The Evolution of the Flagellum
And the Climbing of "Mt. Improbable
D. Pitman, M.D.
The Appearance of Design
modern scientists believe that all living things, with all of their various
parts and systems of function, evolved through a process of random mutation and
natural selection from a common ancestor life form over the course of millions
and even billions of years of time. Of
course, Darwin's famous observations and documentations of various real time
changes in many different creatures, such as the fairly striking differences in
finches in different regions, helped to popularize this concept. Since then, the
interpretations of the geologic column and the fossil record along with many
modern examples of real time evolution, such as the rapid evolution of
antibiotic resistance in bacteria, only seem to confirm the theory of evolution
as something, "more than a theory".
Still, there are some who continue to question the creative potential of such a mindless process. Can random mutation and natural selection really give rise to the amazing complexity and diversity of all living things? For many, the wonders of the natural world and universe, especially when it comes to living things, are so awe inspiring that they intuitively appear as though they were deliberately set in place by an extraordinary brilliant intelligence - even a godlike intelligence. Consider an observation from Keiichi Namba (program director of a team of dedicated scientists working to detail the various steps in flagellar assembly):
"An enormous number of those macromolecules play each role just like purposefully designed machines and maintain the complex network activities."12
counter argument is, of course, that though grand, the universe and all living
things only have the "appearance of design" when, in reality, no
deliberate foresight or planning went into their creation. (
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what about the evolutionary mechanism? Can
a mindless non-directed process, that has absolutely no goal or higher purpose
in mind, really produce the wonders that we see all around us and even within
our own selves? We are told by
popular science that it is all about "baby steps" - the little differences and
changes that add up over time to produce fantastic variation in form and
function. Clearly, if little
changes could be selected in a positive way over time they could obviously add
up to produce bigger and bigger changes until all the diversity and
functional complexity that we see today is the end
result - the result of billions of years of tiny modifications in many diverging
and even converging family trees.
famous British biologist, Richard Dawkins, describes this process as,
"Climbing Mt. Improbable". In
his book Dawkins explains that although the mountain of complexity that now
exists might seem quite improbable to achieve through blind chance, evolution is
not really a process of purely random chance.
Evolution uses random chance to create little steps where each tiny
change is either good or bad or neutral with regard to overall reproductive
through a process popularly known as the "survival of the fittest",
gives increased reproductive fitness to those creatures that have positive
changes and takes away reproductive fitness from those creatures that negative changes. Obviously then, the next generation is going to be more
heavily populated by those creatures with the most beneficial changes. In this
way, the beneficial changes add up over time in each generation so that the mountain
of enormous complexity is scaled one baby step at a time.
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all sounds very good, and quite convincing actually, except perhaps for one
little problem. Natural selection
is limited in that it can only select, in a positive way, for changes that show
improvement in function over what was there before.
As it turns out, many mutational changes (i.e., changes in the underlying
genetic codes of DNA that dictate how a creature is formed) have absolutely no
affect on the function of the organism. Such changes, or mutations, are called
"neutral" with respect to functional selectability.
There is even a "Neutral Theory of Evolution" proposed fairly
recently by Motoo Kimura.
neutral difference is like "spelling" the code for the same
function in a different way. This different spelling still results in producing the
same/equal/equivalent result - as I just did by using three different words that
mean pretty much the same thing. Or, neutral differences may exist between
equally non-meaningful sequences - like the difference between "quiziligook"
and "quiziliguck". Both
are equally meaningless when spoken in most situations - right?
Therefore, neither has more meaningful/beneficial
"fitness" in a given environment as compared with the other.
Obviously then, selection between them would be equal/neutral - i.e.,
why might this be a problem for evolution?
Well, at very low levels of functional complexity (i.e., functions that
require a very short sequence of genetic real estate to be realized) the
ratio of potentially beneficial to non-beneficial sequences is quite high. So,
the numbers of neutral differences between one beneficial sequence and the next
closest beneficial sequence are relatively few.
example, consider the sequence: cat - hat - bat - bad - big - dig - dog.
Here we just evolved from cat to dog where every single character change was
meaningful and potentially beneficial in the right environment. It
is easy to get between every potential 3-character sequence in the English language system
because the ratio between meaningful and non-meaningful in the "sequence
space" of 3-character sequences is only about 1 in 18.
However, this ratio decreases dramatically, exponentially in fact, with
each increase in minimum sequence length. For
example, in 7-character sequence space, the ratio is about 1 in 250,000 - and
that is not even taking into account the "beneficial" nature of a
particular sequence relative to a particular environment/situation.
Still, meaningful 7-character sequences are generally very
interconnected, like a web made up of thin interconnected roads going around the
large pockets of non-meaningful/non-beneficial potential sequences.
However, the exponential decrease in the ratio is obvious and the
implications are clear. For higher and higher level functions, requiring
larger and larger sequences to code for them, the ratio of meaningful to
meaningless becomes so small so quickly that when more than a few dozen
characters are needed the interconnected roadways and bridges that connect
various island-clusters of beneficial sequences start to snap apart. At surprisingly
low levels of functional complexity this process isolates the tiny islands of
beneficial sequences from every other island to such an extent that there is
simply no way to reach these tiny isolated islands except to traverse the gap of
non-beneficial sequences through a process of purely random change(s) over time.
every additional step up the ladder of functional complexity, this gap gets
wider and wider, in an exponential manner, until it is simply uncrossable this
side of trillions upon trillions of years of average time.
Natural selection is simply blind when it comes to crossing such gaps.
Without the guidance of natural selection, this crossing takes exorbitantly
greater amounts of time since the non-beneficial junk sequences of sequence
space must be sorted through randomly before a very rare beneficial
sequence is discovered by sheer luck.
course, some have suggested to me that a single fortuitous insertion mutation,
composed of just the right sequence of multiple characters, could cross a
sizable gap between one island function and another far away island.
Certainly this is true, but the
problem here is that not just any multi-character sequence or insertion will do.
This sequence has to be just right to work for many types of high-level
functions. The odds that such a specific sequence will actually come along are
extremely remote this side of trillions of years of time when the gap reaches
sizes of just a few dozen or so average non-beneficial character differences.
Then, even if the needed sequence did happen to arrive in the genome, it must
be inserted in just the right place for it to work in a beneficial manner for a
particular evolving function. The vast majority of potential insertion
positions would be detrimental or at best neutral with respect to overall
Then, even if the needed sequence did happen to arrive in the genome, it must be inserted in just the right place for it to work in a beneficial manner for a particular evolving function. The vast majority of potential insertion positions would be detrimental or at best neutral with respect to overall function.
then, getting it "right" is not a simple matter. It literally
requires trillions upon trillions of years of average time to cross relatively
small neutral gaps. That's the problem in a nutshell. How is this problem
overcome? ( Back
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Evolving Highly Complex Functions
that the flagellar system, in particular, requires the services of about 50
genes - including the genes for the sensory apparatus (turns the flagellum
clockwise or counterclockwise at a greater or lesser rate depending on the
environment). All of these genes
have been characterized in detail. At
minimum it seems that about 30 different types of proteins (coded for by the
genes) are needed to build the actual structure and another 20 or so are needed
to assist in the building, regulation, and operational control of the flagellum
(appendix). The total number of
fairly specified (specifically arranged for minimum function) characters that
make up all of these protein parts is more than 10,000 amino acid residues (aa)
coded for by about 50 genes. That's
like a good-sized 2,000-word essay.
Now, some have argued that the actual minimum number is less than 50 genes since certain types of bacteria can build useful flagellar systems a bit less than the 30 parts usually listed. The following table lists 21 protein parts shared by the widely different types of bacteria to include Aquifex aeolicus, Bacillus subtilis, Escherichia coli, and Treponema pallidum. 15
MotA, MotB, FliG (C-term)
FliF, FliG (N-term), FliM/N
FlhB, FliQ, FliR, FliP, FliI, FlhA
FlgB, FlgC, FlgG, FliE
Hook and Adapters
FlgE, FlgL, FlgK, FlgD
It seems then that these 21 structural genes are at least close to the required minimum for useful flagellar function. Combined with the other genes needed to assist in the building of the flagellar structure, the bare minimum still seems to be around 35 to 40 unique genes. Clearly then, the flagellar system of motility is very informationally complex. To achieve the motility function the flagellum requires a minimum of several thousand amino acid residues working together in a very specific or "specified" arrangement relative to each other.
Many attempts to explain the stepwise evolution of such an obviously complex system have been proposed. Most are very superficial, leaping over huge evolutionary gaps, involving large changes of multiple proteins, with a wave of the hand. However, there are some better attempts. Perhaps one of the best attempts to explain flagellar evolution is that proposed by Nicholas J. Matzke in this 2003 paper, "Evolution in (Brownian) space: a model for the origin of the bacterial flagellum." 1
the time, Matzke was a geography grad student at the University of California in
Santa Barbara who had obvious passions outside of geography.
In this paper Matzke suggests that the starting point for flagellar
evolution probably began with a type III secretion system (TTSS).
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that the bacterial flagellum is found in both mesophilic, thermophilic,
gram-positive, gram-negative, and spirochete bacteria while TTSS systems are
restricted to a few gram-negative bacteria. Not only are TTSS systems restricted
to gram-negative bacteria, but also to pathogenic gram-negative bacteria that
specifically attack animals and plants . . . which supposedly evolved billions
of years after flagellar motility had already evolved!
Beyond this, when TTSS genes are found in the chromosomes of bacteria,
their GC (guanine/cytosine) content is typically lower than the GC content of
the surrounding genome. Given the fact that TTSS genes are commonly found on
large virulence plasmids (which can be easily passed around between different
bacteria), this is good evidence for horizontal transfer to explain TTSS gene
distribution. Flagellar genes, on
the other hand, are usually split into 14 or so operons, they are not found on
plasmids, and their GC content is the same as the surrounding genome suggesting
that the code for the flagellum has not been spread around by horizontal
So, if anything, it seems like the TTSS system would have evolved from the flagellum (which does in fact contain TTSS system-like subparts, such as a basal body that secretes various non-flagellar proteins - including virulence factors), and not vice versa.
evidence for this comes from the fact that the TTSS system shows little homology
with any other bacterial transport system (at least 4 major ones). Yet,
evolution is supposed to build upon what already exists.
Since the TTSS system is the most complex of the bunch, why didn't it
evolve from one of these less complex systems and therefore maintain some higher
degree of homology with at least one of them? This evidence suggests that the
TTSS system did not exist, nor anything homologous, in the "pre-flagellar
era". It must therefore have
arisen from the fully formed flagellum via the removal of pre-existing
parts - and not the other way around. In fact, several scientists have actually
started promoting this idea in recent literature.2-7
it is so very handy to start one's explanation of a very complex system by
beginning in the middle - or so it might seem at first. Of the 27 or so protein parts utilized in the flagellar
structure, 10 of them are homologues to proteins in the TTSS.
One of these 10 is the "FliI" protein.
FliI is an ATPase that is anchored to the cytoplasmic face of the inner
membrane and probably supplies energy for the synthesis of the export machinery
or transport of secreted proteins, which are selectively captured from the
cytoplasm for the purpose of transport. Then,
there are the proteins that make up the inner membrane transport apparatus and
probably make up the protein-conducting channel.
These include FlhA, FliP, FliQ, FliR, and FlhB.
The flagellar homologue to the MS-ring is made by FliF and the homologue
to the C-ring is made by FliN and FliG. The last protein part, FliH, has an
seems then that most of these 10 flagellar homologues are required for TTSS
function. So, the assumption of an intact proto-TTSS system is a nice little
head start for explaining flagellar evolution. The fact is that the TTSS system
is highly complex in its own right and this only adds to the notion that the
TTSS system did not evolve from a system of lesser complexity, but arose from a
system of much higher complexity (the fully formed flagellum) via a process of
removing pre-established parts - not the addition of new parts.
Obviously, it is much easier to take parts away and maintain lower-level
sub-functions that are already there than to add new parts to lower level
functions to gain beneficial higher-level functions that are, as yet,
to this the fact that some of the homologues between the flagellar system and
the TTSS system are not that homologous. The
FliN in TTSS is only homologous to ~80 C-terminal residues of flagellar FliN
(out of 137aa). There is very little FliG similarity and TTSS FliF is missing
the C- and N-terminal domains that are involved in forming the MS ring. All that
is left of FliF is about 90 out of over 550 amino acid residues.
What this means is that the TTSS system cannot rotate.
Evolving the ability to rotate would involve the addition of a sizable
number of specifically sequenced residues.
short, the function of the TTSS system itself is very difficult to explain using
mindless evolutionary mechanisms. I have yet to see reasonable attempt to
explain how a TTSS system could have evolved with neutral gaps small enough to
be crossed by random mutations of any type.
and other evolutionists approach such problems by suggesting that some as of yet
undiscovered homologue to the flagellar secretory apparatus may one day be
found. Matzke explains:
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.1
origin of this proto-TTSS system begins with the homologue to FliF, a membrane
pore protein complex. FlhB, the protein complex that controls the type of
proteins secreted through the pore, is somehow attached to FliF. FlhA, whose
function is unknown, was also added so that together with FlhB, a passive
general transporter pore could be turned into a substrate specific transporter.
Where FlhB or FlhA might have come from or what other jobs they might
have had is not discussed nor is it clear how their selective abilities would
necessarily have been helpful especially if the wrong proteins were selected for
any case, once the FlhB and FlhA are combined with FliF, some power is needed
for active transport. In comes FliI
to the rescue. The proposal is that
F1-αβ ATPase, a heterohexamer made up alternating α-subunits
(noncatalytic) and β-subunits (catalytic) found in many types of bacteria,
evolved from a common ancestor of FliI (a
homohexamer made up of catalytic subunits and the power source for TTSS) since
FliI shares ~30% homology with the F1 subunit of F1F0-ATP
synthetase. No detailed account of
how this could have happened, mutation by mutation, is given. It is simply
assumed to have happened. Perhaps, given enough faith in evolution as a creative
force, no real detail in needed here?
course, given that FliI can be made, it is easy to get the FliI power source to
attach to the FliF pore - right? Not
so fast. FliI cannot attach to FliF
directly. Another protein called
"FliH" is required to get the FliI ATPase to stick to FliF.
Where FliH came from or how it might have miraculously evolved the
ability to stick to both FliI and FliF in just the right way is not quite clear.
But it gets worse. Another protein
complex, known as "FliJ", is required to interact with the FliI ATPase
and FliH before any flagellar components can be exported.
for active protein export to be achieved by TTSS, three protein complexes are
needed to be arranged just so (FliI, FliH, and FliJ) - and this is just the
imaginary version. The other parts
of the secretory apparatus, FliOPQR, are simply not discussed in Matzke's
"detailed" step-by-step model of flagellar evolution owing to a
"lack of data."
top of this, what about the argument that similarities between proteins of the F1F0-ATP
synthetase and the flagellar type III export apparatus support the notion that
they share a common early ancestor? In
the very same breath Matzke adds, "Individually, the cited similarities are
easily attributable to chance, but together they are at least suggestive."1
This sounds to me like some rather large gaps are at least potentially present
in the proposed pathway already. At
least these gaps are not discussed in any sort of detail by Matzke's model nor
any other model that I am aware of. Those
steps, which seem to require hundreds of fairly specific genetic differences,
are simply passed over with a wave of the hand and the conclusion that,
"The key event in the origin of type III export was the association of a
primitive F1F0-ATP synthetase with a proto-FlhA or FlhB
inside the proto-FliF ring, converting it from a passive to active transporter.
Since little is known about the details of the coupling of ATPase activity to
protein export in Type III export, this step remains speculative."1
- No kidding! And I thought
this was supposed to be a "detailed" discussion of flagellar
evolution? So far, it seems to be
fairly superficial speculation. (
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Although alluded to earlier in this essay, the term "irreducible complexity" was originally defined by Behe as:
"A single system which is composed of several interacting parts that contribute to the basic function, and where the removal of any one of the parts causes the system to effectively cease functioning".16
Behe chose the flagellar motility system as one of his examples of an irreducibly complex system. However, the following is an interesting reaction to Behe's concept of irreducible complexity - suggesting that the flagellar system really isn't irreducibly complex at all:
Mainstream scientists regard this argument as having been largely disproved in the light of fairly recent research. They point out that the basal body of the flagella has been found to be similar to the Type III secretion system (TTSS), a needle-like structure that pathogenic germs such as salmonella use to inject toxins into living eukaryote cells. The needle's base has many elements in common with the flagellum, but it is missing most of the proteins that make a flagellum work. Thus, this system seems to negate the claim that taking away any of the flagellum's parts would render it useless. This has caused [Kenneth] Miller to note that, "The parts of this supposedly irreducibly complex system actually have functions of their own." 17,18
What "mainstream scientists", like Kenneth Miller, don't seem to understand is that all systems of function are irreducibly complex regardless of whether or not a working subsystem can be found within the larger system. The flagellar motility system still requires at least 35-40 genes producing a structure with at least 21 different specifically arranged proteins each requiring a minimum of hundreds of specifically arranged amino acid residues in order for the function of flagellar motility to be realized at all - even a little bit. Just because one or more subsystems may be found within the overall requirements needed to build a flagellar motility system, such as a TTSS system, does not remove the fact that the flagellar system still has a minimum structural requirement that cannot be reduced beyond a high threshold point without complete loss of the flagellar motility function. System reduction may leave the TTSS system intact, since the TTSS system has a much lower minimum threshold structural requirement. However, having the TTSS function in place, does not mean that the flagellar function will also be in place.
Now, one might be able to build higher-level functional systems, systems that require greater and greater minimum structural requirements, with the use of pre-established smaller systems that are already available. However, this potentiality does not remove the fact that higher-level systems have greater minimum size and specificity requirements before they can be realized at all - even a little bit. All types of functions have their own minimum requirements. These minimum requirements are not all the same. And, it is this difference in minimum requirements that makes all the difference.
The real question is, can irreducibly complex systems be built up with the use of what is already in the gene pool? And, if so, is it equally likely to end up with functions at different levels of minimum size and specificity requirements?
It is my position that functional systems that require a minimum of only a few dozen amino acid residues in a fairly specific orientation can be evolved in relatively short order (just a few generations for a colony of a few billion bacteria). However, likelihood that higher and higher level functions will be within reach in such a short amount of time decreases in an exponential manner with each step up this ladder of irreducible functional complexity.
This notion is born out in literature. There are a whole lot of examples of evolution "in action" when it comes to functions that require a minimum of only a few dozen residues or if the residue positions need not be very specified (antibiotic resistance, improved immune system specificity, phage infectivity, etc). However, when it comes to functions that require a minimum of a few hundred fairly specified residues working together at the same time (as in single protein enzymes like lactase, nylonase, etc) the number of examples drops off dramatically and the number of bacterial gene pools that are capable of evolving functions at this level, even in a highly selective environment, also drops off exponentially. When one gets to the level of functions that require just 1,000 fairly specified residues working together at the same time, there simply are no examples of evolution "in action" mentioned in literature - none at all. All that there are, at this point, are stories about how evolutionary mechanisms of random mutation and function-based selection must have done the job. That's it, just stories based on nothing more than assumptions. There are no actual observations of evolution in action beyond this point - not one example. There are also no serious attempts to calculate the odds of evolution happening at such levels within the proposed few billion years of time that the evolution of life has supposedly taken place on this Earth. The paper discussed here, by Matzke, is no exception. Matzke makes no attempt to calculate the odds of evolution crossing any of his proposed steps in the flagellar evolution pathway. He simply relies, as do other mainstream scientists, on the notion that sequence similarities could only be the result of an evolutionary relationship. Statistical calculations concerning the ability of random mutations and function-based selection to actually make it across these proposed steps just doesn't seem to be needed by mainstream scientists. Why calculate the odds when the story seems so good?
Perhaps, just perhaps, there is a little problem with these stories. There seems
to be a linearly expanding gap problem between what is and what might be.
Each step up the ladder of functional complexity results in a linear expansion
of the neutral/non-beneficial gap between what exists in a gene pool and the
closest potentially beneficial genetic sequence(s) in the vastness of
"sequence space". Each linear expansion in gap distance, as
defined by the number of residue changes that would be needed to achieve the new
function, results in an exponential increase in the number of random walk/random
selection steps that would be needed - on average. Of course, this results
in an exponential increase in the average time required to find a new beneficial
functional sequence at higher and higher levels of minimum functional
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The "Little" Steps Up the Mountain
Maztke's 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 binding of proto-FlhA and/or FlhB to FliF. 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 a secreted adhesin (or alternatively an adhesin is coopted by transposition of the secretion recognition sequence), and a later mutation lets it bind to the outer side of the secretin (3a). Oligomerization of the adhesin produces a pentameric 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). Oligomerization 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 loses its binding sites to the axial filament, becoming the proto-P-ring, and the role of outer membrane pore is taken over by the secretin’s lipoprotein chaperone ring, which becomes the proto-L-ring (5b). Perfection of the L-ring and addition of the rod cap FlgJ muramidase domain (which removes the necessity of finding a natural gap in the cell wall) results in 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.1
the TTSS system as a starting point, regardless of the tenuousness of that
hypothesis, the next steps in the evolution of the flagellum should be easy -
right? With just a few residue
changes here and there, the pathway of improved beneficial function should be
made up of neat, closely spaced, steppingstones.
Consider that Matzke's proposed scenario is one of the most detail
descriptions that I have come across - as superficial as it is.
Necessary parts just pop into existence and easily attach to each other
in just the right way. No detailed
discussion concerning the significant modifications that would be required for
such specific attachments to be realized to a beneficial degree is provided.
Matzke's discussion is a gross underestimate of the complexity involved in going
from one beneficial state to the next along his proposed evolutionary pathway.
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usual, the next step, give the existence of a TTSS system, is the addition of a
filament. Matzke and many others
argue that simple protein-based filaments are easy to make - pointing to the
polymerization of hemoglobin in sickle cell patients as a result of a single
point mutation (like changing a single letter in a paragraph and getting a brand
new function). This sort of
thinking understates several rather specific requirements needed to form a useful
filament of any sort.
instance, the parts of a random filament, like those that form sickled
hemoglobin, are very likely to aggregate into clumps or long tangled strands
before they are transported through any sort of pore to the outer surface of the
cell. Obviously that wouldn't be helpful. Also, even if such filament monomers
do make it to the outer surface without clumping up, they have to preferentially
stick to the right place. That requires fairly specific binding features.
What are the odds that such a random filament monomer will also have such
binding features? These odds
translate into an enormous amount of average time.
And, there are a lot of other potential problems for average filament
monomers. What about degradation? What about transport to the channel and the
selectivity of channel uptake? What about sticking to the inside of the channel
and clogging up the pathway? What if the filament ended up forming a solid core
instead of a hollow core? How would
more filament parts get through to get added to the tip? What if the tip was not
capped with a different type of protein that placed each new filament protein
part in the right spot? What are
the odds that just any filament stuck onto the export machinery is going to be
"beneficial" in a given environment?
- even as a "simple" anchoring filament?
not only do the more and more "special" filament parts have to stick
to themselves as well as the secretory apparatus in just the right way,
they must form a filament who's distal tip is able to stick to something else
other than itself and its own host bacterial surface. On top of everything else,
that might be just a bit tricky to achieve. What are the odds that a gene able
to code for such specialized filament proteins will just happen to come along to
be secreted in a specific way by an existing active transport pore?
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"Simple" P Pilus
order to even begin answering this question, let's consider what it takes to
make even the most simple useful bacterial "filament" -
like the P pilus.
P pilus functions as an attachment anchor between bacterial cells and other
cells. It is a thin hollow filament that thins near the tip. On this tip is a
protein that specifically binds to certain types of sugar molecules on certain
types of cells (like kidney cells). Even
though this pilus is about as simple as it gets in real life and even though its
function seems quite mundane, it is coded for by around10 or 11 genes - just as
many genes as code for the obviously complex type III secretory system (TTSS).
The thicker proximal portion is formed by PapA protein parts, the thinner distal
portion by PapE parts, and the very tip by PapG (the specific
"adhesin" that binds to sugars).
There is also an adaptor protein, PapF, that binds PapG to PapE and
another, PapK, that binds PapE to PapA.14 That's a total of 5
different proteins that come together in a very specific order. How is this
it is done with a rather complicated interaction of "chaperone"
proteins. But first, the cell has to make a multiprotein export pathway called
the sec pathway, which dumps cytoplasmic material into the periplasmic space.
The trick for gram-negative bacteria "wanting" to grow a pilus is to
get a filament to penetrate the outer membrane. This takes some fancy
coordination. First, all the pilus subunits are preferentially exported, in an
unfolded state, into the periplasm through the sec pathway where they refold.
However, if left to themselves, they would form disorganized clumps. So,
a chaperone protein, PapD, is required to prevent this clumping problem and to
aid in proper folding conformation with the use of donor strand complementation
(DSC). The filament parts, by themselves, are very unstable and never fold
properly. And, PapD, has no other known function.
Next, the pilus subunit-chaperone complex specifically interacts with a protein channel on the outer membrane known as PapC. This channel is large enough for the tip of the filament to go through, but not the proximal part. PapD, the chaperone, hands off the pilus subunit to PapC, which then aids in its attachment to the growing filament where each subunit contributes a strand to perfectly complete the fold of its neighbor, thereby stabilizing it.14,15
even something as relatively "simple" as building a pilus seems rather
complicated in comparison to the proposed evolutionary step of filament
evolution. It's just very difficult
to make a "useful" filament - it would seem. But, lets say that some
such filament does happen to evolve somehow. How is it going to evolve into a
flagellum? A flagellum needs to be
able to secrete proteins in order to be built.
The problem is, no P-pilus has been shown to secrete proteins - perhaps
because of the small channel size or the lack of an associated energy source for
pumping proteins out. In any case, all such pili are very different from a
flagellum in one very important respect. Such
pili are built from the top down where each new monomer that is added pushes the
existing pilus up and out. Flagella,
on the other hand, are built from the bottom up where each new monomer is added
to the tip as the tip grows outward on the existing flagellum (see animation of
flagellar assembly above).
late Robert Macnab, a former professor of molecular
biophysics and biochemistry at Yale University who also studied flagella, noted
that the mechanism of flagellar assembly is, "a much more sophisticated
process than any of us could have envisaged."8 He went on to
note that, "We think it would not be possible for the system to work with
any significantly lower complexity." 9
FliD cap looks like a pentagon-shaped ring sitting atop the hollow flagellar
filament. Each one of the 5-part FliD pentamer units has a leg-like extension
that points downward and interacts tightly with the filament monomers. However,
there is a slight mismatch.
The cap has 5 legs, but the end of the filament has 5.5 flagellin
subunits in its circumference. So, there is always a little crevice at one spot
between the cap and the filament. The next subunit gets added to the growing
filament at this site. As the new
subunit is added to the open spot, the cap is rotated so that a new spot opens
up adjacent to the one that was just filled. So, as the cap spins round and
round, at 10 rotations per second, new flagellin monomers (FliC) are added
one-at-a-time, 50 per second.8
is most interesting about all of this is that the ends of the flagellin subunits
are unfolded as the travel down the hollow filament tube. One of the reasons for
this is that folded flagellin has a big kink in the middle that makes it too big
to travel through the tube. By themselves, the flagellin subunits cannot fold
properly. So, the FliD cap is required to both fold and place the flagellin
monomers. It other words, it is a type of chaperon protein. In addition, the
hollow area just below the cap is about twice the size as the rest of the tube
and just large enough to allow for the folding of one monomer subunit. The
spinning of the cap combined with favorable protein-protein interactions
provides the energy for this folding process - since there is no ATP involved.8
short, without this highly specialized cap, the flagellin units cannot
self-assemble to form such an orderly filament at all. And, neither the cap
protein nor the flagellin monomers have any other cellular function. Beyond
this, how is it that the cap gets placed in the right position at the tip of the
filament and that no other cap monomers are sent down the tube once this is
done? Again, a specific chaperone
is required for cap assembly and prevention of untimely aggregation.
To counter this argument, the assertion is made that since the FliL and FliK flagellar protein tubular units do not need a cap for proper assembly that the addition of a cap to the system was a late evolutionary modification for improved speed and efficiency.1 One potential problem is that FliL and FliK are only linking proteins. They link the hook-part of the flagellum (FlgE) to the rest of the flagellum (FliC) (see animation above). They do not form flagella by themselves. Even if they did, this would not explain how the flagellin (FliC) units, in particular, could have self-assembled without a cap or how they could have evolved without co-evolution of the very specific FliD cap - involving a large number of highly specified residue changes for minimum selective advantage.
what about the "cap first" hypothesis in which the cap evolves,
because of its adhesive properties, and is improved upon by further evolution of
pilus proteins which extend the cap outward from the cell?
Again, how long would it take to come up with a flagellar protein monomer
specific enough to interact with such a cap in such a complex manner?
explanations get no more detailed than this.
If such evolutionary steps were so easy to cross, they could be easily
tested in the lab. Simply delete
the flagellin FliC gene in a bacterium and see if its decedents will evolve back
the flagellum under the pre-established cap. As far as I am aware, no such
experiments have ever been successful. As
previously mentioned, the same thing is true of bacteria that do not have the
FliD cap gene, like Shigella. These bacteria may have all the other
flagellar genes, but have lost the cap gene - and they can't make a flagellum
nor have they evolved back the cap gene. Why
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Motorizing the Flagellum
Ok, let's say that somehow an early colony of bacteria was in fact able to evolve a proto-TTSS system and a proto-flagellar filament system where each system was independently functional in some beneficial manner. At this point, Matzke argues that it would be a very simple thing to simply stick these two systems together to gain flagellar motility.
In looking into this notion, let's do just a bit of review. Remember that the flagellar motor is indeed broken down into two basic units - the stator and the rotor. The stator is composed of motA and motB subunits (each comprised of approximately 300 residues). The rotor is composed of FliM (~330aa), FliN (~130aa), and FliG (~330aa). All 3 rotor components are involved in flagellar assembly. The C-ring formed by these components acts as a sort of measuring cup that determines the size of the hook filament. What happens is that approximately 120 hook monomers bind to FliM, FliN and FliG, (4 binding sites each). When all the binding sites are filled, all the monomers are released at once and a "hook" segment of a specific length is formed. After the hook monomers leave the C-ring, another protein enters and converts the C-ring from a hook-monomer-secretor to a flagellin-monomer-secretor. The specificity of the C-ring changes with regard to which monomers it accepts.
So, FliG is important in flagellar assembly in that the 200 N-terminal residues of FliG seem to be required. In fact, if one divides up the 331aa of FliG into segments of 10aa each, deletion mutations of segments 11, 13, 16, 17, 20, 21, and 27 result in a lack of adequate flagellum formation and obviously the motility function. Also, those bacteria with 1, 3, 12, 14, 15, 22, 23, and 26 mutations to FliG are completely "nonflagellate".10
This means that Metzke's assertion that FliG, as part of the proto-secretion complex, is "retained only in order to stabilize/support the coadapted secretion complex and the FliF ring, and [is] otherwise vestigial" is complete nonsense. FliG is vital to secretion and has nothing to do with FliF stabilization (FliF has been shown to be quite independently stable). It is just that FliF without FliG cannot form an adequate flagellum.
Of course, the FliG protein (with no significant homologous counterparts by the way) is also the subpart responsible for converting the proton-motive force into torque forces for the rotating motion of the flagellum. The ~100 C-terminal residues seem to be required for this function to be realized. In addition, specific mutations to segments 10, 18, 19, 24, 25, 28, 29 and 31 formed flagella, but were paralyzed.10
As far as the rotary function is concerned, FliM and FliN are responsible for switching the motion from one direction to the other - not for the actual creation of the torque forces. However, FliM and FliN are still necessary for flagellar assembly.
Now, let's talk about FliF (~550aa central MS-ring membrane pore complex) for a minute. FliF has no known homologues outside of TTSS systems (which are thought to have evolved from the flagellar system - not the other way around). Even given its proto-form existence, explaining how a proto-flagellum/filament could get stuck to it in a beneficial manner is quite a challenge. The assembly of even the simplest filaments is quite involved, as described above. Various chaperone proteins are involved in bringing specific monomers into place at just the right time and folding and attaching them together just so. The building of an apparently simple pilus is extremely complex. The building of a hollow flagellum where the flagellum is formed by adding monomers to the distal tip is extraordinarily complicated.
Given these few facts presented so far, I have just a few questions. Matzke suggests that FliG didn't need to evolve with FliF as part of the export apparatus. How is this explained if FliG is currently required for flagellar assembly? If FliG did not evolve with FliF, then wouldn't it need not only to bind strongly to FliF in a manner that overcomes the sheer forces of the spinning FliG, but also in a way that aids in flagellar assembly? Not only does FliG have to bind to FliF, but it also has to have specificity in place for a certain type of filament monomer. I mean, without the flagellin specificity of FliG, the flagellum does not form. When the flagellum is first starting to form in real life, the MS-ring (FliF) and the C-ring (FliG N-term + FliN + FliM) must form first or the flagellum will not form. That seems just a bit hard to explain using evolutionary mechanisms.
Oh, but maybe it would be easier if FliG was already bound to FliF? - If FliG originally evolved with FliF? Then it would already have filament monomer specificity in place and the flagellar filament could already be in place - right? But, how would motA/B bind to FliG in a beneficial manner then? Quite a number of very specific residues have to be aligned just right in order for the proton-motive force of motA/B to be transferred into FliG torque power - and that is in addition to the simple preferential binding of motA/B to FliG + FliF - right?
In short, either way one looks at it there is more that is needed than simple FliF-FliG binding. What good would FliG specificity be for flagellin if it were not bound to FliF first? And, what good would FliG specificity be for motA/B proton-motive force if it were not bound to motA/B first? This specificity would most certainly involve quite a few additional residue position differences starting from the original "proto" forms. And, most likely, these required differences would not be sequentially beneficial in a way where natural selection could guide them along.
Beyond this, a selectable degree of non-covalent binding is not going to happen between FliG and FliF with just one or two correct residue positions in place out of the 46 fairly specific residue positions used to link up FliG with FliF in modern flagella. To overcome the buffeting effects of Brownian motion, the flagellum needs to spin very rapidly (~100-300 rotations/second for 3-4 seconds). This means that a lot of inertial and sheer forces must be overcome to keep FliG connected to FliF. A significant number of the 46 attachment "bolt-like" residues would have to be in place, all at the same time, in order to overcome these sheer forces to any selectable degree. In fact, it is suggested by deletion experiments that only N-terminal segment 4 of FliG can sustain significant change without a complete loss of motility. Mutations in the first 3 N-terminal segments (~30aa) resulted in a complete loss of motility - obviously due to a lack of sufficient binding strength to FliF and/or a lack of ability to aid in the formation of the flagellum.10
However, it just so happens that the genes for FliF and FliG are located right next to each other in the genome. Certain deletion mutations between FliG and FliF result in a fusion protein, a covalently bound FliG/FliF protein that does in fact work fairly well. Clearly a covalent bond is much stronger than a non-covalent bond, so the need for dozens of non-covalent bonds to be in place is removed. Although the covalently bound fusion protein doesn't work as well as the non-covalently bound wild-type system, it works well enough to get the job done.
Because of this ability to covalently bind FliG with FliF, without the need to get dozens of sequences just right, some have told me that this makes it easy to get the two independently beneficial lower level systems (i.e., the motor and the rotor) to bind together to give rise to the much higher level system of flagellar motility. This simply isn't true because of the multifunctional need for FliG in both systems at the same time - as described above. In short, either way one looks at it there is more that is needed than simple FliF-FliG binding. What good would FliG specificity be for flagellin if it were not bound to FliF first? And, what good would FliG specificity be for motA/B proton-motive force if it were not bound to motA/B first? This specificity would most certainly involve quite a few additional residue position differences starting from the original "proto" forms. And, most likely, these required differences would not be sequentially beneficial in a way where natural selection could guide them along.
done with FliF mutations show that a "short C-terminus stretch" of 9
"core" amino acid residues is
required for "flagellar assembly".
Note that this assembly process is going on at a time when the motor is
turned off and no FliG rotation is going on. The authors go on to state that,
"Removal or substitution of up to 10 amino acids immediately upstream of
the core region resulted in a paralyzed flagellum."11
That sounds quite specified. The authors said that
removal or substitution of 10 additional residues resulted in
flagellar paralysis. It seems then
that flagellar rotation requires something structurally specific besides
what flagellar formation required. A total of around 19aa fairly
specified residues of the FliF protein need to be in place for both
flagellar assembly and motility to be realized. (
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Trillions upon Trillions of Years
A non-beneficial gap of just a couple dozen specific residues required at a specific position in the genome may not sound like much at first glance, but such a gap would literally take trillions upon trillions of years of average time for a population of all the bacteria on Earth (~1030 individuals) to cross (see calculation in appendix below). In fact, not a single evolutionary step proposed by Matzke or anyone else has ever been demonstrated to be "crossable" in any laboratory experiment - - not one. Without the ability to test such stories in the laboratory, they are simply not falsifiable and therefore are, by definition, not supported by scientific method. It may seems strange for many to even consider this, but such statements concerning the evolution of complex functions, on the order of flagellar system complexity, are not scientific at all - they aren't even theory. At the very best they are untested and perhaps untestable propositions. Simply put, these "stories" about flagellar evolution are just that - - fairytale stories. And, when examined in closer detail, they don't even look good on paper.
It just seems a bit more complicated than Matzke and other evolutionary scientists seem to be letting on. Consider this most interesting conclusion of Lynn Margulis, also noted in an interesting review of Matzke's work by William Dembski:
"Like a sugary snack that temporarily satisfies our appetite but deprives us of more nutritious foods, neo-Darwinism sates intellectual curiosity with abstractions bereft of actual details -- whether metabolic, biochemical, ecological, or of natural history." (Acquiring Genomes, p. 103.)13
Back to Top )
Matzke, Evolution in (Brownian) space: a model for the origin of the
bacterial flagellum, talkreason.org, 2003 ( http://www.talkreason.org/articles/flagellum.cfm
Sukhan, Tomoko Kubori, James Wilson, and Jorge E. Galán. 2001. Genetic
Analysis of Assembly of the Salmonella enterica Serovar Typhimurium
Type III Secretion-Associated Needle Complex. J. Bacteriology 183:
R. M., 1999. The bacterial flagellum: reversible rotary propellor and type
III export apparatus. J Bacteriology. 181 (23), 7149-7153.
S. Y., 1998. Type III protein secretion in plant and animal pathogenic
bacteria. Annual Reviews in Phytopathology. 36, 363-392.
J. F., 2001. Revisiting the chlamydial type III protein secretion system:
clues to the origin of type III protein secretion. Trends Genet. 17
G. V., Day, J. B. and Ferracci, F., 2001. Type III export: new uses for an
old pathway. Mol Microbiol. 40 (2), 284-293.
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.
R. M., Science 290, p. 2087
Macnab R. M., Bacteria create natural nanomachines, USA Today, 2005 (http://www.USAtoday.com/weather/science/aaas/flagella121500.htm)
May Kihara, Gabriele U. Miller, and Robert M. Macnab, Deletion Analysis of the Flagellar Switch Protein FliG of Salmonella, J. Bacteriol. 2000 June; 182(11): 3022–3028. ( http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=94485 )
Bjorn Grunenfelder, Stefanie Gehrig, and Urs Jenal,Role of the Cytoplasmic C Terminus of the FliF Motor Protein in Flagellar Assembly and Rotation, Journal of Bacteriology, Mar. 2003, p. 1624–1633 Vol. 185, No. 5 ( http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=148050&blobtype=pdf )
All animations presented here are the amazing work of Keiichi Namba et al. of the ERATO Protonic NanoMachine Project ( http://www.npn.jst.go.jp/index.html )
William Dembski, Biology in the Subjunctive Mood: A Response to Nicholas Matzke, personal website, 2003. ( http://www.designinference.com/documents/2003.11.Matzke_Response.htm )
Yvonne M. Lee, Patricia A. DiGiuseppe, Thomas J. Silhavy, and Scott J. Hultgren, P Pilus Assembly Motif Necessary for Activation of the CpxRA Pathway by PapE in Escherichia coli, Journal of Bacteriology, July 2004, p. 4326-4337, Vol. 186, No. 13 ( http://jb.asm.org/cgi/content/full/186/13/4326 )
Special thanks to Mike Gene for the excellent information provided on the topic of flagellar evolution on his website:
( http://www.idthink.net/ )
Behe, Michael (1996). Darwin's Black Box. New York: The Free Press. ISBN 0-684-83493-6
Wikipedia, Irreducible Complexity, last accessed 9/28/06 ( Link )
Miller, Kenneth R. The Flagellum Unspun: The Collapse of "Irreducible Complexity" with reply here (last accessed 9/28/06)
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Calculation of "Trillions upon Trillions of Years"
Take a population of bacteria the size of all the bacteria that currently exist on the entire Earth - about 1e30 bacteria. Let's say that this steady state population produces a new generation at a rate of 20 minutes and has a mutation rate of 1e-8 per codon position - given a genome per bacterium of 10 million codons. How long would it take such a population to find a new beneficial function at the level of 1,000 fairly specified residues?
Well, first we have to calculate the likely gap size. Using an average between the calculations of Yockey and Sauer, the ratio of potential beneficial vs. non-beneficial for 100aa systems is about 1e-40. This creates a ratio for a 1,000aa system of about 1e-40^(1000/100) = 1e-400. So, the average gap size between potentially beneficial sequences at this level would be about 308 residue differences - i.e., 20^308 = 1e400.
At his point, one can calculate the Poisson distribution curve to determine the odds that any particular gap would exist (given the average gap of 308 residue differences). Although extremely unlikely given the Poisson distribution, let's say that our colony has a few closer sequences that just aren't "average" - close enough to be only 50 specific residue changes away from at least one beneficial function at this level of minimum size and specificity. How long would it take to get just 50 specific residue changes?
A gap of 50 specific residue differences from a given 1,000aa sequence means that each of these sequences is surrounded by 1e65 non-beneficial options. But, we have 1e30 bacteria with 1e7 codons each. For arguments sake, lets say that each bacterium has 1e5 sequences of 1,000 codons that are within 50 residue changes of success. This gives us a total population of 1e35 starting points that are within 50 changes of success.
Now, how long will it take to get these 50 needed changes in at least one bacterium in our population? After equilibrium of distribution randomly through sequence space is realized, each one of our starting point sequences must search through a sequence space of 1e65/1e35 = ~1e30 sequences, on average, before success will be realized. With a mutation rate of 1e-8 per codon per generation our 1,000-codon sequence will get mutated once every 1e5 generations. With a generation time of
20 minutes, that one mutational step every 2,000,000 minutes = ~ 4 years. So, with one random walk step every 4 years, it would take 1e30 * 4 = 4e30 years to achieve success - on average (i.e., trillions upon trillions of years).
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