Bacterial Flagellar Motor

Dale Sanders

2 December 2009

Module 711: Molecular Machines

Lecture 7

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 therotor;

• How the flagellar motor might haveevolved.

ReadingReviews:• 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: 7033-7035

• 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 motorrevealed 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 bacterialflagellar motor. Nature 437: 916-919

• Thomas et al. (2006) The three-dimensional structure of the flagellar rotor froma 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 optimalenvironment:

Tactic responses: Chemo-; Photo-;Osmo-; Ero- (O2); Thermo-…. Taxis

• Tactic responses rely onsensors/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 nmdiameter

• 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 whichabout 1% is motor

Bacterial flagellae differ significantly fromeukaryotic counterparts

Eukaryote Prokaryote

Major protein Tubulin Flagellin

Powered by ATP H+ or Na+ flux

Rigidity Undulates Rigid corkscrew

Moves as Linear waves Rotations

Directionality Unidirectional Bidirectional

Speed (m/s) 10 30

Force (pN) 10 35

Efficiency 50% 100%

Cross Section of Filament from X-RayCrystallography

Sowa & Berry (2008) Q Rev Biophys 41: 103

Direction of rotation, runs and tumbles

• Flagellum can rotate clockwise (CW) orcounterclockwise (CCW)

• Directionality controlled by switch at base ofmotor

• 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 tumblingRandom 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 lessfrequent

Signaling and Switching in E. coli inresponse 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 ofthe bacterialflagellum

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 aboutdifferent axes

• L and P rings: in outer membrane (L) andpeptidoglycan coat (P) - “bushing” through whichrotating drive shaft passes

• Socket and S ring: in periplasm

• M ring: a 25 nm diameter disk traversing innermembrane

• C ring: a 45 nm annulus in the cytoplasm

• Studs: 10 membrane particles associated with theM 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 (increceptors)

Roles of Proteins in theSwitch and Motor Complexes

FliN, FliM and FliG

• Mutants giving rise to Che- and Mot-

phenotypes are towards C terminus

• Overexpression of some non-motile allelesleads to restoration of motor function

Conclude FliN has no role in torquegeneration, but probably in stabilization ofstructure

The only 3 genes with mutants giving rise to all 3 phenotypesAll cytosolic and part of the switch complex

FliN

FliM

• Phosphorylated CheY binds to FliM

• Most mutations are Che-

• Overexpression of mutants: some motorfunction

Conclude FliM has no role in torquegeneration, 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 orswitching

• Non-polar residues mutated to polar or Pro destabilizebut don’t affect torque generation

• R279, D286, D287 all conserved, involved in torquegeneration but not H+ transport because replacementwith neutral residues leads to no loss of function.

Conclude FliG directly involved in torque generation, andprobably 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 intransmembraneregions suppressH+ translocation

Berg (2003) Annu Rev Biochem 72: 19

MotA

• If missing protein restored incrementally,observe that torque restored in at least 8equal steps i.e. at least 8 independenttorque generators

Conclude 2 different roles of MotA:

H+ translocation

Utilization of H+ flow to generate torque

Discrete increments in torque generationas MotA is expressed in E. coli followingaddition of inducer IPTG

Berg (2003) Annu Rev Biochem 72: 19

Resurrection traces for the MotA strain (A), the MotABstrain (B), and the chimera strain (C)

Reid S. W. et.al. PNAS 2006;103:8066-8071

11 levels

Levels 2-10

Levels 1-7, 5-10

MotB

• Most periplasmic, anchors motor topeptidoglycan layer

• Intergenic suppressors in MotA, FliG,FliM

• TIRF images of GFP-MotB reveal 22GFP-MotB molecules per motor

Conclude 3 different roles of MotB:

Anchor; Positioning; H+ channel

Leake et al. (2006) Nature 443: 355

TIRF Microscopy of Live GFP-MotB Cells

TIRF Photobleaching in GFP-MotB Cells toEstimate Unitary GFP Fluorescence

Leake et al. (2006) Nature 443: 355

Leake et al. (2006) Nature 443: 355

Demonstration of 22 MotB Molecules Per Motor inGFP-MotB Expressing Cells

Positions of the Proteins

• FliG: Cytoplasmic face of extended Mring

• 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 complexand anchored to peptidoglycan, complex ispart of stator

• FliF an M-ring protein, connects switchproteins to drive shaft: a rotor protein

• FliF-FliG fusions produce a functionalrotor: 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)25-fold symmetry FliGFliG

C ring (contains FliM and FliN):34-fold symmetry

Thomas et al. (2006)

Some additional facts and questions

• 1200 protons must flow through motor forcomplete rotation

• At low turnover rates, can discern 400steps/rotation, i.e. 3H+/step

• What are the detailed structures of themotor proteins??

• Are bacteria “intelligent” in sensing andswimming up gradients??

Putting it all Together…

http://www.fbs.osaka-u.ac.jp/labs/namba/npn/index.html

How Did Bacterial Flagellar MotorsEvolve?

• How could so many proteins co-evolve tointeract and make such a complexstructure?

• A key question of the “Intelligent Design”lobby.

• We don’t know for sure, but answers areemerging…

• Evolved from Type III Secretion Systems

Pallen & Matzke (2006) NatureMicrobiol Rev 4: 784-790

• 41 bacterial genomesanalysed

•24 “core” gene products inthe flagellum (boldface)

• Phylogenetic analysisreveals commonevolutionary history and ingeneral agreement withbacterial phylogeny

Evolution of a MolecularMotor

Liu & Ochman (2007) Proc. Natl. Acad.Sci. USA 104: 7116

A Homology Network ofFlagellar Core Proteins

• Pairwise comparisonssuggest commonevolutionary originsbetween numerous flagellarproteins

• Potentially just a single,duplicated precursorgene??

• Other pairwise “hits” arein 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 therotor

• The notion that flagella might have evolvedfrom protein secretory systems