1. Dr. habil. Kőhidai László
Bacterial chemotaxis
Dr. habil. Kőhidai László
2017.

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

3. { Bacterial flagellum - 12-30nm 5 monotrich 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 April; 64(4): 961–973

7. Gradient Gradient Length of linear path Number of tumblings

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. sugars
dipeptides
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. 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

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

17. 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+

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 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

20. 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

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

22. 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

23. Structure of CheY

24. Structure of ChA - ChY complex

27. 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-

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
P amino acids, non inhibited region
P 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
- 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 which results 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) 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.

33. Characterization of Methyl-Accepting Chemotaxis
proteines (MCP)
MCP1 - Tsr, MCP2 - Tar, MCP3 - Trg, MCP4 - Tap
H kD pI 5.1; H kD pI 5.1; H 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. 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.

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
+ ATTRACTANT
DEMETHYLATION
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. 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

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 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

47. Measurement of bacterial chemotaxis
in 3-channel system