|Bacterial Motility - flagella and nanotechnology|
The diagram above shows a model of a close-up view of the basal components of a bacterial flagella.
These are the wheels at the roots of the flagella as mentioned in the introduction to bacteria, if you have
not read this introduction then click here to learn the basics of bacterial structure. The diagrams below add
some labels to this structure. Note that only a tiny portion of the filament is shown here, since this is the
long helix-like propeller that we saw previously. Remember the flagellum rotates like a propeller to propel
the bacterial cell along, as shown by the arrows in the diagram below.
Above: the flagellum emerges from the bacterial surface or wall (or cell envelope)` which consists of
three main layers: the outer membrane (OM), the periplasm (P) which contains a mesh of very strong
fibrous material called peptidoglycan (or murein) and an inner cytoplasmic membrane (CM). (Click
here to learn about cell membranes). Note how different this arrangement is from an animal cell which
has a single cell membrane rather than this double membrane structure. Beneath these layers is the cell
interior (the intracellular compartment) or protoplast (consisting of cytoplasm and nucleoid) and external
to these layers is the extracellular (external) environment, such as the water the bacterium is swimming
in. (Additional layers may exist outside the OM, including a slime capsule, but we shall look at these
possibilities later). This type of wall structure is particular to some types of bacteria called Gram
negative bacteria, but other wall structures occur, as we shall see later. The root of the flagellum
consists of a series of rings (made of proteins) that anchor the structure in the cell envelope.
Above: the flagellar basal complex with some outer structures (Mot proteins) removed to show the
internal structure of the motor. The labels are shown in the figure below:
Above: the flagellar motor rotates, causing the flagellum to rotate (and the cell to rotate in the opposite
sense). Notice the scale bar: the line illustrates a real-life length of 20 nanometres (20 millionths of a
millimetre!) so we are dealing with a minute machine - a nanomachine! These minute electric motors
evolved by natural means, on Earth, long before human beings existed.
The basal structure consists of a series of rings connected by a rod, the rings are the L ring (embedded in
the lipid bilayer of the outer membrane), the P ring (embedded in the periplasm), the S and the M rings
(the rotating motor or rotor) and the C ring. The L and P rings act as a bushing (a bushing is a ring-like
structure that constrains moving mechanical parts, in this case the rotating rod, and may also be lubricated
to reduce friction). The motor proteins (Mot) conduct electric current carried by positively charged
protons ('positive electricity' as opposed to negative electricity in which the current is carried by negatively
charged electrons as in a metal wire) from the periplasm into the cell cytoplasm. The electric charge is
thought to flow into the M (motor) ring where it is converted into rotary mechanical motion, causing the
M-ring to rotate. The M ring is attached to the rod, causing the rod to rotate. The M ring acts against the
fixed Mot (stator) ring, which rotates slowly in the opposite direction to the M ring - slowly because it is
fixed to the bacterial cell wall and so causes the whole bacterial cell to rotate in a direction opposite to the
M ring and rod. The S ring is now known to be part of the rotor, along with the M ring, and forms a socket
for the rod, but is not part of the stator. Confusion may arise when the stator ring is referred to as the
The rod is attached to the filament via a flexible hook (which acts as a universal joint, transferring rotary
motion to the filament via the hook associated proteins (HAPs)). The filament is actually much too long
to show more than a tiny segment of it in these diagrams, it is about 20 nanometres in diameter, but 10 -
15 micrometres (10 - 15 thousand nanometres) long, which is longer than a typical bacterial cell which is
about 2 micrometres long. The filament is made up of about 30 000 subunits of a protein called flagellin
and is a corkscrew or helix shape. (The flagellin is arranged into typically 11 strands that are twined
together). This shape is important, mutants with straight filaments are immotile - the filament is the
propeller driven by this remarkable microscopic electric motor! (The cell body may contribute to thrust in
some forms in which the body is also helical). The helical filament is hollow, and flagellin is transported
from inside the cell, through the C ring (which has a hole in its centre) and along the filament, in its hollow
core, to its tip to which they are added - the filament constantly grows, as it must do to compensate for
breakages. A cap protein forms the tip and stabilises the filament.
Above a diagram of the bacterial flagellum showing the detailed structure of the flagellum basal complex
(in a Gram negative bacterium). The units of protein FliG (about 25-45 units) form a ring extension to
the M-ring (the M ring is shown in section here). Units of the proteins FliM (about 35 units) and FliN
(about 110 units) form the 45 nm diameter C ring, which together with the M and S rings forms the rotor.
This rotor drives the rod, which is a rotor-shaft connected through the centre of the L and P rings to the
hook. The proteins MotA and MotB form a ring of 10 studs embedded in the cytoplasmic membrane,
forming the stator, which is tethered by connections to the rigid Peptidoglycan layer (PG). The L
and P rings act as bushing.The C ring is made up of the proteins FliM and FliN and the Mot proteins
consist of two subunits: Mot A and MotB. The hook associated proteins HAP3 and HAP1 are also known
as FlgL and FlgK respectively. The rod is made up of a variety of proteins and the protein FliF spans the
region between the M and S rings. It is thought that as protons (H+ or hydrogen nuclei) move through
the Mot ring complex, MotA undergoes a shape change, exerting a torque (rotary force) on FliG which
connects to the M ring.
The basal complex anchors the flagellum into the surface of the cell. There are two structural variations
according to the type of bacterial wall it is anchored in. Gram positive bacteria (stain purple with Gram's
stain) like Bacillus possess a thick peptidoglycan wall (about 80 nm) overlying the bilipid cytoplasmic
membrane (CM). In this case the basal body has three rings, the 26 nm diameter M (membrane) ring
embedded in the CM, the S (supramembrane) ring or socket attached to the inner surface of the
peptidoglycan wall by techoic acids and the C (cytoplasmic) ring. The S ring is an extension of the
M-ring, to which it is attached, and both are composed of the same ring of protein FliF, thus the M and S
rings are sometimes considered to be a single double-ring, the MS ring. A rod (7nm diameter) passes
into the S ring socket and its other (distal) end attaches to the hook. More details of these structures are
Above: illustrating the flow of protons (carrying positive electric charge) across the Mot ring from the
periplasm into the cell, powering the motor.
Back to bacteria intro...
Comment on this article!
More articles on bacterial motility:
Models of flagella rotation - how does the rotor work? Click here to find out!
Learn about motility in the strange spirochaetes which drill through materials (including people!).
Learn about gliding and other alternative mechanisms of locomotion in bacteria.
Download an illustrated essay on bacterial motility and navigation in pdf format: Prokaryotes_motility.
How can something as complex as a bacterial flagellum evolve?
Inside the 'circle' of flagella, driven by their CCW rotation, a CW vortex would be established. At this low
Re we would expect no turbulence, so these are orderly vortices and the central CW vortex will spiral
outward toward us (the central dot indicates the head of its velocity vector coming out of the page
toward us). This core of fluid will be displaced, essentially drilled out of the viscous medium and the 6
flagella will close together to fill the void it leaves behind it (fluid flow from outside the flagella, passing
in-between them to fill this void would not readily occur, since the flows of neighbouring vortices tend to
cancel midway between them) and the flagella will bundle together. Fluid will continue to spiral CW
around the bundle as it rotates CCW, propelling the cell.
We have to ask the question: do the flagella or the cell body generate most of the propulsive thrust? For
a spherical or rod-shaped bacteria powered by a single CCW rotating flagellum the cell body will rotate
CW. This rotation of the cell body will not, by itself, generate thrust since, as already discussed, in order
to generate thrust in a high viscous fluid it is necessary to have time-non-reversible flow, which requires
a helix of definite handedness. Perhaps the spiral S-layer of protein that encases many bacteria
(forming an outer layer around the cell envelope) helps break the symmetry and generate thrust. In
coma (vibrioid) and spiral forms, the cell body will indeed displace fluid in a non-reversible manner and
so contribute to the thrust, effectively drilling its way through the medium.
So, what happens when the flagella rotate CW?
If the bundle remained intact then it would begin to pull the cell backwards by displacing a helix of fluid
toward the cell body. However, the external fluid will flow towards the end of the flagellum bundle and
perhaps this drives the bundle apart by forcing its way between the flagella. If this was so, might the cell
be observed to reverse momentarily before tumbling? This raises the question as to how many flagella
must rotate CW for the bundle to separate. If one flagellum only reverses direction, then its flow would
couple with the neighbouring flagellar vortices.
Consider two neighbouring vortices, one rotating CW and one rotating CCW, as shown below:
Vortices generated by the 6 rotating flagella of Escherichis coli
as viewed from behind the cell looking forward along its
direction of locomotion.
This vortex pair will drive fluid in one direction between them and around them – the two vortices are
coupled together. Viscosity will cause the vortex pair to move in the direction of this flow. Some of the
pairs will try to move toward the centre (which they can not do since other flagella in the bundle will block
their path) and other pairs will tend to move away from the bundle – the bundle will disperse.
In addition to changing rotation sense, the flagella also change pitch and this may also contribute to the
decoupling of the flagella and dispersal of the bundle.
Most bacteria alternate between a single-celled stage and some form of multicellular stage during their
life-cycle. Although bacteria rarely form true multicellular organisms, in which cells are in intimate contact
and communicating directly with their neighbours through cell-to-cell junctions, most do form multicellular
aggregates in which cells are separately embedded in a common slime layer, which is attached to a solid
surface and forming a biofilm. Cells in the biofilm are often immotile and certainly lose their ability to swim
as they shed their flagella. Swarmer cells, however, are a dispersive stage and these may float, swim,
crawl or glide away from the biofilm before eventually settling down, adhering to a surface and
establishing a new microcolony which may eventually develop into a biofilm. Sometimes the flagella
assist in adhesion by sticking to a suitable surface they come into contact with. However, soon after
adhering the swarmer cells lose their flagella and establish a microcolony. Bacteria in both the swarmer
cell stage and the biofilm stage maintain a degree of individuality and both are capable of dividing and
Biofilms also disperse by shedding slimy streamers, containing cells, downstream. Generally, dispersal
is more efficient if the biofilm sits upstream of fluid flow. How do the swarmer cells, which are often
released into the stream some 400 or so micrometres above the surface swim in these currents?
Research has shown that Escherichia coli, at least, has a neat trick. In those strains under study it was
found that swimming Escherichia coli (swarmer cells) preferentially circle to the left until they find a
sheltered crevice in a surface, where flow is less, and they swim upstream to establish a new microcolony
Swarming and crawling
The normal swimming flagellated cells that we have considered so far are described as swarmer cells, or
planktonic cells, distinguishing them from the non-flagellated cells that are integral residents in biofilms.
However, ‘swarming’ is usually used for yet another, though closely related, phenomenon – the mass
migration of colonies of bacteria across a solid surface. This swarming behaviour is regulated by quorum
sensing and in Escherichia coli and Salmonella typhimurium involves a switch to a multinucleoid elongate
filamentous hyperflagellated phenotype. These multinucleoid cells, called filaments, are up to 50
micrometres long and have several nucleoids, dispersed at intervals throughout the filament (nucleoid
division and cell wall growth have continued in the absence of cell division). They move as a colony with
the outer layers spiraling outward with the evacuated space inside the colony becoming filled with new
cells. This can give rise to fast colony expansion at rates up to ~3 mm/s (1 cm/h). Spiral and 2D-
branching patterns of colonial growth result and probably acts to optimize nutrient uptake on a solid
substrate in which diffusion is limited (in a solid sheet that completely covers the surface, many cells may
become starved of nutrients due to competition with their neighbours – similar considerations have shown
to predict the branching growth of sponges in 3D in computer models utilizing the diffusion equation).
The hyperflagellated filaments use their many flagella to crawl over solid surfaces, such as agar in a petri
dish. The flagella still bundle, but the cells do not tumble, instead when the flagella disperse the filament
simply stops and the flagella may reform a bundle of the opposite sense resulting in a reversal of
direction of movement. Synthesis of so many flagella appears to be triggered when the bacteria sense
the presence of a highly viscous medium (such as the surface of an agar plate).
A pair of oppositely rotating vortices couple
together and move downwards in this case.
Under typical conditions the filament (at least in Escherichia coli) is a left-handed super-helix (the
flagellin protein subunits that make up the filament are arranged in a helix which is itself coiled into a larger
helix). Counterclockwise rotation of this helix exerts force on the cell body (due to fluid viscosity resisting
the moving filament) causing it to rotate as it is pushed along. In peritrichously flagellated bacteria,
hydrodynamic forces draw the flagella into a bundle when they rotate counterclockwise. Clockwise rotation
of the filaments causes them to fly apart in the bundles of peritrichous enteric bacteria or pulls the cell in
reverse in polarly flagellated bacteria.
Performance of the bacterial flagella motor
Not all bacteria are motile and of those that are more than half use one or more helical flagella, like the
one we have described above. Compared to other bacterial propulsion systems (which we shall look at
later) the flagella propellers have the advantage of speed. For example, a cell of the bacterium Escherichia
coli is about 2 micrometres (2 thousandths of a millimetre) long and has six flagella that originate from
various points on the cell surface but the filaments come together to form a bundle which propels the cell
at about 20 micrometres per second, or ten body lengths per second! This is a clear advantage that more
than pays back the high cost of flagella - each flagellum contains about 1% of the bacterium's total protein
and requires some 50 genes for its production (about 2 % of the genome of about 2 500 genes). The
flagella enable swarmer cells to disperse from the biofilm (colony) and locate new sites for colonisation.
They enable the bacteria to swim to a source of nutrients and to avoid harmful irritants. The drawback of
flagella is that they require a fluid medium in which to work most effectively, although bacteria can use
them to move across moist surfaces. Flagella also fail to work well in highly viscous media, such as the
muddy ooze at the bottom of ponds, and bacteria may employ different propulsion systems in these
The helical filament rotates in a rigid manner as determined by attaching latex beads along the filaments of
mutants in which the filaments are straight. This ruled out the possibility that they undulate like whips (as
do the flagella of many non-bacterial cells) or that they move by winding and unwinding. More recent
experiments using laser dark-field microscopy have enabled individual flagella to be directly observed
rotating. They can flex, however, by virtue of the flexible hook. When the six flagella of Escherichia coli
rotate counterclockwise (CCW) forces exerted on them by the surrounding water (hydrodynamic forces)
force them to come together into a single bundle, trailing behind the tail end of the cell.
The flagella of Vibrio alginolyticus can rotate at about 1000 revolutions per second (rps) and propel the
cells at up to 116 micrometres per second (compare to the flagellum of Escherichia coli with 270 rps and a
top speed of 36 micrometres per second). However, removal of the filament (which loads the motor) may
increase the engine rotation rate to 200 000 rps!
Although many bacteria have a single falgellum at one end or side of the cell, or perhaps one at each end,
so-called polar flagellation, many also have multiple flagella that entwine together to form a rotating
flagellum bundle. This is the type seen in Escherichia coli and Salmonella typhimurium. These bacteria are
peritrichously falgellated - they have several or many flagella dispersed over the cell's surface. Obviously if
flagellum rotated independently then the cell would go nowhere fast! Instead the many flagella bundle
together at the rear of the cell. In order to turn around the flagella change the sense of their rotation,
rotating in the opposite direction, and this mysteriously causes the flagella bundle to fly apart and the cell
tumbles, randomly changing direction, before the flagella switch rotation direction again and come
together as a bundle and the cell moves off in a new direction. A flagella bundle creates the opportunity for
more locomotive force, since more motors are working together to propel the cell and are perhaps also
useful for moving through more viscous fluids.
What causes flagella to bundle or to fly apart?
It is said that ‘hydrodynamic forces’ cause the flagella to bundle, for example during CCW (counter-
clockwise) rotation in the left-handed flagella of Escherichia coli the flagella bundle together. Recall that a
helix or screw can be either left-handed or right-handed. If you look down the axis of a left-handed helix
from behind and then rotate it CCW then it will move forwards as it rotates. A right-handed helix, on the
other hand, will move forwards when rotating CW (clockwise) when viewed in the same manner.
We propose a model using elemntary fluid mechanics. Perhaps we can picture what could be happening
with a simple diagram. The diagram below is the view we would have looking from behind a bacterium with
6 flagella, like Escherichia coli, as it swims forward straight away from us (into the page). The six left-
handed (LH) flagella rotate CCW. Since these are LH helices, the flagella will drive forward into the page (X
marks the tail-end of their velocity vectors). Remember that we have a very low Reynolds number (Re) for
such a small organism and so the water medium is behaving as a very viscous (thick and sticky) fluid.
The table below summarises the various substructures shown in the figures above and their
Page last updated: 1/9/2013. Details about flagellar bundles, swarming, swarmer
cells and protein structure added. Errata concerning the nature of the stator and
end-cap proteins applied.
Above: a hyperflagfellated bacterial filament (this one is 4 times the normal length at about 8 micrometres
and has 20 flagella). When fully developed this cell may have 100 flagella and measure about 50
micrometres in length!
Evolution in (Brownian) space: a model for the origin of the bacterial flagellum
Copyright 2003 by N. J. Matzke
Version 1.0 (last updated November 10, 2003)
(Update section added September 2006.)
E-mail address: matzke@ATncseweb. (please remove obvious anti-spam modification)
Abstract: The bacterial flagellum is a complex molecular system with multiple components required for functional motility. Such systems are sometimes proposed as puzzles for evolutionary theory on the assumption that selection would have no function to act on until all components are in place. Previous work (Thornhill and Ussery, 2000, A classification of possible routes of Darwinian evolution. J Theor Biol. 203 (2), 111-116) has outlined the general pathways by which Darwinian mechanisms can produce multi-component systems. However, published attempts to explain flagellar origins suffer from vagueness and are inconsistent with recent discoveries and the constraints imposed by Brownian motion. A new model is proposed based on two major arguments. First, analysis of dispersal at low Reynolds numbers indicates that even very crude motility can be beneficial for large bacteria. Second, homologies between flagellar and nonflagellar proteins suggest ancestral systems with functions other than motility. The model consists of six major stages: export apparatus, secretion system, adhesion system, pilus, undirected motility, and taxis-enabled motility. The selectability of each stage is documented using analogies with present-day systems. Conclusions include: (1) There is a strong possibility, previously unrecognized, of further homologies between the type III export apparatus and F1F0-ATP synthetase. (2) Much of the flagellums complexity evolved after crude motility was in place, via internal gene duplications and subfunctionalization. (3) Only one major system-level change of function, and four minor shifts of function, need be invoked to explain the origin of the flagellum; this involves five subsystem-level cooption events. (4) The transition between each stage is bridgeable by the evolution of a single new binding site, coupling two pre-existing subsystems, followed by coevolutionary optimization of components. Therefore, like the eye contemplated by Darwin, careful analysis shows that there are no major obstacles to gradual evolution of the flagellum.
Update, September 2006
This essay has now been cited in the literature (Pallen et al. 2006, Evolutionary links between FliH/YscL-like proteins from bacterial type III secretion systems and second-stalk components of the FoF1 and vacuolar ATPases. Protein Science, 15(4), 935-941 - DOI) and linked from a peer-reviewed article I have just coauthored (Pallen and Matzke 2006, From The Origin of Species to the origin of bacterial flagella. Nature Reviews Microbiology, 4(10), 784-790. Advanced Online Publication on September 5, 2006 - DOI). Therefore, in order to avoid confusion, I will not update the text of this article at this address. I have, however, made some minor formatting changes, and updated the Reader Background page.
While Evolution in (Brownian) Space was admittedly a first attempt, and I was a dedicated enthusiast rather than a professional, I think the model has stood up rather well over the last two and a half years. Writing in 2006, I would still agree with about 90% of the 2003 model. To summarize the major updates I would make:
First, the hypothesis of homology between the Type 3 Secretion System export apparatus and the F1F0-ATPase (and its archaeal and eukaryotic equivalents) has been dramatically strengthened by the findings of two papers, Lane et al. 2006 (Molecular basis of the interaction between the flagellar export proteins FliI and FliH from Helicobacter pylori. Journal of Biological Chemistry, 281(1), 508-17 - DOI), and the aforementioned Pallen et al. 2006. As I predicted in 2003, sequence studies have now confirmed homology between FliH/YscL and F0-b (and its equivalents in other ATPases). They also strongly indicate that F1-delta is homologous to the C-terminal domain of FliH; I did not predict this, but it does further confirm my more general prediction of a strong possibility, previously unrecognized, of further homologies between the type III export apparatus and F1F0-ATP synthetase. However, I would retract some of my more speculative suggestions for ATPase homology to FliJ, FliO, and FliP (FliJ and FliO are apparently not even universally required in flagella). I am still hopeful regarding the suggestions for FliQ and FliR.
Secondly, in the 2003 essay I for the most part assumed that the nonflagellar Type 3 Secretion System (NF-T3SS) was derived from the flagellum, rather than being an outgroup with a sister group relationship. I took this position partially to show that even under this assumption the evidence for evolution was strong, and partially because the evidence seemed to lean slightly in that direction. The parsimony argument of Pallen et al. 2005 and various minor points now have me leaning somewhat towards the view that the flagellar and nonflagellar systems are sister groups, and the NF-T3SS is therefore an outgroup. However, as we note in Pallen and Matzke 2006 the scientific community is split on this question. There are several avenues of investigation that might clarify matters, which I will explore in the future.
Thirdly, the question of which proteins are actually universally essential for flagellum function, and which proteins have homology to other flagellar proteins or nonflagellar proteins, has been systematically reviewed in Table 1 of Pallen and Matzke (2006). I have reposted the table in my blog post on Panda's Thumb. It is important to note that this table is much more conservative than the Matzke 2003 homology suggestions, which ranged from well-established to loose speculation. The homologies in the 2006 table are all well-confirmed by standard BLAST techniques, except for five proteins where homology is based on structural or other similarities. Even for these five, two of the flagllar proteins have other known homologies based on sequence (FliC to FlgL and FliH to YscL), two are not universally essential (FliH and FliJ), and three of the homologies have been repeatedly put forward in the literature (FliC to EspA, FliK to YscP, and FliH/YscL to F0-b+F1-delta and equivalents). In the entire list, only one required protein has a new proposed homology that could be considered speculative (FliG, to MgtE).
Many of the homologous and/or inessential proteins found in Table 1 of Pallen and Matzke 2006 were cited in the 2003 paper, but the 2006 table is an authoritative update and supercedes what is said here. The important overall point, as discussed in my blog post, is that of the 42 proteins in Table 1 of Pallen and Matzke, only two proteins, FliE and FlgD, are both essential and have no identified homologous proteins. This is substantially more impressive than the situation in 2003, and means that the evidence for the evolutionary origin of the flagellum by standard gene duplication and cooption processes is even stronger than in 2003. Important specific updates include: a homolog of FlgA has been confirmed (along the lines that I suggested in 2003); FliG has no homolog in NF-T3SS or the Exb/Tol systems, rather it may be homologous to the magnesium transporter MgtE; and the flagellar filament protein FliC (and its sister FlgL) is probably homologous to EspA and other pilus proteins found in NF-T3SS. I still suspect that all of the axial proteins (including FliE and FlgD) are homologous to each other and therefore to pilus proteins in NF-T3SS, but only the confirmed homologies are reported in Pallen and Matzke 2006.
Finally, if I were doing a revision, I would update the terminology along the lines suggested in Desvaux et al. 2006 (Type III secretion: what's in a name? Trends in Microbiology 14(4), 157-160, April 2006 - DOI). As they point out, the terminological distinction between "flagellum" and "type 3 secretion system" is dubious and artificial, and it is more true to acknowledge that flagella have a type III secretion system. Therefore, there are two known groups of type III secretion systems, flagellar and nonflagellar, abbreviated F-T3SS and NF-T3SS.
There is much more to be said about recent research and its implications for flagellum evolution. For the near future I intend to post my thoughts on this in the new flagellum evolution section of the Panda's Thumb blog.
1.1. A complex contrivance
The bacterial flagellum is one of the most striking organelles found in biology. In Escherichia coli the flagellum is about 10 μm long, but the helical filament is only 20 nm wide and the basal body about 45 nm wide. The flagellum is made up of approximately 20 major protein parts with another 20-30 proteins with roles in construction and taxis (Berg, 2003; Macnab, 2003). Many but not all of these proteins are required for assembly and function, with modest variation between species. Over several decades, thousands of papers have gradually elucidated the structure, construction, and detailed workings of the flagellum. The conclusions have often been surprising. Berg and Anderson (1973) made the first convincing case that the flagellar filament was powered by a rotary motor. This hypothesis was dramatically confirmed when flagellar filaments were attached to coverslips and the rotation of cells was directly observed (Silverman and Simon, 1974). The energy source for the motor is proton motive force rather than ATP (Manson et al., 1977). The flagellar filament is assembled from the inside out, with flagellin monomers added at the distal tip after export through a hollow channel inside the flagellar filament (Emerson et al., 1970). The flagella of E. coli rotate bidirectionally at about 100 Hz, propelling the rod-shaped cell (dimensions 1x2 μm) 10-30 μm/sec. The flagella of other species, powered by sodium ions rather than hydrogen ions, can rotate at over 1500 Hz and move cells at speeds of several hundred μm/sec. The efficiency of energy conversion from ion gradient to rotation may approach 100% (DeRosier, 1998). The bacterial flagellum is now one of the best understood molecular complexes, although numerous detailed questions remain concerning the function of various protein components and the exact mechanism of torque generation. However, the origins of this remarkable system have hardly been examined. This article will propose a detailed model for the evolutionary origin of the bacterial flagellum, along with an assessment of the available evidence and proposal of further tests. That the time is ripe for a serious consideration of this question is discussed below.
1.2. An evolutionary puzzle
Biologists find it almost inescapable to compare the bacterial flagellum to human designs: DeRosier remarks, More so than other structures, the bacterial flagellum resembles a human machine (DeRosier, 1998). The impression is heightened by electron micrograph images (Figure 1) reminiscent of a engine turbine (e.g., Whitesides, 2001), and the scientific literature on the flagellum is filled with analogies to human-designed motors. There is no shortage of authorities willing to express mystification on the question of the evolutionary origin of flagella. In a 1978 review, Macnab concluded,
The basic puzzle is that the flagellum is made up of dozens of protein components, and deletion experiments show that the flagellum will not assemble and/or function if any one of these components is removed (with some exceptions). How, then, could this system emerge in a gradual evolutionary fashion, if function is only achieved when all of the required parts are available?
1.3. Theory: the evolution of systems with multiple required components
The standard answer to this question was put forward by Darwin. Mivart (1871) argued that the incipient stages of useful structures could not have evolved gradually by variation and natural selection, because the intermediate stages of complex systems would have been nonfunctional. Darwin replied in the 6th edition of Origin of Species (Darwin, 1872) by emphasizing the importance of change of function in evolution. Although Darwins most famous discussion of the evolution of a complex system, the eye, was an example of massive improvement of function from a rudimentary ancestor (Salvini-Plawen and Mayr, 1977; Nilsson and Pelger, 1994), Darwin gave equal weight to examples of functional shift in evolution. These included the complex reproductive devices of orchids and barnacles, groups with which he was particularly familiar (Darwin, 1851, 1854, 1862). Intricate multi-component systems such as these could not have originated by gradual improvement of a single function, but if systems and components underwent functional shift, then selection could have preserved intermediates for a function different from the final one. The equal importance of improvement of function and change of function for understanding the evolutionary origin of novel complex systems has been similarly emphasized by later workers (Maynard Smith, 1975; Mayr, 1976). Recent studies give cooption of structures a key role in the origin of feathers (Prum and Brush, 2002), and novel organs (Pellmyr and Krenn, 2002); Mayr (1976) gives many other examples. Computer simulations also show the importance of cooption for the origin of complex systems with multiple required parts (Lenski et al., 2003).
Do these common insights from classical, organismal evolutionary biology help us to understand the solution to the puzzle Macnab put forward regarding the origin of flagellum? Cooption at the molecular level is in fact as well-documented at it is at the macroscopic level (Ganfornina and Sanchez, 1999; Thornhill and Ussery, 2000; True and Carroll, 2002). It has been implicated in origin of ancient multi-component molecular systems such as the Krebs cycle (Melendez-Hevia et al., 1996) as well as the rapid origin of multi-component catabolic pathways for abiotic toxins that humans have recently introduced into the environment, such as pentachlorophenol (Anandarajah et al., 2000; Copley, 2000), atrazine (de Souza et al., 1998; Sadowsky et al., 1998; Seffernick and Wackett, 2001), and 2,4-dinitrotoluene (Johnson et al., 2002); many other cases of catabolic pathway evolution exist (Mortlock, 1992). All of these systems absolutely require multiple protein species for proper function. Even for some molecular systems equaling the flagellum in complexity, reasonably detailed reconstructions of evolutionary origins exist. Generally these are available for systems which originated relatively recently in geological history, which are well-studied due to medical importance, and where phylogeny is relatively well resolved; examples include the vertebrate blood-clotting cascade (Doolittle and Feng, 1987; Hanumanthaiah et al., 2002; Jiang and Doolittle, 2003) and the vertebrate immune system (Muller et al., 1999; Pasquier and Litman, 2000).
Thornhill and Ussery (2000) summarized the general pathways by which systems with multiple required components may evolve. They delineate three gradual routes to such systems: parallel direct evolution (coevolution of components), elimination of functional redundancy (scaffolding, the loss of once necessary but now unnecessary components) and adoption from a different function (cooption, functional shift of components); a fourth route, serial direct evolution (change along a single axis), could not produce multiple-components-required systems. However, Thornhill and Usserys analysis did not distinguish between the various levels of biological organization at which these pathways might operate. The above-cited literature on the evolution of complex molecular systems indicates that complex systems usually originate by a key shift in function of an ancestral system, followed by an intensive period of improvement of the originally crudely functioning design. At the level of the system, cooption is usually the key event in the origin of the modern system with the function of interest. However, a great deal of the complexity in terms of numbers of parts is added to the system after origination. These accessory parts get added by duplication and cooption of novel genes (for reviews of gene duplication in evolution, see Long, 2001; Chothia et al., 2003; Hooper and Berg, 2003) and/or duplication and subfunctionalization (Force et al., 1999) of genes already involved in the crudely-functioning system. Cooption of whole subsystems, linking them to the core system, may also occur.
Therefore, improvement of function at the system level might be implemented by cooption at the level of a protein or subsystem. Change of function at the system level might occur without any lower level cooption of new components. Thornhill and Usserys four routes can be reduced to the two major pathways proposed by Darwin: improvement of current function (optimization) and shift of function (cooption). Cooption remains its own category, while the other three routes (serial direct evolution, parallel direct evolution, and elimination of functional redundancy) can be considered as three versions of functional improvement, with the lower-level components undergoing optimization, coevolutionary optimization, or loss, respectively. This conceptual framework is basically equivalent to the patchwork model for the evolution of metabolic pathways (Melendez-Hevia et al., 1996; Copley, 2000), where components are recruited from diverse sources and functional improvement or functional shift might occur at any organizational level, e.g. system, subsystem, protein, or protein domain.
1.4. Constructing and testing evolutionary models
In order to explain the origin of a specific system such as the flagellum, the general theory discussed above must be combined with the available evidence in order to produce a detailed, testable model. Detail in evolutionary scenarios makes them more testable, not less: Cavalier-Smith argues that Specifying transitional stages in considerable detail is not unwarranted speculation, but a way of making the ideas sufficiently explicit to be more easily tested and rigorously evaluated (Cavalier-Smith, 2001b). Obviously detailed cannot mean that every mutation and substitution event be recorded for events that occurred billions of years ago this is impossible. A detailed evolutionary model should reduce a puzzling event like the origin of the flagellum into a series of events that occur by well-understood processes.
In an ideal model, the origin of every protein component will fulfill three criteria. First, a putative ancestral protein with a different function (a homolog that can reasonably be suspected to precede the flagellum) should be identified. Second, the cooption of the protein should occur by a reasonably probable mutation event -- e.g., a mutation produces a single new binding site enabling one protein to act on another. Initially this new complex functions crudely, but can gradually be perfected by coevolutionary optimization of the two proteins. Third, the selective regime favoring retention of the coopted protein should be identified. Each of these three criteria encourages further testing against new data. Hypothesized homologies can be assessed by new data, for example by detailed sequence analysis or the comparison of protein structures. The plausibility of mutational steps can be investigated by examination of similar mutations observed today; and the selective forces invoked can be assessed by study of analogies and by mathematical modeling. Furthermore, an evolutionary model might have testable implications for other fields: for example, if a biological system is hypothesized to be derived from a homologous system, similarities in mechanism between the two systems would be suspected. The fact that we do not have all of the data that we would like, and that uncertainty is high, are not problems unique to evolutionary models; rather, these problems are commonplace in any advancing science. For example, many contradictory models have been published for the mechanism of motor action in the flagellum, and most (or all) of them must be wrong, but this has not stopped anyone from proposing new models (Schmitt, 2003). Science is advanced by proposing and testing hypotheses, not by declaring questions unsolvable.
2.1. Modern flagella
The canonical flagellum of E. coli is shown in Figure 2. Descriptions of the structural components are given in Table 1. Cytoplasmic components involved in regulation and assembly, as well as the chemotaxis components, are listed in Table 2. Excellent overviews of flagellar function and assembly are available elsewhere (Berg, 2003; Macnab, 2003) and so will not be discussed further here.
Table 1: Structural components of the E. coli flagellum. Based on recent reviews (Berg, 2003; Macnab, 2003); figures in parentheses represent suggestions made in this paper. Components with an asterisk (*) are not included in the final structure.
Table 2: Components of the E. coli regulation/assembly and chemotaxis systems. Cytoplasmic components based on Berg (2003) and Macnab (2003), chemotaxis components based on Eisenbach (2000).
2.2. Previous attempts to explain flagellar origins
2.2.1. Short discussions
Occasional examples of very general suggestions about the evolutionary origin of flagella can be found in the literature, for example in discussions of how various aspects of the chemotaxis system are optimized (Berry, 2000); in the suggestion that prokaryote flagella may have been a relatively late invention, after biofilms and microbial mats had become well-developed and crowding on surface habitats became a problem (Stoodley et al., 2002); or in the alleged common ancestry of archaeal and bacterial flagella (Harshey and Toguchi, 1996). Archaeal and bacterial flagella were indeed once thought to be homologous (Jones et al., 1987), but they are actually totally distinct motility systems (Jarrell et al., 1996; Faguy and Jarrell, 1999; Thomas et al., 2001). Although both kinds of flagella rotate and are superficially similar, archaeal flagella are fundamentally different in many respects (Table 3). In archaeal flagella, the filaments are thinner, lack a central channel, and subunits are added from the base rather than the tip. Forward movement is typically attained by clockwise rather than counterclockwise motion. Additionally, archaeal flagella are probably powered by ATP rather than protonmotive force (suggested by homologies of FlaI to PilT/U (Jarrell et al., 1999; Thomas et al., 2001; Merz and Forest, 2002, although the literature is contradictory: Bardy et al. (2003) assert that archaeal flagella use protonmotive force, but cite no supporting evidence). Finally, the homologies of the two flagella to nonflagellar secretion systems are different. The bacterial and archaeal flagella are therefore a classic case of analogy, not homology (Faguy et al., 1994; Jarrell et al., 1996; Bayley and Jarrell, 1998; Faguy and Jarrell, 1999; Thomas et al., 2001; Thomas et al., 2002; Bardy et al., 2003). However, the misperception persists in the assumption that the flagella (Harshey and Toguchi, 1996; Campos-Garcia et al., 2000; Rizzotti, 2000) or their basal bodies (Cavalier-Smith, 2002a, 2002c) are homologous. On the other hand, the chemotaxis systems are indeed homologous, and are shared with nonflagellar motility systems as well (Faguy and Jarrell, 1999; Koretke et al., 2000).
Table 3: Some microbial motility systems. Several more mysterious systems (the perhaps cytoskeleton-based motilities of Mycoplasma and Spiroplasma; Trachtenberg et al., 2003) have been excluded. Prokaryotes undoubtedly have additional motility systems that have not yet been discovered. Only one eukaryote system, the cilium or eukaryotic flagellum, is included in the table, because it is often confused with the prokaryote systems even though it is totally distinct. Many other eukaryote motility systems, not relevant here, are not listed. Data gathered from many sources (Young et al., 1999; Eisenbach, 2000; McBride, 2001; Thomas et al., 2001; Bardy et al., 2003; Youderian et al., 2003).
A slightly more detailed attempt at explaining the origin of the bacterial flagellum was made by de Duve (1995), who apparently got the bacterial flagellum confused with the completely different eukaryotic cilium (also known as the eukaryotic flagellum or undulipodium in an interminable terminological dispute; see Corliss, 1980; Margulis, 1980; Cavalier-Smith, 1982). He suggested that the flagellum, which he acknowledges is rotary, was somehow descended from a simpler ATP-powered filament-bending motor. In a more reasonable vein, de Duve then gave a brief scenario for the gradual origin of chemotactic behavior from random swimming, but was again puzzling in postulating that essentially fully functional, bidirectional-switching flagella with specific positioning on the cell surface existed before the signal transduction system was coupled to the flagellum. What the purpose of switching would be without a chemotaxis system was not explained. De Duve furthermore stated that these well-developed but non-chemotactic flagella gave little advantage until they were chemotactically enabled, leaving unexplained the selective reason for the origin of the whole nearly-complete system in the first place.
Finally, Goodenough (1998; 2002) offers a short account deriving a flagellum from a proton-transducing membrane channel. She postulates that a coopted protein increased the efficiency of proton transport, and rotated the channel as a by-product. Later binding of a filament to the outside of this rotating channel produced primitive motility which increased food gathering ability. However, the original function of proton transport (which, uncoupled to another process, would simply de-energize the cytoplasmic membrane) is not specified. In her 2002 account Goodenough suggested that a fibrous protein binding to the F1F0-ATP synthetase produced the proto-flagellum. Presumably she meant that the proto-filament would bind to the distal side of a c-subunit of F0. As recent work indicates that F0-c and F1-εγ rotate inside the F0-ab and F1-αβδ complex (Weber and Senior, 2003), Goodenoughs suggestion is not immediately impossible, but suffers difficulties similar to those discussed for Rizzotti (2000), below.
2.2.2. Cavalier-Smith (1987)
Cavalier-Smith is one of the few who has proposed detailed hypotheses for the origin of many fundamental features of eukaryotes and prokaryotes (Cavalier-Smith, 1987a, 1987b, 2001a, 2002b, 2002a, 2002c). He bases his work on a refreshingly clearly-stated philosophy for reconstructing the origin of complex systems, advocating a holistic approach considering environment, organism, mutation, and selection all together and emphasizing testability (Cavalier-Smith, 2001a). Although Cavalier-Smith has addressed the origin of the eukaryotic cilium on several occasions (Cavalier-Smith, 1978, 1982, 1987b, 2002b), Cavalier-Smiths only treatment of the origin of the bacterial flagellum is found in a 1987 article (Cavalier-Smith, 1987a). He makes two suggestions: first, that a mutant version of an outer membrane protein pore formed a tubular polymer extending through the outer membrane into the extracellular medium. Linking this to proton-conducting proteins in the cytoplasmic membrane provided the primitive motor. In this scheme, spirochete axial filaments were derived from regular flagella. His second suggestion was that flagella evolved from gliding motility systems, which are also widespread and powered by protonmotive force. Some early models of gliding motility postulated a spirochete-like mechanism, with rotating filaments in the periplasmic space, and on this basis spirochetes might represent a transitional stage. Motility would develop from rotating filaments first used just to stir the fluid in the periplasmic space and increase diffusion of nutrients. On either scenario, the rotary mechanism existed from the beginning of the evolutionary sequence, and the first crude motility function would have been selected for because it increased random dispersal, useful in overcrowded regions depleted in nutrients. Much of the complexity could have post-dated the original crudely functioning motility.
Cavalier-Smith was hampered by the relatively primitive state of knowledge at the time, and he conceded that the actual evolutionary process must have been much more complicated than his suggestions. The linkage between the filament and motor is very complex, mediated by about ten proteins, and the filament subunits are secreted through the base of the flagellum via a type III export pathway, rather than via a type II pathway as might be expected for a protein derived from an outer membrane pore; type III virulence systems do utilize an outer membrane secretin secreted by the type II pathway, and the flagella P- and L-ring proteins FlgI and FlgH are similarly secreted via the type II pathway (Macnab, 2003). A secretin might therefore be more likely posited as the source for FlgH; this will be discussed in more detail below.
Regarding the postulated homology between gliding motility and the axial filaments of spirochetes, today it is apparent that gliding motility is not a matter of rotating periplasmic filaments. Two mechanisms for gliding motility have been clearly identified (Merz and Forest, 2002; Bardy et al., 2003). First, the social gliding of Myxococcus xanthus occurs via retraction of type IV pili, sometimes also called twitching motility (Merz and Forest, 2002). Second, the adventurous motility of M. xanthus is driven by the secretion of a polysaccharide gel (slime) via the junctional pore complex; a similar complex is found in gliding cyanobacteria. The mechanism of the gliding motility of Cytophaga and Flavobacterium is still a matter of speculation (McBride, 2001), but may involve a ratchet structure and slime secretion (Bardy et al., 2003). These latter forms of gliding motility inspired the comparison between flagella and gliding motility as they are powered by protonmotive force, and beads attached to the cell surface of Cytophaga will rotate (Eisenbach, 2000). Thus, it is occasionally suggested (Cavalier-Smith, 2002a), even in textbooks (e.g. Campbell, 1993), that flagella and gliding motility are homologous, and the gliding motility apparatus may be some version of the flagellum basal body without the flagellar filament. As our understanding of slime-related gliding motility is still limited (the relevant genes are still being identified, much less detailed mechanism or structure), the possibility of any connection between type III protein secretion and polysaccharide secretion is difficult to evaluate. However, the study of gliding motility bears close watching: the recent discovery of homology between M. xanthus gliding motility proteins AglS/AglV to TolR and of AglR/AglX to TolQ (Youderian et al., 2003) which are in turn homologs of the flagellar motor proteins MotA and MotB (Cascales et al., 2001) suggests that there may be a common mechanism for coupling proton flow to motility. If the general similarity between the junctional pore complex and type III secretion systems (Spormann, 1999; Merz and Forest, 2002) turns out to be more than skin deep, then the common descent of gliding motility and flagella from an ancestral motility organelle will have to be seriously considered. Cavalier-Smiths suggestion that stirring the periplasmic fluid may have been a precursor to primitive motility is similar to Rizzottis main suggestion and will be discussed in the next section.
2.2.3. Rizzotti (2000)
The only major recent attempt at explaining the origin of the flagellum is that of Rizzotti (2000), which, like Goodenough, proposes that the flagellum was derived from the F1F0 ATP synthetase. The initial appeal of this hypothesis derives from the spate of recent comparisons between the flagellum and ATP synthetase as proton-driven, rotary motors (Block, 1997; Boyer, 1997; Khan, 1997; Sabbert and Junge, 1997; Berg, 1998; Oplatka, 1998a, 1998b; Berry, 2000; Walz and Caplan, 2002), sometimes leading to the suggestion of homology (Oster and Wang, 2003). These comparisons go back at least to Cox et al.s (1984) proposal that the ATP synthetase had a rotary mechanism, and continued through the testing and refinement of this hypothesis (Mitchell, 1985; Sabbert and Junge, 1997; Weber and Senior, 2003), followed by the conclusive demonstration of rotation by direct observation of an actin filament tethered to the gamma subunit of F1-ATPase (Noji et al., 1997). A relationship between the F1F0 ATP synthetase and the flagellum is further suggested by homology between the flagellar ATPase FliI and the β subunit of F1-ATPase, indicated by ~30% sequence similarity (Albertini et al., 1991; Vogler et al., 1991). The α and β subunit ATP synthetase subunits are themselves paralogous, with only the β subunit retaining catalytic activity (Gogarten et al., 1989; Gogarten and Kibak, 1992).
In a creative scenario (Figure 3), Rizzotti imagined that an accidental insertion in the middle of the F1-γ subunit created a short filament outside the cytoplasmic membrane, between the membrane and the cell wall. As the synthetase subunits rotated, this protofilament served to mix the nearby fluid, increasing the diffusion of molecules in and out of the cell. This provided sufficient selective benefit to retain the mutation. Production of a more sophisticated mixing instrument occurred via duplication and modification of the mutant γ subunit, so that branches of the filament extended above the cell wall. In the process, the ε and δ subunits were lost, along with ATPase activity, resulting in a proton-powered stirring mechanism with incipient motility function. From here, a process of optimization ensued. Selection first favored random motion of the cell that further improved nearby fluid mixing and diffusion. More powerful motility followed by extension of the filament and by duplications of the proton-transmitting proteins of the stator (in this scenario, derived from the c subunit of the F0 structure). The F1-αβ complex apparently became the rotor inside the stator ring. Rizzotti concluded by discussing a number of other steps that must have happened along the way, although the order is not specified. However, it seems that he considered the origin of the export apparatus a relatively late event. Rizzotti hypothesized that once the central cavity became large enough, a secretion complex (presumably a type III export apparatus already functioning elsewhere) was patched in at the base of the rotor, allowing the secretion of a more complex filament.
Rizzotti argued that bacteria with a single membrane were simpler and therefore probably ancestral to gram-negative bacteria with both an inner and outer membrane. He hypothesized that the outer membrane arose as an alimentary adaptation from extensions of the inner membrane. The L- and P-rings arose as the developing outer membrane encroached on the flagellum (gram positive bacteria, lacking outer membranes, have no requirement for the L- and P-rings and lack them altogether). Rizzotti discounted the alternative scenario, whereby the flagellum arose in a bacterium already possessing a double membrane, because he deemed the simultaneous origin of the rings and filament too difficult.
This scenario is considerably more detailed than any other available, but remains vague on the specific origin of almost all of the proteins that make up the flagellum. Although Rizzotti does make use of some interesting similarities between the flagellum and ATP synthetase, and he is able to come up with a proposal that includes rotary motion from the beginning, there are major flaws which shall be discussed shortly. Before the critique, however, it is worth noting that Rizzottis scenario has been cited by Cavalier-Smith (2001a) as well as others (Rosenhouse, 2002), apparently for lack of anything better.
Rizzottis suggestion that stirring might be a primitive function of a proto-flagellum is intuitively appealing, but intuition is a poor guide to life at a low Reynolds number (Purcell, 1977; Vogel, 1994; Purcell, 1997). Bacteria live in a world dominated by Brownian motion, where viscous forces overwhelm inertia and small molecules spread much faster by diffusion than by bulk movement of fluid. The scale at which moving fluid (stirring) or moving through fluid (swimming) will increase diffusion into the cell is determined by comparing the time for transport by diffusion (td) versus the time for transport by bulk flow such as stirring (ts) (Purcell, 1977). For diffusion, the average time td for transport of a particle a distance l, with diffusion coefficient D is (Berg, 1993):
while the corresponding time for bulk flow transport via stirring (ts) is approximately (Purcell, 1977):
that is, the distance l divided by the fluid velocity v induced by stirring. Stirring works only if the transport time using stirring is less than the transport time from simple diffusion:
The ratio in equation (5) gives the Péclet number, Pé, which must be greater than unity for bulk flow to have substantial impact on diffusion (Vogel, 1994). For a typical small molecule (e.g. sucrose) in water, D=10-10 m2s-1. For a typical-length bacterium (1 μm) moving fluid past itself with the swimming velocity of a typical fully functional flagellum (30 μm/s), Pé = 0.06 << 1 (Vogel, 1994). For Rizzottis primitive stirrer, Pé would be even lower. As Purcell (1977) noted, in the world of low Reynolds number, stirring isnt any good. Bacteria that do induce currents for their benefit (e.g., Thar and Kuhl, 2002) probably succeed because of the large number of bacteria cooperating in the effort, in effect increasing body size. Another postulated function of primitive motility, swimming for the sake of running into more molecules, also does not work: Purcell calculated that a bacterium would have to swim 700 μm/sec in order to gather only 10% more food molecules. Thus, if diffusion of molecules into the cell is the only matter of concern, a bacterium will do just as well by sitting still as it will by stirring or swimming. The reason bacteria swim is not to increase diffusion but to find locations with a higher local concentration of nutrient molecules (Purcell, 1977; Berg, 1993; Vogel, 1994). Purcells argument breaks down in situations where the uptake rate parameter, a, representing the fraction of available molecules being consumed each second, is greater than 1 s-1. However, a typical value for a is 0.01, where uptake is considered negligible (Dillon et al., 1995; Mitchell, 2002). Thus, fundamental physical considerations make the hypothesized stirring filament an unlikely intermediate.
Additional difficulties with Rizzottis model exist. While it is unrealistic to expect sequence similarity to give evidence for the ancestry of every component of the 3+ billion year old flagellum, considering the time lapse and large nature of some of the changes that must be postulated on any scenario, a scenario certainly should not contradict those homologies that have been identified. The Rizzotti scenario (Figure 3) implies homology between the synthetase F1-αβ subunits and FliF/FliG (the flagellar rotor), but the homology that inspired the scenario is between F1-αβ and FliI (the ATPase that energizes export of rod, hook, and filament). Similarly, Rizzotti (2000) implies that the F0-c subunit is homologous with the flagellar motor proteins MotAB, but sequence homology has instead been discovered homology between MotAB and a phylogenetically widespread family of proteins that couple protonmotive force to diverse membrane transport processes. These homologs, namely ExbBD (Kojima and Blair, 2001) and TolQR (Cascales et al., 2001), provide a simpler and much more direct ancestor for MotAB. The homologies could be explained by invoking additional independent cooption events, but this would require a rather more complex scenario than that presented by Rizzotti.
As Rizzottis scenario fails on the twin tests of homology and a simple model of stirring at a low Reynolds number, it is now time to see if Rizzotti can be improved upon. It should be noted that although published proposals about flagellar evolution are very limited, the topic is a popular one as the flagellum is the icon of the antievolutionary Intelligent Design movement. Therefore several of the ideas proposed here have been previously raised in informal debates about flagellar evolution. Miller (2003, 2004) and Musgrave (2004) review this aspect of the debate in detail, and Musgrave proposes a model that is similar in outline to that presented here, although his account is more general.
3. The Model
3.1. Phylogenetic context and assumed starting organism
The paradigm for prokaryote phylogeny, if there is one, is the universal rRNA tree. This shows a number of widely separated bacterial lineages, with archaea and eukaryotes separated from them all by a very long branch. This tree is unrooted, and many possible rootings have been proposed in the literature. As these are the most remote and difficult phylogenetic events it is possible to study, and as there is by definition no outgroup to life in general, the debate can be expected to continue for some time. For current purposes the most important point is that flagella are widespread across the bacterial phylogenetic tree, with losses in various taxa and no clearly primitive nonflagellate taxa. It is therefore assumed that flagella evolved near the base of the bacterial tree.
Rizzotti (2000) and others (e.g., Koch, 2003) have suggested that the last common ancestor of bacteria was gram positive. However, the very general consideration that most of the bacterial phyla are gram negative, including the many different taxa that come out as basal on different analyses, weighs against this hypothesis. Therefore, we shall side with Cavalier-Smith, who argues that the last common ancestor was gram-negative. He has put forward the most detailed model for the origin of bacteria and the double membrane (Cavalier-Smith, 2001a, 2002a). The model thus begins with a generic double-membraned, gram-negative bacterium. Whether or not archaea are an outgroup to extant bacteria (the most common opinion), or a relatively late group derived from actinobacteria (high G+C content gram-positive bacteria), in turn derived from endobacteria (low G+C-content gram-positives) and cyanobacteria (Cavalier-Smith, 2002a) shall be left unresolved, although implications of flagellar evolution for Cavalier-Smiths scheme will be highlighted. The present model will begin with a reasonably complex bacterium, already possessing the general secretory pathway and type II secretion system, as well as signal transduction, a peptidoglycan cell wall, and F1F0-ATP synthetase. As these components are ubiquitous, almost certainly predating the cenancestor, whereas many bacteria (perhaps 50% of species) lack flagella entirely, this seems plausible. These assumptions are consistent with Cavalier-Smiths position that the cenancestor was a bacterium similar in complexity to modern bacteria (Cavalier-Smith, 2001a, 2002a). Cavalier-Smith (2002a) hypothesizes that chlorobacteria may be the most basal offshoot of the tree and be primitively nonflagellate.
3.2. Starting point: protein export system
3.2.1. Type III secretion systems
The model begins with a hypothetical primitive type III export apparatus. As terminology is sometimes inconsistently used, following Hueck (1998), the term secretion is reserved for the transport of proteins from the cytoplasm to the cell surface or the extracellular medium. Export refers to the transport of proteins from the cytoplasm to the periplasmic space. An export system plus a mechanism to cross the outer membrane forms a secretion system. Bacteria make use of a number of distinct secretion systems, reviewed as a group elsewhere (Hueck, 1998; Thanassi and Hultgren, 2000a; van Wely et al., 2001). Six major well-characterized secretion systems (Figure 4a, Figure 5) are reviewed by Thanassi and Hultgren (2000a). These are: (1) autotransporters (Henderson et al., 1998), (2) the chaperone/usher pathway (Thanassi et al., 1998), (3) type I secretion or the ATP-binding cassette (ABC) transporter (Buchanan, 2001), (4) type II secretion or general secretory pathway (Pugsley, 1993; Sandkvist, 2001; Cao and Saier, 2003), (5) type III secretion systems of flagellar export and some infectious systems (Hueck, 1998; Cornelis and Van Gijsegem, 2000), and (6) type IV secretion (Christie and Vogel, 2000; Christie, 2001), homologous to type II secretion, conjugation pili, twitching motility systems, and archaeal flagella (Jarrell et al., 1996; Bayley and Jarrell, 1998; Sandkvist, 2001; Peabody et al., 2003). It is likely that systems will be added to the list in time.
About 10 well-conserved protein species make up the core of the type III export apparatus, which is used to export the axial components of bacterial flagella (rod, hook, filament, adaptor, and cap proteins). In 1994 it was discovered that homologs of these proteins are also used to secret virulence factors in a diverse array of proteobacterial pathogens, such as Yersinia pestis, Salmonella typhimurium, Pseudomonas aeruginosa and enteropathogenic E. coli (Hueck, 1998). The term type III secretion system is commonly used to refer to the virulence systems, but here it will be used to denote the class of secretion systems that make use of the type III export pathway. This includes the two currently known members (virulence and flagellar secretion systems) and any unknown homologs.
The existence of a nonflagellar type III export apparatus falsifies the argument that flagellar components are useless if they are not part of a fully functioning flagellum. One answer to Macnabs (1978) query, What advantage could derive from a preflagellum (meaning a subset of its components) is now obvious: a subset of flagellar components could serve as an export system. Thus, the model for the origin of flagella begins with the hypothesis of a primitive type III export system. This hypothesis, however, requires justification on several grounds in order to ameliorate obvious objections.
3.2.2. Are nonflagellar type III secretion systems derived from flagella?
The fact that known nonflagellar type III secretion systems are restricted to proteobacteria, and that these systems are mostly virulence systems specializing on eukaryotes (which are probably far younger than flagella), lead Macnab (1999) as well as others (He, 1998; Kim, 2001; Plano et al., 2001) to conclude that the flagellar pathway is probably the older one, and that type III virulence systems are derived from flagella. Although some apparently avirulent type III secretion systems have been discovered (e.g., in the legume symbiote Rhizobium; see Marie et al., 2001), and the phylogenetic distribution of type III secretion systems has been widened somewhat by their discovery in Chlamydiales (Kim, 2001), these data still support the conclusion that type III virulence systems are derived eukaryote-interaction systems, rather than phylogenetically basal homologs. Phylogenetic analysis of type III secretion systems seemed to confirm the case (Nguyen et al., 2000). Aizawa (2001) was one of the few dissenting opinions, arguing that flagella and virulence systems might have diverged in parallel from a common nonflagellar ancestor, pointing out that there are bacteria that parasitize or prey on other bacteria, a point with some merit although predatory bacteria are poorly studied (Guerrero et al., 1987).
Nguyen et al.s (2000) conclusion has recently been challenged by Gophna et al. (2003), who demonstrated with phylogenetic trees of FlhA, FliI, FliP, and FliO homologs that type III virulence system sequences do not nest within flagellar sequences. This supports the view that the two systems diverged from a common ancestor, which could plausibly have been a type III export system functioning in a nonflagellar, nonpathogenic context. However, Gophna et al. (2003) are not able to exclude the possibility that virulence systems evolve more rapidly, or that the frequent lateral transfer of type III virulence system genes (Nguyen et al., 2000; Gophna et al., 2003) might have increased the rate of sequence divergence. Gophna et al. also cite for support the progressionist notion that evolution disfavors events such as the simplification of complex systems like the flagellum, a dubious proposition in modern evolutionary theory, especially considering the common evolutionary trend of simplification in pathogens and parasites. As long as known nonflagellar type III secretion systems are phylogenetically restricted and only function as specialized systems for eukaryote penetration, the suspicion will remain that they are derived from flagella. For the purposes of the current discussion it will be assumed that type III virulence systems are derived, although they still give valuable insights about the possible traits of a hypothetical ancestral type III secretion system.
3.2.3. An ancestral type III secretion system is plausible
If type III virulence systems are derived from flagella, what is the basis for hypothesizing a type III secretion system ancestral to flagella? The question would be resolved if nonflagellar homologs of the type III export apparatus were to be discovered in other bacterial phyla, performing functions that would be useful in a pre-eukaryote world. That such an observation has not yet been made is a valid point against the present model, but at the same time serves as a prediction: the model will be considerably strengthened if a such a homolog is discovered. For the moment, it is easy enough to explain the lack of discovery of such a homolog on the basis of lack of data. Knowledge of microbial diversity is quite poor (Whitman et al., 1998): far less than 1% of bacteria extant in a particular environment are readily culturable (Hayward, 2000). Cultivation-independent surveys of prokaryote diversity based on environmental rRNA sequencing commonly discover deeply-branching microbes previously unknown to science (DeLong and Pace, 2001), and that certain groups are unexpectedly ubiquitous (Karner et al., 2001). In addition, only a fraction of cultured microbes have been studied in any substantial biochemical or genetic detail, and this subsample is heavily skewed towards pathogens and convenient model organisms. Of the ~112 complete bacterial genomes sequenced as of July 2003 (http://www.ncbi.nlm.nih.gov/PMGifs/Genomes/eub_g.html), at least two-thirds are pathogens, mutualists, or commensals of multicellular eukaryotes. Many of the free-living bacteria that have been sequenced are extremophiles or are used in industrial applications.
Even with such a skewed dataset, a general argument for the plausibility of a primitive type III export system can be constructed on the basis of analogy. Each of the six secretion systems described above has been coopted to serve diverse functions by prokaryotes (Table 4). The thoroughness of some of the observed convergences is remarkable notably, all of the systems have been adapted for eukaryotic virulence, five secrete surface structures, at least four are used for adhesion, three or four form pili, and two perform motility-related functions. That pili and adhesion often play a role in virulence in well-studied organisms is not particularly significant, as such functions are useful in free-living contexts as well (Kennedy, 1987). The overall picture is that any secretion system that exists will sooner or later get coopted for diverse functions, including virulence, in various lineages. The commonality of the virulence function in known systems almost certainly reflects human interests rather than the situation in the wild.
Table 4: Convergent functions of well-characterized prokaryote secretion systems. Other secretion systems are known to exist: e.g., curli fimbriae based on the extracellular nucleation/precipitation pathway (Smyth et al., 1996; Wu and Fives-Taylor, 2001; Chapman et al., 2002) and slime secretion (Merz and Forest, 2002). Others undoubtedly remain to be discovered.
It might be objected that with so many available secretion systems, postulating the existence of an additional system is superfluous. However, many bacteria have multiple secretion systems. An illustrative case is Pseudomonas aeruginosa, which has all of the above-listed systems (Bitter, 2003). Furthermore, many bacteria will have two or more copies of certain types of secretion systems, with mildly to strongly divergent functions: e.g., E. coli can have both P-pili and type 1 pili (Thanassi and Hultgren, 2000a); Salmonella and Yersinia have two type III virulence systems each (Cornelis and Van Gijsegem, 2000); and Pseudomonas aeruginosa has at least two type II secretion systems and probably two kinds of type IV pili (Bitter, 2003).
3.2.4. The origin of a primitive type III export system
Type III virulence systems have well-conserved homologs of the following flagellar components (Plano et al., 2001): FliF (the membrane-embedded MS-ring); FlhA, FlhB, FliP, FliQ, FliR (integral membrane export components inside the MS-ring); FliI and FliH (ATPase and regulator); and FliG and FliM/N (the switch complex). The primitive type III secretion system would not necessarily have had all of the components that are conserved in the possibly derived virulence systems. In particular, if the type III virulence systems are derived, the homologs of the switch complex proteins (FliN/M, FliG) are probably retained only in order to stabilize/support the coadapted secretion complex and FliF ring, and are otherwise vestigial.
FliF is fundamentally a membrane pore and so its origin must lie with the origin of transport proteins in general, a question explored by Saier (2003). FlhA and FlhB are larger than FliOPQR, and have large cytoplasmic C-terminal domains that appear to bind the export substrates. FlhA interacts with FliF and the soluble components of the type III secretion system but its exact function is unknown. FlhB plays a key role in determining whether rod/hook or filament axial proteins are secreted, and therefore controls the length of the hook by a poorly-understood mechanism (Macnab, 2003). Substrate switching would not have been a necessary feature of a primitive type III secretion system, but perhaps the association of proto-FlhA and/or FlhB with the proto-FliF pore turned it from a somewhat general passive transporter into a substrate-specific passive transporter. One of the differences between type II and type III secretion systems is that type II systems recognize their substrates by a N-terminal signal peptide that is removed during transport. The signal sequences for type III secretion substrates are also in the N-terminal regions but they are not cleaved (Büttner and Bonas, 2002). Perhaps this difference allowed the primitive type III secretion system to export an important substrate on a different control circuit independent of the sec pathway, and this finer control was the selective basis for the retention of the system.
3.2.5. The relationship between type III export and the F1F0-ATP synthetase
That a phylogenetically basal type III export apparatus must have existed is supported by several additional facts. As discussed previously, the protein that powers protein export in type III secretion, FliI, has long been considered homologous to the F1 subunit of F1F0-ATP synthetase on the basis of about 30% amino acid identity to the active F1-β subunit (Albertini et al., 1991; Vogler et al., 1991; Gogarten et al., 1992). The F1-αβ ATPase is a heterohexamer made up of alternating α-subunits (noncatalytic) and β-subunits (catalytic). This pattern is shared by all bacteria and is also found in the archaeal A-ATP synthase and eukaryote V-ATP synthase, so F1-α and F1-β are thought to have diverged before the cenancestor (Gogarten and Kibak, 1992). FliI, on the other hand, probably consists of a homohexamer of catalytic subunits (FliIs hexameric nature was only recognized very recently: Blocker et al., 2003; Claret et al., 2003). It diverges before the F1-α and F1-β split in sequence similarity trees, and thus probably also diverged prior to the cenancestor (Gogarten and Kibak, 1992). However, it is more similar to the F1 subunits than the more distantly related hexameric ATPases such as the RNA/DNA helicase termination factor rho (Boyer, 1997), and therefore Gogarten and Kibak (1992) conclude that the FliI family diverged specifically from a primitive F1-ATPase prior to the cenancestor. There is not similar evidence that flagella specifically evolved before the cenancestor, so this is a point in favor of the primitive type III export system hypothesis.
In light of the long-established homology between FliI and F1-αβ, it is surprising that there have been few searches for further homologies between the F1F0-ATP synthetase and type III export system. Sequence similarity searches do not turn up significant hits, but considering the timespan and divergence in function this is not necessarily surprising. As discussed above, homology between the F1F0-ATP synthetase and flagellum is commonly suggested, but explicit protein-protein homologies are never proposed, and the assumption that the rotational mechanisms of the two systems are homologous implies a quite radical transformation of ATP synthetase components. However, several recent discoveries suggest specific homologies that are much more conservative than those implied by previous workers. First, FliH forms a (FliH)2FliI heterotrimer with FliI (Minamino and Macnab, 2000; Minamino et al., 2001). FliH has an elongated shape (Minamino et al., 2001), and both FliI and FliH are soluble cytoplasmic components that associate intrinsically with the membrane and with lipid vesicles (Auvray et al., 2002). If the FliH2 homodimer associates with the FliI6 complex in vivo, all of this begins to look suspiciously similar to the association (Figure 4b) between the F1F0-ATP synthetase F1-α3β3 and F0-b subunits: two elongated F0-b subunits form a dimer and interact with F1-α3β3. In F0-b it is the N-terminal region that associates with the membrane, and the C-terminal region with the N-terminal regions of F1-α3β3 (Boyer, 1997; Weber and Senior, 2003). In FliH it is known that the C-terminal region associates with N-terminal region of FliI (Gonzalez-Pedrajo et al., 2002), but the region responsible for membrane association is undetermined (Auvray et al., 2002); F0-b FliH homology would predict that the FliH N-terminus associates with the membrane. Although BLAST searches on FliH only return F0-b as a non-significant hit, a search of NCBIs CDART (Geer et al., 2002) based on FliH does retrieve F0-b as a result with similar domain architecture (using the default e-value cutoff of 0.01), another point in favor of the hypothesis of homology. Jackson and Plano (2000) report that the Yersinia pestis FliH homolog YscL (corresponding to SctL/HrpE in Figure 4a) has low but significant sequence similarity with the e subunit of the archaeal ATPase of Methanococcus jannaschii and the e subunit of the vacuolar ATPase of Desulfurococcus spp.; these subunits are the homologs of the b subunit of the F1F0-ATP synthetase. Thus the present scenario predicts that careful multiple alignment of FliH sequences with bacterial F0-b and the corresponding archaeal and eukaryotic homologs (all of which would be equally related to FliH) will confirm homology.
Can further homologies between flagella and the F1F0-ATP synthetase be discerned? In the F1F0-ATP synthetase, an F1-δ monomer associates with the proximal end of F1-α3β3 and F0-b2. In the type III export apparatus, it is FliJ that interacts with FliI and FliH2. FliJ seems to be required for the export of all flagellar components, and so has been interpreted as a general chaperone in the cytoplasm (Macnab, 2003). However, this observation is equally well explained if FliJ is a required part of a FliI6FliH2 complex essential for export. Both FliJ and F1-δ have a similar size and N-terminal binding sites to the N-terminal regions of FliI/F1-α. There may also be a structural similarity: FliJ has a high probability of exhibiting an N-terminal α-helical coiled-coil arrangement (Macnab, 2003), using sequence-based predictions (Lupas et al., 1991, method implemented at http://www.ch.embnet.org/software/COILS_form.html). F1-δ has several conserved α-helices at its N-terminal binding site to F1 (Weber et al., 2003b). Although predictions do not generally yield a high probability of coiled-coil structure for F1-δ, a cursory non-exhaustive sampling of orthologs shows that at least one FliJ protein does not show a high probability prediction of coiled-coil structure either (Buchnera aphidicola, accession no. P57179) while at least one F1-δ protein does (Rhodopseudomonas blastica, accession no. P05437). It appears that the C-terminal region of F1-δ associates with the C-terminal region of F0-b2, although the details remain to be worked out (Weber and Senior, 2003). Regarding the FliJ-FliH2 interaction, Fraser et al. (2003) favor a model where FliJ interacts with the N-terminal region of FliH2, but their data (Gonzalez-Pedrajo et al., 2002) shows that deletions in either the N-terminus (perhaps the region that associates with the membrane) or middle (dimerization region) of FliH preclude FliJ binding; thus failure of FliJ binding could be due to general malformation of FliH2 due to the failure of FliH to dimerize (middle deletion) or associate with the membrane (N-terminal deletion). Homology between F1-δ and FliJ would predict that FliJ-FliH interaction is actually mediated through the C-terminal regions of each, but that the association may be rather weak, as it is between F0-b2 and F1-δ (Weber and Senior, 2003).
Similarities in F1F0-δca, the integral membrane proteins FliPQR of the type III export apparatus, and the proteins SecFEY of type II secretion proteins were pointed out by Aizawa (2001), who calls these triplets the proto-channel and suggests homology. His evidence is of a general nature (calculated similarities in molecular size, aliphatic index, instability index, and isoelectric point) and so cannot be accepted uncritically. In particular, it is no longer thought that F1-δ (or its eukaryote homolog OSCP) is associated with the membrane or ATP synthetase stalk (Weber et al., 2003a), and the evidence discussed above points to a different homology for F1-δ. However, the proposed matches between FliQ--F0-c and FliR--F0-a are decent in terms of protein size and also the number of transmembrane helices of the respective proteins (Table 5). And surprisingly, extrapolating the homology hypothesis to match the two remaining type III secretion components (FliO and FliP) to the two remaining synthetase components (F1-ε and F1-γ, respectively) also seems to provide plausible matches in terms of size. When the similarities between F1F0-ATP synthetase and type III export components are tabulated (Table 5), it is apparent that that each component of the F1F0-ATP synthetase can be matched to a component of the type III export apparatus with a similar size and topology, as far as evidence is available (the function and structure of the flagellar proteins FliOPQR are poorly understood).
Table 5: Similarities between proteins of the F1F0-ATP synthetase and the flagellar type III export apparatus that may suggest homology. Protein size is the length in amino acids for E. coli. TMH = Transmembrane helices. Little detailed information on FliOPQR is available, the topologies listed are the predictions of Minamino and Macnab (1999). Data taken from several sources: general ATP synthase component information (Boyer, 1997, updated by later references); FliI--F1-β homology (Gogarten et al., 1992; N-terminal F1-α to N-terminal F1-δ interaction (Weber et al., 2003a); FliIHJ (Minamino and Macnab, 2000; Minamino et al., 2001; Auvray et al., 2002; Minamino et al., 2002; Macnab, 2003). The membrane-associating region of FliH is not determined (Auvray et al., 2002), but the C-terminal region interactions appear similar to the C-terminal interactions for F0-b (see text), so an N-terminal association with the membrane seems likely.
Individually, the cited similarities are easily attributable to chance, but together they are at least suggestive. Although detectable sequence similarity may be too much to hope for given the already very low similarity between FliI--F1-α3β3 and FliH--F0-b, the postulated homologies would be further testable by Aizawas technique. Table 5 also shows that there are some apparent dissimilarities. Notably, while both F0-c and FliQ have 2 transmembrane helices, the loop between the helices is exposed to the cytoplasm in F0-c (Birkenhager et al., 1999), while the loop between the helices in FliQ was predicted to be periplasmic (Ohnishi et al., 1997); a reversal of this finding would support the homology hypothesis. The weakest case for homology is between F1-ε and FliO; FliO is predicted (Ohnishi et al., 1997) to have a single transmembrane helix, while the structure of F1-ε has been solved (Wilkens and Capaldi, 1998) as a two-domain protein that binds to the stalk. However, both proteins tolerate substantial variability; F1-ε functions with large deletions (Wilkens and Capaldi, 1998) and clear homologs of FliO have not even been identified in type III virulence systems (Gophna et al., 2003).
The hypothesis that the entirety of a primitive F1F0-ATP synthetase may have been coopted in toto into a primitive gated pore (proto-FliF and proto-FlhA/B) is certainly provocative; it would explain at a stroke the origin of most of the type III export apparatus and provide a phylogenetically basal precursor to the flagellum even though clearly basal type III secretion systems remain undiscovered. The complex would fit well in the FliF ring; using the stoichiometry of FlhA2FlhB2 proposed by Macnab (2003), and the equivalent stoichiometry of an ATP synthetase for the other integral membrane components, FliO1P1Q~12R1, the total number of transmembrane helices is 60, well within the approximate MS-ring capacity of about 70 transmembrane alpha-helices (Fan et al., 1997). Fan et al. estimate <3 copies of FliR per flagellum, which is consistent with the ATP synthase homology hypothesis, but also estimate 4-5 copies for FliP, which is not, so if the ATP synthetase hypothesis is true it would be expected that the FliP finding is in error.
Macnab (1999) called the homology between FliI and F1-αβ inexplicabl[e]. However, there may be a relatively simple explanation. If the postulated homology between the ATP synthetase and type III export is correct, then the key event in the origin of type III export was the association of a primitive F1F0-ATP synthetase with a proto-FlhA or FlhB inside the proto-FliF ring, converting it from a passive to active transporter. Since little is known about the details of the coupling of ATPase activity to protein export in Type III export, this step remains speculative. Probably motion in the synthetase was linked to a conformational change in FlhA and/or FlhB, with the proton pumping function of the synthetase lost soon afterwards. Currently there are several documented associations between FlhAB and the rest of the type III export apparatus (Macnab, 2003). These associations include proteins in both the F0 and F1 regions of the type III export apparatus. FlhA or FlhB may thus take over some of the linker role that is played by F0-b in the ATP synthetase and (on the homology hypothesis) by FliH in the type III export apparatus; this would help to explain why FliH is not absolutely required for successful construction of flagella, and FliH null mutants can be compensated by mutations in FlhA and FlhB (Minamino et al., 2003).
Other possible hypotheses for the origin of the type III export apparatus are not currently ruled out, such as the idea that much of apparatus is descended from a passive channel and that only a portion of the F1F0-ATP synthetase was coopted to power transport, or that there is an ancient, obscured homology between the various secretion systems. Alternatives are currently disfavored because they are more complex and explain the origin of fewer components. However, even if FliI remains the only confirmed homolog to the F1F0-ATP synthetase, general considerations indicate that the evolution of an export system is not very difficult. A diversity of export systems of varying complexity exist, and there is a functional continuum of membrane complexes ranging from single proteins and passive pores through to active, gated export systems, indicating that there are no major evolutionary puzzles to solve. The cataloguing and categorizing of transport proteins is already yielding insights into their origin (Saier, 2003).
The ATP synthetase homology hypothesis has the advantage of numerous testable implications for the structure and function of FliHIJOPQR. The ATP synthetase is relatively well-understood; structures have been determined for most of the components and a number of sophisticated techniques for studying the complex as a whole have been developed. If the homology hypothesis is correct, then similar structures would be expected for the corresponding type III export components, and many of the techniques applied to the ATP synthetase should apply to the export apparatus. It is worth noting in passing that if a significant portion of the type III export apparatus is indeed homologous to the ATP synthetase, then it becomes fairly likely that the rotary flagellum contains within it a second rotary motor powering protein export. This is a fairly incredible notion, but would merely be the latest in a long line of surprising discoveries yielded by the study of the flagellum. This possibility might mean that the proto-flagellar secretion system was rotating from the start (echoing the rotation-early hypotheses of Cavalier-Smith, Goodenough, and Rizzotti), although this is not a necessary postulate for the rest of the scenario to proceed.
3.3. Type III secretion system
For the remainder, the hypothesis of a primitive type III export system will be taken for granted. This complex would have transported proteins manufactured in the cytoplasm into the periplasmic space. If secretins were already available from the type II secretion system, as they probably were given the universal distribution of type II secretion, then from the start the type III export system would have been a primitive kind of type III secretion system, as small proteins could diffuse in the periplasmic space until they found an outer membrane pore and diffused out. Digestive proteases or antibiotic molecules are likely candidates for the secreted proteins. Alternatively, the export system could have originally secreted proteins destined for the periplasm, and later cooption of a secretin converted the export system into a secretion system.
The association of an outer membrane channel with the type III export apparatus would improve the efficiency of secretion. This advantage would increase as exported substrates became larger, because the peptidoglycan cell wall only allows the diffusion of globular proteins with a size less than about 50 kDa (Young, 2001); as protein size increased, diffusion would be increasingly impeded. Once a new single-step secretion channel was available it would be possible to secrete larger proteins and proteins that would be harmful if left to wander about the periplasmic space. These are selective forces that would favor the spread and diversification of the channel, after its origin as an efficiency-improving measure.
Outer membrane secretins have been coopted repeatedly by various versions of the secretion systems discussed above (Hueck, 1998; Thanassi, 2002; Bitter, 2003); if the type III virulence system is derived from the flagellum, it probably originated in part by replacing the flagellar L- and P-ring proteins with a secretin. The first association of a secretin with a primitive type III export apparatus was probably mediated by the simultaneous cooption of a secretin and its outer membrane lipoprotein chaperone (Dailey and Macnab, 2002). Both of these proteins are secreted by the type II secretion pathway. The channel to the extracellular medium could be recruited in a single step if a mutation caused the secretin to associate with the type III export apparatus. The secretin appears to cross both the cell wall and outer membrane in the Hrp pilus, and to associate with the FliF homolog (SctJ/HrcJ; Figure 4a) in the cytoplasmic membrane (Blocker et al., 2003), so having two ring proteins (the L- and P-rings in flagella) does not appear to be a prerequisite for secretion. Thus the double ring may have been a later addition to the system, perhaps even coinciding with the early stages of improvement of the proto-flagellum and the loss or modification of the secretin (see below).
3.4. Origin of a type III pilus
In the model, flagellin and all of the proteins of the axial structure FlgBCFG (rod), FlgE (hook), FlgKL (adaptor), FlgD and FliD (caps), in addition to FliC (flagellin) -- are descended from a common ancestral pilin secreted from the primitive type III secretion system. All of these proteins are placed in the axial protein family (Homma et al., 1990a; Hirano et al., 2001). Homma et al. (1990a) put the rod, hook, and first adaptor (FlgK) proteins into a closely-related subfamily. The divergence of the axial filament family probably occurred mostly after the origin of a functioning protoflagellum; this will be discussed in a later section. First, the origin of the pilus must be considered.
The diversity of surface structures based on secretion systems was documented in Table 4; modern flagella retain many of these functions (Moens and Vanderleyden, 1996). To expand on a likely function of a primitive pilus, successfully adhering to a surface can be a problem for a floating bacterium: at a low Reynolds number, the boundary layer near a surface can be a significant barrier (Vogel, 1994). A bacterium can increase its chances of attachment by secreting adhesins with an affinity for the desired surface, ensuring successive attachment if it happens to get near a surface (e.g., the adhesins secreted by autotransporters, independent of pili (Henderson et al., 1998)). It can increase its chances still further either by putting the adhesin at the end of a filament (e.g., the PapG adhesin located at the tip of the P pilus fibrillum (Sauer et al., 2000); the flagellar cap FliD of Pseudomonas aeruginosa doubles as an adhesin (Scharfman et al., 2001)) and/or by making the filament adhesive along its whole length, which is a common occurrence in modern bacterial flagella as well as many other surface structures (Kennedy, 1987; Moens and Vanderleyden, 1996; Fernandez and Berenguer, 2000; Giron et al., 2002). Probably any filament, adhesins or no, will have some utility in attaching to inorganic surfaces, simply by expanding effective size and surface area available for adhesion. Even in the absence of specific adhesins, charge, hydrophobicity, and/or van der Waals forces can be exploited for more general surface adhesion (Vogel, 1988), particularly at the small scale of bacteria.
Three hypotheses present themselves as to how the ancestral pilus originated: filament-first, cap-first, and modified filament-first. The latter hypothesis combines the best features of the filament-first and cap-first hypotheses.
3.4.1. Filament-first hypothesis
One way that the kinds of pili described above could get their start is by simple polymerization of a surface adhesin. The adhesin would have inherited from its ancestor the ability to bind with the outer membrane channel and with the extracellular substrate; all that would have to be added is self-binding capability. The plausibility of this step is attested by several facts: first, structures made up of multiple copies of the same subunit are biochemically ubiquitous, and the evolution of large multimeric complexes has in many instances been traced back to simpler ancestors, e.g., AAA ATPases (Mocz and Gibbons, 2001). Second, polymerization into a filament or tubule via mutation is a quite common event: sickle-cell hemoglobin, derived by only a single substitution from regular hemoglobin, forms not only self-assembling polymers but dynamic polymers (Mitchison, 1995). In fact, Mitchison (1995) argues that evolution can start with just about any protein fold and produce a self-assembling polymer.
An alternative to polymerizing an adhesin is to postulate that a gene for a pre-existing filament-forming protein was coopted by transposition of the promoter and N-terminal signal sequence of an already secreted protein. Support for this second possibility might be found in homology between flagellin and a modern filament-forming protein. Homology between flagellin and actin has been proposed. Harris and Elder (2002) cite an 8/13 amino acid sequence match between flagellin and actin in the N-terminal region, but this could easily be due to chance. Novikova et al. (2000) found that flagellar filaments co-precipitate with rabbit skeletal myosin, and that flagellin and F-actin compete for myosin binding, but this might be explained by a general similarity of filaments rather than homology. The search for flagellin-actin homology is somewhat misguided in any case, because actin is a eukaryotic protein, so ancient prokaryote actin homologs such as FtsA (Mitchison, 1995) would be more appropriate subjects. Similarly, what should be sought is not the homolog of flagellin but the homolog of the entire axial protein family. Given the large divergence of flagellin from the more conserved rod and hook members of the family (Homma et al., 1990a), any relationships outside of this family are bound to be difficult to detect.
A simple assumption is that the first filament was a chain of monomers, probably in an open helix. Longer filaments are presumably better for adherence than short filaments, and thus selection for adhesion can be expected to favor longer filaments. Once a polymer filament of reasonable length has been built, however, there may be a difficulty in extending it. The problem does not arise if filament subunits are added at the base, as occurs in type IV pili: type IV pili are based on type II secretion systems, which use a two-step process to transport proteins. First, proteins are exported into the periplasm, and then they are pushed out through a secretin, perhaps via a plunger-type mechanism involving a pseudopilus (Thomas et al., 2001). However, the type III secretion system exports proteins from the cytoplasm in one step. By exporting a number of individual subunits, a short filament binding to the outer membrane pore can be formed, but its possible length will be severely limited by the decreasing chances of successfully adding monomers to the receding distal tip. This problem might be overcome in a gradual manner by modifications to the open helix, so that it better corralled the monomers as they exited the secretin. Each mutation that brought the turns of the helix closer together would decrease the rate of monomer escape, and allow the extension of the filament. A tube with closed or nearly closed walls would be the optimal solution, and selection for rigidity (necessary for very long filaments) would also favor the closed tube.
The result would be something rather like the modern type III virulence pili, which appear to have far less complex axial structures than flagella. Indeed, despite several investigations it has yet to be determined that the Hrp pilus has any axial components (rod-like, hook-like, etc.) apart from the main protein of the pilus, HrpA (MxiH/PrgI in Shigella/Salmonella; Blocker et al., 2003; Aizawa (2001) tentatively suggests a few others). The extracellular portion of the filament seems to extend continuously into the secretion complex (Fernandez and Berenguer, 2000) whereas in flagella there is a distinction between filament, hook, and rod.
It might be objected at this point that the flagellum requires the cap (FliD) in order to chaperone the flagellin subunits into place at the elongating tip of the filament; without it, they diffuse away and are lost (Blocker et al., 2003). The hook has its own temporary cap (FlgD), and it has been suggested, but not proven (Hirano et al., 2001; Berg, 2003; Macnab, 2003), that the rod has a cap protein as well (FlgJ). However, the necessity of the cap for successfully assembling subunits is ambiguous. Flagellin will self-assemble into filaments in vitro (Hirano et al., 2001). No cap has been identified in any type III virulence systems (Blocker et al., 2003), and although PrgJ has been suggested as a possible cap for the Salmonella needle (Sukhan et al., 2003), the evidence is indeterminate as Sukhan et al. could not detect PrgJ in sheared-off needles and did not detect it at needle tips using immunoelectron microscopy (they therefore suggest that PrgJ may be a basal component). The polar flagellum of Vibrio grows normally without the cap (Bardy et al., 2003), probably because it is sheathed by an extension of the cell membrane (McCarter, 2001) that constrains the subunits. Finally, even in the canonical E. coli flagellum the adaptor proteins FlgK and FlgL are added without any capping structure (Macnab, 2003), leading Macnab (2003) to argue that capping structures are perhaps best viewed as a means of increasing efficiency of addition rather than as an absolute requirement. On this view, the cap could be a relatively late evolutionary addition to the pilus structure, originating by pentamerization of a pilus subunit and initially improving speed and efficiency of pilus assembly. Later co-adaptation between filament and cap subunits would make it a more-or-less required feature.