Module 711: Molecular Machines
Lecture 7
Bacterial Flagellar Motor
Dale Sanders
2 December 2009
Aims:
By the end of the lecture you should understand…
• The overall structure of the flagellum;
• The significance of “running” and
“tumbling”;
• The structures and roles of the subunits;
• How proton transport might power the
rotor;
• How the flagellar motor might have
evolved.
Reading
Reviews:
• Berg, HC (2003) The rotary motor of bacterial flagella. Annu. Rev.
Biochem. 72: 19-54
• Blair DF (2006) Fine struture of a fine machine. J. Bacteriol. 188: 70337035
• Sowa, Y & Berry, RM (2008) Bacterial flagellar motor. Q. Rev. Biophys.
41: 103-132
• Minamino, T et al. (2008) Molecular motors of the bacterial flagella. Curr.
Opin Struct. Biol. 18: 693-701
Papers:
• Suzuki et al. (2004) Structure of the rotor of the bacterial flagellar motor
revealed by electron cryomicroscopy and single-particle image analysis. J. Mol.
Biol. 337: 105-113
• Sowa et al. (2005) Direct observation of steps in rotation of the bacterial
flagellar motor. Nature 437: 916-919
• Thomas et al. (2006) The three-dimensional structure of the flagellar rotor from
a clockwise-locked mutant of Salmonella entrica serovar Typhimurium. J.
Bacteriol. 188: 7-39-7048
Bacterial Swimming
>50% of bacterial species are motile
• Possess flagella(e):
Monotrichous (single flagellum at one
end)
Amphitrichous (one at each end)
Peritrichous (all over)
• Most work on Salmonella or E.coli
(peritrichous with 10 flagellae);
• Swimming keeps in optimal
environment:
Tactic responses: Chemo-; Photo-;
Osmo-; Ero- (O2); Thermo-…. Taxis
• Tactic responses rely on
sensors/receptors
Swimming Bacteria:
Real and Reconstructed Images
http://www.fbs.osaka-u.ac.jp/labs/namba/npn/index.html
Analysing Flagellar Rotation in Tethered Cells
Leake et al. (2006) Nature 443: 355
Basic properties of swimming in E.coli
• Flagellae are 10 – 20 m long, 10 –20 nm
diameter
• Flagellum rotates like outboard motor
• Typically, rotation rate is 270 revs s-1
(16,000 rpm)
• In some species, rotation is 100,000 rpm
• Flagellar assembly has Mr = 109, of which
about 1% is motor
Bacterial flagellae differ significantly from
eukaryotic counterparts
Major protein
Powered by
Rigidity
Moves as
Directionality
Speed (m/s)
Force (pN)
Efficiency
Eukaryote
Prokaryote
Tubulin
ATP
Undulates
Linear waves
Unidirectional
10
10
50%
Flagellin
H+ or Na+ flux
Rigid corkscrew
Rotations
Bidirectional
30
35
100%
Cross Section of Filament from X-Ray
Crystallography
Sowa & Berry (2008) Q Rev Biophys 41: 103
Direction of rotation, runs and tumbles
• Flagellum can rotate clockwise (CW) or
counterclockwise (CCW)
• Directionality controlled by switch at base of
motor
• CCW rotation: results in straight-line runs:
Last for about 1 s as helix screws through medium
Moves 10 – 20 body-lengths
• CW rotation: results in tumbling
Random changes in direction of swimming
Helical flagellar filaments fly apart
• If no gradient: random movement: runs, tumbles,
runs in random new direction
• If a gradient: Runs are longer, tumbles less
frequent
Signaling and Switching in E. coli in
response to a chemoattractant
• No attractant:
Chemoreceptors in periplasm unoccupied;
Methylaccepting chemotactic proteins
(MCPs) demethylated
 CheA protein phosphorylated
 CheY protein phosphorylated
 Interacts with switch protein FliM
 Clockwise rotation
 Tumbling
• Attractant:
Chemoreceptors occupied
Tumbling less frequent: runs towards source
Overall structure of
the bacterial
flagellum
Berg (2003) Annu Rev Biochem 72: 19
A More Detailed Visualisation of the Important Motor Proteins
Sowa & Berry (2008) Q Rev Biophys 41: 103
Overall architecture
• Filament
• 2 junctional proteins
• Hook: flexible to permit filament to rotate about
different axes
• L and P rings: in outer membrane (L) and
peptidoglycan coat (P) - “bushing” through which
rotating drive shaft passes
• Socket and S ring: in periplasm
• M ring: a 25 nm diameter disk traversing inner
membrane
• C ring: a 45 nm annulus in the cytoplasm
• Studs: 10 membrane particles associated with the
M and C rings
Flagellar Genes
>50 genes encode flagellum and chemosensing proteins
Genes are named after null phenotypes of mutants:
• Fla-: no complete flagellum
3 loci (flg, flh, fli)
40 genes involved in structure, regulation,
assembly
• Mot-: complete flagellum, no rotation
2 genes: motA, motB
• Che-: rotation, but no reversal or chemotaxis
12 genes involved in chemotaxis (inc
receptors)
Roles of Proteins in the
Switch and Motor Complexes
FliN, FliM and FliG
The only 3 genes with mutants giving rise to all 3 phenotypes
All cytosolic and part of the switch complex
FliN
• Mutants giving rise to Che- and Motphenotypes are towards C terminus
• Overexpression of some non-motile alleles
leads to restoration of motor function
Conclude FliN has no role in torque
generation, but probably in stabilization of
structure
FliM
• Phosphorylated CheY binds to FliM
• Most mutations are Che• Overexpression of mutants: some motor
function
Conclude FliM has no role in torque
generation, but is important in switching
FliG
331 residues, with Che- alleles mapping towards middle,
Mot- in C-terminal half
• N terminal not necessary for rotation, assembly or
switching
• Non-polar residues mutated to polar or Pro destabilize
but don’t affect torque generation
• R279, D286, D287 all conserved, involved in torque
generation but not H+ transport because replacement
with neutral residues leads to no loss of function.
Conclude FliG directly involved in torque generation, and
probably in switching
•
MotA and MotB
• 4 and 1 transmembrane spans respectively
Number mutations
Nonmotile phenotypes
Severe phenotypes
Mild phenotypes
Each is part of H+ channel
Mutations in
transmembrane
regions suppress
H+ translocation
Berg (2003) Annu Rev Biochem 72: 19
MotA
• If missing protein restored incrementally,
observe that torque restored in at least 8
equal steps i.e. at least 8 independent
torque generators
Conclude 2 different roles of MotA:
H+ translocation
Utilization of H+ flow to generate torque
Discrete increments in torque generation
as MotA is expressed in E. coli following
addition of inducer IPTG
Berg (2003) Annu Rev Biochem 72: 19
Resurrection traces for the MotA strain (A), the MotAB
strain (B), and the chimera strain (C)
11 levels
Levels 2-10
Levels 1-7, 5-10
Reid S. W. et.al. PNAS 2006;103:8066-8071
MotB
• Most periplasmic, anchors motor to
peptidoglycan layer
• Intergenic suppressors in MotA, FliG,
FliM
• TIRF images of GFP-MotB reveal 22
GFP-MotB molecules per motor
Conclude 3 different roles of MotB:
Anchor; Positioning; H+ channel
TIRF Microscopy of Live GFP-MotB Cells
Leake et al. (2006) Nature 443: 355
TIRF Photobleaching in GFP-MotB Cells to
Estimate Unitary GFP Fluorescence
Leake et al. (2006) Nature 443: 355
Demonstration of 22 MotB Molecules Per Motor in
GFP-MotB Expressing Cells
Leake et al. (2006) Nature 443: 355
Positions of the Proteins
• FliG: Cytoplasmic face of extended M
ring
• FliM & FliN: Part of C ring
• MotA & MotB: Components of 11 studs
Stoichiometries:
FliG: 25-45 (?26: Sowa et al, 2005)
FliM: 35
FliN:  110
MotA and MotB: (4 x 11) and (2 x 11)
Components of the Stator and Rotor
• Since MotB part of MotA/MotB complex
and anchored to peptidoglycan, complex is
part of stator
• FliF an M-ring protein, connects switch
proteins to drive shaft: a rotor protein
• FliF-FliG fusions produce a functional
rotor: thus FliG part of rotor too.
Rotor-Stator Interactions at the Subunit Level
Sowa & Berry (2008) Q Rev Biophys 41: 103
Two Views of the Rotor plus Model for the Stator (Cryo-EM Study)
Stator (MotA, MotB)
8 complexes, but could accommodate 2 or 3 more
M ring (FliF)
FliG
25-fold symmetry
FliG
C ring (contains FliM and FliN):
34-fold symmetry
Thomas et al. (2006)
Some additional facts and questions
• 1200 protons must flow through motor for
complete rotation
• At low turnover rates, can discern 400
steps/rotation, i.e. 3H+/step
• What are the detailed structures of the
motor proteins??
• Are bacteria “intelligent” in sensing and
swimming up gradients??
Putting it all Together…
http://www.fbs.osaka-u.ac.jp/labs/namba/npn/index.html
How Did Bacterial Flagellar Motors
Evolve?
• How could so many proteins co-evolve to
interact and make such a complex
structure?
• A key question of the “Intelligent Design”
lobby.
• We don’t know for sure, but answers are
emerging…
• Evolved from Type III Secretion Systems
Pallen & Matzke (2006) Nature
Microbiol Rev 4: 784-790
Evolution of a Molecular
Motor
• 41 bacterial genomes
analysed
•24 “core” gene products in
the flagellum (boldface)
• Phylogenetic analysis
reveals common
evolutionary history and in
general agreement with
bacterial phylogeny
Liu & Ochman (2007) Proc. Natl. Acad.
Sci. USA 104: 7116
A Homology Network of
Flagellar Core Proteins
• Pairwise comparisons
suggest common
evolutionary origins
between numerous flagellar
proteins
• Potentially just a single,
duplicated precursor
gene??
• Other pairwise “hits” are
in protein secretion systems
Liu & Ochman (2007) Proc. Natl. Acad.
Sci. USA 104: 7116
Summary:
We have covered…
• The overall structure of the flagellum;
• The significance of “running” and
“tumbling”
• The structures and roles of the subunits;
• How proton transport might power the
rotor
• The notion that flagella might have evolved
from protein secretory systems