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

5

{

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” rotor

S L P 27 nm 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 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 a ‘primitive’ memory refer to the presence of of cells

periplasmatic binding/transport molecules sugars chemotaxis receptpors dipeptides amino acids 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 O C C O O

residues for methylation 8 db szignal transmitter domain in basal activity

O O

Methylation of Asp chemotaxis receptor

C O O C C O O

methyltransferase

O O C O O

CH 3

methylesterase

C C O O

CH 3

Repellent molecule CheW , CheB-P

200 ms

CheA CheA-P CheA-P + CheY

Mg 2+

CheY-P + CheZ

Mg 2+

CheY-P+ CheA

CW rotation „tumbling”

CheY + P

i

Attractant molecule CheA - activity CheY-P - amount direction of H

+

transport in the motor region of flagellum is reversed

CCW rotation „swimming”

dipeptides

T

ap galactose ribose

T

rg Leu, Ser

T

sr Ni 2+ , Asp

T

ar CheR CheB-P CheB CheA CheA-P

MOTOR

CheW CheY CheY-P CheZ

Ser Asp Maltose MalE Ribose D-Gal RbsB MglB Dipeptide DppA

Tsr Tar Trg Tap

Gases

Aer MotA =MotB

-m

CheB

+P

CheA CheW

+P

CheY

-P

CheZ

+m

CheR

FliG FliM FliN m = methylation P = phosphorylation

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

NH 3 CheD H 2 O Methanol CheB Homocyst CheW CheA -CH 3 CheR SAM CheV -P -P ADP ATP CheY H 2 O P Pi Sink P CheY Motor app CheC CheX FliY CheZ Pi

Structure of CheY

Structure of ChA - ChY complex

Significant flagellar proteins of bacteria

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

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 substilis

ban 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 Mg 2+ Mg 2+ is essential for activation of CheY; results the release of salt bond Lys109 - Asp 57 which makes possible the phosphorylation

N Che A (kb. 650 AA) P1 P2 P3 P4 P5 H

Phosphorylation RR-bdg. Dimer Catal. CheW rec bdg.

C Che Y (kb. 120 AA) N DD D T/S K

Mg 2+ bdg. Phosphorylation Catal.

C

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 Attractant

MCP

-

CH 3

-

CH 3

rapid

CARRIER

-

CH 3 CARRIER

-

CH 3

slow

Methanol + CARRIER

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 E.coli CheB +

ATTRACTANT DEMETHYLATION

Bac.subst. CheB E.coli MCP +

ATTRACTANT 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 CheY-P  Bac.subst. CheY-P E.coli CheZ 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 Ca 2+ on the bacterial chemotaxis

 38kD, Ca 2+ -binding protein is detectable  Ca 2+ channel blockers (e.g. verapamil, LaCl 3 ) 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