Bacteria - pili
Above: a pilus projecting from the surface of a Gram negative bacterial cell. 'Pilus' is Latin for 'hair' (plural pili)
and describes not so much hair-like bacterial appendages, but thin rod-like appendages that some bacteria
have. Whether or not bacteria possess pili depends upon both species and strain and environmental waste
resources in producing them, but will rapidly switch on their synthesis if environmental signals suggest they will
be needed. For example, one function of pili is adhesion to surfaces - thus bacteria inside the human intestine
may produce pili in order to adhere to the host's cells and only swarmer cells may produce them. Swarmer cells
are dispersal stages, they swim off to find new surfaces to colonise. Once on a surface the bacteria usually
produce a multicellular biofilm or 'slime city' which will give off new swarmer cells.

Bacteria may possess no pili, one pilus, a few pili, or they may be clothed in hundreds of pili, giving them a hairy
appearance (in which case the pili are sometimes called fimbriae, singular fimbria, meaning 'fringe' due to their
appearance under the microscope).
The diagram at the top of the page shows a pilus emerging through the cell envelope into the external medium.
Notice that the pilus is depicted as being made up of many spheres, these are the subunits, each one is a
molecule of protein called pilin (fimbrin). Pili are about 1-2 micrometres long and 2-8 nanometres wide, so they
are long and thin with the exact diameter depending upon pilus type. Notice the rings that form holes in the
membranes through which the pilus projects - these represent complex and not well understood export and
assembly systems (proteins) that transport the pilin subunits from inside the cell to the cell surface and
assemble them into the pilus, which may grow as needed. Different pili types utilise different export and
assembly systems.

Pili that function in adhesion
Some bacteria are swarmer cells, swimming around by means of flagella (usually) until they locate a suitable
substrate on which to feed and establish a new colony or
biofilm. To colonise a surface the swarmer cell has to
adhere to the surface and prevent itself from being washed away. This surface might, for example, be a stone
or decomposing organic body in a pond etc, or it may be the gut or other compartment inside a host animal.
Bacteria can adhere in all sorts of ways: by their cell surface, or via sticky threads which may occur at one end
of the cell or all over its surface, via the slime capsule and other surface structures. Often adhesion occurs by
means of appendages which protrude from the cell surface, such as the flagella, or by pili.

Not all bacteria have flagella, the bacterium in the picture above has no flagella and is non-motile but eventually
it settles and makes contact with the surface (if it had flagella it may have actively swam toward the surface)
and once its pili touch the surface they stick to it, because their ends are sticky. We say that the pili (or more
specifically their sticky ends) function as
adhesins (biological glues). The pili may then retract to bring the
bacterium closer to the surface, enabling other parts to adhere, such as the outer envelope or slime capsule
that surround the cell, and stabilising or strengthening the adhesion. The bacterium will continue to grow and
reproduce and may form a biofilm or layer of multicellular slime that covers the surface. You may have removed
an object from a pond to find that it feels slimy - that is why it is covered by a biofilm.

If the liquid is flowing, for example if the bacteria are colonising the human bladder then they will be subject to
quite forceful fluid flows, then the bacteria must hold on tightly to avoid being washed away and put back into
suspension. The bacteria may be quite forcefully pulled and pushed about. To resist these forces the pili may
act as
springs, dampening the forces of flowing liquid on the bacterium and absorbing the shocks. Inside the
human body, quite a chemical war may be waged between the human host and potentially harmful bacteria as
the body may attempt to chemically block the pili of the bacteria to prevent them adhering or it may allow them
to adhere to cells which then gobble them up. Some bacteria use their pili to adhere and trigger the host cell to
eat them but they then escape being destroyed and live inside the host cell! Pili can also be used to stick
bacterial cells to one another, forming microcolonies.

The function of pili in DNA transfer

The picture below shows two bacteria joined by a single large pilus called an F-pilus. This pilus will retract,
pulling the two bacterial cells close together and enabling one of them, the donor, to pass DNA to the recipient.
The diagrams below illustrate the detailed stages in this DNA transfer process, which is called conjugation.
conjugation mechanism

Once the pilus adheres to a target cell (an F-minus cell lacking the conjugation plasmid) it retracts until the two cells dock and form a mating bridge along which DNA is transferred (A). DNA transfer is also thought to occur through the extended pilus itself, but this is a precarious affair as the cells may be pulled apart by turbulence; the mating bridge is a more stable connection. Conjugation also occurs (indeed more frequently) in colonies and biofilms where the cells are encased in a slime matrix and perhaps attached to a solid surface, which creates a more stable environment for DNA transfer.

One strand of the double-stranded plasmid DNA is nicked and this free strand transferred to the recipient cell (B, C).

Each cell now synthesises the missing complementary strand of plasmid DNA, so that both now contain complete copies of the plasmid (D). Any genes encoded by the plasmid will be acquired by the recipient, such as genes for virulence, antibiotic resistance and synthesis of conjugation pili to spread the plasmid to more recipients.

If the plasmid DNA is integrated into the host chromosome then during transfer some of the host's chromosomal DNA may also be transferred (conjugation will not last long enough to transfer a copy of the whole chromosome however) such that other genes, apart from those carried by the plasmid, may also be transferred. This makes conjugation a powerful survival mechanism for bacteria, since all manner of useful genes may be horizontally transferred, even between different species. Bacteria have been known to acquire antibiotic resistance by this mechanism.

Plasmids may carry genes that impart virulence to pathogenic strains of bacteria, for example in Vibrio cholerae.

Conjugation is clearly beneficial to the F-plasmid, since its genes get replicated and is also useful to the bacteria. The F-plasmid often contains useful genes that deal with stressful situations that have recently arisen, for will
spread this resistance to other bacteria. Since the F-plasmid can integrate into the host's chromosomal DNA it
can, upon leaving, take away copies of host genes with it, these genes may be of use to the recipient cell. This
conjugation process is the closest that bacteria get to having sex - bacteria have no gender and they do not
reproduce sexually, but they can exchange and mix-up their genetic material in this way.

plasmid is a small circular DNA molecule which transfers copies of itself from one bacterial host to another,
they are small mobile, self-replicating genetic elements. Some plasmids can exist in multiple copies within a
single cell, but only a single F-plasmid may occur in each host bacterial cell.

The function of pili in export and import

Another way that bacteria can take on-board new genetic information is by taking up bits of DNA from their
environment - DNA released by dead or damage cells nearby. This DNA can then be incorporated into the cell's
own DNA and the whole process is called (natural)
transformation. Pili (of type IV) have been implicated in this
process. These pili appear to bind to DNA and then retract, bringing the DNA into the cell. They have hollow
channels and the possibility remains that DNA is transported through the pilus channel. Other protein systems,
apart from pili, may also accomplish DNA uptake. (In artificial transformation, bacteria that do not naturally take
up DNA are made to do so, for example by applying electric shocks which open up transient channels in the cell
envelope through which external DNA can enter). Not all bacteria can take up DNA, and those that can are
described as competent.

Some bacteria use special pili to inject DNA into host cells, for example, the bacterium
is a plant pathogen that forms crown galls on trees and it uses its single T-pilus (formed at one
end of the cell) to inject some of its DNA (from a plasmid called the Ti-plasmid) into host plant cells. This DNA
effectively takes over the host cell and causes it to proliferate, forming a gall which houses, protects and
nourishes the bacteria. Initially the T-pilus binds to the host cell and then coils up to bring the cells closer
together, stabilising the connection, and then DNA is transferred from the bacterium to the host plant cells
through a protein bridge assembled between the two cells - a process similar to conjugation.

In these examples, although the pili are hollow tubes, they do not appear to be the channels for transport
between cells, rather their function is to adhere to a target cell and then retract, bringing the two cells together
for the formation of stronger contacts and a bridge for DNA transfer. However, the role of pili in transporting
materials along their internal channels cannot be ruled out.

The function of pili in locomotion

Twitching Motility – Type IV Pili. This form of motility occurs, for example, in Pseudomonas aeruginosa,
Neisseria gonorrhoeae and some Escherichia coli strains. Motility occurs in short, intermittent jerks of a few
micrometers. A moist surface is required for twitching motility and cells must be within several microns of each
other. The pili are polar, 6 nanometres in diameter and up to 4 micrometres long and occur at one or both
poles. These pili actively extend and retract. These pili are also involved in conjugation, bacteriophage
infection, biofilm formation and transformation. At least 35 genes are involved in twitching motility and speeds of
up to one micrometre per second are attained. Pili are also involved in attachment and invasion of host cells.

Myxobacteria travel by gliding across solid surfaces in swarms that co-operatively digest macromolecular
food, including prey cells and other bacteria. Myxobacteria aggregate when nutrients become scarce. These
aggregations produce multicellular fruiting bodies that release dormant myxospores. These bacteria have large
genomes, that of
Myxococcus xanthus, for example, is some 9.5 mbp mbp = mega base pairs, or million base
pairs; a base pair is a unit of information stored in DNA, rather like a byte is a unit of information stored by a
computer). There are two myxococcal gliding systems, the S system or 'social' gliding system and the A system
or 'adventurous' gliding system. The A system operates in single, isolated cells. Gliding speeds are about 0.4
micrometres per second.

S system relies upon type IV pili tethering and retraction to pull the cells along. The gliding is smooth and
possibly some pili extend as others retract. The
A system operates over drier surfaces and does not require
pili. Chain-like strands, grouped into bands, wrapped helically around the cell, occur in the periplasm. In
Myxococcus fulvus, three or more of these chains are combined into ribbons. The subunit rings have an outer
diameter of about 12-16 nanometres (12.6 to 15.6 nanometres) joined by elongated linkers 2.8 nanometres
wide and 9.9-11.9 nanometres long. The linkers are arranged in two parallel rows, so that there is one pair of
linkers between each pair of rings.

Pili as electric nanowires

Nanowires are pili (often type IV) that conduct electricity (as flowing electrons) that may be as thin as 3
nanometres in diameter. For example, the bacterium
Geobacter sulfurreducens (or G. sulphurreducens) grows
on metallic surfaces, such as iron deposits, and pili conduct electrons from inside the cell to the iron outside the
cell. The metal functions as what is called a terminal electron acceptor in respiration. In humans (and most
animals, fungi and plants) the terminal electron acceptor is oxygen. Electrons are removed from oxidised fuels
(oxidation is loss of electrons), such as sugars, inside cells during respiration. These electrons are then
combined with oxygen (from the air you breathe in) to make water (the oxygen is reduced to water, since
reduction is gain of electrons). Without a terminal electron acceptor, the flow of electrons stops and respiration
stops and the supply of energy from fuels also stops. Some bacteria grow in places where there is no or too
little oxygen for respiration, or where other chemicals that will do the job are more abundant, indeed oxygen is
poisonous to many bacteria.

If the terminal electron acceptor is an insoluble solid, then it cannot be easily mobilised and transported into the
cell. The solution is to leave it outside the cell and to send the electrons to it. The pili conduct electrons from
the respiratory system (from the electron transport system) onto the final electron acceptor.
This makes
nanowires among the smallest known electrical wires and they were developed by nature long
before human beings discovered electricity
! Nanowires are another great bacterial invention and an
example of nature's own nanotechnology.
Pov-Ray model of bacterium with pili
Pov-Ray model of bacterium with pili
Above (and left, up close): a Pov-Ray computer model
of a Pseudomonas bacterium, with seven polar flagella
and 600 pili. This 'hairy' looking cell can be said to be
fimbriated ('fringed' with hairs) and the pili referred to
as fimbriae. Bacterial cells may contain almost any
number of pili, but between 100 and 1000 are typical
may be present as a single copy).

Pili are more-or-less straight structures and
more-or-less flexible or brittle.
Curli are related
appendages which are similar to pili but form coils.
Pov-Ray model of Pseudomonas

The model below illustrates the structure of basal structures associated with a type four pilus (T4P or type IV pilus). T4P have a variety of important functions, including motility, attachment uptake of DNA from the environment (transformation) and colony and biofilm formation. These pili are also important in pathogenic bacteria. Pilin, as a characteristic bacterial protein, triggers and immune response. indeed, the human (mammalian) immune system has special receptors to recognise so-called pathogen-asdsociated molecular patterns (PAMPs) such as toll-like receptors (TLRs). Pilin is one PAMP, flagellin (the main protein in the bacterial flagellum filament) is another. Pili are important virulence factors: that is they help pathogens to cause disease. First and foremost, they are important in colonising a new host, since they allow bacteria to rapidly adhere to a surface inside the body before being swept out. for example, pili are crucial virulence factors to uropathogenic Escherichia coli (uropathogenic = causing urinary tract infections) which is the main species causing (about 80%) of urinary tract infections in humans. These bacteria possess type I pili (T1P) with a specific adhesin molecule called FimH at the tip of each type-one pilus, which binds specifically to sugar molecules (mannose) on the surface of epithelial cells lining the urinary tract. FimH forms catch bonds with the target sugar, a type of mechanochemical bond which is able to resist the pulling forces of flowing fluid (urine) which would otherwise wash the bacteria out of the urinary tract. T4P have adhesins (minor pilin proteins) distributed along their length, allowing bacterial cells to also bind to one-another by interlocking their pili.

Pov-Ray model of Type 4 Pilus

Bacteria, such as Pseudomonas aeruginosa (an opportunistic pathogen which can cause disease in those with weakened immune systems) can use their T4P in two modes of locomotion. The T4P are located at both poles of the rod-shaped bacterium. In the first mode, with the cell lying horizontal upon a surface, the pili at one pole extend, attach, then retract to pull the cell along. This type of crawling is much slower than swimming by means of flagella but neverthless allows the bacteria to cover good distances and may be directed towards food sources, though is more often considered to be twitching motility in which movement is somewhat erratic, the net effect being to disperse the bacteria out from the colony centre along the surface. That is twitching motility serves in dispersion. In the second mode of twitching motility, the bacterium uses its pili to pull itself upright, standing on one end the pili are then used more like 'legs' as the bacterium walks along the surface. This movement is erratic as pili apparently compete with one-another in a 'tug-of-war' sometimes pulling the cell one way, sometimes in another direction. This mode is good for covering a surface in exploratory walks.

Pov-Ray model of Type 4 Pilus

The protein machinery at the base of the T4P is responsible for manufacuring (secreting) the filament of the pilus and in pilus extension and retraction. The model above is (loosely) based on the model proposed by Chang et al. (2016, Science 351: 1165-1173) based on electron microscopical studies of mutants of Myxococcus xanthus (each mutant lacking one of the components). Growth/extension occurs when new pilin protein subunits are added at the base of the pilus and retraction occurs when subunits are removed from the base. In Gram negative bacteria (like Pseudomona aeruginosa) the cell envelope consists of three principle layers (additional layers may or may not be present in some strains/species): the outermost is the outer membrane (OM) and the innermost the inner membrane (IM). sandwiched in between these two layers is the periplasmic gel containing a layer of tough but flexible peptidoglycan (P) (a peptide crosslinked polysaccharide). The main filament of the pilus is made of PilA (cyan) which passes through a protein ring (PilQ, pink) embedded in the OM. This OM ring consists of 12 subunits of PilQ protein and includes 36 beta domains (3 per subunit) which anchor the structure in the peptidoglycan layer. There are several; rings in the periplasm, through which the pilus passes, the upper ring consists of the proteins PilQ and TsaP, the middle ring of PilP and TsaP, the lowermost of PilN and PilO. the pilus is anchored to a dimer of PilM which adds new PilA subunits to the extending pilus. In order to add these subunits to the required positions of the filament it is suggested that the PilM dimer must rotate about its axis. The energy for this rotation and pilus assembly comes from ATP hydrolysis.

Pov-Ray model of Type 4 Pilus
Pov-Ray model of Type 4 Pilus