. Detection of MCP-fluorescence in diverse 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 CheRE Adaptation to attractants

42. 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

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

44. 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

45. Effect of Ca2+ on the bacterial chemotaxis • 38kD, Ca2+-binding protein is detectable • Ca2+ channel blockers (e.g. verapamil, LaCl3) • disturbs chemotaxis

46. 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

47. Measurement of bacterial chemotaxis in 3-channel system