Download thebacterialflagellum

Helsinki University of Technology
Laboratory of Computational Engineering
S-114.500 Basics for Biosystems of the Cell
Fall 2004
6.1.2005
ASSIGNMENT REPORT
THE BACTERIAL FLAGELLUM
Sebastian Köhler
55017P
Index
INDEX
1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 The Flagellum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1 The Engine and Hook. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2 The Propeller. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 The Swimming of a Peritrichous Bacterium. . . . . . . . . . . . . . . . . . . . . .
4 The Structure of Flagellin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 The Formation of the Flagellar Filament . . . . . . . . . . . . . . . . . . . . . . . .
5.1 Self-Assembly and the Export System. . . . . . . . . . . . . . . . . . . . . .
5.2 The Cap Complex. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6 References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
2
3
3
4
6
7
9
9
9
12
1. Introduction
1
INTRODUCTION
The dutch scientist Antonie van Leeuwenhoek was the first to describe bacteria in
the late 17th century [1]. He manufactured microscopes and discovered small
animals in his well water. He described them as “little eels, or worms, lying all
huddled up together and wriggling” [2]. The bacteria he was observing was
spirillum, probably Spirillum Volutans. However, Leeuwenhoek did not see its
flagellum; the propulsion system of bacteria.
The bacterial flagellum was first seen on Chromatium okenii by the german
naturalist and zoologist Christian Ehrenberg about 150 years later in 1836. In 1872
Ferdinand Cohn also saw it on S. Volutans [2].
The bacterial flagellum is a large organelle consisting of over 20 different
proteins, some of them present in only a couple of ten molecules, while others form
huge complexes consisting of tens of thousands of molecules. The flagellum looks
like a long thin string protruding from the bacterium. Different species have
different numbers and arrangements of flagella, largely affecting the way in which
the bacterium swims in a viscous fluid. Bacteria are classified into four groups
based on the flagellum: Monotrichous, Amphitrichous, Lophotrichous and
Peritrichous.
Amphitrichous
Picture 1. A collage of pictures of the four groups of bacteria classified according
to the number and arrangement of flagella [3].
These groups are illustrated in picture 1. Monotrichous bacteria have a single
flagellum. If the flagellum is located at either end of the bacterium it has a
monotrichous polar distribution. An amphitrichous bacterium has one flagellum at
both ends of the body. Lophotrichous bacteria have several flagella in a close
formation at one end of the oblong body and if the flagella are evenly distributed
over the whole body, then the bacterium is called peritrichous.
In this assignment report the structure and composition of the bacterial
flagellum will be discussed, as well as the means by which a peritrichous bacterium
can swim and change direction of movement. Furthermore, the self-assembly of the
2
2. The Flagellum
long, stringlike propeller will be discussed. This report is based on the material
stated in the References - section.
2
THE FLAGELLUM
The propulsion system of bacteria, or the flagellum as it is called, consists of three
parts as illustrated in picture 2. The basal body embedded in the cell wall constitutes
the engine. A short “hook” or “universal joint” is attached to the engine and a long
helical filament: the propeller.
Picture 2. The structure of the bacterial flagellum. The stators (green) and the rotor (red)
embedded in the cell membrane constitute the engine. The protruding hook (dark orange)
is followed by the propeller (lighter orange).
2.1
THE ENGINE AND HOOK
The rotary engine consists of a rotor and stators. It is approximately 30 nm in
diameter. Picture 3 gives a more detailed view of the engine, and as can be seen, the
stators are made of two proteins, MotA and MotB, anchored to the inner membrane.
Two other proteins, S ring and M ring, form the rotor inside the cylindrical stator
complex. There are also many other proteins attached to the rotor, essential to the
operation of the engine.
Torque is generated between the rotor and the stator by the flow of protons
from the outside to the inside of the cell. This is a result of a concentration gradient
between the bacterium and its environment, probably evoked by the cell’s
metabolism. As the protons flow across the cell’s membrane through the stators,
3
2. The Flagellum
which are proton channels, it is thought [2] that a part of the protein MotA moves or
undergoes a conformational change, which exerts a force on the protein FliG. FliG
is attached to the rotor and therefore this would cause the rotor to rotate.
The engine is fully reversible and can reach speeds of ~20000 rpm [4] by
itself, but with an attached filament (propeller) it only reaches 200 – 1000 rpm [5].
Picture 3. A detailed picture of the structure of the engine and hook. [4]
The hook is a joint between the engine and propeller. It is attached to the rotor
through a series of different proteins and allows the helical filament to point away
from the cell. The hook is approximately 55 nm long and consists of a single
protein, FlgE. The hook, like the propeller, which will be discussed next, is hollow.
2.2
THE PROPELLER
The propeller, a helical filament, is a hollow tube 20 nm thick and 10 – 15 µm long
[4]. It is composed of a single protein: flagellin, or FliC, as it is also called. There
are about 30000 flagellin subunits forming the filament. At the end of the filament
there is a cap protein complex. Its importance, as well as the tube’s hollowness, will
be explained later.
The helical filament consists of 11 protofilaments of flagellin subunits twisted
together to form the compact structure. There are about 11 subunits per 2 turns. The
protofilaments, however, come in two different types, resulting in two different
conformations of a straight filament (all 11 protofilaments are of the same type): Rtype (right-handed twist) and L-type (left-handed twist).
4
2. The Flagellum
A study [4] of these two straight filaments revealed that the repeat distance
along the protofilament in the L- and R-type straight filament differ with 0,8 Å (see
picture 5).
Picture 4. A detailed picture of the structure of the helical filament,
which is the propeller. [4]
Picture 5. Comparison between the repeat
distance along the protofilament in the L- and
R-type straight filaments. [4]
Picture 6. 3D electron density maps of the Land R-type straight filaments. Resolution ~10
Å. Scale bar, 100 Å. [4]
The repeat distance in the L-type is 52,7 Å and 51,9 Å in the R-type. This means
that the flagellin subunits are more thightly packed in the R-type protofilaments.
5
3. The Swimming of a Peritrichous Bacterium
There are no wild-type bacteria with straight filaments, since these would not be
very effective propellers. A more effective propeller is supercoiled, i.e. it twists
around itself like a corkscrew. The study of the two straight filaments is the key to
understanding what causes the supercoiling of the filaments in wild-type bacteria.
3
THE SWIMMING OF A
PERITRICHOUS BACTERIUM
As previously mentioned, a peritrichous bacterium has flagella all over its body,
protruding in different directions. A question now arises: How can it move in a
specific direction and how does it change direction?
The helical filament of each flagellum consists of different numbers of L- and
R-type protofilaments, and since the flagellin subunits are more dense in the R-type
protofilaments, these are also slightly shorter. This causes the supercoiling of the
flagellar filament. Depending on the combination of different protofilaments there
are two helical forms of supercoiled filament: left- and right-handed. A normal
filament of wild-type flagellin, has two R-type protofilaments and nine L-type
protofilaments if it is left-handed. The right-handed form is obtained by switching
all R-type protofilaments to L-type, and vice versa [6].
Picture 7. The swimming motion of the peritrichous bacterium. The
movement alternates between two modes: “run” and “tumble”. [7]
When the bacterium moves forward it is said to “run”. In this mode several
left-handed flagellar filaments are bundled together generating the force which
allows the bacterium to move in a specific direction. During this mode of
movement the engine of each flagellum in the bundle rotates counter clockwise, as
seen from outside the cell. To change direction the bacterium quickly reverses the
rotors of the flagella. This sudden twist causes the left-handed supercoiled filaments
to be transformed into right-handed filaments. This, in turn, causes the bundle to
rapidly fall apart.
Now the bacterium is said to be in “tumble” mode. The individual flagella
cannot move the bacterium in any given direction, but the resulting erratic
movement alters the orientation of the bacterium. When the engines are once again
6
4. The Structure of Flagellin
reversed it is headed in a new direction (see picture 8). The average run lasts for
about 1 s and the tumble for only about 0,1 s [2].
Picture 8. The swimming pattern of a peritrichous bacterium. [4]
In the next section we’ll take a look at how the filament can switch between
its left- and right-handed forms as quickly as it does.
4
THE STRUCTURE OF FLAGELLIN
In order to find out how the switching between different supercoiled filaments is
accomplished, the atomic structure of flagellin (the monomer of the filament) had to
be defined. This was done by Namba et al. [6]. They crystallized a fragment of
Salmonella flagellin by clipping of peptides from the carboxy- and aminoterminal
ends. Then, with an improved cryocrystallographic technique, they obtained an
atomic model at 2.0 Å resolution.
Picture 9. The structure of the fragment of Salmonella flagellin. The Cα backbone is to the left
and all hydrophobic side-chains are displayed with the backbone to the right. [6]
This fragment consists of three distinct domains, labeled D1, D2 and D3. The
fragment looks like a boomerang, or a pair of airplane wings, each about 70 Å long
7
4. The Structure of Flagellin
and about 20 Å wide at the center. An interesting part of the flagellin fragment is
the β-hairpin (140-160) in domain D1. Two parallel distorted β-turns are linked at
the tip by a crossed loop (146-153).
The atomic model of the flagellin fragment was docked into an electron
density map of the R-type straight filament (see picture 10) and the fit was almost
perfect. The docking revealed that the flagellin subunits in a single protofilament
are connected through interactions between domain D1 (near the β-hairpin) of the
upper subunit and domain D1 (upper part) and a small part of domain D2a of the
lower subunit.
Picture 10. Docking of the atomic model of flagellin into
an electron density map of the filament. [6]
Because the L-type protofilament repeat is only 0,8 Å longer than the R-type,
the conformational change between these forms are relatively small. To study the
nature of the conformational change Namba et al. used a model of three consecutive
protofilament subunits. The top subunit was fixed and then the lower subunit was
pulled down to see what would happen in the middle subunit. The lower subunit’s
Cα backbone was translated 0,1 Å at each step and energy minimization was
carried out. Up to a dislocation of 4,5 Å the middle subunit stretched elastically, but
after this point over the next 0,2 Å a sudden conformational change was observed in
the β-hairpin in domain D1. This has lead to the belief that it is, in fact, the βhairpin which is responsible for the to distinct conformations of the protofilament
and that it is also the switch which alters the protofilament at subångström
precision. A more extensive model simulation is still needed to prove that this is
actually true.
8
5. The Formation of the Flagellar Filament
5
THE FORMATION OF THE
FLAGELLAR FILAMENT
5.1
SELF-ASSEMBLY AND THE EXPORT
SYSTEM
The bacterial flagellum is formed from the inside of the cell outward, by a process
called self-assembly. In self-assembly the component proteins interact
spontaneously without the aid of enzymes or other factors. In this section the selfassembly of the flagellar filament will be described.
Not until the basal body and the FliF ring has been formed at the inner
membrane (see picture 11) does the self-assembly of the hook and the filament
begin. This is carefully regulated by genetic control; for example, no FliC proteins
are made until the basal body is complete. It is believed that a protein export system
is attached to the FliF ring and that it selectively exports flagellar proteins into the
hollow tube by using the energy of ATP hydrolysis [8]. The hollow tube of the
hook and the filament is approximately 30 Å wide [8].
Picture 11. The bacterial flagellum with the protein
export system attached to the FliF ring. The export
system exports flagellin subunits through the central
channel to the distal end of the flagellar filament. [8]
5.2
THE CAP COMPLEX
In order for the growing process of the filament at its distal end to proceed, a
protein cap complex has to stay attached to the growing end. The cap, made of the
protein HAP2 (Hook Associated Protein 2), is an assembly promoter, and the
flagellin subunits which travel through the central channel polymerize underneath
it. The cap complex is vital to the self-assembly, without it the flagellin subunits
9
5. The Formation of the Flagellar Filament
would simply leak out of the hollow filament. A dimer of the cap complex is shown
in picture 12.
Picture 12. A dimer of the cap complex. (A)
Electronmicroscope image. Scale bars: left long 290
Å; left short 100 Å; bottom 120 Å.
(B) Solid surface representation of the 3D density
map. [8]
Picture 13. (B) Electron cryomicrographs of the
cap-filament complex. (C) Averaged image of
the cap-filament complex. The inner tube and
the plate of the cap complex can clearly be
seen. Scale bar, filament diameter: 230 Å. [8]
As picture 12B shows, the cap complex is a pentameric plate with 5 leg-like
anchor domains. The plate is 120 Å wide and 25 Å thick. Picture 13C shows that
there is a cavity right beneath the pentameric plate of the cap complex. This cavity
inside the filament is roughly 40 Å wide and 70 Å deep [8]. All domains of the
folded flagellin protein, except domain D2, are small enough to pass through the
hollow tube through the hook and the filament. This suggests that the flagellin
subunit is not completely folded when the export system inserts it into the central
channel. This cavity seems to have the right size to allow a flagellin subunit to fold
without interference into its final conformation before it is polymerized into the
filament.
The five legs of the cap complex are also anchored inside the cavity. This
leaves five gaps between the plate of the cap and the filament. These gaps are of
varying shapes and sizes (see picture 14C), but only one is large enough to allow a
flagellin subunit to fit into it. This is the binding site for the next flagellin protein.
As the flagellin binds to its proper place in the growing filament it forces the cap
complex to rotate into the next energetically stable and equivalent position to the
current one. This is accomplished by the “walking” of the anchor legs inside the
cavity. Thus only one flagellin binding site is left open for the next protein subunit.
10
5. The Formation of the Flagellar Filament
Picture 14. Electron density maps of the cap
complex and the tip of the flagellar filament.
(A) Top view of the cap complex attached to
the filament. (B) Side view of the filament.
(C) Five views of the gaps between the plate
and the filament in the directions labeled 1-5 in
(A). Notice the inverted L gap in the first view.
(D) Cross-section of the filament. The cavity
beneath the plate of the cap complex is clearly
visible. (E) Contoured map of the central
section of the cylindrically averaged density.
[8]
Picture 15. The cap complex rotates as new flagellin subunits are inserted at the
open binding site, leaving a new site open for the next subunit. [8]
11
6. References
6
REFERENCES
[1]
[2]
Bra Böckers Lexikon, Vol 14, Bokförlaget Bra Böcker, 1987
Physics today on the Web: Howard C. Berg, Motile Behavior of Bacteria,
http://www.aip.org/pt/jan00/berg.htm
Mary Johnson, Mechanisms of Bacterial Motility,
http://www.indstate.edu/thcme/micro/flagella.html
Keiichi Namba, Dynamic Aspects of the Bacterial Flagellum,
Macromolecular Architecture, 2004; 333-344
Wikipedia - The Free Encyclopedia, http://www.wikipedia.com
Samatey F, Imada K, Nagashima S, Vonderviszt F, Kumasaka T, Yamamoto
M, Namba K, Structure of the bacterial flagellar protofilament and
implications for a switch for supercoiling, Nature, 2001; 410, 331-337
http://microbiology.okstate.edu/faculty/demed2/lecture_notes/
cell%20biologyppt.html
Yonekura K, Maki S, Morgan DG, DeRosier DJ, Vonderviszt F, Imada K
and Namba K, The Bacterial Cap as the Rotary Promoter of Flagellin
Self-Assembly, Science, 2000; 290, 2148-2152
[3]
[4]
[5]
[6]
[7]
[8]
12