Ultrastructure of Bacteria: Cell Walls and flagella

The cytoplasm of prokaryotic cells maintains a high concentration of dissolved solutes that
creates significant osmotic pressure—about 2 atm (203 kPa). To withstand these pressures and
prevent bursting (cell lysis) most cells of Bacteria have a layer outside the cytoplasmic
membrane called the cell wall. Besides protecting against osmotic lysis, cell walls also confer
shape and rigidity on the cell. The walls of cells of Bacteria contain a rigid polysaccharide called
peptidoglycan that confers structural strength on the cell. Peptidoglycan is found in all Bacteria
that contain a cell wall, but it is not present in the cell walls of Archaea or Eukarya.

Overall structure of peptidoglycan. G, N-acetylglucosamine; M, N-acetylmuramic

acid. Glycosidic bonds confer strength on peptidoglycan in the X direction whereas
peptide bonds confer strength in the Y direction.

Three of the amino acids are not found in proteins: D-glutamic acid, D-alanine, and meso-
diaminopimelic acid. The presence of D-amino acids protects against degradation by most
peptidases, which recognize only the L-isomers of amino acid residues.

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Overview of the Gram-Positive Cell Wall
Many gram-positive bacteria produce acidic molecules called teichoic acids embedded in their
cell wall. Teichoic acids are composed of glycerol phosphate or ribitol phosphate with attached
molecules of glucose or D-
alanine (or both). Individual
alcohol molecules are then
connected through their
phosphate groups to form
long strands, and these are
then covalently linked to
peptidoglycan. Teichoic acids
also function to bind divalent
metal ions, such as Ca2+ and
Mg2+ prior to their transport
into the cell. Some teichoic
acids are covalently bonded to
membrane lipids rather than to
peptidoglycan, and these are
called lipoteichoic acids.

Archaeal Cell Walls

The cell walls of certain methane-producing Archaea (methanogens) contain a molecule that is
remarkably similar to peptidoglycan, a polysaccharide called pseudomurein. The backbone of
pseudomurein is formed from alternating repeats of N-acetylglucosamine and N-
acetyltalosaminuronic acid. Pseudomurein also differs from peptidoglycan in that the glycosidic
bonds between the sugar derivatives are β-1,3 instead of β-1,4, and the amino acids are all of the
L- stereoisomer.
Peptidoglycan can be destroyed by lysozyme, an enzyme that cleaves the glycosidic bond
between N-acetylglucosamine and N-acetylmuramic acid. This weakens the peptidoglycan and
can cause cell lysis. Lysozyme is present in human secretions including tears, saliva, and other
bodily fluids, and functions as a major line of defense against bacterial infection. The final step
in peptidoglycan synthesis is transpeptidation. Transpeptidation forms the peptide cross-links
between muramic acid residues in adjacent glycan chains. Transpeptidation reaction is inhibited
by the antibiotic penicillin. When penicillin bound to transpeptidase proteins (FtsI in E. coli), the
proteins are inactivated. If transpeptidation is blocked in growing cell, the continued activity of
autolysins so weakens the peptidoglycan that the cell eventually bursts.

A bacterium can have one flagellum or two or many flagella. Bacteria with a single polar
flagellum located at one end, or pole, are said to be monotrichous. Bacteria with two flagella, one
at each end, are amphitrichous Bacteria with two or more flagella at one or both ends are
lophotrichous Bcateria with flagella all over the surface are peritrichous. Bacteria without
flagella are atrichous. Cocci rarely have flagella.
Transmission electron microscope studies have shown that the bacterial flagellum is composed
of three parts: 1. The longest and most obvious portion is the filament, which extends from the
cell surface to the tip. 2. The basal body is embedded in the cell envelope; 3. A short, curved
segment, the hook, links the filament to its basal body and acts as a flexible coupling.

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The basal body is the most complex part of a flagellum. The basal bodies of E. coli and most
other typical Gram-negative bacteria have four rings: L, P, MS, and C, which are connected to a
central rod. The L, P, and MS rings are embedded in the cell envelope, and the C ring is on the
cytoplasmic side of the MS ring. Typical Gram-positive bacteria have only two rings: an inner
ring connected to the plasma membrane and an outer one probably attached to the peptidoglycan.

monotrichous lophotrichous peritrichous

amphitrichous

The filament is a hollow, rigid cylinder constructed of subunits of the protein flagellin, which
ranges in molecular mass from 30,000 to 60,000 daltons, depending on the bacterial species. The
filament ends with a capping protein. Some bacteria have sheaths surrounding their flagella. For
example, Vibrio cholerae flagella have lipopolysaccharide sheaths.

Flagellar Movement
The filament of a bacterial flagellum is in the shape of a rigid helix, and the cell moves when this
helix rotates like a propeller on a boat. The flagellar motor can rotate very rapidly. When bacteria
are in an aquatic environment, flagellar rotation results in two types of movement: a smooth
swimming movement often called a run, which actually moves the cell from one spot to another,
and a tumble, which serves to reorient the cell. Often, the direction of flagellar rotation
determines whether a run or a tumble occurs. For many bacteria with monotrichous, polar

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flagella, counterclockwise roation results in a run. When rotation is reversed, the cell tumbles.
Peritrichously flagellated bacteria operate in a somewhat similar way.

The motor that drives flagellar rotation is located at the base of the flagellum. Torque generated
by the motor is transmitted to the hook and filament. The motor is composed of two
components: the rotor and the stator. In typical Gram-negative bacteria, the rotor is composed of
the MS ring and the C ring. The stator is composed of the proteins MotA and MotB, which form
a channel through the plasma membrane.
The power used by most flagellar motors is a difference in charge and pH across the plasma
membrane. This difference is called the proton motive force (PMF) that is largely created by the
metabolic activities of organisms [electron transport chain (ETC)]. The channels created by the
MotA and MotB proteins allow protons to move across the plasma membrane from the outside to
the inside. Thus the protons move down the charge and pH gradient. This movement releases
energy that is used to rotate the flagellum.
Rotation of the flagellum occurs at the expense of the proton motive force and it is thought that
rotation is imparted to the flagellum by a type of “proton turbine” process. In this model, proton
translocation through the Mot complex drives rotation of the flagellum, with about 1200 protons
being translocated per each rotation of the flagellum. Protons flowing through the Mot proteins
exert electrostatic forces on helically arranged charges on the rotor proteins. Alternating
attractions between positive and negative charges on the rotor as protons flow though the Mot
proteins then cause the entire basal body to rotate. Rotational speed of the flagellum is set by the
proton flow rate through the Mot proteins, which is a function of the intensity of the proton
motive force.

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Flagella biosynthesis

A flagellar filament grows from its tip. The MS ring is synthesized first and inserted into the
cytoplasmic membrane. Then other anchoring proteins are synthesized along with the hook
before the filament forms. Flagellin molecules synthesized in the cytoplasm pass up through a 3-
nm channel inside the filament and add on at the terminus to form the mature flagellum. A
protein “cap” is present at the end of the growing flagellum. Cap proteins assist flagellin
molecules that have diffused through the filament channel to assemble in the proper fashion at
the flagellum terminus. Self-assembly is the spontaneous formation of a complex structure from

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its component molecules without the aid of special enzymes or factors. The basal body is a
specialized version of the type III protein secretion system observed in typical Gram-negative
bacteria. Type III secretion systems have a needlelike structure through which proteins are
secreted. In the flagellar type III secretion system, the filament replaces the needle. Individual
flagellin subunits are transported through the hollow filament. When the subunits reach the tip,
they spontaneously aggregate under the direction of a protein called the filament cap; thus the
filament grows at its tip rather than at the base. Filament synthesis, like S-layer formation, is an
example of self-assembly.

Endospores
Endospores are highly differentiated cells that are extremely resistant to heat, harsh chemicals,
and radiation. Endospores function as survival structures and enable the organism to endure
unfavorable growth conditions. Endospores are easily dispersed by wind, water, or through the
animal gut, and hence endospore-forming bacteria are widely distributed in nature. The
endospore-forming bacteria Bacillus and Clostridium are common in soil and the best-studied
representatives.
Endospores are impermeable to most dyes, so occasionally they are seen as unstained regions
within cells that have been stained with basic dyes such as methylene blue. To stain endospores,
special stains and procedures must be used. In the classical endospore-staining protocol, the stain
malachite green is used and is infused into the spore with steam.

Endospore Structure and Features

The endospore contains many layers absent from the vegetative cell. The outermost layer is the
exosporium, a thin protein covering. Moving inward there are several spore coats, composed of
layers of spore-specific proteins. Below the spore coat is the cortex, which consists of loosely
cross-linked peptidoglycan, and inside the cortex is the core, which contains the core wall,
cytoplasmic membrane, cytoplasm, nucleoid, ribosomes, and other cellular essentials.
Endospores contain large amounts of calcium (Ca2+), most of which is complexed with
dipicolinic acid. The calcium–dipicolinic acid (DPA) complex forms about 10% of the dry
weight of the endospore and functions to bind free water within the endospore, helping to
dehydrate the developing endospore. In addition, the DPA complex inserts between bases in
DNA, which helps stabilize DNA against heat denaturation. The endospore core also contains
high levels of small acid-soluble spore proteins (SASPs) that are synthesized only during the
sporulation process. SASPs bind tightly to DNA in the core and protect it from potential damage
from ultraviolet radiation, desiccation, and dry heat. In addition, SASPs function as a carbon and
energy source for the outgrowth of a new vegetative cell from the endospore during germination.

Endospore Formation and Germination

Sporulation in Bacillus subtilis
The endospore formation is a complex process that involves asymmetric division of the
cytoplasm to yield a large mother cell and a smaller forespore (prespore), followed by
engulfment of the forespore by the mother cell, and construction of additional layers of spore
coverings. The secret of the success of endospore formation lies in the altruistic behaviour of
the mother cell, which uses all of its resources to endow the forespore with resources,
particularly protective layers, thereby maximizing the chances of survival for the mature spore.

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In B. subtilis, sporulation is controlled by phosphorelay, posttranslational modification of
proteins, numerous transcription initiation regulatory proteins, and alternate sigma factors.
Initiation of sporulation is controlled by the protein Spo0A, a response regulator protein that is
part of a phosphorelay system. The sensor kinases protein kinA senses nutrient starvation and
trigger sporulation by autophosphorylation. The phosphoryl group is sequentially transferred to
Spo0F followed by Spo0B and finally to Spo0A. The phosphorylated "master regulator" Spo0A
controls the expression of over 500 genes needed for sporulation.
Once a cell of B. subtilis commits to sporulation, endospore development is controlled by four
different σfactors, two of which, σF and σG, activate genes needed inside the developing
endospore (called the forespore) and two of which, σE and σK , activate genes needed in the
mother cell surrounding the forespore. The sporulation signal, transmitted through Spo0A,
activates σF in the forespore (σF is already present in the forespore but is inactive because it is
bound by an anti-sigma factor). Once free, σF is active and can bind to RNA polymerase and
promote transcription of genes whose products are needed for the next stage of sporulation.
These include the gene encoding the sigma factor σG and the genes for proteins that cross into
the mother cell and activate σE. Active σE is required for transcription of yet more genes inside
the mother cell, including the gene for σK. The sigma factors σG (in the forespore) and σK (in
the mother cell) are required for transcription of genes needed even later in the sporulation
process. Eventually, the many spore coats and other unique structures typical of the endospore
are formed, and the mature endospore is released.

The Bacillus subtilis cells at the onset of sporulation secrete extracellular killing factors that lyse
sibling nonsporulating cells that have not developed immunity to these toxins. This killing
releases nutrients from the dead cells into the starved medium that the surviving sporulating cells
can feed on, and thus this behavior was termed cannibalism.

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Molecular mechanisms of cannibalism
The killing of nonsporulating cells is controlled by two independent gene clusters: skf for
sporulation killing factor, and sdp for sporulation delaying protein. Both clusters were
identified as positively regulated by Spo0A, the master regulator of sporulation, and they are
highly expressed at the onset of sporulation. The skf operon is responsible for the production and
extracellular export of the killing factor which lyse the sister cells. The sdp operon synthesizes
the protein responsible for delaying sporulation. The eight‐gene skf operon contains structural
gene that is directly involved in the production of the exported killing factor. The operon is also
able to confer resistance to the cells that produce the killing factor. Thus, it is demonstrated that
some cells in a population of B. subtilis initiates synthesis of the killing factor and export it to the
medium. The cell that does not initiate the synthesis is vulnerable to the killing factor and is
lysed. Thus, cells those are able to cannibalize can delay endospore formation at the expense of
the sister cells.

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