Bacterial chemotaxis
Dr. habil. Kőhidai László
2012.
Diverse swimming
behaviours of
chemotaxis and
their interpretation
regarding
concentration
gradients and cell
size
Bacterial flagellum - 12-30nm
monotrich
lopotrich
peritrich
Main composing protein:
 flagellin (53.000)
 pentahelical structure
 fast regeneration (3-6 min.)
5
{
Structure of basal body of bacterial
flagellum
flagellum
{
22.5 nm
„hook”
L
rotor
P
S
M
stator
27 nm
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
Length of linear path
Number of tumblings
Gradient
Length of linear path
Number of tumblings
E. coli
E. coli
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
Composition of Asp receptor
ligand binding domain
O
C
O
O residues for
C
O methylation
8 db
szignal transmitter domain
in basal activity
Methylation of Asp chemotaxis receptor
O
C
O
O
C
O
methyltransferase
O
C
O
O
C
O
O
C
O
CH3
methylesterase
O
C
O
CH3
Repellent molecule
CheW , CheB-P
200 ms
CheA
CheA-P
CheA-P + CheY
Mg2+
CheY-P + CheZ
Mg2+
CheY-P+ CheA
CW rotation
„tumbling”
CheY + Pi
Attractant molecule
CheA - activity
CheY-P - amount
direction of H+ transport in the
motor region of flagellum is reversed
CCW rotation
„swimming”
dipeptides
Tap
galactose
ribose
Trg
Leu, Ser
Ni2+, Asp
Tsr
Tar
CheR
CheB-P
CheB
CheA
CheA-P
MOTOR
CheW
CheY
CheY-P
CheZ
MotA =MotB
Ser
Tsr
Asp
-m
CheB
Tar
Maltose MalE
Ribose RbsB
D-Gal
MglB
Dipeptide DppA
Gases
Trg
Tap
+P
CheA
CheW
+P
CheY
-P
FliG
FliM
FliN
CheZ
+m
CheR
Aer
m = methylation
P = phosphorylation
Repellent molecules
CH3
CheB-P
CheB
CheA
Che A-P
CheY-P
CheY
CheA
Receptor
Effector
NH3
Homocyst
CheD
H2O
Methanol
CheR
-CH3
CheW
CheV -P
ADP
CheA -P
CheB
SAM
ATP
H2O
CheY
P- Pi
Sink
CheZ
P- CheY
CheC
CheX
Motor app FliY
Pi
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-
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)
P1
N
P2
P3
P4
P5
C
H
Phosphorylation RR-bdg. Dimer
Catal.
CheW
rec bdg.
Che Y (kb. 120 AA)
N
DD
Mg2+ bdg.
D
T/S
K
Phosphorylation
C
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
CARRIER-CH3
MCP-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
E.coli CheRE
Adaptation to repellents
Adaptation to attractants
Chemotaxis - Evolution
Methyl-esterases CheB
Homology:
Bac.subst. MCP
+ ATTRACTANT
E.coli CheB
DEMETHYLATION
Bac.subst. CheB
+ ATTRACTANT
E.coli MCP
DEMETHYLATION
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
Bac.subst.
E.coli
CheY-P
CheY
positive chemotaxis
positive chemotaxis
Bac.subst.
- CheY-P
- Chey-P
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
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

Measurement of bacterial chemotaxis
in 3-channel system