- Introduction
- Background
- The Model
- Conclusions
- Acknowledgements
- References
- Figure legends and tables
1. Introduction
The bacterial flagellum is one of the most striking devices
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) approximately 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
minor components and the exact mechanism of torque generation. However, the origins of this remarkable
device 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.
Biologists find it almost inescapable to compare the
bacterial flagellum to human designs: "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,
As a final
comment, one can only marvel at the intricacy, in a simple bacterium, of the
total motor and sensory system which has been the subject of this review and
remark that our concept of evolution by selective advantage must surely be an
oversimplification. What advantage could derive, for example, from a
"preflagellum" (meaning a subset of its components), and yet what is the
probability of "simultaneous" development of the organelle at a level where it
becomes advantageous?" (Macnab, 1978).
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
Darwin's 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 in a gradual,
linear fashion, but if systems and components underwent functional shift, then
intermediates could have been selected for a function different from the final
one. The equal importance of
improvement of function and change of function in 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.
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 Ussery's 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 Ussery's 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.
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: "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, which initially functions crudely but which can then
undergo standard microevolutionary optimization. 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, e.g., structures. The plausibility of mutational steps can be
investigated by examination of similar mutations observed today; and the
selection 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 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.
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.
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).
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), Goodenough's 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-Smith's 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.
Either way, the rotary mechanism existed from the beginning of the
evolutionary sequence. On either
scenario, 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.
Gliding via the ratchet structure of Cytophaga and Flavobacterium
is more mysterious, but may also involve 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-Smith's suggestion that stirring the periplasmic fluid may have
been a precursor to primitive motility is similar to Rizzotti's 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 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 Rizzotti's scenario has been cited by Cavalier-Smith (2001a) as well as others (Rosenhouse,
2002), apparently for lack of anything better.
Rizzotti's 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):
(1)
while the corresponding time for
bulk flow transport via stirring (ts) is approximately (Purcell, 1977):
(2)
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:
(3)
(4)
(5)
The ratio in equation 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 with the swimming velocity of a typical fully functional
flagellum (30 μm/s), Pé = 0.06 << 1 (Vogel, 1994). For Rizzotti's
primitive stirrer, Pé would be even lower. As Purcell (1977) noted, in the world of low Reynolds number,
"stirring isn't any good". Bacteria
that do induce currents for their benefit (e.g., Thar and Kuhl, 2002) 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
concentration of nutrient molecules (Purcell,
1977; Berg, 1993; Vogel, 1994). Purcell's
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 Rizzotti's 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 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 Rizzotti's 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. Musgrave (2004) reviews this aspect of the debate in detail, and
proposes a model that is similar in outline to that presented here, although
his account is more general.
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 practically every possible rooting has been
proposed in the literature somewhere.
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 clear that flagella evolved
near the base of the bacterial tree.
Rizzotti (2000) and others (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 has put forward the
most detailed model for the origin of bacteria and the double membrane (Cavalier-Smith, 2001a, 2002a), and begin 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-Smith's scheme will be highlighted. The present model will begin with a
reasonably complex gram-negative 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, and as many bacteria (perhaps 50% of
species) lack flagella entirely, this seems plausible. These assumptions are consistent with
Cavalier-Smith's 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.
3.2.1. 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 method 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). 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 Macnab's (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 gene trees of FlhA, FliI,
FliP, and FliO that type III virulence system sequences do not nest within
flagellar systems. 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 very 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. This
view is strengthened by the fact that type III virulence systems have homologs
of proteins like FliG, which only have an obvious function in the flagellar
motor and may be essentially vestigial in type III virulence systems. 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,
2001 #176). 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,
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.
It might be objected that with so many available secretion
systems, postulating the existence of an additional system is superfluous. However, the case of Pseudomonas aeruginosa,
which has all of the above-listed systems except for type IV secretion (Bitter, 2003) proves that having multiple secretion systems can be
useful. 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 FliN/M (the switch complex). The primitive type III secretion system
would not necessarily had all of the components that are conserved in the
possibly derived virulence systems. In
particular, the homologs of the switch complex proteins (FliN/M, FliG) are
probably retained in type III virulence systems 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-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
(FliI's 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 no 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. Several recent discoveries seem
suggestive of further homology. 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 PSI-BLAST searches on FliH only return F0-b
as a non-significant hit, a search of NCBI's CDART database based on FliH does
retrieve F0-b as a result with similar domain architecture, another
point in favor of the hypothesis of homology.
In the 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 short 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 the 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) and middle (dimerization
region) of FliH preclude FliJ binding.
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 the type III secretion integral membrane
proteins FliPQR, the type II secretion proteins SecFEY, and F1F0-δca
were pointed out by Aizawa (2001), who calls these triplets the "proto-channel" and
suggests homology. His evidence is of a
very general nature (similarities in aliphatic index and other features) 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. And
surprisingly, a leap into speculation, matching FliP to F1-γ
and FliO to F1-ε, also seems to provide plausible matches. 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).
Individually, the cited similarities are easily attributable
to chance, but together they are at least suggestive. 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 entire 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 system and provide a
phylogenetically basal precursor to the flagellum even though basal type III
export systems remain undiscovered. The
complex would fit well in 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, FliO1P1Q12R1,
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 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 probably the key event in the origin of type III export was the
association of the F1F0-ATP synthase 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, this
step remains speculative. Probably
motion in the ATPase 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 that there is a functional continuum of
membrane complexes ranging from single proteins and passive pores through to
active, gated export systems, indicates 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 rotary motor powering protein export. This is a fairly incredible notion, but
would merely be the latest in a long line of surprises 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.
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 their ubiquity, 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
increasing 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 secretion system,
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 very old 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 without help with the pilus FliF homolog in the
cytoplasmic membrane (Blocker et al., 2003), so a P-ring-like protein does not appear to be a
prerequisite for secretion. Thus the
P-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 of the
secretin (see below).
In the model, flagellin and all of the proteins of the axial
structure – FlgBCFG (rod), FlgE (hook), FlgKL (adaptor), FlgD and FliD (cap),
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 an 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 (Novikova et al., 2000; Harris and Elder, 2002), and ancient actin homologs such as FtsA are present
in prokaryotes (Mitchison, 1995), but the evidence is weak. Harris and Elder (2002) cite an 8/13 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. Given the large
divergence of flagellin from the more conserved rod and hook members of the
axial filament 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), and 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 species 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 "standard" 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.
3.4.2. Cap-first hypothesis
An alternative to gradual formation of a hollow pilus would
be to start with the cap. In Pseudomonas
aeruginosa, FliD serves not only as a cap protein, but also as an
adhesin. P. aeruginosa infects
the human respiratory system by adhering to mucins. FliD mutants were found to be nonadhesive, which could occur
either because FliD is a necessary adhesin, or because the flagellar filament
fails to assemble without the cap.
However, Arora et al. (1998) found that FliC null mutants retained adhesive
ability. This implies that it is FliD
specifically which serves as the adhesin, and not the whole flagellar filament,
a conclusion supported by additional lines of evidence (Arora et al., 1998;
Scharfman et al., 2001). On the filament-first
hypothesis, an adhesin attached to the outer membrane secretin mutated to form
a polymeric chain. The fact that caps
can be adhesive, however, suggests an alternative hypothesis. Instead of a mutation forming a polymeric
chain, the mutant adhesin formed an oligomer that associated with the distal
rim of the outer membrane secretin, approximately covered the mouth of the
secretin, and allowing more adhesin monomers to be packed into the available
space. In this case, a pentamer ring
was approximately the right size. Once
this was established, however, the utility of the adhesive "secretin cap" would
be further improved if it could be extended away from the surface of the cell. This could occur by mutation of a duplicate
cap protein that formed a slightly wider ring.
This ring would associate with the base of the cap, but the size
mismatch would allow the insertion of more subunits, forming a short pilus in
one step. On this view, the primitive
pilus would derive its structure of ~5 subunits per turn from the pentameric
cap, rather than the reverse. The
channel inside the pilus would be descended from the hole in expanded
ring.
3.4.3. Modified filament-first hypothesis
The filament-first hypothesis has the disadvantage of
explaining the addition of distal subunits to the filament before a tube
structure could evolve. The cap-first
hypothesis has the difficulty that a pentameric proto-cap covering the surface
of the secretin pore might impede the secretion of other substrates. The difficulty is not insurmountable, as
secreted substrates might escape from beneath the sides of the cap, or
alternatively might knock loose the cap, which is then replaced by continually
secreted cap protein (both mechanisms may operate in modern flagella). However, these problems are easy to solve if
the hypothesized adhesin pentamer were to initially form a ring atop the
secretin, instead of a cap. Secretion
of diverse substrates could then continue unabated without continual secretion
of the adhesin protein. From here, a
proto-pilus could easily be formed by a mutant adhesin that polymerized the
ring into a tube. This hypothesis is
simpler and more appealing because it combines the advantages of the two
previous scenarios: a pilus initially assembled by simple mechanisms without a
cap, but having a well-formed tube structure from the start, and allowing the
uninterrupted secretion of other type III secretion system substrates. On this hypothesis, the proto-cap would
again be a late addition (a modified pilus protein) increasing assembly
efficiency.
It is interesting to reflect on the surprise many
researchers felt when the mechanism of flagellar filament construction – adding
subunits at the tip rather than the base – was discovered. It has been called "astonishing" and
"somewhat bizarre" (Macnab and
DeRosier, 1988). However,
with the modified filament-first hypothesis in hand, the decidedly unintuitive
method of filament assembly used by the flagellum can be seen as a product of
the constraint of building a pilus from the starting point of type III
secretion. That flagella can also be
built perfectly well from the base is shown by the archaeal flagellum and its
type IV secretion-based system. But
unlike the type IV secretion system, which has periplasmic ATPases and other
outer membrane-associated active transport components, the type III system had
no mechanism of powered outer membrane transport available to build on.
3.4.4. Improvements on the type III pilus
Once a primitive pilus has evolved, a number of rapid
improvements can be expected. First
would be optimization of the pilin protein for its new role, under selection
for increased strength, minimizing breakage, increased speed of assembly, etc. Addition of the cap and subsequent
coevolution of the pilin and cap subunits could have occurred fairly early,
particularly as the pili became very long and assembly times became
significant. Pilus lengthening would be
selected for because it increases "reach" and adhesive surface area. That lengthening is a trivial matter of
regulation is shown by various lab-produced mutants that exhibit lengthened
hooks, needles, etc. due to simple mutations.
Type III pili might have reached the length of flagellar filaments (10
μm) long before motility originated; flagella and Hrp pili are of
comparable length (He and Jin, 2003, figure 1).
Soon the type III secretion system would become a specialized
pilus-secretion apparatus, and pili would be adapted for a variety of more
strenuous uses. For example, pili might
be used as stalks to elevate the bacterium above the surface, in order to
better access light, a particular concentration of molecules, or escape
competition from bacteria on the surface, all functions of attachment
organelles today (Dyer, 2003). Duplication
and modification of the pilin protein would allow greater functional
flexibility, such as adhesion to different substrates and the production of
certain kinds of pili based on environmental stimuli. One important modification that might have occurred after these
trends were well along would be to strengthen the pilus attachment to the cell
by extending the filament down into the secretion system to attach to the
export apparatus embedded in the cytoplasmic membrane. This would have been effected by cooption of
a duplicate pilin. The core tubule
structure of the flagellar rod, hook, and filament is constructed exclusively
by the N- and C-terminal domains of the axial proteins; the middle domains are
placed on the outside of the tubule, and in flagellin are highly modifiable and
often dispensable (Cohen-Krausz and
Trachtenberg, 2003). Therefore,
the "proto-rod" probably originated by loss of the outer domains, assuming that
the extracellular pilus had them for adhesion or structural purposes. This duplication event would create the
ancestors of the rod/hook subfamily and flagellin. Initially, one cap protein could chaperone the assembly of both
structures, but as they diverged, the cap protein would be duplicated as well
to allow specialization on each protein, assuming that modern flagellar rods
have cap proteins, as has been suggested (FlgJ; Berg, 2003; Macnab, 2003).
It is not clear that modern type III virulence pili make use
of rodlike proteins at all (the filament may simply extend all the way from the
cytoplasmic export apparatus out into the extracellular space), so it is also
possible that differentiation of filament and rod proteins occurred later and
that attachment to the export apparatus occurred via modification of the pilin
subunit. However, the hypothesis that
the duplication was early helps to explain the high divergence between
flagellin and the rod/hook subfamily, as well as explaining how the filament
became attached to the export apparatus instead of the outer membrane secretin
(although some attachment to the secretin may have remained; see below). Another protein, FliE, serves as the adaptor
protein between FliF (the MS-ring) and FlgB (the proximal rod protein). FliE homologs have not yet been detected in
type III virulence systems, so the utility of a FliE-like adaptor in nonmotile
systems is ambiguous. Here it will be
assumed that it was a relatively late, post-motility addition that strengthened
the attachment between the MS-ring and the rod. Investigation of the attachment mechanisms of modern type III
pili to the secretion system may shed light on the relative likelihood of these
possibilities.
3.5.1. The selective advantage of undirected motility
Even with a complex pilus in place, the modern flagellum
could not have originated in a single step.
It is hypothesized that the first, very crude motility function was
random dispersal. The function was
probably not stirring or gathering more food by more rapid movement, because of
the previously-discussed constraints of life at a low Reynolds number. Dispersal, on the other hand, is both a
ubiquitous adaptation in biology and rather undemanding in terms of
motility. Vogel (1994) reports that passive dispersal (i.e., unguided
dispersal by wind or current) is found in every phylum of animals and division
of plants. For creatures such as
bacteria, some dispersal will occur without any adaptations whatsoever: random
physical events can dislodge them from the substrate, and Brownian motion and
larger-scale turbulence and flow will move them about. However, dispersal is not always a good
thing for a bacterium. Since a
bacterium existing in a spot is most likely descended from a successfully
reproducing bacterium also at that spot, and therefore the environment is
conducive to reproduction, we might expect that the best choice for a bacterium
would be to stay where its at, rather than gambling everything on a rather
literal leap into the blue. On the
other hand, this logic will rapidly break down because any environment
conducive to replication will soon become filled to the brim with bacteria, at
which point competition for nutrients, space, light, etc. will become
severe. In such a situation there are
numerous potential responses (spore formation, killing fellow bacteria by
secreting antibiotics, etc.), but one of them is clearly dispersal (Dusenbery, 1998; Stoodley et al., 2002).
The best general strategy would be for the bacterium to
"decide" whether or not to disperse based on environmental cues: if life is
good, stay put, but if resources are scarce, go somewhere else. In fact, this is basically what regulates
the production of flagella in modern bacteria.
In E. coli, the master operon (class 1) for flagella encodes FlhC
and FlhD. These proteins activate the
genes for flagellar biosynthesis in the next operon (class 2), but are
repressed in high glucose conditions, where nutrients are plentiful and movement
is pointless. If conditions
deteriorate, the level of cyclic AMP in the cell increases, and the expression
of FlhDC is activated by cAMP and the catabolite repressor/activator protein
(CAP) (Berg, 2003). Dispersal
processes and evolutionary stable dispersal strategies are tractable to
mathematical and computer modeling, resulting with a large literature (e.g., Gandon and Roussett, 1999; Lebreton et al., 2000; Mathias et al., 2001; Poethke and Hovestadt,
2002), although most of it is not aimed specifically at
bacteria (for exceptions see Kreft et al., 2001; Kerr et al., 2002). All that is
needed for the argument to proceed at this point is that dispersal be a widely
beneficial behavior. However, the basic
dynamics of random bacterial diffusion are so simply described (Berg, 1993) that a short investigation of what kind of bacterium
might be expected to evolve random dispersal is irresistible.
Even dead or otherwise nonmotile bacteria have a non-trivial
diffusion coefficient: a sphere with a radius r will have a diffusion
coefficient Dsphere of (Berg,
1993, eqn. 4.13):

(6)
For a dead bacterium with a
diameter of 2 μm, Dsphere = 2.1x10-9 cm2/sec. The average time t it will take to
diffuse a distance x is given by (Berg,
1993, eqn. 1.10):
(7)
For the above bacterium, this means that it can be expected
to passively diffuse its 2 μm diameter in ~9.3 sec, purely by Brownian
motion. However, because Brownian
motion is a random walk, after each 'step' the diffusing particle has an equal
chance of going in any direction; the overall drift is zero. A large number of particles placed at one
location will gradually spread out in all directions by diffusion, but the mean
location of the population will not change from the starting point unless
additional forces come into play. The
fact that average diffusion time increases with the square of the distance
means that diffusion becomes an increasing poor way to travel as the dispersal
distance increases. The 2 μm
nonmotile bacterium, above, takes an average of 9 seconds to diffuse one body
length, but to diffuse 100 body lengths takes not 100 times as long (15
minutes) but 10,000 times as long (26 hours).
These figures are somewhat misleading as they completely ignore
turbulence and flow (perfume molecules would take a month to diffuse across a
room if diffusion was the only relevant process; Berg, 1993), but beneath the laminar boundary layer, very near a
surface, these forces will be reduced (Vogel,
1994).
Therefore, one very crude way for
a bacterium adhered to a surface to disperse is to cut loose its adhesins
and/or adhesive pili, and see how far it can move by passive dispersal. At least one such dispersal mechanism has
been documented (Coutte et al., 2003). If the
bacterium can manage to rise substantially in the boundary layer, flow or
turbulence may carry it some distance, at which point it can re-secrete
adhesive pili and attach to a new surface.
It will probably do better if it starts out at the top of long pilus;
similar dispersal-enhancing mechanisms are well known (fruiting bodies in
bacteria such as Myxococcus; Caulobacter stalks).
Under what conditions would it be
advantageous to enhance the effects of Brownian motion by adding crude active
motility? Usefully, Dusenbery (1997; 1998) has derived an equation allowing the calculation of
the relative utility of active and passive dispersal for organisms at a low
Reynolds number. Dusenbery assumes that
the organisms are spherical and that they swim at a velocity of 10 body
lengths/sec, a common value at widely varying scales (Dusenbery, 1996). Taking into
account the fact that rotational Brownian motion will keep any cell from
swimming in a straight line, the ratio of the diffusion coefficient with
motility (Dm) to the diffusion coefficient without it (D0)
is (Dusenbery, 1997; Table 2):
(8)
where r is again the radius
and u is the cell's swimming velocity in radii/sec (here, 20). Dusenbery calculates that a bacterium would
have to have a diameter of at least 0.64 μm in order to double its
diffusion coefficient for the purpose of undirected dispersal. Small bacteria have a very high diffusion
coefficient even without motility, and for small bacteria Brownian rotation is
so severe that swimming straight for any distance is impossible. Therefore, for very small bacteria, active
swimming with flagella is pointless; random Brownian motion is as good as it
gets (other swimming methods may be successful, e.g. the linear motor of Spiroplasma;
Trachtenberg et al., 2003). This has
the obvious implication that flagella cannot have evolved in very small
bacteria; Dusenbery surveyed bacteria genera and found that the smallest motile
genus had a diameter of 0.8 μm.
Similar minimum size constraints were found for motility with
chemotaxis, phototaxis, and thermotaxis (Dusenbery,
1997).
Equation (8) can be modified by converting
the relative swimming velocity u (in radii/sec) into the absolute
swimming velocity v (in cm/sec):
(9)
Equation (9) can be used to estimate the
minimum swimming velocity required for a protoflagellum to substantially
increase the diffusion coefficient of a cell.
Dm/D0 was calculated for cells ranging from 0-8
μm in diameter, with absolute swimming velocities of 0.1, 1, and 10
μm/sec (see Figure 6). Some
advantage to diffusion would result from motility for any values of Dm/D0. However, because the construction and
movement of filaments has some cost, we have followed Dusenbery in setting the
cutoff for "selectable motility function" at doubling the passive diffusion
coefficient (Dm > 2D0). As can be seen from Figure 6, Dusenbery's result is approximately reproduced
(swimming velocity here is absolute rather than relative to cell size, so
slightly different input values were used): Dm/D0
is doubled for a ~0.6 μm bacterium swimming at 10 μm/sec.
However, a crudely functioning
protoflagellum cannot be expected to push a bacterium at 10 μm/sec. The important result shown in Figure 6 is that even very slow absolute swimming velocities
can result in a significant improvement in the diffusion coefficient for large
bacteria. Swimming at 1/10 the velocity
of E. coli is advantageous to dispersal in a bacterium of ~2 μm
diameter, and swimming at 1/100 the velocity of E. coli is advantageous
to the dispersal of a bacterium with a diameter of ~6 μm. Two factors contribute to this pattern: for
larger bacteria, passive diffusion is markedly slower, increasing the relative
advantage of swimming. Similarly,
rotational diffusion is also slower for larger bacteria, but this factor
enhances the efficacy of swimming as swimming runs will take longer to be
randomly reoriented.
There are additional reasons to
think that the protoflagellum may have originated in a large bacterium. Similar beneficial scaling applies if
swimming velocity is considered in terms of body lengths/second: Dusenbery's
0.64 μm bacterium has to swim at 10 body lengths/second in order to beat
diffusion, but a 6 μm bacterium need only swim 0.17 body lengths/second in
order to achieve a benefit. A
consideration of carbon budgets also points this direction: for a
(hypothetical) very small flagellated bacterium, diameter 0.4 μm,
producing 10 peritricious flagella, each ten times the body length of the cell,
would cost 50% of the cell's carbon (Mitchell,
2002). However,
for a 1 μm cell, the relative cost of 10 flagella of proportional size is
only about ~2% of cell carbon. For the
6 μm diameter cell discussed above, it is ~0.2%. 6 μm diameter bacteria are well within the usual size range
of bacteria (Dusenbery, 1997). For a
moderately large bacterium, the costs of crude, poorly functioning flagella are
trivial, while the benefits in terms of dispersal are substantial. The exponential nature of the relationships
is such that moderate violations of the input assumptions will not greatly
change the qualitative results (Dusenbery,
1997); at some moderately large size the costs of
primitive motility become low and the benefits high.
3.5.2. Primitive flagella
The flagellar motor is made up of
two proteins, MotA and MotB. MotB binds
to the peptidoglycan cell wall, allowing the complex to serve as a stator. MotB (and perhaps MotA) also forms a proton
conducting channel. Although the exact
mechanism of motor function is still mysterious, with many proposed models (Berg, 2003; Schmitt, 2003), energy from the translocation of proteins in the
vicinity of MotB is somehow transformed into mechanical energy to move the
rotor. Probably this occurs by
conformational change in MotA, which then binds reversibly with the rotor
protein FliG, causing rotation.
Speaking very metaphorically, FliG appears to act like the teeth of a
gear, converting (in one model) the power stroke of MotA into rotary
motion. FliG is mounted on the central
MS-ring (FliF). Also attached to the
MS-ring (perhaps mostly but not exclusively via FliG) are the switch proteins
FliM and FliN. FliM contains a receptor
domain for the phosphorylated chemotaxis protein CheY-P, and the binding of
CheY-P induces some kind of conformational change in FliM, FliN, and FliG that
results in switching the direction of motor rotation from counterclockwise to
clockwise. This in turn results in a short
'tumble' which reorients the cell, and then the flagellum returns to
counterclockwise rotation.
Even given the minimal costs and
substantial selective benefits of crude motility, how could the sudden origin
of the rotary motor complex be mutationally possible? The basic answer is that the ancestors of the motor proteins were
already fully formed and serving other functions in the cell. It was recently discovered (Cascales et al., 2001; Kojima and Blair, 2001) that the flagellar motor proteins MotAB have
nonflagellar homologs: ExbBD and TolQR (Figure
4c). These
proteins share significant sequence similarity and all form ion channels that
energize work at a distance by a third protein; ExbBD and TolQR energize outer
membrane transport via action on TonB and TolA, respectively, while MotAB
energize flagellar motion via action on FliG.
The recently discovered homologs involved in Myxococcus gliding
systems (Youderian et al., 2003) will likely add another instance, although no
detailed studies of their function have been performed. The nonflagellar MotAB homologs are
phylogenetically widely distributed, found in proteobacteria, cyanobacteria, Aquifex,
and even in archaea (Kojima and
Blair, 2001). These facts
led Kojima and Blair to note that these proteins "could perform work in
contexts other than (and simpler than) the flagellar motor," and they conclude
that "ancestral forms of the MotA/MotB
complex might have arisen independently of any part of the rotor."
In order to form the motor-rotor
interface, however, the origin of a third protein, FliG, must also be accounted
for. No nonflagellar homologs of FliG
have been discovered (except in type III virulence systems), perhaps not surprisingly
given the peculiar function of this molecule and the radical change it must
have undergone, whatever its ancestral function. The structure of the middle and C-terminal domains of FliG has
been resolved (Brown et al., 2002), and is primarily made of alpha helices. Alpha helices are ubiquitous in proteins, so
FliG is not necessarily structurally bizarre, despite its unusual
function. Three general possibilities
present themselves for the origin of the FliG-MotAB complex. (1) TolQR homologs were coopted via a
mutation that allowed them to bind directly to FliF. FliG was a later addition that enhanced motility by improving
binding between the MS-ring and MotA, and gradually took over the interface
function completely. (2) Proto-FliG was
bound to FliF before the cooption of MotAB for some other reason, perhaps a
stabilization or structural function similar to that served by the derived FliG
homologs in type III virulence systems.
Mutant TolQR homologs then bound to proto-FliG. (3)
FliG was coopted simultaneously with MotAB, because it originated
as a fragment of a TolA homolog that ancestrally interacted with a TolQ
homolog. The third hypothesis is the
simplest and most direct pathway; the only novel interaction would be the binding
of the proto-FliG to FliF; binding to the proto-MotA would be inherited. This is less demanding than postulating the
re-engineering of the interface between a TolQ homolog and its substrate (a
feature of both hypotheses #1 and #2), and does not require postulating an
independent cooption of FliG from an unknown source. The hypothesis also has the advantage of being testable via
determination of the structures of TonB or TolA and investigation of their
interactions with ExbBD and TolQR.
On any of the hypotheses, it is
not absolutely necessary that crude motility be an immediate product; a coopted
TolQRA-like complex could first have associated with the type III secretion
system to enhance or help to control protein transport. All that would initially be required for the
very weak motility postulated above would be a slow rotation (other things
being equal, a swimming velocity of 1/100th that of E. coli
would imply a rotation rate similarly reduced; E. coli flagella rotate
at 100 Hz, so perhaps the protoflagellum rotated at ~1 Hz). In addition to the motility advantages
discussed above, wiggling and twisting would probably help to dislodge the
bacterium from the surface and from nearby bacteria; just such a function of
certain type IV bundle-forming pili has been observed in E. coli (Knutton
et al., 1999).
A possible objection here is that
the ancestral pilus cannot be expected to have been freely rotating, preadapted
for the addition of a motor complex; the rod might have been bound to the
peptidoglycan cell wall via the P-ring, making motility impossible. However, this assumes that a P-ring existed
at this point, a dubious assumption if a secretin can bridge both the cell wall
and cell membrane. Additionally, it is
actually not clear that pili are commonly rigidly fixed to the cell wall. It would be very interesting to know if type
III and other pili, when attached to a substrate, allow a bacterium to rotate
via Brownian motion in a fashion similar to motor-disabled bacteria with their
flagella attached to coverslips. Similar
observations would be useful for membrane-embedded structures such as outer
membrane secretins. One selective
reason that might favor freely-rotating pili prior to the evolution of motility
is, again, adhesion. A curved pilus
that is allowed to rotate via Brownian motion can continually explore more area
around a cell (particularly a large, slowly diffusing, slowly rotating cell)
than a rigidly attached pilus, increasing the chances of finding a
substrate.
A final possible objection is
that if the pilus was perfectly straight, then rotating it would not produce
motion. While this is true, it is
irrelevant because modern pili are not always straight or stiff (Bullitt and Makowski, 1998, Figures 2-4; Honma
and Nakasone, 1990; Yamashiro et al.,
1994), the interconversion of pilus shapes is mutationally
trivial, and there are many more ways to build a curved, helical filament than
a straight one from protein subunits.
Additionally, for the pilus rotated by Brownian motion postulated above,
some curvature and helical character would be a requirement in order for the
pilus to explore a larger area than a fixed pilus. The exact shape of the protoflagellum is not crucial in the drag-intensive
world of the low Reynolds number; Purcell (1997) has calculated that any number
of peculiar rotating shapes can swim with varying efficiency (and efficiency is
in fact basically energetically irrelevant at this scale; Purcell, 1977; Berg, 1993). Purcell (1977) notes: "Turn anything – if it isn't perfectly
symmetrical, you'll swim."
3.5.3. Loss of outer membrane secretin
Unlike other secretion systems (type I-IV, including type
III virulence systems), the type III secretion systems of bacterial flagella do
not actually have an outer membrane secretin.
The protein that plays the role of an outer membrane pore, FlgH, is
actually a lipoprotein that Dailey and Macnab (2002) suggest is homologous to the Salmonella type
III secretion system protein InvH. InvH
is a lipoprotein required for the insertion of the secretin InvG (SctC in
Hueck's (1998) unified nomenclature) into the outer membrane (Crago and Koronakis, 1998). Such a
mechanism is very common for outer membrane secretin assembly; in the type II
secretion system of Klebsiella oxytoca, the outer membrane lipoprotein
PulS binds 1:1 with the secretin protein PulD, preventing periplasmic
degradation and helping the localized assembly of the secretin into the outer
membrane. Twelve PulS proteins probably
form a ring about the 12-PulD secretin pore (Bitter,
2003).
These observations suggest a hypothesis for the origin of
the flagellar L-ring: it is not derived from an outer membrane secretin, as
would be naively assumed based on its position in the outer membrane. Rather, the FlgH L-ring may be derived from
the lipoprotein that was the chaperone for the secretin of the primitive type
III secretion system. Why, then, was
the secretin lost? An obvious
possibility is that it slowed the rotation of the protoflagellum. On the present model, the primitive type III
pilus originally bound to the outer membrane secretin, and later the channel
was extended down to the MS-ring in the cytoplasmic membrane. However, some association or binding might
have remained between the outer membrane secretin and the filament. If this was the case, it probably would not
have mattered for a pili rotating by Brownian motion or rotating at low Hz in
the protoflagellum. The outer membrane
can tolerate some rotational motion of embedded components, just as membranes
tolerate the lateral diffusion of proteins.
Some modern flagella are even covered by the membrane, such as the polar
flagella of Vibrio, although here a special sheath evidently prevents
tearing during rotation at 1500 Hz.
However, as rotation speeds increased, the risk of outer membrane
tearing would increase. A simple
solution to this problem could be to delete the secretin entirely; the outer
lipoprotein ring would take over the role of outer membrane pore, but would not
interact with the filament and would provide a bearing between the filament and
outer membrane. The P-ring protein
(FlgI) and its chaperone (FlgA) might have been added after this point by
cooption from another secretion system (perhaps FlgI is a highly modified
secretin itself). If one step in the
evolution of the bacterial flagellum was the loss of the outer membrane
secretin, it would be a classic example of evolutionary "scaffolding" (Thornhill and Ussery, 2000) at the molecular level, the only instance identified
in this evolutionary model. The
hypothesis would be strengthened if lipoproteins other than FlgH formed rings
in the outer membrane in other secretion systems; thus far, lipoprotein has not
been found in isolated secretin complexes although the ring structure is
suspected (Bitter, 2003).
3.5.4. Refinements
For a bacterium, a sphere is the
optimal shape for maximizing dispersal (Dusenbery,
1998). It can thus
be surmised that the cell was a coccus, like some flagellated bacteria today (Zaar
et al., 2003). In such a
cell, the surface positioning of a flagellum is irrelevant – one place is as
good as another on a sphere, so no positioning mechanism is required. Since the model postulates that random
dispersal was the original function of flagella, the first, crudely functioning
protoflagellum lacked many parts that are important in modern flagella. First, chemotaxis and switching are not
required for dispersal, so neither the Che proteins nor the switch complex
would have been required. If switching
is not required and the protoflagellar filament is sticking straight out at a
random position on spherical cell surface, then the hook region is similarly
dispensible. Once functional motility
was even marginally established, however, there would be rapid selection for
improvements. These might have occurred
in more or less any order, or concurrently, so they will be discussed
topically.
3.5.5. Chemotaxis and switching
The "flagellar" chemotaxis genes (Eisenbach, 2000; Table
2, this paper) are in fact not specific to flagella;
the same system is coupled to diverse motility systems, including archaeal
flagella and twitching motility (Faguy
and Jarrell, 1999; Bardy et al., 2003).
Furthermore, homologous components are tied to all manner of cellular
responses to the environment; the central two-component signal transduction
system (consisting of the histidine kinase CheA and the response regulator CheY
in flagellar chemotaxis) is ancient, found in all three domains, and used for
diverse functions. Their evolution is
discussed by Koretke et al. (2000). Another
major set of chemotaxis components, the membrane bound methyl-accepting
chemotaxis proteins (MCPs) which are the receptors for attractants and
repellants, have a similarly wide set of homologs with diverse functions (Zhulin
et al., 2003). More could
be said about details, as there are substantial variations between the
chemotaxis systems of various organisms (Eisenbach,
2000; Kirby et al., 2001), but for the purposes of this paper it will be
assumed that some sort of sensory transduction system preceded the origin of
the flagellum, and that one of the response regulators was the ancestor of
CheY. In modern flagella, a worsening
in conditions results in the increasing phosphorylation of CheY into CheY-P.
In E. coli, switching
rotation from counterclockwise (CCW) to clockwise (CW) causes the cell to
tumble, reorienting it to swim in a new direction. The probability of switching is increased by the binding of
CheY-P to FliM. If concentrations of
attractants are increasing during a run, CheY-P decreases and switching is
suppressed, and thus favorable runs tend to last longer. If repellents are increasing, CheY-P
increases, switching is promoted, and thus unfavorable runs are shortened. The cell uses this method to bias its random
walk, imposing an overall drift towards regions with higher concentrations of
attractants (Berg, 1993). However,
for bacteria with a diameter larger than 1.4 μm, run-and-stop or
run-and-reverse strategies are more energetically favored than the
run-and-tumble strategy, due to the larger costs of actively rotating a large
cell (Mitchell, 2002). As a result
the run-and-tumble strategy, while common in model organisms, is far from
universal. Rhodobacter sphaeroides
swims with a single, stop-start flagellum, with no reversing (Shah and Sockett, 1995; Shah et al., 2000). Passive
rotation via Brownian motion reorients the stopped cell. This is but one of many variations on
switching (Eisenbach, 2000), but probably resembles the most primitive version.
Explaining the origin of the
switch complex, which couples the chemotaxis system to flagellar rotation,
requires an examination of the domain structure and interactions of the switch
proteins. FliN and FliM, which make up
the C-ring, are homologs. FliN is
homologous to the C-terminal domain of FliM, and as a result the two proteins
probably occupy similar positions in the C-ring, perhaps alternating in a 3
FliN:1 FliM pattern, which approximately matches their stoichiometry (Mathews
et al., 1998; see also Figure
2, this paper).
FliM also has a N-terminal domain with no counterpart in FliN that is
the actual CheY-P receptor. CheY-P
binds to the receptor domain, increasing the probability of a switch to CW
rotation (Eisenbach, 2000) via an unknown mechanism involving interactions
between FliM/N and FliG (Mathews et al., 1998). The
receptor domain is homologous to the single-domain chemotaxis protein CheC of Bacillus
subtilis (Kirby et al., 2001). CheC binds
reversibly to the Bacillus C-ring, and is released when it binds to
CheY-P. CheC has not been found in E.
coli, but homologs are found in many early-branching bacteria, as well as
archaea. A cladogram generated for CheC
and the FliM CheC-like domain shows that CheC is phylogenetically basal (Kirby
et al., 2001).
A pre-existing sensory
transduction system could be coupled to flagellar rotation in a single step on
the hypothesis that a FliN-like protein existed for some nonflagellar cellular
response purpose, serving as a receptor for CheC. The exact function of modern CheC is not known, but it appears to
interact with CheA, CheD, and McpB, which form a receptor complex (Kirby
et al., 2001). CheC may
also have a FliM-like function via interaction with the C-ring (Szurmant et al., 2003). The
ancestor of FliN might therefore be found among the other proteins that CheC
interacts with. On the model, a
mutation in this FliN-like protein created a proto-FliN that bound to FliG,
slowing or jamming the motor. The
reversible binding of CheC to proto-FliN, however, happened to alleviate this
effect by changing the conformation of proto-FliN. CheY-P binding to CheC would result in the dephosphorylation of
CheY-P and the release of CheC from proto-FliN, resulting in the
slowed-rotation behavior. Chemotactic
behavior would thereby originate by a single mutation (all other interactions
would be inherited), which could then be followed by gradual improvements in the
initial crude function. This hypothesis
is more economical than supposing that FliN originated for some role in
structural support or enhancing export, and was later coopted to a switching
function via the binding of CheC, although this remains a possibility as FliN
is retained in type III virulence systems for some purpose. The first hypothesis suggests that the
homolog of FliN will be found within sensory transduction systems as one of the
proteins that CheC or a CheC homolog interacts with; it is hard to know where
to look with the latter hypothesis. The
considerable variations in the C-ring of bacteria may yield further hints, as
major variations on chemotaxis and the switch complex are known; for example, Aquifex
aeolicus lacks the traditional chemotaxis system as well as FliM; Bacillus
spp. have FliY (a FliM-FliN fusion protein; Bischoff and Ordal, 1992; Celandroni et al., 2000) rather than FliN.
In any case, the fusion of CheC-like and FliN-like proteins would
produce the FliM seen in most bacteria.
Hypothesizing a detailed pathway
explaining how a stop-start switch complex could be converted into the other
varieties of switching will depend on detailed knowledge of motor mechanism;
many models of the flagellar motor have been proposed and the question is far
from settled (Berg, 2003). However, if
proton-induced conformational changes in MotA induce some kind of poweroke
against FliG, followed by release of the FliG binding site and a return stroke
to the original position, then perhaps the answer is fairly simple. If the conformational change in the switch
complex shifts the FliG binding site up or down relative to MotA, then perhaps
the difference between "forward" and "reverse"
is just the difference between MotA-FliG binding on the forward power
stroke or the return stroke.
Although a detailed analysis will
not be performed here, the transition between random dispersal and dispersal +
chemotaxis is quite gradual; adding just a small amount of directional drift to
the random walk of bacteria allows gradual migration towards nutrient gradients
and away from toxins or waste products.
The advantages of directional drift over random diffusion are
exponential (Berg, 1993), and the costs in terms of extra carbon consumption
are trivial compared to the already small costs of building a flagellum in the
first place.
3.5.6. Hook and additional axial components
It seems likely that the hook (FlgE) and the four rod
proteins (FlgBCFG) are all duplicates of an ancestral rod protein; their
sequence relationships have been described in detail elsewhere (Homma
et al., 1990a; Homma et al.,
1990b). Whether
phylogeny can be expected to correlate well with sequence similarity in this
case is somewhat debatable, as adjacent axial components will tend to have
relatively similar structural roles and signal sequences. However, it is apparent that "adaptor"
proteins (FlgK and FlgL between the hook and filament; FlgG between the hook
and the rest of the rod) must have originated after the duplication of the
major components; for example, as the proto-hook and proto-filament proteins
began to specialize in their particular roles, the mismatches between the
subunits would become increasingly troublesome, limiting further
divergence. However, duplication and
modification of hook and flagellin proteins to produce adaptor proteins (a FlgE
copy producing FlgK, and a FliC copy producing FlgL) would allow both tighter
binding between hook and filament, and would remove the constraints on
specialization of the major structures.
These duplications need not have happened simultaneously; with a
moderate amount of divergence, one adaptor might do (e.g., FlgG is the adaptor
between the rod and hook), with a second being added as divergence
continued. This form of protein
subfunctionalization (Force et al., 1999) can probably explain the rest of the axial proteins
as well: the hook (FlgE) might well have originated as an adaptor between the
proto-rod and proto-filament in the very early flagellum. As FlgE specialized for the hook role,
adaptors for the filament (FlgK) and rod (FlgG) would have been produced from
copied hook proteins. The reason that
the flagellum has three proximal rod proteins (FlgBCF) is not clear, but may
have something to do with assembly checkpoints and coordinating the addition of
the P- and L-rings at the appropriate moment.
FlgB is the proximal rod component, interfacing with FliF via FliE (Berg, 2003); the relative order of FlgC and FlgF has not been
determined, but perhaps one assembles while the P-ring is being assembled
around it, and the other assembles coincident with the L-ring. These components are highly conserved across
all known bacterial flagella, probably because of co-adapted interactions
between the components, but their dispensability for building a basic filament
appears to be shown by type III virulence systems, where no rod homologs have
yet been discovered (Blocker et al., 2003) although the pilus protein shares similarities with
axial proteins (Aizawa, 2001;
Blocker et al., 2003; Cordes et al., 2003). Thus the
coordinated assembly of rod and the P- and L-rings could have been a relatively
late innovation. The origin of FliE,
the adaptor between the basal rod component FlgB and the FliF MS-ring, was
probably a very early event, occuring just after the origin of motility, in
order to strengthen the association between the mismatched symmetries of FliF
and the proto-rod. Since FliE is
exported via the type III pathway and is quite small (11 kDa), perhaps it
originated as a fragment of the proto-rod protein.
The hook capping protein FlgD
probably originated as a duplicate of the putative rod capping protein (FlgJ),
in a manner similar to the divergence of the rod cap and filament cap discussed
previously. However, E. coli
FlgJ has a C-terminal muramidase domain in addition to an N-terminal portion
that interacts with the rod proteins. This domain shows homology to other
muramidases and so was probably coopted in order to speed up flagellar assembly
by boring a hole through the cell wall.
This was probably a late addition; even today muramidase activity is not
absolutely required for successful flagellar assembly, probably because the
assembling rod has a chance of finding a suitable gap in the peptidoglycan on
its own (Hirano et al., 2001). Some
bacteria lack the muramidase domain (Rhodobacter) or FlgJ (gram-positive
bacteria) entirely (Hirano et al., 2001). An
investigation of the assembly of the SctC ring in type III virulence systems,
and similar structures in other systems, might shed light on just what the
muramidase is for, as its requirement has not yet been reported for other
secretion systems. Perhaps positioning
of the primitive type III pilus and protoflagellum was originally determined by
the ability of the P-ring to find a sufficient gap to insert itself in;
association of the secretin and proto-FliF with the P-ring brought the
secretory structure together. Having a
dedicated muramidase in the modern flagellar pathway might simply enable
flagella production on demand, at any predetermined spot, whether or not a
sufficient hole in the peptidoglycan is already available.
3.5.7. Modern variations
The model has arrived at something like the common ancestor
of all currently known bacterial flagella.
Some of the variant flagella, such as those found in gram-positive
bacteria, spirochetes, Aquifex, or Rhodobacter, might in fact be
early offshoots of flagellar evolution.
This is not required on the current model, but an improved understanding
of bacterial phylogeny may change the situation. In any case, most of these variants are probably derived (Cavalier-Smith, 2002a), as are many other minor variations that are known (Eisenbach, 2000; Bardy et al., 2003).
The detailed evolutionary model described above is
summarized in Figure 7. The role
that various evolutionary processes played in the model can now be roughly
quantified. Only one major shift of
function occurred at the system level, the transition from a pilus to a
protoflagellum. All of the other
changes in system function can be seen as minor modifications of a basic
function; if these are enumerated (export-->secretion-->adhesion-->pilus,
and dispersal-->taxis),
then four minor shifts of function occurred.
In all cases a "shift" in function is actually more accurately described
as an addition of function at the system level, as previous functions are
maintained. At the level of subsystems
(consisting of two or more proteins), the cooption events can be tabulated:
subsystem cooption was invoked for the origin of the core export apparatus,
FlgIHA+outer membrane secretin, the adhesin ancestral to the axial protein
family, the motor complex, and the chemotaxis/switch complex; a total of five
subsystem cooption events. In each of
these cases, cooption occurred by the mutation of one protein to link the two
systems, followed by the duplication and integration of the subsystem proteins
into the major system. Except for the
major transition between pilus and motility, subsystem cooption was associated
with improvements of system function rather than major changes in system
function. At the gene level,
duplication events within the core system were invoked 11 times for
origin of 12 axial proteins from one, and an additional time for the divergence
of FliN and FliM. None of these events
requires postulating functional shift at the subsystem or system levels. Addition of a new domain with novel
functionality was identified twice (FliN+CheC-->FliM, rod
cap+muramidase-->FlgJ),
although it probably occurred in additional instances where homologies are
currently more vague. Loss of a
component (the outer membrane secretin of the primitive type III secretion
system) occurred only once, even though some components that are ancient on the
model (e.g., FliH) are apparently not absolutely required in modern flagella (Minamino et al., 2003). All other
changes at all levels were matters of gradual improvement of function, i.e.
optimization and co-adaptation of components.
Even at this early stage of development, the model gives decent estimate
of the relative importance of various evolutionary processes involved in the
origin of complex biochemical systems.
Even the present extended treatment has left out detailed
discussion of the origin of the chemotaxis and regulatory proteins listed in Table 2. However,
many of these proteins have homologs functional in different systems, and the
chaperones of axial proteins might have originated by duplication in a fashion
similar to the axial proteins themselves.
The evolution of the organization of flagellar genes and operons also
deserves attention, although the precise organization found in modern bacteria
is probably not essential (Kalir et al., 2001).
Biological evidence supporting the model is summarized in Table 6, in terms of extant analogs to the hypothesized
intermediates and nonflagellar homologs of system components. Of the 30 major structural components listed
in Table 1, 12 are axial proteins and probably share a common
(unidentified) ancestor, a hypothetical type III pilin subunit. Of the remaining 18 components, four (FliI,
MotA, MotB, and FliM) have well-accepted nonflagellar homologs based on
significant sequence similarity.
Suggestive evidence of homology exists for seven components, FliHJOPQR
(with components of ATP synthetase), and the lipoprotein FlgH (with lipoprotein
chaperones of secretins). On the basis
of interactions with other components with identified nonflagellar homologs,
homologies can be postulated, with little current supporting evidence, for
three components, FlgIA (with other secretin-associated proteins secreted by
the type II secretion system), and FliG (with a fragment of a TolA
homolog). Finally, five components
(FliF, FlhA, FlhB, FliN, and the ancestor of the axial proteins) have no
identified potential homologs, although nonflagellar ancestral functions are
not difficult to postulate. The type
III virulence system contains homologs of most of these proteins (probably including
an axial protein; Cordes et al., 2003), but as discussed previously its phylogenetic
position is controversial.
At this early stage of investigation this mixed bag should
not be surprising. Structural
information (which is conserved even when sequence similarity is lost) is not
available for most of the proteins, and current sampling of bacterial genomes
is not very balanced. However, the
homologies postulated provide opportunities to test the model with future
observations: if the model presented here is correct, then it is expected that
nonflagellar homologs for most flagellar proteins will be found serving the
suggested functions, in the suggested systems.
Similarly, the model can be falsified by discovery of homologies in unexpected
locations: for example, if the proteins of the flagellar basal body are
discovered to be homologous to the junctional pore of gliding motility rather
than a primitive type III secretion system, then the entire model would be
overthrown and replaced by a model relating these two systems.
The proposed analogies (Table
6) provide another set of tests of the model. Each of the systems proposed as analogies to
stages in flagellar evolution is a piece of evidence that the selective forces
invoked in the model are common; the fact that the functions of secretion,
adhesion, pilus formation, and motility appear to be related in analogous
systems lends support to the model, which postulates transitions between these
functions. The model would be weakened
if the proposed analogies, mostly based on well-studied laboratory organisms,
were found to actually be rare in free-living prokaryotes. On the other hand, the discovery of further
similar analogs will strengthen the model – for example, it is expected that
many additional components of the archaeal flagellum will be determined to be
homologous to type IV secretion. The
conclusions of the simple cost-benefit model proposed here can also be tested
via analogs. Calculations indicated that
the cost-benefit tradeoff is strongly in favor of motility, even very crude
motility, in a moderately large bacterium.
It would therefore be expected that, in an experimental environment
where dispersal is advantageous, selection would favor the retention of even
severely impaired (but still motile) flagella for large bacteria, while
similarly impaired flagella would be selected against in small bacteria. Similarly, attempts to evolve crude motility
in the lab (or re-evolve motility after the deletion of a crucial component)
would only work if large bacteria are used.
As there are experimental conditions where it is selectively
advantageous for bacteria to lose motility (Velicer et al., 2002), a careful consideration of the microbial
environment would be required.
The present model has several implications for the evolution
of other prokaryote motility systems.
The conclusions of the cost/benefit analysis, that stirring is an unlikely
intermediate function, and that even crude motility is advantageous for
dispersal in large bacteria, will apply to the evolution of any type of
flagellar-like motility in prokaryotes (the tiny Spiroplasma are
apparently motile, but use a radically different system). However, these conclusions cannot be
generalized to the evolution of the eukaryotic cilium, as many eukaryotes have
reached the size where stirring and swimming become useful feeding behaviors (Vogel, 1994). Although
detailed information on mechanism and homologies is not yet available, gliding
motility and archaeal flagella probably both originated via evolutionary
processes analogous to the present model, by cooption of pre-existing secretion
systems. This basic idea has already
been proposed for archaeal flagella (Bayley
and Jarrell, 1998). The fact
that archaeal and bacterial flagella are completed unrelated appears to weaken
Cavalier-Smith's (2002a) argument that archaea are derived; however, if type
IV secretion systems can be found in gram positive bacteria then a plausible
ancestor for archaeal flagella would exist in Cavalier-Smith's scheme. Presumably, standard bacterial flagella
could not be adapted to hyperthermophillic, hyperacidic conditions (Cavalier-Smith, 2002a), and the archaeal cenancestor was forced to
re-evolve a completely new form of flagellum.
It is sometimes alleged that the construction of
evolutionary models amounts to nothing more than the telling of "just-so
stories." However, the putative
originators of this criticism, Gould and Lewontin (1979), only attacked scenarios that were untestable or
untested. They particularly focused
their criticism of "adaptive storytelling" on cases where the adaptive function
of the trait in question was highly dubious, such as human sacrifice (Gould and Lewontin, 1979). Their point
was that some traits might be explained by processes other than selection. They never argued that systems like the
bacterial flagellum, where function, complexity, and adaptiveness are obvious,
might have an explanation not involving the extended action of natural selection.
A related objection to evolutionary modeling is that it is
armchair theorizing, unrelated to the practical concerns of the present
day. However, an examination of recent
discoveries of nonflagellar homologs of flagellar components shows that this is
not the case. The recognition of
homology between flagella and type III virulence systems has contributed
greatly to an understanding of the latter, which are implicated in many
diseases of humans, livestock, and crops (Hueck,
1998; Cornelis and Van Gijsegem, 2000; Büttner and Bonas, 2002; Blocker et al., 2003; He and Jin, 2003). Similarly,
the homology between ion channels and flagellar motor proteins contributes to
the understanding of the still-mysterious mechanism of the flagellar motor (Schmitt, 2003; Zhai et al., 2003). In the case
of the present model, the hypothesis of more extensive homology between the F1F0-ATP
synthetase and the type III export apparatus, if true, has important
implications, as the integral membrane components are the most poorly understood
portion of the flagellum and type III virulence systems (Macnab, 2003).
A final advantage of constructing an evolutionary model is
that it encourages the synthesis of data, relating the discoveries of
specialist subfields in a coherent framework.
Such a framework is a prerequisite for more detailed evolutionary
investigations, providing research questions and hypotheses to test, and
challenging dissenters to come up with better models. Until now a detailed evolutionary model had never been seriously
attempted for the bacterial flagellum, and even this fairly basic survey has
yielded several discoveries that were not obvious at the outset. The bacterial flagellum (and prokaryote
motility systems in general) probably arose in large, coccus-shaped bacteria
that were essentially modern in terms of complexity. It is not necessary to suppose that the flagellum co-evolved with
the cell wall and membranes before the last common ancestor of life. This would be a much more difficult event to
study in any case. The previously
accepted homologies between flagellar components and nonflagellar systems (such
as for FliI and MotAB) are not the strange anomalies they appear to be when
viewed in isolation, rather they fit well into a gradual model of flagellar
evolution, and give clues as to where further homologies may be
discovered. Cooption of preexisting
subsystems are the key events of interest in the model; gene duplications
within the system primarily add complexity after the origin of the protoflagellum,
and other processes, such as domain-swapping and the loss of "scaffolding"
components, are relatively minor players.
Finally, in light of the organized complexity and apparent "design" of
the flagellum, the very fact that a step-by-step Darwinian model can be
constructed that is plausible and testable significantly weakens the suggestion
that extraordinary explanations might be required.
This work could not have been accomplished without help from
numerous individuals, who supplied ideas, references, encouragement, and
helpful comments. First, Ian Musgrave
and his work on this topic were inspirational, and he was a helpful
discussant. Pete Dunkelberg, Matt
Inlay, Alan Gishlick, Mike Hopkins, Wesley Elsberry, John Wilkins, Pim van
Meurs, and many others gave help in terms of editing and informal
discussions. The initial inspiration
was another paper, on the evolution of biological complexity, written for a
course taught Jim Proctor, although the present paper ended up going far beyond
the original topic.
Aizawa,
S. I., 2001. Bacterial flagella and type III secretion systems. FEMS Microbiol
Lett. 202 (2), 157-164., doi:10.1016/S0378-1097(01)00301-9.
Albertini, A. M., Caramori, T.,
Crabb, W. D., Scoffone, F. and Galizzi, A., 1991. The flaA locus of Bacillus subtilis is part of a large
operon coding for flagellar structures, motility functions, and an ATPase-like
polypeptide. J Bacteriol. 173 (11), 3573-3579.
Anandarajah, K., Kiefer, P. M.,
Donohoe, B. S. and Copley, S. D., 2000. Recruitment of a double bond isomerase
to serve as a reductive dehalogenase during biodegradation of
pentachlorophenol. Biochemistry. 39 (18), 5303-5311., doi:10.1021/bi9923813.
Arora, S. K., Ritchings, B. W.,
Almira, E. C., Lory, S. and Ramphal, R., 1998. The Pseudomonas aeruginosa flagellar cap protein, FliD, is responsible
for mucin adhesion. Infect Immun. 66 (3), 1000-1007.
Auvray, F., Ozin, A. J., Claret, L.
and Hughes, C., 2002. Intrinsic membrane targeting of the flagellar export
ATPase FliI: Interaction with acidic phospholipids and FliH. Journal of
Molecular Biology. 318 (4), 941-950., doi:10.1016/S0022-2836(02)00172-9.
Bardy, S. L., Ng, S. Y. and Jarrell,
K. F., 2003. Prokaryotic motility structures. Microbiology. 149 (Pt 2),
295-304., doi:10.1099/mic.0.25948-0.
Bayley, D. P. and Jarrell, K. F.,
1998. Further evidence to suggest that archaeal flagella are related to
bacterial type IV pili. J Mol Evol. 46 (3), 370-373.
Berg, H. C., 1993. Random Walks in
Biology. Princeton University Press, Princeton.
Berg, H. C., 1998. Keeping up with
the F1-ATPase. Nature. 394 (6691), 324-325., doi:10.1038/28506.
Berg, H. C., 2003. The rotary motor
of bacterial flagella. Annu Rev Biochem. 72, 19-54.,
doi:10.1146/annurev.biochem.72.121801.161737.
Berg, H. C. and Anderson, R. A.,
1973. Bacteria swim by rotating their flagellar filaments. Nature. 245 (5425),
380-382.
Berry, R. M., 2000. Theories of
rotary motors. Philos Trans R Soc Lond B Biol Sci. 355 (1396), 503-509.,
doi:10.1098/rstb.2000.0591.
Birkenhager, R., Greie, J.-C.,
Altendorf, K. and Deckers-Hebestreit, G., 1999. F0 complex of the Escherichia coli ATP synthase . Not all
monomers of the subunit c oligomer are involved in F1 interaction.
Eur J Biochem. 264 (2), 385-396., doi:10.1046/j.1432-1327.1999.00652.x.
Bischoff, D. S. and Ordal, G. W.,
1992. Identification and characterization of FliY, a novel component of the Bacillus subtilis flagellar switch
complex. Mol Microbiol. 6 (18), 2715-2723.
Bitter, W., 2003. Secretins of Pseudomonas aeruginosa: large holes in
the outer membrane. Arch Microbiology. 179 (5), 307-314.,
doi:10.1007/s00203-003-0541-8.
Block, S. M., 1997. Real engines of
creation. Nature. 386 (6622), 217-219., doi:10.1038/386217a0.
Blocker, A., Komoriya, K. and
Aizawa, S. I., 2003. Type III secretion systems and bacterial flagella:
Insights into their function from structural similarities. Proc Natl Acad Sci U
S A. 100 (6), 3027-3030., doi:10.1073/pnas.0535335100.
Boyer, P. D., 1997. The ATP synthase
- A splendid molecular machine. Annu Rev Biochem. 66, 717-749.,
doi:10.1146/annurev.biochem.66.1.717.
Broome-Smith, J. K. and Mitsopoulos,
C., 1999. Overview: Transport of molecules across microbial membranes -- a
sticky situation to get to grips with, in: Broome-Smith, J. K., Baumberg, S.,
Stirling, C. J. and Ward, F. B. (Eds.), Transport of molecules across microbial
membranes, General Society for Microbiology, Cambridge University Press,
Cambridge, UK, pp.1-14.
Brown, P. N., Hill, C. P. and Blair,
D. F., 2002. Crystal structure of the middle and C-terminal domains of the
flagellar rotor protein FliG. Embo J. 21 (13), 3225-3234.,
doi:10.1093/emboj/cdf332.
Buchanan, S. K., 2001. Type I
secretion and multidrug efflux: transport through the TolC channel-tunnel. Trends
in Biochemical Sciences. 26 (1), 3-6., doi:10.1016/S0968-0004(00)01733-3.
Bullitt, E. and Makowski, L., 1998.
Bacterial adhesion pili are heterologous assemblies of similar subunits.
Biophys J. 74 (1), 623-632.
Büttner, D. and Bonas, U., 2002. Port
of entry – the type III secretion translocon. Trends Microbiol. 10 (4),
186-192., doi:10.1016/S0966-842X(02)02331-4.
Campbell, N. A., 1993. Biology.
Benjamin/Cummings, Redwood City, CA.
Campos-Garcia, J., Najera, R.,
Camarena, L. and Soberon-Chavez, G., 2000. The Pseudomonas aeruginosa motR gene involved in regulation of
bacterial motility. FEMS Microbiol Lett. 184 (1), 57-62.,
doi:10.1016/S0378-1097(00)00019-7.
Cao, T. B. and Saier, M. H., Jr.,
2003. The general protein secretory pathway: phylogenetic analyses leading to
evolutionary conclusions. Biochim Biophys Acta. 1609 (1), 115-125.,
doi:10.1016/S0005-2736(02)00662-4.
Capaldi, R. A. and Aggeler, R.,
2002. Mechanism of the F1-F0-type ATP synthase, a
biological rotary motor. Trends in the Biochemical Sciences. 27 (3), 154-160.,
doi:10.1016/S0968-0004(01)02051-5.
Cascales, E., Lloubes, R. and
Sturgis, J. N., 2001. The TolQ-TolR proteins energize TolA and share homologies
with the flagellar motor proteins MotA-MotB. Mol Microbiol. 42 (3), 795-807.,
doi:10.1046/j.1365-2958.2001.02673.x.
Cavalier-Smith, T., 1978. The
evolutionary origin and phylogeny of microtubules, mitotic spindles and
eukaryote flagella. Biosystems. 10 (1-2), 93-114.
Cavalier-Smith, T., 1982. The
evolutionary origin and phylogeny of eukaryote flagella. Symposia of the
Society for Experimental Biology. 35 (5896), 465-493.
Cavalier-Smith, T., 1987a. The
origin of cells: a symbiosis between genes, catalysts, and membranes. Cold
Spring Harbor Symposia on Quantitative Biology. 52 (6111), 805-824.
Cavalier-Smith, T., 1987b. The
origin of eukaryotic and archaebacterial cells. Annals of the New York Academy
of Sciences. 503, 17-54.
Cavalier-Smith, T., 2001a. Obcells
as proto-organisms: membrane heredity, lithophosphorylation, and the origins of
the genetic code, the first cells, and photosynthesis. Journal of Molecular
Evolution. 53 (4-5), 555-595., doi:10.1007/s002390010245.
Cavalier-Smith, T., 2001b. Early
evolution: from the appearance of the first cell to the first modern organisms
(review). Quarterly Review of Biology. 76 (2), 233.
Cavalier-Smith, T., 2002a. The
neomuran origin of archaebacteria, the negibacterial root of the universal tree
and bacterial megaclassification. Int J Syst Evol Microbiol. 52, 7-76.
Cavalier-Smith, T., 2002b. The
phagotrophic origin of eukaryotes and phylogenetic classification of Protozoa.
Int J Syst Evol Microbiol. 52, 297-354.
Cavalier-Smith, T., 2002c. Origins
of the machinery of recombination and sex. Heredity. 88, 125-141.,
doi:10.1038/sj.hdy.6800034.
Celandroni, F., Ghelardi, E.,
Pastore, M., Lupetti, A., Kolsto, A. B. and Senesi, S., 2000. Characterization
of the chemotaxis fliY and cheA genes in Bacillus cereus. FEMS Microbiol Lett. 190 (2), 247-253.,
doi:10.1016/S0378-1097(00)00343-8.
Chapman, M. R., Robinson, L. S.,
Pinkner, J. S., Roth, R., Heuser, J., Hammar, M., Normark, S. and Hultgren, S.
J., 2002. Role of Escherichia coli
curli operons in directing amyloid fiber formation. Science. 295 (5556),
851-855., doi:10.1126/science.1067484.
Chothia, C., Gough, J., Vogel, C.
and Teichmann, S. A., 2003. Evolution of the protein repertoire. Science. 300
(5626), 1701-1703., doi:10.1126/science.1085371.
Christie, P. J., 2001. Type IV
secretion: intercellular transfer of macromolecules by systems ancestrally
related to conjugation machines. Mol Microbiol. 40 (2), 294-305.,
doi:10.1046/j.1365-2958.2001.02302.x.
Christie, P. J. and Vogel, J. P.,
2000. Bacterial type IV secretion: conjugation systems adapted to deliver
effector molecules to host cells. Trends Microbiol. 8 (8), 354-360.,
doi:10.1016/S0966-842X(00)01792-3.
Claret, L., Calder, S. R., Higgins,
M. and Hughes, C., 2003. Oligomerization and activation of the FliI ATPase
central to bacterial flagellum assembly. Mol Microbiol. 48 (5), 1349-1355., doi:10.1046/j.1365-2958.2003.03506.x.
Cohen-Krausz, S. and Trachtenberg,
S., 2003. The structure of the helically perturbed flagellar filament of Pseudomonas rhodos: implications for the
absence of the outer domain in other complex flagellins and for the flexibility
of the radial spokes. Mol Microbiol. 48 (5), 1305-1316.,
doi:10.1046/j.1365-2958.2003.03466.x.
Copley, S. D., 2000. Evolution of a
metabolic pathway for degradation of a toxic xenobiotic: the patchwork
approach. Trends in Biochemical Sciences. V25 (N6), 261-265.,
doi:10.1016/S0968-0004(00)01562-0.
Cordes, F. S., Komoriya, K.,
Larquet, E., Yang, S., Egelman, E. H., Blocker, A. and Lea, S. M., 2003.
Helical structure of the needle of the type III secretion system of Shigella flexneri. J Biol Chem. 278
(19), 17103-17107., doi:10.1074/jbc.M300091200.
Corliss, J. O., 1980. Objection to
"undulipodium" as an inappropriate and unnecessary term. Biosystems.
12 (1-2), 109-110.
Cornelis, G. R. and Van Gijsegem,
F., 2000. Assembly and function of type III secretory systems. Annu Rev
Microbiol. 54, 735-774., doi:10.1146/annurev.micro.54.1.735.
Coutte, L., Alonso, S., Reveneau,
N., Willery, E., Quatannens, B., Locht, C. and Jacob-Dubuisson, F., 2003. Role
of adhesin release for mucosal colonization by a bacterial pathogen. J Exp Med.
197 (6), 735-742., doi:10.1084/jem.20021153.
Cox, G. B., Jans, D. A., Fimmel, A.
L., Gibson, F. and Hatch, L., 1984. Hypothesis. The mechanism of ATP synthase.
Conformational change by rotation of the beta-subunit. Biochim Biophys Acta.
768 (3-4), 201-208.
Crago, A. M. and Koronakis, V.,
1998. Salmonella InvG forms a
ring-like multimer that requires the InvH lipoprotein for outer membrane
localization. Mol Microbiol. 30 (1), 47-56.,
doi:10.1046/j.1365-2958.1998.01036.x.
Dailey, F. E. and Macnab, R. M.,
2002. Effects of lipoprotein biogenesis mutations on flagellar assembly in Salmonella. J Bacteriol. 184 (3),
771-776., doi:10.1128/JB.184.9.2460-2464.2002.
Darwin, C., 1851. A Monograph of the
Sub-class Cirripedia, with Figures of all the Species. The Lepadidae; or,
Pedunculated Cirripedes. Ray Society, London.
Darwin, C., 1854. A Monograph of the
Sub-class Cirripedia, with Figures of all the Species. The Balanidae (or
Sessile Cirripedes); the Verrucidae, etc. Ray Society, London.
Darwin, C., 1862. On the various
contrivances by which orchids are fertilised. J. Murray, London.
Darwin, C., 1872. The origin of
species by means of natural selection. John Murray, London.
de Duve, C., 1995. Vital Dust: Life
as a cosmic imperative. Basic Books, New York.
de Souza, M. L., Seffernick, J.,
Martinez, B., Sadowsky, M. J. and Wackett, L. P., 1998. The atrazine catabolism
genes atzABC are widespread and highly conserved. J Bacteriol. 180 (7),
1951-1954.
DeLong, E. F. and Pace, N. R., 2001.
Environmental diversity of bacteria and archaea. Systematic Biology. 50 (4),
470-478., doi:10.1080/106351501750435040.
DeRosier, D. J., 1998. The turn of
the screw: the bacterial flagellar motor. Cell. 93 (1), 17-20.
Dillon, R., Fauci, L. and Gaver, D.,
3rd, 1995. A microscale model of bacterial swimming, chemotaxis and substrate
transport. J Theor Biol. 177 (4), 325-340., doi:10.1006/jtbi.1995.0251.
Doolittle, R. F. and Feng, D. F.,
1987. Reconstructing the evolution of vertebrate blood coagulation from a
consideration of the amino acid sequences of clotting proteins. Cold Spring
Harbor Symposia on Quantitative Biology: The Evolution of Catalytic Function.
LII, 869-874.
Durand, E., Bernadac, A., Ball, G.,
Lazdunski, A., Sturgis, J. N. and Filloux, A., 2003. Type II protein secretion
in Pseudomonas aeruginosa: the
pseudopilus is a multifibrillar and adhesive structure. J Bacteriol. 185 (9),
2749-2758., doi:10.1128/JB.185.9.2749-2758.2003.
Dusenbery, D. B., 1996. Life At
Small Scale: The Behavior of Microbes. Scientific American Library, New York.
Dusenbery, D. B., 1997. Minimum size
limit for useful locomotion by free-swimming microbes. Proc Natl Acad Sci U S
A. 94 (20), 10949-10954., doi:10.1073/pnas.94.20.10949.
Dusenbery, D. B., 1998. Fitness
landscapes for effects of shape on chemotaxis and other behaviors of bacteria.
J Bacteriol. 180 (22), 5978-5983.
Dyer, B. D., 2003. A Field Guide to
Bacteria. Cornell University Press, Ithaca.
Eisenbach, M., 2000. Bacterial
chemotaxis, Nature Encyclopedia of Life Sciences, Nature Publishing Group,
London., doi:10.1038/npg.els.0001251.
Emerson, S. U., Tokuyasu, K. and
Simon, M. I., 1970. Bacterial flagella: polarity of elongation. Science. 169
(941), 190-192.
Faguy, D. M. and Jarrell, K. F.,
1999. A twisted tale: the origin and evolution of motility and chemotaxis in
prokaryotes. Microbiology. 145 (Pt 2), 279-281.
Faguy, D. M., Jarrell, K. F., Kuzio,
J. and Kalmokoff, M. L., 1994. Molecular analysis of archael flagellins:
similarity to the type IV pilin-transport superfamily widespread in bacteria.
Can J Microbiol. 40 (1), 67-71.
Fan, F., Ohnishi, K., Francis, N. R.
and Macnab, R. M., 1997. The FliP and FliR proteins of Salmonella typhimurium, putative components of the type III
flagellar export apparatus, are located in the flagellar basal body. Mol
Microbiol. 26 (5), 1035-1046.
Fernandez, L. A. and Berenguer, J.,
2000. Secretion and assembly of regular surface structures in Gram-negative
bacteria. FEMS Microbiol Rev. 24 (1), 21-44., doi:10.1016/S0168-6445(99)00026-1.
Force, A., Lynch, M., Pickett, F.
B., Amores, A., Yan, Y. L. and Postlethwait, J., 1999. Preservation of
duplicate genes by complementary, degenerative mutations. Genetics. 151 (4),
1531-1545.
Francis, N. R., Sosinsky, G. E.,
Thomas, D. and DeRosier, D. J., 1994. Isolation, characterization and structure
of bacterial flagellar motors containing the switch complex. J Mol Biol. 235
(4), 1261-1270., doi:10.1006/jmbi.1994.1079.
Fraser, G. M., Gonzalez-Pedrajo, B.,
Tame, J. R. and Macnab, R. M., 2003. Interactions of FliJ with the Salmonella
Type III Flagellar Export Apparatus. J Bacteriol. 185 (18), 5546-5554.,
doi:10.1128/JB.185.18.5546-5554.2003.
Gandon, S. and Roussett, F., 1999.
Evolution of stepping-stone dispersal rates. Proc R Soc Lond B Biol Sci. 266
(1437), 2507-2513.
Ganfornina, M. D. and Sanchez, D.,
1999. Generation of evolutionary novelty by functional shift. Bioessays. V21
(N5), 432-439.
Giron, J. A., Torres, A. G., Freer,
E. and Kaper, J. B., 2002. The flagella of enteropathogenic Escherichia coli mediate adherence to
epithelial cells. Mol Microbiol. 44 (2), 361-379.,
doi:10.1046/j.1365-2958.2002.02899.x.
Gogarten, J. P. and Kibak, H., 1992.
The bioenergetics of the last common ancestor and the origin of the eukaryotic
endomembrane system. Biosystems. 28 (1-3), 131-153.
Gogarten, J. P., Kibak, H.,
Dittrich, P., Taiz, L., Bowman, E. J., Bowman, B. J., Manolson, M. F., Poole,
R. J., Date, T. and Oshima, T., 1989. Evolution of the vacuolar H+-ATPase:
implications for the origin of eukaryotes. Proc Natl Acad Sci U S A. 86 (17),
6661-6665.
Gogarten, J. P., Starke, T., Kibak,
H., Fishman, J. and Taiz, L., 1992. Evolution and isoforms of V-ATPase
subunits. J Exp Biol. 172, 137-147.
Gonzalez-Pedrajo, B., Fraser, G. M.,
Minamino, T. and Macnab, R. M., 2002. Molecular dissection of Salmonella FliH,
a regulator of the ATPase FliI and the type III flagellar protein export
pathway. Mol Microbiol. 45 (4), 967-982., doi:10.1046/j.1365-2958.2002.03047.x.
Goodenough, U., 1998. The Sacred
Depths of Nature. Oxford University Press, New York.
Goodenough, U., 2002. Of flagella
and outboard motors, accessed online. URL:
http://www.metanexus.net/archives/printerfriendly.asp?archiveid=7143.
Gophna, U., Ron, E. Z. and Graur,
D., 2003. Bacterial type III secretion systems are ancient and evolved by
multiple horizontal-transfer events. Gene. 312, 151-163.,
doi:10.1016/S0378-1119(03)00612-7.
Gould, S. J. and Lewontin, R. C.,
1979. The spandrels of San Marco and the Panglossian paradigm: A critique of
the adaptationist programmme. Proceedings of the Royal Society of London,
Series B. 205 (1161), 581-598.
Guerrero, R., Esteve, I.,
Pedrós-Alió, C. and Gaju, N., 1987. Predatory bacteria in prokaryotic
communities. Annals of the New York Academy of Sciences. 503, 238-250.
Hanumanthaiah, R., Day, K. and
Jagadeeswaran, P., 2002. Comprehensive analysis of blood coagulation pathways
in Teleostei: evolution of coagulation factor genes and identification of
zebrafish factor VIIi. Blood Cells, Molecules, and Diseases. 29 (1), 57-68.
Harris, R. J. and Elder, D., 2002.
Actin and flagellin may have an N-terminal relationship. J Mol Evol. 54 (2),
283-284., doi:10.1007/s00239-001-0067-0.
Harshey, R. M. and Toguchi, A.,
1996. Spinning tails: homologies among bacterial flagellar systems. Trends
Microbiol. 4 (6), 226-231., doi:10.1016/0966-842X(96)10037-8.
Hayward, C. A., 2000.
Microorganisms, Nature Encyclopedia of Life Sciences, Nature Publishing Group,
London., doi:10.1038/npg.els.0000460.
He, S. Y., 1998. Type III protein
secretion in plant and animal pathogenic bacteria. Annual Reviews in
Phytopathology. 36, 363-392., doi:10.1146/annurev.phyto.36.1.363.
He, S. Y. and Jin, Q., 2003. The Hrp
pilus: learning from flagella. Curr Opin Microbiol. 6 (1), 15-19.,
doi:10.1016/S1369-5274(02)00007-3.
Henderson, I. R., Navarro-Garcia, F.
and Nataro, J. P., 1998. The great escape: structure and function of the
autotransporter proteins. Trends Microbiol. 6 (9), 370-378.,
doi:10.1016/S0966-842X(98)01318-3.
Hirano, T., Minamino, T. and Macnab,
R. M., 2001. The role in flagellar rod assembly of the N-terminal domain of Salmonella FlgJ, a flagellum-specific
muramidase. J Mol Biol. 312 (2), 359-369., doi:10.1006/jmbi.2001.4963.
Homma, M., DeRosier, D. J. and
Macnab, R. M., 1990a. Flagellar hook and hook-associated proteins of Salmonella typhimurium and their
relationship to other axial components of the flagellum. J Mol Biol. 213 (4),
819-832.
Homma, M., Kutsukake, K., Hasebe,
M., Iino, T. and Macnab, R. M., 1990b. FlgB, FlgC, FlgF and FlgG. A family of
structurally related proteins in the flagellar basal body of Salmonella typhimurium. J Mol Biol. 211
(2), 465-477.
Honma, Y. and Nakasone, N., 1990.
Pili of Aeromonas hydrophila:
purification, characterization, and biological role. Microbiol Immunol. 34 (2),
83-98.
Hooper, S. D. and Berg, O. G., 2003.
On the nature of gene innovation: duplication patterns in microbial genomes.
Mol Biol Evol. 20 (6), 945-954., doi:10.1093/molbev/msg101.
Hueck, C. J., 1998. Type III protein
secretion systems in bacterial pathogens of animals and plants. Microbiol Mol
Biol Rev. 62 (2), 379-433.
Jarrell, K. F., Bayley, D. P.,
Correia, J. D. and Thomas, N. A., 1999. Recent excitement about the archaea.
Bioscience. 49 (7), 530-541.
Jarrell, K. F., Bayley, D. P.,
Correia, J. D. and Thomas, N. A., 2000. Archaeal flagella, Nature Encyclopedia
of Life Sciences, Nature Publishing Group, London.,
doi:10.1038/npg.els.0000386.
Jarrell, K. F., Bayley, D. P. and
Kostyukova, A. S., 1996. The archaeal flagellum: a unique motility structure. J
Bacteriol. 178 (17), 5057-5064.
Jiang, Y. and Doolittle, R. F.,
2003. The evolution of vertebrate blood coagulation as viewed from a comparison
of puffer fish and sea squirt genomes. Proc Natl Acad Sci U S A. 100 (13),
7527–7532., doi:10.1073/pnas.0932632100.
Johnson, G. R., Jain, R. K. and
Spain, J. C., 2002. Origins of the 2,4-dinitrotoluene pathway. Journal of
Bacteriology. 184 (15), 4219-4232., doi:10.1128/JB.184.15.4219-4232.2002.
Jones, W. J., Nagle, D. P., Jr. and
Whitman, W. B., 1987. Methanogens and the diversity of archaebacteria.
Microbiol Rev. 51 (1), 135-177.
Kalir, S., McClure, J., Pabbaraju,
K., Southward, C., Ronen, M., Leibler, S., Surette, M. G. and Alon, U., 2001.
Ordering genes in a flagella pathway by analysis of expression kinetics from
living bacteria. Science. 292 (5524), 2080-2083., doi:10.1126/science.1058758.
Kennedy, M. J., 1987. Role of
motility, chemotaxis, and adhesion in microbial ecology. Annals of the New York
Academy of Sciences. 503, 260-273.
Kerr, B., Riley, M. A., Feldman, M.
W. and Bohannan, B. J., 2002. Local dispersal promotes biodiversity in a
real-life game of rock-paper-scissors. Nature. 418 (6894), 171-174.,
doi:10.1038/nature00823.
Khan, S., 1997. Rotary chemiosmotic
machines. Biochim Biophys Acta. 1322 (2-3), 86-105.
Kim, J. F., 2001. Revisiting the
chlamydial type III protein secretion system: clues to the origin of type III
protein secretion. Trends Genet. 17 (2), 65-69.,
doi:10.1016/S0168-9525(00)02175-2.
Kirby, J. R., Kristich, C. J.,
Saulmon, M. M., Zimmer, M. A., Garrity, L. F., Zhulin, I. B. and Ordal, G. W.,
2001. CheC is related to the family of flagellar switch proteins and acts
independently from CheD to control chemotaxis in Bacillus subtilis. Mol Microbiol. 42 (3), 573-585.,
doi:10.1046/j.1365-2958.2001.02581.x.
Knutton, S., Shaw, R. K., Anantha,
R. P., Donnenberg, M. S. and Zorgani, A. A., 1999. The type IV bundle-forming
pilus of enteropathogenic Escherichia
coli undergoes dramatic alterations in structure associated with bacterial
adherence, aggregation and dispersal. Mol Microbiol. 33 (3), 499-509.,
doi:10.1046/j.1365-2958.1999.01495.x.
Koch, A. L., 2003. Were
Gram-positive rods the first bacteria? Trends Microbiol. 11 (4), 166-170.,
doi:10.1016/S0966-842X(03)00063-5.
Kojima, S. and Blair, D. F., 2001.
Conformational change in the stator of the bacterial flagellar motor.
Biochemistry. 40 (43), 13041-13050., doi:10.1021/bi011263o.
Koretke, K. K., Lupas, A. N.,
Warren, P. V., Rosenberg, M. and Brown, J. R., 2000. Evolution of two-component
signal transduction. Mol Biol Evol. 17 (12), 1956-1970.
Kreft, J. U., Picioreanu, C.,
Wimpenny, J. W. and van Loosdrecht, M. C., 2001. Individual-based modelling of
biofilms. Microbiology. 147 (Pt 11), 2897-2912.
Lebreton, J., Khaladi, M. and
Grosbois, V., 2000. An explicit approach to evolutionarily stable dispersal
strategies: no cost of dispersal. Math Biosci. 165 (2), 163-176.,
doi:10.1016/S0025-5564(00)00016-X.
Long, M., 2001. Evolution of novel
genes. Curr Opin Genet Dev. 11 (6), 673-680.,
doi:10.1016/S0959-437X(00)00252-5.
Lupas, A., Van Dyke, M. and Stock,
J., 1991. Predicting coiled coils from protein sequences. Science. 252 (5010),
1162-1164.
Macnab, R. M., 1978. Bacterial
motility and chemotaxis: the molecular biology of a behavioral system. CRC Crit
Rev Biochem. 5 (4), 291-341.
Macnab, R. M., 1999. The bacterial
flagellum: reversible rotary propellor and type III export apparatus. J
Bacteriol. 181 (23), 7149-7153.
Macnab, R. M., 2003. How bacteria
assemble flagella. Annu Rev Microbiol. 57, 77-100.,
doi:10.1146/annurev.micro.57.030502.090832.
Macnab, R. M. and DeRosier, D. J.,
1988. Bacterial flagellar structure and function. Can J Microbiol. 34 (4),
442-451.
Manson, M. D., Tedesco, P., Berg, H.
C., Harold, F. M. and Van der Drift, C., 1977. A protonmotive force drives
bacterial flagella. Proc Natl Acad Sci U S A. 74 (7), 3060-3064.
Margulis, L., 1980. Undulipodia,
flagella and cilia. Biosystems. 12 (1-2), 105-108.
Marie, C., Broughton, W. J. and
Deakin, W. J., 2001. Rhizobium type
III secretion systems: legume charmers or alarmers? Curr Opin Plant Biol. 4
(4), 336-342., doi:10.1016/S1369-5266(00)00182-5.
Mathews, M. A., Tang, H. L. and
Blair, D. F., 1998. Domain analysis of the FliM protein of Escherichia coli. J Bacteriol. 180 (21), 5580-5590.
Mathias, A., Kisdi, E. and Olivieri,
I., 2001. Divergent evolution of dispersal in a heterogeneous landscape.
Evolution Int J Org Evolution. 55 (2), 246-259.
Maynard Smith, J., 1975. The Theory
of Evolution. Cambridge University Press.
Mayr, E., 1976. The emergence of
evolutionary novelties, Evolution and the diversity of life, Harvard University
Press, Cambridge, pp.88-113.
McBride, M. J., 2001. Bacterial
gliding motility: multiple mechanisms for cell movement over surfaces. Annu Rev
Microbiol. 55, 49-75., doi:10.1146/annurev.micro.55.1.49.
McCarter, L. L., 2001. Polar
flagellar motility of the Vibrionaceae. Microbiol Mol Biol Rev. 65 (3),
445-462, table of contents., doi:10.1128/MMBR.65.3.445-462.2001.
Mecsas, J. J. and Strauss, E. J.,
1996. Molecular mechanisms of bacterial virulence: type III secretion and
pathogenicity islands. Emerg Infect Dis. 2 (4), 270-288.
Melendez-Hevia, E., Waddell, T. G.
and Cascante, M., 1996. The puzzle of the Krebs citric acid cycle: assembling
the pieces of chemically feasible reactions, and opportunism in the design of
metabolic pathways during evolution. J Mol Evol. 43 (3), 293-303.
Merz, A. J. and Forest, K. T., 2002.
Bacterial surface motility: slime trails, grappling hooks and nozzles. Curr
Biol. 12 (8), R297-303., doi:10.1016/S0960-9822(02)00806-0.
Minamino, T., Gonzalez-Pedrajo, B.,
Kihara, M., Namba, K. and Macnab, R. M., 2003. The ATPase FliI can interact
with the type III flagellar protein export apparatus in the absence of its regulator,
FliH. J Bacteriol. 185 (13), 3983-3988.
Minamino, T., Gonzalez-Pedrajo, B.,
Oosawa, K., Namba, K. and Macnab, R. M., 2002. Structural properties of FliH,
an ATPase regulatory component of the Salmonella
type III flagellar export apparatus. J Mol Biol. 322 (2), 281-290.,
doi:10.1016/S0022-2836(02)00754-4.
Minamino, T. and Macnab, R. M.,
1999. Components of the Salmonella
flagellar export apparatus and classification of export substrates. J
Bacteriol. 181 (5), 1388-1394.
Minamino, T. and Macnab, R. M.,
2000. FliH, a soluble component of the type III flagellar export apparatus of Salmonella, forms a complex with FliI
and inhibits its ATPase activity. Mol Microbiol. 37 (6), 1494-1503.,
doi:10.1046/j.1365-2958.2000.02106.x.
Minamino, T., Tame, J. R., Namba, K.
and Macnab, R. M., 2001. Proteolytic analysis of the FliH/FliI complex, the
ATPase component of the type III flagellar export apparatus of Salmonella. J Mol Biol. 312 (5),
1027-1036., doi:10.1006/jmbi.2001.5000.
Mitchell, J. G., 2002. The energetics
and scaling of search strategies in bacteria. The American Naturalist. 160 (6),
727-740., doi:10.1086/343874.
Mitchell, P., 1985. Molecular
mechanics of protonmotive F0F1 ATPases. Rolling well and
turnstile hypothesis. FEBS Lett. 182 (1), 1-7.
Mitchison, T. J., 1995. Evolution of
a dynamic cytoskeleton. Philos Trans R Soc Lond B Biol Sci. 349 (1329),
299-304.
Mivart, S. G., 1871. Genesis of
Species. Macmillan, London.
Mocz, G. and Gibbons, I. R., 2001.
Model for the motor component of dynein heavy chain based on homology to the
AAA family of oligomeric ATPases. Structure (Camb). 9 (2), 93-103.,
doi:10.1016/S0969-2126(00)00557-8.
Moens, S. and Vanderleyden, J.,
1996. Functions of bacterial flagella. Crit Rev Microbiol. 22 (2), 67-100.
Mortlock, R. P. (Ed.), 1992. The
Evolution of Metabolic Function. CRC Press, Boca Raton Fla.
Muller, W. E., Blumbach, B. and
Muller, I. M., 1999. Evolution of the innate and adaptive immune systems:
relationships between potential immune molecules in the lowest metazoan phylum
(Porifera) and those in vertebrates. Transplantation. 68 (9), 1215-1227.
Musgrave, I., 2004. Evolution of the
Bacterial Flagellum, in: Young, M. and Edis, T. (Eds.), Why Intelligent Design
Fails: A Scientific Critique of the Neocreationism, forthcoming from Rutgers
University Press, Piscataway, N. J.
Nguyen, L., Paulsen, I. T., Tchieu,
J., Hueck, C. J. and Saier, M. H., Jr., 2000. Phylogenetic analyses of the
constituents of Type III protein secretion systems. J Mol Microbiol Biotechnol.
2 (2), 125-144.
Nilsson, D.-E. and Pelger, S., 1994.
A pessimistic estimate of the time required for an eye to evolve. Proceedings
of the Royal Society of London Series B: Biological Sciences. 256, 53-58.
Noji, H., Yasuda, R., Yoshida, M.
and Kinosita, K., Jr., 1997. Direct observation of the rotation of F1-ATPase.
Nature. 386 (6622), 299-302., doi:10.1038/386299a0.
Nougayrede, J. P., Fernandes, P. J.
and Donnenberg, M. S., 2003. Adhesion of enteropathogenic Escherichia coli to host cells. Cell Microbiology. 5 (6), 359-372.,
doi:10.1046/j.1462-5822.2003.00281.x.
Novikova, N. A., Levitsky, D. I.,
Metlina, A. L. and Poglazov, B. F., 2000. Interaction of bacterial flagellum
filaments with skeletal muscle myosin. IUBMB Life. 50 (6), 385-390.,
doi:10.1080/152165400300089457.
Ohnishi, K., Fan, F., Schoenhals, G.
J., Kihara, M. and Macnab, R. M., 1997. The FliO, FliP, FliQ, and FliR proteins
of Salmonella typhimurium: putative
components for flagellar assembly. J Bacteriol. 179 (19), 6092-6099.
Oplatka, A., 1998a. Are rotors at
the heart of all biological motors? Biochem Biophys Res Commun. 246 (2),
301-306., doi:10.1006/bbrc.1998.8424.
Oplatka, A., 1998b. Do the bacterial
flagellar motor and ATP synthase operate as water turbines? Biochem Biophys Res
Commun. 249 (3), 573-578., doi:10.1006/bbrc.1998.8969.
Oster, G. and Wang, H., 2003. Rotary
protein motors. Trends Cell Biol. 13 (3), 114-121.,
doi:10.1016/S0962-8924(03)00004-7.
Pasquier, L. D. and Litman, G. W.
(Eds.), 2000. Origin and Evolution of the Vertebrate Immune System (Series:
Current Topics in Microbiology and Immunology). Springer, Berlin.
Pei, Z., Burucoa, C., Grignon, B.,
Baqar, S., Huang, X. Z., Kopecko, D. J., Bourgeois, A. L., Fauchere, J. L. and
Blaser, M. J., 1998. Mutation in the peb1A locus of Campylobacter jejuni reduces interactions with epithelial cells and
intestinal colonization of mice. Infect Immun. 66 (3), 938-943.
Pellmyr, O. and Krenn, H. W., 2002.
Origin of a complex key innovation in an obligate insect-plant mutualism. Proc
Natl Acad Sci U S A. 99 (8), 5498-5502., doi:10.1073/pnas.072588699.
Plano, G. V., Day, J. B. and
Ferracci, F., 2001. Type III export: new uses for an old pathway. Mol
Microbiol. 40 (2), 284-293., doi:10.1046/j.1365-2958.2001.02354.x.
Poethke, H. J. and Hovestadt, T.,
2002. Evolution of density- and patch-size-dependent dispersal rates. Proc R
Soc Lond B Biol Sci. 269 (1491), 637-645., doi:10.1098/rspb.2001.1936.
Prum, R. O. and Brush, A. H., 2002.
The evolutionary origin and diversification of feathers. Q Rev Biol. 77 (3),
261-295., doi:10.1086/341993.
Pugsley, A. P., 1993. The complete
general secretory pathway in gram-negative bacteria. Microbiol Rev. 57 (1),
50-108.
Purcell, E. M., 1977. Life at low
Reynolds number. American Journal of Physics. 45, 3-11.
Purcell, E. M., 1997. The efficiency
of propulsion by a rotating flagellum. Proc Natl Acad Sci U S A. 94,
11307-11311., doi:10.1073/pnas.94.21.11307.
Rizzotti, M., 2000. Early Evolution:
From the appearance of the first cell to the first modern organisms. Birkhäuser
Verlag, Boston.
Rosenhouse, J., 2002. Probability,
optimization theory, and evolution. Evolution. 56 (8), 1721-1722.
Sabbert, D. and Junge, W., 1997.
Stepped versus continuous rotatory motors at the molecular scale. Proc Natl
Acad Sci U S A. 94 (6), 2312-2317., doi:10.1073/pnas.94.6.2312.
Sadowsky, M. J., Tong, Z., de Souza,
M. and Wackett, L. P., 1998. AtzC is a new member of the amidohydrolase protein
superfamily and is homologous to other atrazine-metabolizing enzymes. J
Bacteriol. 180 (1), 152-158.
Saier, M. H., Jr., 2003. Tracing
pathways of transport protein evolution. Mol Microbiol. 48 (5), 1145-1156.,
doi:10.1046/j.1365-2958.2003.03499.x.
Salvini-Plawen, S. V. and Mayr, E.,
1977. On the evolution of photoreceptors and eyes. Evolutionary Biology. 10,
207-263.
Sandkvist, M., 2001. Biology of type
II secretion. Mol Microbiol. 40 (2), 271-283.,
doi:10.1046/j.1365-2958.2001.02403.x.
Sauer, F. G., Barnhart, M.,
Choudhury, D., Knight, S. D., Waksman, G. and Hultgren, S. J., 2000.
Chaperone-assisted pilus assembly and bacterial attachment. Curr Opin Struct
Biol. 10, 548-556., doi:10.1016/S0959-440X(00)00129-9.
Scharfman, A., Arora, S. K.,
Delmotte, P., Van Brussel, E., Mazurier, J., Ramphal, R. and Roussel, P., 2001.
Recognition of Lewis x derivatives present on mucins by flagellar components of
Pseudomonas aeruginosa. Infect Immun.
69 (9), 5243-5248., doi:10.1128/IAI.69.9.5243-5248.2001.
Schmitt, R., 2003. Helix rotation
model of the flagellar rotary motor. Biophys J. 85 (2), 843-852.
Seffernick, J. L. and Wackett, L.
P., 2001. Rapid evolution of bacterial catabolic enzymes: a case study with
atrazine chlorohydrolase. Biochemistry. 40 (43), 12747-12753.,
doi:10.1021/bi011293r.
Shah, D. S., Perehinec, T., Stevens,
S. M., Aizawa, S. I. and Sockett, R. E., 2000. The flagellar filament of Rhodobacter sphaeroides: pH-induced
polymorphic transitions and analysis of the fliC gene. J Bacteriol. 182 (18),
5218-5224., doi:10.1128/JB.182.18.5218-5224.2000.
Shah, D. S. and Sockett, R. E.,
1995. Analysis of the motA flagellar motor gene from Rhodobacter sphaeroides, a bacterium with a unidirectional,
stop-start flagellum. Mol Microbiol. 17 (5), 961-969.
Silverman, M. and Simon, M., 1974.
Flagellar rotation and the mechanism of bacterial motility. Nature. 249 (452),
73-74.
Smyth, C. J., Marron, M. B., Twohig,
J. M. and Smith, S. G., 1996. Fimbrial adhesins: similarities and variations in
structure and biogenesis. FEMS Immunol Med Microbiol. 16 (2), 127-139.,
doi:10.1016/S0928-8244(96)00074-0.
Spormann, A. M., 1999. Gliding
motility in bacteria: insights from studies of Myxococcus xanthus. Microbiol Mol Biol Rev. 63 (3), 621-641.
Stoodley, P., Sauer, K., Davies, D.
G. and Costerton, J. W., 2002. Biofilms as complex differentiated communities.
Annu Rev Microbiol. 56, 187-209., doi:10.1146/annurev.micro.56.012302.160705.
Sukhan, A., Kubori, T. and Galán, J.
E., 2003. Synthesis and localization of the Salmonella
SPI-1 type III secretion needle complex proteins PrgI and PrgJ. J Bacteriol.
185 (11), 3480-3483., doi:10.1128/JB.185.11.3480-3483.2003.
Szurmant, H., Bunn, M. W.,
Cannistraro, V. J. and Ordal, G. W., 2003. Bacillus
subtilis hydrolyzes CheY-P at the location of its action: the flagellar
switch. J Biol Chem. 14, 14.
Thanassi, D. G., 2002. Ushers and
secretins: channels for the secretion of folded proteins across the bacterial
outer membrane. J Mol Microbiol Biotechnol. 4 (1), 11-20.
Thanassi, D. G. and Hultgren, S. J.,
2000a. Multiple pathways allow protein secretion across the bacterial outer
membrane. Curr Opin Cell Biol. 12 (4), 420-430.,
doi:10.1016/S0955-0674(00)00111-3.
Thanassi, D. G. and Hultgren, S. J.,
2000b. Assembly of complex organelles: pilus biogenesis in gram-negative
bacteria as a model system. Methods. 20 (1), 111-126., doi:10.1006/meth.1999.0910.
Thanassi, D. G., Saulino, E. T. and
Hultgren, S. J., 1998. The chaperone/usher pathway: a major terminal branch of
the general secretory pathway. Curr Opin Microbiol. 1 (2), 223-231.,
doi:10.1016/S1369-5274(98)80015-5.
Thar, R. and Kuhl, M., 2002.
Conspicuous veils formed by vibrioid bacteria on sulfidic marine sediment. Appl
Environ Microbiol. 68 (12), 6310-6320., doi:10.1128/AEM.68.12.6310-6320.2002.
Thomas, N. A., Bardy, S. L. and
Jarrell, K. F., 2001. The archaeal flagellum: a different kind of prokaryotic
motility structure. FEMS Microbiol Rev. 25 (2), 147-174.,
doi:10.1016/S0168-6445(00)00061-9.
Thomas, N. A., Mueller, S., Klein,
A. and Jarrell, K. F., 2002. Mutants in flaI and flaJ of the archaeon Methanococcus voltae are deficient in
flagellum assembly. Mol Microbiol. 46 (3), 879-887.,
doi:10.1046/j.1365-2958.2002.03220.x.
Thornhill, R. H. and Ussery, D. W.,
2000. A classification of possible routes of Darwinian evolution. J Theor Biol.
203 (2), 111-116., doi:10.1006/jtbi.2000.1070.
Trachtenberg, S., Gilad, R. and
Geffen, N., 2003. The bacterial linear motor of Spiroplasma melliferum BC3: from single molecules to swimming
cells. Mol Microbiol. 47 (3), 671-697.,
doi:10.1046/j.1365-2958.2003.t01-1-03200.x.
True, J. R. and Carroll, S. B.,
2002. Gene co-option in physiological and morphological evolution. Annu Rev
Cell Dev Biol. 18, 53-80., doi:10.1146/annurev.cellbio.18.020402.140619.
van Wely, K. H., Swaving, J.,
Freudl, R. and Driessen, A. J., 2001. Translocation of proteins across the cell
envelope of Gram-positive bacteria. FEMS Microbiol Rev. 25 (4), 437-454.,
doi:10.1016/S0168-6445(01)00062-6.
Velicer, G. J., Lenski, R. E. and
Kroos, L., 2002. Rescue of social motility lost during evolution of Myxococcus xanthus in an asocial
environment. J Bacteriol. 184 (10), 2719-2727.
Vogel, S., 1988. Life's devices: the
physical world of animals and plants. Princeton University Press, Princeton,
NJ.
Vogel, S., 1994. Life in Moving
Fluids. Princeton University Press, Princeton, NJ.
Vogler, A. P., Homma, M., Irikura,
V. M. and Macnab, R. M., 1991. Salmonella
typhimurium mutants defective in flagellar filament regrowth and sequence
similarity of FliI to F0F1, vacuolar, and archaebacterial ATPase subunits. J
Bacteriol. 173 (11), 3564-3572.
Walz, D. and Caplan, S. R., 2002.
Bacterial flagellar motor and H(+)/ATP synthase: two proton-driven rotary
molecular devices with different functions. Bioelectrochemistry. 55 (1-2),
89-92., doi:10.1016/S1567-5394(01)00162-1.
Weber, J., Muharemagic, A., Wilke-Mounts,
S. and Senior, A. E., 2003a. F1F0-ATP synthase. Binding
of delta subunit to a 22-residue peptide mimicking the N-terminal region of
alpha subunit. J Biol Chem. 278 (16), 13623-13626., doi:10.1074/jbc.C300061200.
Weber, J. and Senior, A. E., 2003.
ATP synthesis driven by proton transport in F1F0-ATP
synthase. FEBS Lett. 545 (1), 61-70., doi:10.1016/S0014-5793(03)00394-6.
Weber, J., Wilke-Mounts, S. and
Senior, A. E., 2003b. Identification of the F1-binding surface on
the delta-subunit of ATP synthase. J Biol Chem. 278 (15), 13409-13416.,
doi:10.1074/jbc.M212037200.
Whitesides, G. M., 2001. The once
and future nanomachine. Scientific American. 285 (3), 78-83.
Whitman, W. B., Coleman, D. C. and
Wiebe, W. J., 1998. Prokaryotes: The unseen majority. Proc Natl Acad Sci U S A.
95, 6578-6583., doi:10.1073/pnas.95.12.6578.
Wilkens, S. and Capaldi, R. A.,
1998. Solution structure of the epsilon subunit of the F1-ATPase
from Escherichia coli and
interactions of this subunit with beta subunits in the complex. J Biol Chem.
273 (41), 26645-26651., doi:10.1074/jbc.273.41.26645.
Wu, H. and Fives-Taylor, P. M.,
2001. Molecular strategies for fimbrial expression and assembly. Crit Rev Oral
Biol Med. 12 (2), 101-115.
Yamashiro, T., Nakasone, N., Honma,
Y., Albert, M. J. and Iwanaga, M., 1994. Purification and characterization of Vibrio cholerae O139 fimbriae. FEMS
Microbiol Lett. 115 (2-3), 247-252., doi:10.1016/0378-1097(94)90022-1.
Youderian, P., Burke, N., White, D.
J. and Hartzell, P. L., 2003. Identification of genes required for adventurous
gliding motility in Myxococcus xanthus
with the transposable element mariner. Mol Microbiol. 49 (2), 555-570.,
doi:10.1046/j.1365-2958.2003.03582.x.
Young, G. M., Schmiel, D. H. and
Miller, V. L., 1999. A new pathway for the secretion of virulence factors by
bacteria: the flagellar export apparatus functions as a protein-secretion
system. Proc Natl Acad Sci U S A. 96 (11), 6456-6461.,
doi:10.1073/pnas.96.11.6456.
Young, K. D., 2001. Peptidoglycan,
Nature Encyclopedia of Life Sciences, Nature Publishing Group, London.,
doi:10.1038/npg.els.0000702.
Zaar, A., Fuchs, G., Golecki, J. R.
and Overmann, J., 2003. A new purple sulfur bacterium isolated from a littoral
microbial mat, Thiorhodococcus drewsii
sp. nov. Arch Microbiology. 179 (3), 174-183.
Zhai, Y. F., Heijne, W. and Saier,
M. H., 2003. Molecular modeling of the bacterial outer membrane receptor
energizer, ExbBD/TonB, based on homology with the flagellar motor, MotAB.
Biochim Biophys Acta. 1614 (2), 201-210., doi:10.1016/S0005-2736(03)00176-7.
Zhulin, I. B., Nikolskaya, A. N. and
Galperin, M. Y., 2003. Common extracellular sensory domains in transmembrane
receptors for diverse signal transduction pathways in bacteria and archaea. J
Bacteriol. 185 (1), 285-294., doi:10.1128/JB.185.1.285-294.2003.

Figure 1:
Composite electron micrograph of the flagellum basal body and hook, produced by
rotational averaging (Francis et al., 1994). The motor
proteins and export apparatus (included in Figure
2) do not survive the extraction procedure and so are
not shown. Image courtesy of David DeRosier, reproduced with permission.

Figure 2: Schematic diagram
of a typical bacterial flagellum, shown in cross-section. The names of substructures are given in
bold, and the names of the constituent proteins are given in regular type,
including approximate stoichiometry (see Table
1). The depiction
of the flagellar axial protein complex (rod, hook, filament) and MS-, P-, and
L-rings is based on composite electron micrographs (see DeRosier, 1998). The
depictions of the other proximal components are based on specific published models:
FliM/N C-ring (Mathews et al., 1998), the position of MotA, MotB, and FliG (Brown
et al., 2002), and the hexameric complex of FliI (Blocker
et al., 2003; Claret et al., 2003). The
position of FliJ is a guess based on its interaction with FliH and FliI (Macnab, 2003). The
depiction of FliH is based on studies of its structure and interaction with
FliI (Minamino and Macnab, 2000;
Minamino et al., 2001; Minamino et al., 2002) and on the homology of FliH to the F0-b
subunit of ATP synthetase, postulated in this paper (see text). Apart from FliH and FliI, the structure and
stoichiometry of the rest of the type III export apparatus are obscure.

Figure 3: Rizzotti's (2000)
scenario for the origin of a proto-flagellum from an F1F0
ATP synthetase, via a "stirring filament." Rizzotti only used three subunits of the synthetase, F1-αβ
(white), F0-c (grey), and F1-γ (black). (a) F1F0 ATP
synthetase (for a more complete depiction, see Figure 4b). (b) An
insertion in the γ subunit creates a stirring filament. (c) A proto-flagellum created by extension
of the stirring filament. F1-αβ
becomes the rotor, F0-c the stator, and F1-γ the
filament. Rizzotti assumes a
gram-positive bacterium. After Rizzotti
(2000), Figure 4.4.

Figure 4: Systems with
components homologous to flagellar components.
(a) Hrp pilus of Pseudomonas spp. For components with well-documented homology to flagellar
components, the name according to the unified nomenclature for type III
secretion systems proposed by Hueck (1998) is given (Sct: Secretion and Cellular Translocation)
first, followed by the currently accepted name for the Hrp protein. The name of the flagellar homolog is shown
in brackets. (b) The F1F0-ATP
synthetase shown to scale, based on Capaldi and Aggeler (2002). The F1-α
and β subunits are homologous to each other and to FliI (Gogarten et al., 1992). Further
possible homologies are discussed in the text. (c) The Tol-Pal system, similar
to the Exb-TonB system. TolA is
homologous to TonB, and TolQR, ExbBD, and MotAB are homologs (Cascales et al., 2001). The 4:2
stoichiometry for MotAB is favored in recent models (Schmitt, 2003; Zhai et al.,
2003).

Figure 5: Various secretion
systems of prokaryotes. (a) Type I secretion system, a single-step transporter,
substrates are recognized by an uncleaved C-terminal sequence. OMP, outer membrane channel-forming protein;
MFP, membrane fusion protein; ABC, ATP-binding cassette exporter. (b) Three
sec-dependent secretion systems: (b1) Autotransporter. (b2) Chaperone/usher
pathway and P pilus. (b3) Type II secretion. (c) Type IV secretion, also
sec-dependent. (d) The archaeal
flagellum, with several components homologous to type IV secretion. Based on several sources (Jarrell
et al., 2000; Thanassi and Hultgren, 2000a; Büttner and Bonas, 2002;
Thanassi, 2002; Bardy et al., 2003). Another
nucleotide may be substituted for ATP in some cases. See Table 4 for description of the functions of the systems.

Figure 6: Relative diffusion
advantage of motility (Dm/D0) as a function
of cell size and absolute swimming velocity, plotted on a log scale. Calculations were made for three swimming
velocities. Thin line: 0.1
μm/sec; Medium line: 1 μm/sec. Thick line: 10 μm/sec (typical E. coli
swimming velocity). The horizontal line represents Dm/D0
= 2, the point at which active motility doubles the diffusion
coefficient.

Figure 7: Summary of the evolutionary model for the
origin of the flagellum, showing the six major stages and key
intermediates. White components have
identified or reasonably probable nonflagellar homologs; grey components have
either suggested but unsupported homologs, or no specific identified homologs,
although ancestral functions can be postulated. The model begins with a passive, somewhat general inner membrane
pore (1a) that is converted to a more substrate-specific pore (1b) by addition
of proto-FlhA and/or FlhB. Interaction
of an F1F0-ATP synthetase
with FlhA/B produces an active transporter, a primitive type III export
apparatus (1c). Addition of a secretin
which associates with the cytoplasmic ring converts this to a type III
secretion system (2). A mutated secretion
substrate becomes an adhesin (or alternatively an adhesin is coopted by
transposition of the secretion recognition sequence), and binds to the outer
side of the secretin (3a).
Pentamerization of the adhesin produces a ring, allowing more surface
adhesins without blocking other secretion substrates (3b). Polymerization of this ring produces a tube,
a primitive type III pilus (4a; in the diagram, a white axial structure is
substituted for the individual pilin subunits; all further axial proteins are descended
from this common pilin ancestor).
Pentamerization of a pilin produces the cap, increasing assembly speed
and efficiency (4b). A duplicate pilin
that loses its outer domains becomes the proto-rod protein, extending down
through the secretin and strengthening pilus attachment by association with the
base (4c). Further duplications of the
proto-rod, filament, and cap proteins, occurring before and after the origin of
the flagellum (6) produce the rest of the axial proteins; these repeated
subfunctionalization events are not shown here. The protoflagellum (5a) is produced by cooption of TolQR homologs
from a Tol-Pal-like system; perhaps a portion of a TolA homolog bound to FliF
to produce proto-FliG. In order to
improve rotation, the secretin is lost and the pore role taken over by its
lipoprotein chaperone ring, which becomes the proto-L-ring (5b). Addition of the P-ring, perhaps itself a
modified secretin, and the rod cap FlgJ muramidase domain, removes the
necessity of finding a natural gap in the cell wall (5c). Finally, binding of a
mutant proto-FliN, probably a CheC receptor, to FliG, couples the signal
transduction system to the protoflagellum, producing a chemotactic flagellum
(6); fusion of proto-FliN and CheC produces FliM. Each stage would obviously be followed by gradual coevolutionary
optimization of component interactions.
The origin of the flagellum is thus reduced to a series of mutationally
plausible steps.
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.
|
Position
|
Secretion pathway
|
operon class
|
Size
(a. a.)
|
Stoichiometry (approx)
|
Function
|
Integral
membrane components
|
|
|
|
|
|
FliF
|
Inner
membrane
|
sec
|
2
|
552
|
26
|
Rotor/housing
|
FlhA
|
Center
of FliF ring
|
sec?
|
2
|
692
|
2?
|
Protein
export
|
FlhB
|
Center
of FliF ring
|
sec?
|
2
|
382
|
2?
|
Hook
length control
|
FliO
|
Center
of FliF ring
|
sec?
|
2
|
121
|
1?
|
Type
III protein export
|
FliP
|
Center
of FliF ring
|
sec?
|
2
|
245
|
(1?)
|
Type
III protein export
|
FliQ
|
Center
of FliF ring
|
sec?
|
2
|
89
|
1?
|
Type
III protein export
|
FliR
|
Center
of FliF ring
|
sec?
|
2
|
261
|
1?
|
Type
III protein export
|
|
|
|
|
|
|
|
Membrane-associated
components
|
|
|
|
|
|
FliI
|
Cytoplasm
side of membrane
|
---
|
2
|
457
|
(6?)
|
Type
III protein export
|
FliH
|
Cytoplasm
side of membrane
|
---
|
2
|
235
|
(2?)
|
Type
III protein export
|
FliJ
|
Cytoplasm
side of membrane
|
---
|
2
|
147
|
(1?)
|
Type
III protein export
|
|
|
|
|
|
|
|
Rotor/switch
complex
|
|
|
|
|
|
FliM
|
Cytoplasm
side of membrane
|
self-assembly
|
2
|
334
|
37?
|
Rotor/switch
|
FliN
|
Cytoplasm
side of membrane
|
self-assembly
|
2
|
137
|
110
|
Rotor/switch
|
FliG
|
Cytoplasm
side of membrane
|
self-assembly
|
2
|
331
|
26
|
Rotor/switch
|
|
|
|
|
|
|
|
Rings
|
|
|
|
|
|
|
FlgI
|
Peptidoglycan
cell wall
|
sec
|
2
|
365
|
26
|
Bushing
|
FlgH
|
Outer
membrane
|
sec
|
2
|
232
|
26?
|
Bushing
|
FlgA
|
Periplasmic
space
|
sec
|
2
|
219
|
?
|
Assembly
of P-ring
|
|
|
|
|
|
|
|
Axial
proteins
|
|
|
|
|
|
FliE
|
Periplasmic
space
|
Type III
|
2
|
104
|
9?
|
Driveshaft
|
FlgJ
|
Periplasmic
space
|
Type III
|
2
|
313
|
5?
|
Rod
cap (?)
|
FlgB
|
Peptidoglycan
cell wall (P)
|
Type III
|
2
|
138
|
6?
|
Driveshaft
|
FlgC
|
Peptidoglycan
cell wall (P)
|
Type III
|
2
|
134
|
6
|
Driveshaft
|
FlgF
|
Peptidoglycan
cell wall (P)
|
Type III
|
2
|
251
|
6
|
Driveshaft
|
FlgG
|
Extracellular
|
Type III
|
2
|
260
|
26
|
Driveshaft
|
FlgE
|
Extracellular
|
Type III
|
2
|
402
|
~130
|
Universal
joint
|
FlgD
|
Extracellular
|
Type III
|
2
|
231
|
5?
|
Hook
cap
|
FlgK
|
Extracellular
|
Type III
|
3a
|
547
|
11
|
Linker
|
FlgL
|
Extracellular
|
Type III
|
3a
|
317
|
11
|
Linker
|
FliC
|
Extracellular
|
Type III
|
3b
|
498
|
~20000
|
Filament
|
FliD
|
Extracellular
|
Type III
|
3a
|
468
|
5
|
Filament
cap
|
|
|
|
|
|
|
|
Motor
proteins
|
|
|
|
|
|
MotA
|
Inner
membrane/cytoplasm
|
sec
|
3b
|
295
|
32?
|
Motor/stator
|
MotB
|
Inner membrane/peptidoglycan
|
sec
|
3b
|
308
|
16?
|
Stator
|
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).
Protein
|
Position
|
operon class
|
Stoichiometry (approx)
|
Function
|
Cytoplasmic
|
|
|
|
FlhC
|
Cytoplasm
|
1
|
--
|
Master
regulator for class 2 operons
|
FlhD
|
Cytoplasm
|
1
|
--
|
Master
regulator for class 2 operons
|
FlhE
|
Cytoplasm
|
2
|
--
|
?
|
FliK
|
Cytoplasm,
binds to FlhB
|
2
|
--
|
Hook-length
control
|
FliL
|
Cytoplasm
|
2
|
--
|
?
|
FliA
|
Cytoplasm
|
2
|
--
|
Sigma
factor for class 3 operons
|
FlgM
|
Cytoplasm
|
3a
|
--
|
Anti-sigma
factor
|
FlgN
|
Cytoplasm
|
3a
|
--
|
FlgK,
FlgL-specific chaperone
|
FliS
|
Cytoplasm
|
3a
|
--
|
FliC-specific
chaperone
|
FliT
|
Cytoplasm
|
3a
|
--
|
FliD-specific
chaperone
|
|
|
|
|
|
Chemotaxis
|
|
|
|
Methyl-accepting chemotaxis proteins (MCPs)
|
|
(copy # / cell)
|
|
aer
|
Inner
membrane
|
--
|
150
|
Oxygen
receptor
|
tap
|
Inner
membrane
|
--
|
150
|
Dipeptide
receptor
|
tar
|
Inner
membrane
|
--
|
900
|
Amino
acid receptor
|
tsr
|
Inner
membrane
|
--
|
1600
|
Amino
acid receptor
|
trg
|
Inner
membrane
|
--
|
150
|
Receptor
for sugar-binding proteins
|
Signal transduction
|
|
|
|
CheW
|
Inner
side of IM (bound to MCP)
|
--
|
3000
|
Attaches
MCP to CheA
|
CheA
|
Inner
side of IM (bound to MCP)
|
--
|
3000
|
Histidine
protein kinase (HPK)
|
CheY
|
Cytoplasm
|
--
|
3000-17500
|
Response
regulator
|
Response regulation
|
|
|
|
CheZ
|
Cytoplasm
|
--
|
1200
|
Response
regulator; phophatase
|
CheB
|
Cytoplasm
|
--
|
1700
|
Response
regulator; methylesterase
|
CheR
|
Cytoplasm
|
--
|
850
|
Methyltransferase
|
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).
|
“Flagella”
|
Gliding motility
|
Other
|
|
Bacterial
flagellum
|
Archaeal
flagellum
|
Eukaryotic
cilium (flagellum)
|
Retractable type IV pilus
|
Junctional pore complex
|
Ratchet structure
|
oarlike spicules
|
Example
|
E.
coli flagellum
|
Halobacterium
salinarum
flagellum
|
Animal
sperm flagella
|
M. xanthus S-motility
|
M. xanthus A-motility
|
Cytophaga
gliding
|
Synech-ococcus swimming
|
Length (um)
|
10-15
|
10-15
|
60-1,000+
|
variable
|
N/A
|
N/A
|
150 nm
|
Diameter (nm)
|
20-23
|
10-14
|
300-1000
|
6
|
pore: 14-16 nm
|
?
|
?
|
Major structural protein(s)
|
FliC
(flagellin)
|
FlaA1/2,
FlaB1/2/3
|
tubulin
MTs (microtubule)
|
PilA
|
N/A
|
N/A
|
spicule
|
Structural protein count
|
18-20
|
~12
|
~250
|
~13 (+)
|
10+
|
~8 (+)
|
1 (?)
|
Number of genes
|
30-50
|
12+
|
250+
|
35+
|
30+
|
8+
|
?
|
Structure
|
stiff
naked filament, left-handed helix, rarely glycosylated
|
stiff
naked filament, right-handed helix, often glycosylated
|
flexible,
complex axoneme of MTs usually in “9+2” arrangement
|
stiff naked filament
|
slime secretion
|
probably slime secretion
|
stiff spicules in cell
surface
|
Major motor molecule
|
MotA,
MotB
|
FlaI
(?)
|
dynein
|
PilT/PilU (?)
|
AglS/V/R/X (?)
|
?
|
?
|
Motor energy source
|
protonmotive
force
|
ATP
(?)
|
ATP
|
ATP
|
proton-motive force
|
proton-motive force
|
proton-motive force
|
Motor mechanism
|
rotation
at the base
|
rotation
at the base
|
axoneme
bending
|
filament retraction
|
slime secretion
|
slime secretion (?)
|
Ca2+-induced
paddles (?)
|
Assembly
|
subunits
secreted through channel inside filament, subunits added to distal tip
|
cleaved
signal sequence, subunits added at base, no central channel in the filament
|
addition
of tubulin from an MT organizing center
|
cleaved signal sequence,
subunits added to the base
|
N/A
|
N/A
|
?
|
Major homologies
|
type
III secretion, membrane transport proteins (TolQR)
|
type
IV pili, type II secretion
|
cytoskeleton,
centriole, mitotic apparatus
|
archaeal flagella, type II
secretion
|
membrane transport proteins
(TolQR)
|
junctional pore complex (?)
|
junctional pore complex (?)
|
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.
Secretion
system
|
Virulence
effectors and other secreted proteins (Thanassi
and Hultgren, 2000a)
|
Adhesion (Thanassi and Hultgren, 2000b)
|
Nonmotile
surface structures (Fernandez and
Berenguer, 2000)
|
Motility
(Bardy et al., 2003)
|
Autotransporter
(sec-dependent)
|
Neisseria
gonorrhoeae IgA1 protease (Thanassi
and Hultgren, 2000a)
|
adhesins (Henderson et al., 1998)
|
|
|
Chaperone/Usher
(sec-dependent)
|
P-pilus of
uropathogenic E. coli (Thanassi
and Hultgren, 2000a)
|
FimH adhesin
(Thanassi and Hultgren, 2000a), PapG adhesin (Fernandez
and Berenguer, 2000)
|
Type 1 pili
(fimbriae) of pathogenic and nonpathogenic E. coli
|
|
Type I
(sec-independent)
|
E. coli α-hemolysin; proteases; also
antibiotic secretion (Buchanan,
2001)
|
PEB1 in C..
jejuni (Pei et al., 1998)
|
S-layer
proteins of e.g. Caulobacter crescentus (Broome-Smith
and Mitsopoulos, 1999)
|
|
Type II (sec)
|
Enteropathogenic
E. coli (EPEC) and enterotoxigenic E. coli (ETEC) (Mecsas and Strauss, 1996); cholera toxin (Fernandez
and Berenguer, 2000)
|
Type II
pseudopilus (Xcp secreton in P.aeruginosa; Durand et al., 2003)
|
Type II
pseudopilus (Durand et al., 2003); S-layer proteins (Fernandez and Berenguer, 2000)
|
|
Type III
(sec-independent)
|
Type III
virulence systems of Yersinia, Salmonella, etc. (Hueck, 1998)
|
Flagella
double as adhesion organelles (Moens
and Vanderleyden, 1996; Giron et al., 2002); type III virulence systems (Hueck, 1998); intimin (Nougayrede
et al., 2003)
|
Type III
pili: Pseudomonas Hrp pilus (He
and Jin, 2003), Shigella needle complex (Blocker et al., 2003)
|
Bacterial
flagella (Macnab, 2003)
|
Type IV
(sec-dependent)
|
Effector
proteins of Bordetella pertussis and many other pathogens (Christie and Vogel, 2000; Christie, 2001)
|
Enteropathogenic
E. coli (EPEC) type IV bundle-forming pilus (BFP; Knutton et al., 1999); PilC (Fernandez
and Berenguer, 2000)
|
Type IV pili,
conjugation T-DNA transfer pili (Christie,
2001)
|
Twitching
motility from retractable type IV pili; Archaeal flagella (Bardy et al., 2003)
|
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.
Protein
|
Size
|
Protein
Interactions
|
Membrane
interactions
|
Notes
|
other
|
self
|
F1-α,
F1-β
FliI
|
495, 450
457
|
F0-b2,
F1-δ, F1-γ
FliH2,
FliJ, ?
|
heterohexamer
homohexamer
|
associates
associates
|
F1-β
and FliI have ATPase activity, ~30% sequence similarity proves homology
|
F1-γ
FliP
|
279
245
|
F1-αβ,
F0-c
?
|
monomer
?
|
? (F0
portion)
4-5 TMH
|
C-term
associates with inside of F1
Possible
C-terminal sequence in cytoplasm
|
F1-δ
FliJ
|
171
147
|
F1-α,
F0-b
FliI, FliH
|
monomer
?
|
0
0
|
N-term
α-helices bind with F1-αβ N-term
N-term
α-helices bind with FliI N-term
|
F1-ε
FliO
|
126
121
|
F1-β,
F1-γ
?
|
monomer
?
|
0
1 TMH
|
Structure (Wilkens and Capaldi, 1998), no TMH
N-term
cytoplasmic
|
F0-a
FliR
|
270
261
|
F0-b,
F0-c
?
|
monomer
?
|
6 TMH
5-6 TMH
|
N-term
cytoplasmic
N, C (?), 2
middle loops cytoplasmic
|
F0-b
FliH
|
153
235
|
F1-αβ,
F1-δ
FliI, FliJ
|
dimer
dimer
|
N-term
N-term (?)
|
Similar
domain architecture (CDART);
Both F0-b and FliH elongated, C-terms bind with FliI/F1-αβ
|
F0-c
FliQ
|
72
89
|
F0-a,
F1-αβγ
?
|
~12-mer
?
|
2 TMH
2 TMH
|
Loop between
helices cytoplasmic
Loop between
helices periplasmic
|
Table 6: Functions and analogs at each stage of the
presented model. See Figure 7 and text for further details.
Stage
|
Function of
core system
|
Analogs
|
1. Primitive type III export system and precursors
|
ß=
= = ===== Export
ß
= = = = = = = = = ============ Secretion
ß= = = = = = ========= Adhesion
ß=======Dispersal
ßTaxis
|
Passive inner membrane pores
Gated pores
Export systems
(e.g. sec system)
|
2. Primitive type III secretion system
|
Secretion systems (Table
4, Figure
5)
|
3. Surface adhesin
|
Outer membrane adhesins (Table
4)
|
4. Type III pilus
|
Pili (Table
4, Figure
5b,c)
|
5. Protoflagellum
|
Random dispersal mechanisms (Vogel,
1994)
Dispersal
by modern flagella (Dusenbery,
1997)
|
6. Flagellum
|
Motility
systems (Table 3, Figure
5d)
|