Rotation of Flagella

1
Adler, 1969
2
Howard Berg in Austin, 1998
3
Outline

• 1. Flagella EM images suggested the rotation
  idea to Berg.
• 2. Support for this idea came from published
  experiments in the literature where
• bivalent antibodies and flagellotropic phages
  blocked motility.
• 3. A specific nut bolt model was proposed for
  how phages interfered with motility.
• 4. Three requirements of this model rotation,
  groove pattern and direction were
• satisfied.
• 5. We learn about filament structure
  polymorphisms, handedness and grooves.
• 6. Direct demonstration of rotation came from
  flagellar tethering experiments.

4
Rotation of Flagella
Berg Anderson, 1973 Silverman Simon,
1974 Samuel et al., 1999
5
What is known (as of 1973)

• Bacteria are motile (1676-Leewenhoek)
• Bacteria can do chemotaxis (1880-Pfeffer/Engelman
  1969-Adler)
• Bacteria have flagella (1909-Reichart-darkfield
  microscopy)
• Motility is dependent upon flagella (1915)
• Flagella appear helical in nature (1960s)

6
Flagella are helical
Bar 1mm
How do they work?
Primitive muscles?
7
M-ring
F. C. Neidhardt. E. coli and Salmonella. 2nd
ed. ASM Press. 1999.
8
Without fantasy, science would have nothing to
test. Without testing, fantasy would be
unchallenged.
Kenneth Boulding
9
Mechanical torque is possible

• M-ring could spin freely in the cytoplasmic
  membrane while anchored to the rod
• The top edge of the M-ring mayform cross-bridges
  with peptidoglycan to produce torque
• It would only take three muscular cross-bridges
  to produce enough power to spin the flagella at
  50 Hz
• (Hz unit of frequency 1 cycle/sec)

10
Two models of motility
Rotate
Flex
11
How can one experimentally distinguish helical
wave vs rotation?
12
Two models of motility
Rotate
Flex
13
Cross-linking flagella affects motility

• Univalent a-flagellin (up to 105/cell) has no
  affect on motility (S. typhimurium or E. coli),
  however, as few as 200 bivalent a-flagellin per
  cell will immobilize half of the cells.

Bivalent a-flagellin has no effect on P.
fluorescens, which has only one flagellum, until
cells begin to link together.
14
c-phage sensitivity is dependent on flagella

• For the phage to infect, motility is required
  i.e. flagella must be active
• Phage bind to Salmonella flagella, stop motility
• Even inactive (irradiated) phage can render
  Salmonella immobile
• Phage binding does not affect the EM morphology
  of the flagella
• A single Bacillus phage can render all the
  flagella non-functional
• ?

Bergs conclusion evidence that flagella rotate.
Bulky phage body prevents filaments from
rotating past each other
15
c-phage sensitivity is dependent on active
flagella

• Berg and Anderson suggest nut and bolt theory
  for phage infection (EM images of full phages at
  tip and empty ones at base of flagellum/irradiated
  phages infect/ phages infect only motile cells)
• Samuel et. al. expand this theory in 1999 to
  suggest that flagella must spin CCW to be
  sensitive

16
Packing of subunits in the flagellar filament
Berg Anderson Phage might use the grooves
between these rows as threads, moving down like
a nut on a bolt, or like children sliding down a
helical ladder.
17
Samuel et al. Testing the Nut and Bolt model

• Three mechanical requirements of the model
• Flagellar rotation
• Correct direction of rotation
• Correct pattern of grooves on filament surface

18
H. C. Berg. 2003. The rotary motor of bacterial
flagella. Annu. Rev. Biochem. 72 19-54.
F. C. Neidhardt. E. coli and Salmonella.
2nd ed. ASM Press. 1999.
Theory predicts 12 different polymorphic forms
(f0 through f11 eg. 10L 1 R .10R 1L etc.),
of which two are straight (pure L and pure R)
and the others are left or right handed. 9 have
been experimentally observed. Transformations
between these forms can be driven by changes in
protein structure (e.g. mutation in the fliC
gene), by changes in pH or ionic environment, or
by mechanical twist.
19
Flagella are helical
Normal left-handed- f2
Curly 1 right-handed
pH 7.0
pH 4.5
Bar 1mm
20
Single filament undergoing polymorphic
transformations from Turner et al., 2000
http//www.rowland.org/labs/bacteria/movies_ecoli.
html ( 4 5)
21
H. C. Berg. 2003. The rotary motor of bacterial
flagella. Annu. Rev. Biochem. 72 19-54.
F. C. Neidhardt. E. coli and Salmonella.
2nd ed. ASM Press. 1999.
Polymorphism is important to the nut and bolt
model, because it predicts that susceptibility
to phage depends of filament surface structure,
not on bacterial motility.
22
Samuel et al. Testing the Nut and Bolt model

• Three mechanical requirements of the model
• Flagellar rotation
• Correct direction of rotation
• Correct pattern of grooves on filament surface

23
Evidence that rotation is required

• A fliC mutant (no filament) is resistant to phage
• A motA mutant is also resistant, but become
  sensitive when MotA is supplied
• Polyhook mutant (hook 1µM long) is sensitive at
  high phage concentration, unless deleted for MotA

24
Samuel et al. Testing the Nut and Bolt model

• If rotation drives c phage then sensitivity
  should depend on
• Flagellar rotation
• Correct direction of rotation
• Correct pattern of grooves on filament surface

25
Direction of rotation is important
When CheY concentration is low (CCW), cells are
sensitive (?) When CheY concentration is high
(CW) cells become resistant ()
26
Samuel et al. Testing the Nut and Bolt model

• If rotation drives c phage then sensitivity
  should depend on
• Flagellar rotation
• Correct direction of rotation
• Correct pattern of grooves on filament surface

27
H. C. Berg. 2003. The rotary motor of bacterial
flagella. Annu. Rev. Biochem. 72 19-54.
F. C. Neidhardt. E. coli and Salmonella.
2nd ed. ASM Press. 1999.
f0, f1, and f2 are sensitive, but f5/6 and f11
are resistant
28
Tethered cells suggest flagellar rotation
(Silverman and Simon)

• Anti-hook antibodies were used to tether cells
  either to each other or the glass slide
  observed pairs of counter rotating cells

29
Rotation of cell tethered to glass slide
http//www.rowland.org/labs/bacteria/movies_tether
ed.html
Movie 4 rotation of bead
Rotation of cell tethered to another cell
30
Swimming Rhodobacter
http//www.rowland.org/labs/bacteria/movies_rsphe.
html
Uniqueness is a commodity in great abundance in
the biological world
Stephen J. Gould
31
There is a wonderful thing about science you
get such an enormous return from such a
trifling investment of fact. Mark Twain
32
Mechanisms/organs that have evolved several
times, independently Eyes, Sound production,
Echo-location, Venomous sting, winged flight.
Evolved only once Flagellar rotation, Language
Never evolved Radiowave transmission or
reception .
33
Questions to ponder
How is the activity of bivalent antibody similar
to that of phage in blocking motility? What is
more important for c infection motility or
flagella rotation? What is the relationship
between polymorphic forms of flagella and c
infection ? Why were two cells linked through
their hooks via a bivalent antibody,
counter-rotating?