Bio 119
Motility and Chemotaxis
7/12/11
BOM-12:
4.13
4.15
9.7
16.16.1
Flagella and Motility
Cell Motion as a Behavioral Response: Microbial Taxes
Regulation of Chemotaxis
Motility of Spirochetes
p. 96
p. 102
p. 235
p. 477
Text Review Questions: Chap. 4: #16, 18 Chap. 9: #5
4.13
Flagella and Motility
Motion, or movement, may not be true MOTILITY:
Convection
Brownian Motion
Adhesion to moving objects
Buoyancy: Gas Vesicles
Ameboid Motility does not ocurr in Bacteria or Archaea
Bacterial Flagella
Bacterial “flagella” are mis-named because they are rigid; not a flexible, whip-like structure as are
eucaryotic flagella.
Filament: Up to 15 um length
20 nm diameter
3 nm channel
Polar
Lophotrichous
Perithrichous
Sheathed (endoflaglla) vs Unsheathed
Vibrio sp.
unsheathed peritrichous flagella
and sheathed polar flagellum
1 of 9 Bio 119
Motility and Chemotaxis
7/12/11
Flagellar Structure
Flagella is assembled from 40-50
different proteins.
Flagellar baseplate is structurally
complex and similar in all flagellated
bacteria except for absence of L ring
in Gram-positive species.
Adherents of "Intelligent Design" use
the bacterial flagellum as an example
of "irreducible complexity".The
flagellar "filament" is composed of
many copies of a single protein,
"flagellin". Flagellin Protein Subunit of
Salmonella is a protein of 494 amino
acids
3D Structure of Flagellin
2 of 9 Bio 119
Motility and Chemotaxis
7/12/11
Flagellar Movement
The baseplate of the bacterial flagellum is the only 'wheel' in biology, it is a 'motor' that converts
energy stored in the electrochemical proton gradient into mechanical work (rotation).
Flagellar Synthesis
Flagellar filaments are self-assembled at distal end from single protein monomers which diffuse
through hollow core. Structure (wavelength, amplitude sense) and mechanical properties of filament
are determined by flagellin sequence and chemical environment during self-assembly.
Cell Speed
Flagellar swimming is a luxury, there is a high energy cost in terms of drain on proton gradient.
Approx. 1,000 H+ per revolution. Swimming is suppressed when cells are in poor nutritional status.
Water is a viscous medium to a particle as small as a bacterial cell; when flagellar motor stops, cell
stops immediately-no coasting.
Rotation velocity = 100 rpm approx.
Swimming velocity = 25-100 um/sec approx.
Energy cost = 1,000 protons/revolution/flagellum
4.15
Cell Motion as a Behavioral Response: Chemotaxis and Phototaxis
Chemotaxis
Temporal Gradient vs Spatial Gradient
More than 60 genes are required for flagellar swimming and chemotaxis in E. coli. Approximately 40
dozen of these are for structural components of the flagellum and baseplate. The rest are involved
with chemotactic behavior.
Tactic responses in E. coli are accomplished by a biased random walk composed of alternating
episodes of "running" and "tumbling". The random walk is biased by decreasing the probability of
twiddling when the direction of swimming is favorable.
I SO TR OP I C
C HE MO ATTR AC TANT
C O NC E NTR ATIO N
3 of 9 Bio 119
Motility and Chemotaxis
7/12/11
The transition between running and twiddling is caused by change in direction of flagellar rotation.
d
ccw
cw
ccw
Morphology of flagella during running (a and b) and twiddling (c
and d).
a and c are darkfield light photomicrographs.
b and d are diagrammatic representations.
In b, motors are running CCW and flagellar filaments are CCW
throughout their length; therefore filaments form a bundle.
In d reversal of motors to CW rotation induces a transient
heteromorphic state in which the filaments are CW proximally
and CCW diatally; this disrupts bundle.
In an isotropic environment, rotation is already CCW biased; therefore run times are longer than
twiddle times.
1 SECOND RUNNING
CCW rotation; flagellar filaments in CCW state form bundle at
pole of cell
0.1 SECOND TUMBLING
CW rotation initiates transition from CW state of filament
beginning at the base of the filament. In this transient
heteromorphous state the filament bundle is dispersed
Average Duration
Motor Direction (from outside)
Helical Conformation of Filament
Filament Interaction
RUN
1 to several seconds
CCW
LH
Bundle
TUMBLE
Fraction of second
CW
LH -> RH
Independent
4 of 9 Bio 119
Motility and Chemotaxis
7/12/11
Measuring Chemotaxis
Phototaxis
Other Taxes
9.7
Regulation of Chemotaxis
Step One: Response to Signal
Methyl-accepting chemotaxis proteins (MCP's) in E. coli
TSR
TAR
TRG
TAP
serine/temperature/pH/hydrophobic amino acids
aspartate / maltose (+malE) / heavy metals
ribose (+rbsB) / galactose (+mglB)
dipeptides (+dpp)
All are transmembrane signal transducing proteins. All are constitutive.
Periplasmic domain of MCP binds attractants/repellents directly, or complexed with PBP’s.
Cytoplasmic domain regulates autophosphorylation of CheA in response to ligand (chemoattractant)
binding, subject to modulation by methylation of cytoplasmic domain.
5 of 9 Bio 119
Motility and Chemotaxis
7/12/11
CheA
A “sensor kinase”. cheA autophosphorylation using ATP substrate is regulated by MCP. This in turn is
regulated by chemoattractant binding and by methylation of MCP.
Chemoattractant binding decreases cheA phosphorylation rate. (Note that Fig. 8.27 misleadingly
suggests the opposite.)
Methylation increases cheA phosphorylation rate.
cheA-P phosphorylates both CheY (fast) and CheB (slow).
Che W
“Mediates” interaction of MCP with CheA
Step Two: Controlling Flagellar Rotation
Tumbles are generated by inducing reversal of rotation from CCW to CW.
CheY
When phosphorylated, cheY-P interacts with flagellar motor via fli proteins in baseplate to to induce
tumble. Note typo on p. 226 (fla for fli).
CheZ
Dephosphorylates cheY at constant rate.
Step Three: Adaptation
CheR
Methylates cytoplasmic domain of MCP’s at constant rate.
cheB
When phosphorylated, cheB-P demethylates MCP’s.
Note that the text does not identify a component for de-phosphorylation of cheB-P.
6 of 9 Bio 119
Motility and Chemotaxis
7/12/11
Summary of Signal Transduction and Adaptation
A molecular model of chemotaxis must account for both response and adaptation.
Chemoattractant binding
signals a suppression of
motor reversal by a
complex sensory
transduction mechanism
involving products of genes
cheA, cheW, cheY and
cheZ and fli. Mutaions in
of these genes produce a
phenotype in which cells
motile but are nonchemotactic to all
substances. The product of
cheY interacts directly with
flagellar baseplate to
promote ccw to cw
reversal. Binding of
chemoattractants to
chemoreceptors reduces
interaction and therefore
extends the length of runs.
any
are
the
this
Adaptation to higher
concentrations of
chemoattractant is
accomplished by increasing
the
methylation of the
chemoreceptor on the
cytoplasmic side of the
membrane by decreasing
the
activity of the
methylesterase (cheB).
Thus, the chemosensorchemoattractant binding equilibrium on the periplasmic side of the membrane is a function of the
immediate attractant concentration. The methylation state of the chemosensor on the inside of the
membrane indicates what the binding equilibrium was a short time in the past. This is the molecular
basis of the MEMORY that allows the cell to sense a temporal change in attractant concentration.
In a nutshell:
Attractant Concentration
Attractant Binding to MCP
Methylation of MCP
Tumble Generation
LOW
+
RESPONSE
HIGH
+
-
ADAPTATION
HIGH
+
+
+
7 of 9 Bio 119
Motility and Chemotaxis
7/12/11
16.16.1 Motility of Spirochetes
Spirochete motility is variant of flagellar motility designed to facilitate migration through semisolid
media such as tissue. The flagella (“endoflagella”) are assembled between the plasma membrane and
the outer membrane (i.e. in the periplasmic space). I believe that the “Outer Sheath” referred to in
the text is equivalent to the Outer Membrane of other Gram-Negative Bacteria.
Spirochete motility allows migration through semisolid environments (such as human tissue, in the
case of Treponema palladium).
8 of 9 Bio 119
Motility and Chemotaxis
7/12/11
STUDY QUESTIONS:
1.
Draw a thumbnail sketch of a flagellar baseplate from a Gram-negative baterium. Show the
cytoplasmic membrane, peptidoglycan, outer membrane, the L ring, the P ring, the MS ring
and the C ring. Also show the location of fli and mot proteins. Finally, indicate the path of
protons (H+).
2.
What would be the phenotype of an E. coli strain in which mutation had inactivated the
methyl-accepting chemotactic protein "Tar"? i.e. What characteristics would you use to
distinguish this mutant from wild-type, and from other types of motility/chemotaxis mutants?
3.
How do bacterial flagella differ from eukaryotic flagella?
4.
What makes an E. coli switch from running to twiddling?
5.
How does the mechanism of Spirochete motility differ from flagellar swimming?
6.
Match each of the listed proteins (1-8) with the appropriate function/s (A-K).
1. CheA
_____
A. binds attractants/repellents
2. CheB
_____
B. bound to MCP and CheA; book does not give a function
3. CheR
_____
C. dephosphorylates CheY
4. CheW
_____
5. CheY
_____
6. CheZ
_____
D. methylates MCP at constant rate
E. phosphorylated form interacts with flagellar baseplate (Fli) to
generate tumbles
F. phosphorylated form demethylates MCP
7. Fli
_____
G. phosphorylates CheB
8. MCP
_____
H. phosphorylates CheY
I. regulates the phosphorylation of CheA
J. sensor kinase; autophosphorylates using ATP as substrate
K. switches flagellar baseplate rotation
9 of 9