Bacteria or microbes belong to the

prokaryotic world but still are known

to be the most diverse and important
group of organisms.

Two main groups of prokaryotes:

Bacteria
Archaea
 Cell Shape & Arrangement:

Major cell morphologies commonly

encountered in case of bacteria
are mainly of two types:

Spherical or ovoid cell shape-

Coccus (plural Cocci)

Two Cocci- Diplococci

Cocci in chains- Streptococci
Cocci in bunches- Staphylococci
Square group of Cocci- Tetrad
Cubical arrangement of Cocci-
Sarcina
 Cell Shape & Arrangement:

The other common shape is Rod or cylindrical cell-

Bacillus (plural Bacilli). They vary greatly with respect
to length and width ratio. Coccobacilli being too short
resemble the Cocci.
Two Bacilli together- Diplobacilli
Bacilli in chains- Streptobacilli
 Cell Shape & Arrangement:

Other than Bacilli and Cocci many different

shapes of bacteria are often found in nature.

Comma shaped bacteria- Vibrio

Spiral shaped bacteria- Spirilla (Singular
Spirillum)
Flexible Spirilla- Spirochete
Bacteria containing hyphae that collectively form
mycellium- Actinomyces
Bacteria that forms a bud- Hyphomicrobium sp.
 Size of bacteria:

 Bacteria vary in size greatly.

 E. coli is a rod of about 1.1 to 1.5 μm wide by 2 to 6

μm long.

 Smallest bacteria- Nanobacteria (0.2 to less than

0.05 μm)
Mycoplasma (bacteria lacking cell wall)- 0.3 μm
Spirochete- 500 μm in length
Thiomargarita namibiensis- 750 μm cell width (largest
bacteria)

 Some bacteria are pleomorphic, being variable in shape

and lacking a single characteristic form.
o There are significant advantages of
smallness. Small cells have large
surface area relative to cell volume
than do large cells.

o Small cells have higher surface-to-

volume ratio.

o The S/V ratio affects cell’s biology,

evolution and many other aspects of
biology.

o The higher S/V ratio of smaller

cells supports a faster rate of
nutrient exchange per unit cell volume
o Bacterial cells are surrounded by several
layers and the innermost cell envelope is the
Plasma Membrane that encloses the
cytoplasm.

o Plasma Membrane (PM) is very important

because it separates the cytoplasm from its
environment and if the membrane is broken,
the integrity of the cell is destroyed, the
cytoplasm spills into the environment and the
cell dies.
o The plasma membrane is a thin membranous
barrier that surrounds the cell and separates
the cytoplasm from the environment.

o Plasma Membrane (PM) is a selectively

permeable membrane. It allows particular ions
and molecules to pass either into or out of the
cell, while preventing the movement of others.
Thus the plasma membrane prevents the loss of
essential components through leakage while
allowing the movement of other molecules.
Fluid-Mosaic Model:
The most widely accepted model for membrane structure is the
fluid mosaic model of Singer and Nicholson, which proposes
that membranes are lipid bilayers within which proteins float. The
model is based on studies of eukaryotic and bacterial membranes, and
was established using a variety of experimental approaches, including
transmission electron microscopy (TEM) and atomic force
microscopy. Cell membranes are very thin structures, about 5 to 10
nm thick, that look like two dark lines on either side of a light
interior when imaged by TEM. This characteristic appearance is
evidence that the membrane is composed of two sheets of molecules
arranged end-to-end. Cleavage of membranes by freeze-etching, a
technique that allows the microscopist to see fine detail, exposes the
proteins lying within the membrane lipid bilayer.
The chemical nature of membrane lipids is critical to
their ability to form bilayers. Most membrane-
associated lipids, the phospholipids are amphipathic:
they are structurally asymmetric, with polar and
nonpolar ends. The polar ends interact with water and
are hydrophilic; the nonpolar hydrophobic ends are
insoluble in water and tend to associate with one
another. In aqueous environments, amphipathic lipids
can interact to form a bilayer. The outer surfaces of the
bilayer are hydrophilic, whereas hydrophobic ends are
buried in the interior away from the surrounding water.
The plasma membrane is very dynamic: the lipid
composition varies with environmental temperature in
such a way that the membrane remains fluid during
growth. For example, bacteria growing at lower
temperatures have more unsaturated fatty acids in their
membrane phospholipids; that is, there are one or more
double covalent bonds in the long hydrocarbon chain. At
higher temperatures, their phospholipids have more
saturated fatty acids-those in which the carbon atoms are
connected only with single covalent bonds.
Bacterial membranes usually differ from eukaryotic membranes in
lacking sterols (steroid-containing lipids) such as cholesterol.
However, many bacterial membranes contain sterol-like
molecules called hopanoids.
Hopanoids are synthesized from the same precursors as steroids,
and like the sterols in eukaryotic membranes, they probably
stabilize the membrane.
Hopanoids are also of interest to ecologists and geologists: the
total mass of hopanoids stored in sediments is as much as the total
mass of organic carbon in all living organisms and evidence exists
that hopanoids have contributed significantly to the formation of
petroleum.
Two types of membrane proteins have been identified based on their ability to
be separated from the membrane.

Peripheral membrane proteins are loosely connected to the membrane and

can be easily removed. They are soluble in aqueous solutions and make up
about 20 to 30% of total membrane protein.

The remaining proteins are integral

membrane proteins. These are not
easily extracted from membranes
and are insoluble in aqueous
solutions when freed of lipids.
Integral membrane proteins, like
membrane lipids, are amphipathic;
their hydrophobic regions are
buried in the lipid while the
hydrophilic portions project from
the membrane surface.
• Integral membrane proteins carry out some of the
most important functions of the membrane. Many are
transport proteins used to move materials either into or
out of the cell. Others are involved in energy-conserving
processes, such as the proteins found in electron
transport chains. Those integral membrane proteins with
regions exposed to the outside of the cell enable the cell
to interact with its environment.

• Integral membrane proteins function in transportation

and signaling processes.
Archaeal membranes are composed primarily of lipids that differ from
bacterial and eukaryotic lipids in following ways:

1. Archaeal lipids lack true fatty acid side chains and instead, the side
chains are composed of repeating units of the hydrophobic five-carbon
hydrocarbon isoprene. Thus the hydrocarbons are branched. This affects
the way the lipids pack together, which in turn affects the fluidity of the
membrane and its permeability. This is especially important for extremophilic
archaea for which membrane fluidity and permeability could be compromised
by extreme conditions.
2. In contrast to the lipids of Bacteria and Eukarya in which ester linkages bond the fatty
acids to glycerol, the lipids of Archaea contain ether bonds between glycerol and their
hydrophobic side chains The cytoplasmic membrane of Archaea can be constructed of either
glycerol diethers, which have 20-carbon side chains (the 20-C unit is called a phytanyl
group), or diglycerol tetraethers, which have 40-carbon side chains.
3. Archaeal membrane is formed by lipid monolayer instead of
a lipid bilayer membrane. In contrast to lipid bilayers, lipid
monolayer membranes are extremely resistant to heat
denaturation and are therefore widely distributed in
hyperthermophiles, prokaryotes that grow best at temperatures
above 80°C.
1. The plasma membrane acts as a permeability barrier:
The cytoplasm is a solution of salts, sugars, amino acids, nucleotides, and
many other substances. The hydrophobic portion of the PM is a tight barrier
to diffusion of these substances. Although some small hydrophobic molecules
pass the PM by diffusion, polar and charged molecules do not diffuse but
instead must be transported. Even a substance as small as a proton cannot
diffuse across the membrane. One substance that does freely pass the
membrane in both directions is water, a molecule that is weakly polar but
sufficiently small to pass through phospholipid molecules in the lipid bilayer
but movement of water across the PM is accelerated by transport proteins
called aquaporins.
2. Transport:
PM not only functions as a permeability barrier but also allow movement of
nutrients into the cell. Bacteria uptake nutrients from their environment by
different transport mechanisms: passive diffusion, facilitated diffusion,
primary and secondary active transport and group translocation.

Passive Diffusion-
Passive diffusion, often called simple diffusion, is the process by which
molecules move from a region of higher concentration to a region of
lower concentration (i.e., the molecule moves down the concentration
gradient)
Facilitated Diffusion-
Bacteria employ a variety of transport proteins in their uptake mechanism. During facilitated
diffusion, substances move across the PM with the assistance of transport proteins that are
either channels or carriers.
Primary and Secondary Active Transport-
Active transport is the transport of solute molecules to higher
concentration (against concentration gradient) with the
input of metabolic energy. Three types of active transport are
primary active transport, secondary active transport, and group
translocation.

Primary active transport is mediated by carriers and uses energy

provided by ATP hydrolysis. They are uniporters, i.e., they
move a single molecule across the PM. Example- ATP-binding
cassette transporter (ABC transporter)
Secondary active transport
couples the potential
energy of ion gradients
to transport substances
without modifying them.
They are cotransporters,
i.e., symporters. When
they move in opposite
direction then they are
called antiporters.
Group Translocation-
In this process a molecule is chemically modified and is brought into the
cell. Example- phosphoenolpyruvate:sugar phosphotransferase
system (PTS).
o Cell wall is the layer that lies just
outside the plasma membrane.

o The walls of bacteria have a rigid

layer that is primarily responsible
for giving strength to the bacterial
cell.
o Prokaryotic cell wall is
made up of a polymer
Peptidoglycan (murein).
The polymer is composed
of N-acetylglucosamine
(NAG) and N-
acetylmuramic acid
(NAM).

o Murein is only present

in bacteria but not in
Archaea or Eukarya.
o After Christian Gram developed the Gram
stain in 1884, it soon became evident that
most bacteria could be divided into two major
groups based on their response to the Gram-
staining procedure. Gram-positive bacteria
stained purple, whereas Gram-negative bacteria
were pink or red. The true structural
difference between these two groups did not
become clear until the advent of the
transmission electron microscope.
 The feature common to nearly all bacterial cell walls is
the presence of a polysaccharide, peptidoglycan, which
forms an enormous meshlike structure often referred to
as the peptidoglycan sacculus.

 Peptidoglycan is composed of many identical subunits.

Each subunit within the sacculus contains two sugar
derivatives, N-acetylglucosamine (NAG) and N-
acetylmuramic acid (NAM), and several different
amino acids.
• The amino acids form a short peptide consisting of four
alternating D- and L-amino acids; the peptide is connected to the
carboxyl group of NAM.

• The amino acids include L-alanine, D-alanine, D-glutamic

acid, and either lysine or the structurally similar amino acid
analog, meso-diaminopimelic acid (DAP).

• These constituents are connected to form a repeating structure,

the glycan tetrapeptide.
Three of the amino acids are not found in proteins: D-
glutamic acid, D-alanine, and meso-diaminopimelic acid
(DAP).

The presence of D-amino acids protects against degradation by

most peptidases, which recognize only the L-isomers of amino
acid residues.

The peptidoglycan sacculus is formed by linking the sugars of

the peptidoglycan subunits together to form a strand; the strands
are then cross-linked to each other by covalent bonds formed
between the peptides extending from each strand.
The peptidoglycan strand is helical,
and the peptides extend out from the
backbone in different directions.
Many bacteria cross-link the strands
by connecting the carboxyl group of
the D-alanine at position 4 directly to
the amino group of diaminopimelic
acid (position 3) of the other strand.
Other bacteria use a peptide
interbridge instead.
 Helps to maintain cell shape.

 Protects the cell from osmotic lysis. The

peptidoglycan layer is strong and elastic and hence able
to stretch and contract in response to osmotic
pressure.

 Protects the cell from toxic substances.

 In pathogens it can also contribute to pathogenicity.

 Periplasmic space is the space
between the plasma membrane and
outer membrane in gram negative
bacteria, it is also present in gram
positive bacteria between plasma
membrane and cell wall.
Cell Wall of Gram Positive
bacteria:

o In gram positive bacteria 90%

of the cell wall is peptidoglycan.

o Although some bacteria have

only a single layer of
peptidoglycan surrounding the
cell, many gram-positive bacteria
have several sheets of
peptidoglycan stacked one upon
another.

o Periplasmic space is so narrow

that it is often not visible.
Cell Wall of Gram Positive bacteria:

o Other than peptidoglycan, cell walls of gram positive

bacteria also contain large polymers like Teichoic acids. These
are polymers of glycerol or ribitol joined by phosphate groups.

o The teichoic acids are covalently connected to peptidoglycan

or to plasma membrane lipids; in the latter case, they are
called lipoteichoic acids. Teichoic acids extend to the surface
of the peptidoglycan. They are negatively charged and help
give the cell wall its negative charge.

o Though function of teichoic acids is not very clear but it is

evident from studies that they help to create and maintain
the structure of cell envelope and protect the cell from
harmful substances. They also function to bind Ca2+ and Mg2+
for eventual transport into the cell.
Cell Wall of Gram Negative bacteria:

o The peptidoglycan layer is very thin (10%) in gram negative

bacteria and lies within the periplasmic space.

o The periplasmic space is 30-70 nm wide.

o The outer membrane lies outside the thin peptidoglycan

layer. It is linked to the cell by Braun's lipoprotein, the most
abundant protein in the outer membrane. Other than
phospholipid and protein the outer membrane also contains
polysaccharide. The lipid and p[olysaccharide are linked in the
outer membrane to form a complex and hence the outer
membrane is also called Lipopolysaccharide (LPS) layer.
o LPS consists of three parts:

1. Lipid A-The lipid A, not a typical glycrol lipid, contains two

glucosamine sugar derivatives, each with three fatty acids and phosphate or
pyrophosphate attached. The fatty acids of lipid A are embedded in the outer
membrane, while the remainder of the LPS molecule projects from the
surface..

2. Core polysaccharide- The core polysaccharide consists of

ketodeoxyoctonate (KDO), various seven-carbon sugars (heptoses), glucose,
galactose, and N-acetylglucosamine and is joined to lipid A.

3. The O side chain- The O side chain or O antigen is a

polysaccharide chain extending outward from the core. It has several peculiar
sugars and varies in composition between bacterial strains.
(1) It contributes to the negative charge on the bacterial surface because the
core polysaccharide usually contains charged sugars and phosphate.
(2) It helps stabilize outer membrane structure because lipid A is a major
constituent of the exterior leaflet of the outer membrane.
(3) It helps create a permeability barrier.
(4) The geometry of LPS are thought to restrict the entry of bile salts,
antibiotics, and other toxic substances that might kill or injure the
bacterium.
(5) LPS also plays a role in protecting pathogenic bacteria from host defenses.
The O side chain of LPS is also called the O antigen because it elicits an
immune response by an infected host.
(6) Gram-negative bacteria that are pathogenic for humans and other mammals
include species of Salmonella, Shigella, and Escherichia, among many others,
and some of the intestinal symptoms these pathogens elicit are due to toxic
outer membrane components. Toxicity is associated with the LPS layer, in
particular, lipid A. The term endotoxin refers to this toxic component of
LPS.
The difference between Gram-positive bacteria and Gram-negative bacteria is due to
the physical nature of their cell walls. If the cell wall is removed, Gram positive
bacteria stain Gram negative. During the Gram staining procedure, bacteria are first
stained with crystal violet, a dye with a positive charge that is attracted to the
bacterial cell's net negative charge, and next treated with iodine. Iodine is a mordant
that interacts with the crystal violet, forming an insoluble complex and thus
promoting dye retention. When bacteria are treated with ethanol in the
decolorization step, the alcohol is thought to shrink the pores of the thick
peptidoglycan found in the cell walls of Gram-positive bacteria, causing the
peptidoglycan to act as a permeability barrier that prevents loss of crystal violet. Thus
the crystal violet-iodine complex (CV-I complex) is retained and the bacteria
remain purple, even after the addition of a second dye. In contrast, the peptidoglycan
in Gram-negative cell wall is very thin, and has larger pores. Alcohol treatment also
extracts enough lipid from the outer membrane to increase the cell wall„s porosity
further. For these reasons, alcohol more readily removes the crystal violet-iodine
complex, decolorizing the cells. The counterstain safranin, also a dye with a net
negative charge, easily stains the decolorized cells so that they appear red or pink.
 Microbes have several mechanisms for responding to changes in
osmotic pressure. This pressure arises when the concentration of
solutes inside the cell differs from that outside.

 In those cases, additional protection is provided by the cell wall.

 The protective nature of peptidoglycan is most clearly demonstrated

when bacterial cells are treated with lysozyme or penicillin. The
enzyme lysozyme attacks peptidoglycan by hydrolyzing the bond
that connects N-acetylmuramic acid with N-acetylglucosamine.

 Penicillin works by a different mechanism. It inhibits the enzyme

transpeptidase, which is responsible for making the cross-links
between peptidoglycan chains. If bacteria are treated with either of
these substances while in a hypotonic solution, they lyse.
 Treatment of typical Gram-positive
bacteria with lysozyme or penicillin
results in the complete loss of the cell
wall, and the cell becomes a protoplast.

When typical Gram-negative bacteria

are exposed to lysozyme or penicillin,
the peptidoglycan sacculus is destroyed,
but the outer membrane remains.
These cells are called spheroplasts.
 L-form or Cell-wall deficient (CWD) bacteria are the
strains of bacteria which are basically devoid of cell wall.
 They were first isolated in 1935 by Emmy Klieneberger-
Nobel, who named them “L-form” after the Lister Institute in
London where she was working.
 L-form was discovered in the cultures of Streptobacillus
monoliformis.
 Treatment of bacterial cell with penicillin or lysozyme
generally give rise to non-viable organisms i.e., they do not
multiply. However, if such cells can grow and divide then
they are called L-forms.
Two types of L-form bacteria can be there:

Stable L-form bacteria:- those which do

not revert back to their normal form since they completely
lack peptidoglycan.
Unstable L-form bacteria:- those which
revert back to cell wall containing state when inducing
stimulus (penicillin) is removed. Such forms usually have
small amounts of residual peptidoglycan that serves as
primer for building cell wall
 L-forms may produce chronic infections in the host.

They may persist in protective regions of the body.

 Since L-forms are relatively resistant to antibiotics,

they are difficult to treat.

 Their reversion to normal form can result in relapse

of infection.
Although most prokaryotes cannot survive in nature without
their cell walls, some do so naturally. These include the
mycoplasmas, a group of pathogenic bacteria that cause
several infectious diseases of humans and other animals, and the
Thermoplasma group, species of Archaea that naturally lack cell
walls. These bacteria are able to survive without cell walls
because they either contain unusually tough cytoplasmic
membranes or because they live in osmotically protected
habitats such as the animal body. Most mycoplasmas have sterols
in their cytoplasmic membranes, and these probably function to
add strength and rigidity to the membrane as they do in the
cytoplasmic membranes of eukaryotic cells.
While some L-forms form spontaneously, others
are inducible. Since they lack cell wall, they don‟t
have a definite shape. L-forms resemble
mycoplasma in morphology and type of growth on
agar plates (“Fried-egg colony”), but while
mycoplasma completely lack cell wall and have
sterols in their membrane, the L-forms may have
reminiscent of cell wall but do not have sterols in
their membrane.
• The cell walls of certain methanogenic Archaea contain a
molecule that is remarkably similar to peptidoglycan, a
polysaccharide called pseudomurein.

• The backbone of pseudomurein is composed of alternating

repeats of N-acetylglucosamine (also found in peptidoglycan)
and N-acetyltalosaminuronic acid; the latter replaces the
Nacetylmuramic acid of peptidoglycan.

• 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.
The most common cell wall in species of Archaea is the
paracrystalline surface layer, or S-layer. S-layers consist of
interlocking protein or glycoprotein molecules that show an
ordered appearance when viewed with the electron microscope.

The paracrystalline structure of S-layers is arranged to yield

various symmetries, such as hexagonal, tetragonal, or
trimeric, depending upon the number and structure of the
protein or glycoprotein subunits of which they are composed.

S-layers have been found in almost all major groups of Archaea and
also in several species of Bacteria.
The cell walls of some Archaea, for example the methanogen
Methanocaldococcus jannaschii, consist only of an S-layer. Thus, S-
layers are themselves sufficiently strong to withstand osmotic
bursting. However, in many organisms S-layers are present in
addition to other cell wall components, usually polysaccharides.
However, when an S-layer is present along with other wall
components, the S-layer is always the outermost wall layer, the
layer that is in direct contact with the environment.

The S-layer functions as aselective sieve, allowing the passage of

low-molecular-weight solutes while excluding large molecules and
structures (such as viruses). The S-layer may also function to retain
proteins near the cell surface
• Certain microbes resist to take the stain used in gram process due
to the presence of waxy substances in their cell wall. The cell
wall of these bacteria have high lipid (60%), in particular mycolic
acid- a group of branched chain hydroxylipids, which prevent
dye from readily binding to the cell.

• Mycolic acids are localized in the inner leaflet of the cell wall
either covalently bound or loosely associated with
arabinogalactan polymers.They maintain a rigid cell shape.

• Mycolic acid is found in Mycobacterium tuberculosis,

Mycobacterium leprae, Nocardia sp. etc.
• The staining process of acid fast bacilli utilizes heat and
phenol to drive basic fuchsin into the cells. Once basic
fuchsin has penetrated the cell wall, they are not readily
decolourized by acid-alcohol, and thus said to be acid-fast.

• The technique was developed by Ziehl and later on modified

by Neelsen in 1883 and hence also called Ziehl-Neelsen
technique.

• When the smear is stained with CF, it stabilizes the lipoidal

material present in the cell wall but application of heat helps
further penetration of CF through the cell wall and enters
cytoplasm.
Procedure:
Bacterial smear is prepared in a clean grease free slide and then air dried and
heat-fixed

The slide is flooded with carbol fuchsin

Slide is placed on a water bath ( 60ºC), overheating is avoided. The slide is kept
for 5 min

Excess stain is washed off with water

3% (v/v) acid-alcohol is added to the slide, and kept for 5 min

The slide is then washed with water and then flooded with methylene
blue/malachite green and kept for 1-2 min

The slide is then dried in air and observed under microscope

Many prokaryotes secrete slimy or sticky materials
on their cell surface which may either consist of
polysaccharides or proteins. These are not
considered as part of the cell wall because they do
not confer significant structural strength on the cell.
Depending on their type and chemistry of these
layers, different names are given to them like
capsule, slime layer etc.
• Capsules are thick, rigid, well organized surface layer
that is not easily washed off. Capsules are organized in a
tight matrix that excludes small particles. Capsules
typically adhere firmly to the cell wall and some are even
covalently linked to peptidoglycan.

• Capsules are mostly composed of polysaccharides, but

some are constructed of other materials, e.g; Bacillus
anthracis has a proteinaceous capsule made up of poly-D-
glutamic acid.

• Capsules are easily visible under light microscope when

negative stains or specific capsule stains are used.
 Capsule of Acinetobacter
observed by phase-contrast
microscopy after negative
staning with India Ink

Capsule of Klebsiella pneumoniae

TEM of Rhizobium trifolii capsule
 1% crystal violet stain is applied to a non-heat fixed smear. At this point,
the cell and the capsular material will take up the dark color. As the capsule
is non-ionic, the primary stain adheres to the capsules without binding to it.
Since capsule is water soluble, so 20% copper sulphate, rather than water, is
used to wash the purple primary stain out of the capsular material without
removing the stain that is bound to the cell wall. At the same time it acts as a
counter stain as it is absorbed into the decolorized capsular material. The
capsule will now appear light blue in contrast to the deep purple color of the
cell.

Examples of capsule producing bacteria:

Klebsiella pneumoniae, Streptococcus
pneumoniae, Clostridium perfringens etc.
 Polysaccharide layers have several functions in bacteria. Surface
polysaccharides assist in the attachment of microorganisms to
solid surfaces. For example pathogenic microorganisms that enter
the animal body by specific routes usually do so by first binding
specifically to surface components of host tissues, and this binding is
often mediated by bacterial cell surface polysaccharides.

 Capsules help pathogenic bacteria resist phagocytosis by host

phagocytes.

 In addition, because outer polysaccharide layers bind a significant

amount of water, it is likely that these layers play some role in
resistance of the cell to desiccation.
• A Slime layer is a thin zone of diffuse, unorganized
material that can be deformed easily.

• Slime layers flexible and loosely attached. This

layer does not exclude particles and hence is not
easily observed by light microscope.

• It is usually composed of polysaccharides but is

not as easily observed by light microscopy.
 Slime layers help the microorganism to
adhere to solid surfaces.

 They help in biofilm formation.

 Gliding bacteria often produce slime,

which in some cases has been shown to
facilitate motility.
The term glycocalyx refers to a
layer consisting of a network of
polysaccharides extending from
the surface of the cell. The term can
encompass both capsules and slime
layers because they usually are
composed of polysaccharides. The
glycocalyx aids in attachment to solid
surfaces, including tissue surfaces in
plant and animal hosts.
Bacteria must constantly respond to their
changing environments. Sometimes, it is
advantageous to attach to a surface. Other
times, it is better to move toward or away from
something in the environment. Many bacteria
have structures that extend beyond the cell
envelope and are involved in either attachment
to surfaces or motility. In addition, these
external structures can function in protection
and horizontal gene transfer. Several are
discussed in this section.
Many bacteria have fine filamentous hair-like appendages
which are even thinner than flagella and extend from the
surface of a cell. These are usually called fimbriae (singular:
fimbria) or pili (singular: pilus).

Both are composed of proteins.

A cell can be covered by upto 1000 fimbriae but they can only
be seen under electron microscope.

Fimbriae are slender tubes composed of helically arranged

protein subunits. They are 3-10 nm in diameter and several
micrometers long. Fimbriae contain adhesins which attach
them to the substratum so that the bacteria can avoid shear
forces.
Bacteria which have
fimbriae:

Salmonella,
Neisseria gonorrhoeae,
E. coli

Functions:
• Fimbriae enable cells to stick to surfaces,
including animal tissues in case of pathogenic
bacteria.
• They help to form pelicles (thin sheets of cells
on a liquid surface) or biofilms on surfaces.
Pili are similar to fimbriae, but are typically longer and only
one or a few pili are present on the surface of a cell. They can
best be seen under the electron microscope.

Pili are made up of pilin protein.

Different types of pili are found in bacterial cells like type IV

pili, sex pili etc.

Functions:
• Sex pili facilitate genetic exchange through the process of conjugation.
• Help in the adhesion of pathogens to specific host tissues and invasion.
• Type IV pili assist cells in adhesion but also allow for an unusual form of
cell motility called twitching motility.
Bacteria which have fimbriae:
Streptococcus pyogenes,
Neisseria gonorrhoeae,
E. coli
Bacteria contain another most
important cell surface appendage,
called flagella (singular: flagellum).
The flagellar structure is complex
and they are thicker than fimbriae
and pili. Flagella are mainly the
locomotory structure of bacteria.
Flagella are long thin thread-like locomotor appendages
extending outward from the plasma membrane and cell
wall. Their one end is attched to the cell and the other
end is free.

Bacterial flagella are slender, rigid structures about 20

nm across and up to 20 μm long. Flagella are so thin
they cannot be observed directly with a bright-field
microscope but must be stained with techniques
designed to increase their thickness. The detailed
structure of a flagellum can only be seen in the electron
microscope.
 Bacterial species often differ in their patterns of flagella distribution,
and these patterns are useful in identifying bacteria.

Monotrichous bacteria
(‘trichous’ means hair) have one
flagellum; if it is located at an
end, it is said to be a polar
flagellum.

Example: Pseudomonas, Vibrio

cholerae
Amphitrichous bacteria
(‘amphi’ means at both ends)
have a single flagellum at both
ends.

Example: Lactobacillus
Lophotrichous bacteria
(‘lopho’ means ‘tuft’) have a
cluster of flagella at one or
both ends.

Example: Spirilla
Peritrichous bacteria
(‘peri’ means ‘around’) have
flagella spreaded all around
the cell surface.

Example: Proteus vulgaris

Flagella are not straight but helical.

The filament of a bacterial flagellum is composed of

many copies of a protein called flagellin. Flagellin is
highly conserved in amino acid sequences in species of
bacteria.

A flagellum is composed of several components and

moves by rotation, much like a propeller of a boat
motor.

Transmission electron microscope shows the

ultrastructure of flagella.
A flagellum is composed of three parts.

(1) Filament:- The longest and most obvious

portion which extends from the cell surface to the
tip.
(2) The basal body:- this is embedded in the cell
envelope; and
(3) Hook:- a short, curved segment, the hook,
links the filament to its basal body and acts as a
flexible coupling.
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.
The hook and basal body are quite different from the filament.
Slightly wider than the filament, the hook is made of different
protein subunits.

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 (attached to LPS layer), P (attached to
peptidoglycan layer), and MS (cytoplasmic membrane) rings
are embedded in the cell envelope, and the C (cytoplasm) 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.
There are many other proteins present in a
flagellum. Surrounding the inner ring and
anchored in the cytoplasmic membrane are a
series of proteins called Mot proteins. A final
set of proteins, called the Fli proteins,
function as the motor switch, reversing the
direction of rotation of the flagella in
response to intracellular signals.
• The flagellum is a tiny rotary motor.
Rotary motors contain two main
components: the rotor and the stator.
• In the flagellar motor, the rotor consists
of the central rod and the L, P, C, and MS
rings. Collectively, these structures make
up the basal body.
The stator consists of the Mot proteins
that surround the basal body and function
to generate torque.

• Rotation of the flagellum is imparted by the basal body. The energy required for
rotation of the flagellum comes from the proton motive force. Proton movement
across the cytoplasmic membrane through the Mot complex drives rotation of the
flagellum. Protons flowing through channels in the Mot proteins exert electrostatic
forces on helically arranged charges on the rotor proteins. Attractions between
positive and negative charges would then cause the basal body to rotate as protons
flow though the Mot proteins.
• 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 flagella,
counter-clockwise rotation results in a run. When rotation is reversed, the
cell tumbles.
 The movement of cells toward chemical attractants or away from chemical
repellents is called chemotaxis. Attractants and repellents are detected by
chemoreceptors, proteins that bind chemicals and transmit signals to other
components of the chemosensing system. The chemosensing systems are very
sensitive and allow the cell to respond to very low levels of attractants.

 The chemotactic behaviour of bacteria has been studied by tracking

microscope, which contains a moving stage that automatically keeps an individual
bacterium in view. In the absence of a chemical gradient, bacteria move
randomly, switching back and forth between a run and a tumble.
During a run, the bacterium swims in a straight or slightly curved line.
After a few seconds, the bacterium stops and tumbles. The tumble randomly
reorients the bacterium so that it often is facing in a different direction. Therefore
when it begins the next run, it usually goes in a different direction. In contrast,
when the bacterium is exposed to an attractant, it tumbles less frequently
(or has longer runs) when travelling toward the attractant.
 The plasma membrane and everything within is called the
protoplasm.

 The cytoplasm is the material bounded by the plasma

membrane and it constitutes the major part of the protoplast.

 For many years bacterial cells were thought of bags of water

in which structures like inclusions, ribosomes, nucleoid and
plasmids float. But the discovery of bacterial cytoskeletal
proteins developed the concept of bacterial cytoskeleton
which is much simpler than eukaryotic cytoskeleton and help
to organize the cytoplasm.
Eukaryotic cytoskeletal proteins include actin,
tubulin and many other classes of proteins.
Homologues of all three types of eukaryotic
proteins have been identified in bacteria. The
bacterial cytoskeletal proteins are structurally
similar to their eukaryotic counterparts and carry
out similar functions: they participate in cell
division, localize proteins to certain sites in the
cell, and determine cell shape. In addition, some
cytoskeletal proteins appear to be unique to
bacteria.
Cytoskeleton is best studied in:

E. coli
Bacillus subtilis and
Caulobacter crescentus
The best studied bacterial cytoskeletal proteins are FtsZ,
MreB, and CreS (also known as crescentin).

 FtsZ- a homologue of the eukaryotic protein tubulin. It

forms a ring at the center of a dividing cell and is
required for the formation of the septum that will
separate the daughter cells.
 MreB- an actin homologue. Their major function is to
determine cell shape in bacilli, they are not found in
cocci.
 CreS- is a homolog of lamin and keratin and is
responsible for the curved shape of C. crescentus.
Inclusions are very common in bacterial cells.
They are formed by aggregation of substances
that may either organic and inorganic. Inclusions
can take the form of granules, crystals or globules
and some time amorphous. Some inclusions are
free in the cytoplasm and some are enclosed by
single membranous layer. They perform varied
functions in the cell like storage compounds,
energy reserves etc.
Bacterial storage polymers include:

 Carbon storage polymers

 Polyphosphate and sulfur granules
 Cyanophycin
 Microcompartments
 Magnetic storage inclusion
 Gas vacuoles
Carbon storage polymers:
1. PHB:- Carbon is often stored as
polyhydroxyalkanoate (PHA) granules. Among
different types of PHAs most common is the
polyhydroxybutyrate/polyhydroxybutyric acid
(PHB). This is a lipid which is formed by β-
hydroxybutyrate units. The monomers of PHB are
bonded by ester linkage to form the polymer and then
the polymer aggregates into granules. PHB granules
are known to be surrounded by a single-layered shell
composed of proteins and a small amounts of
phospholipids. PHB can be stained with Sudan black.
PHB:- The monomer in the polymer
is not only hydroxybutyrate (C4) but
can vary in length from as short as
C3 to as long as C18. they function as
carbon and energy source.
2. Glycogen:- Another carbon storage polymer. It
is a polymer of glucose units composed of long
chains formed by α-(1→4) glycosidic bonds and
branching chains connected to them by α-(1→6)
glycosidic bonds. Glycogen is a storehouse of both
carbon and energy. Glycogen is produced when
carbon is in excess.
Polyphosphate and sulfur granules:
Polyphosphate granules:-
Many microorganisms
accumulate inorganic
phosphate (PO43-) in the form
of granules of polyphosphate.

Polyphosphate granules are also called volutin or

metachromatic granules. Metachromatic because
they sometimes show metachromatic effect, that is,
they appear red or different shades of blue when
stained with methylene blue or toluidine blue.
Polyphosphates:- These granules
can be degraded and used as sources
of phosphate for nucleic acid and
phospholipid biosyntheses and in
some organisms can be used to make
the energy-rich compound ATP.
Phosphate is often a limiting
nutrient in natural environments.
Thus if a cell happens upon an
excess of phosphate, it is
advantageous to be able to store it as
polyphosphate for future use.
Sulfur granules:- Many gram-negative prokaryotes can oxidize
reduced sulfur compounds, such as hydrogen sulfide (H2S). The
oxidation of sulfide is linked to either reactions of energy
metabolism (chemolithotrophy) or CO2 fixation (autotrophy). In
either case, elemental sulfur (S0) may accumulate in the cell in
microscopically visible globules.
However, as the reduced sulfur source
becomes limiting, the sulfur in the granules
is oxidized to sulfate (SO42-), and the
granules slowly disappear as this reaction
proceeds. Interestingly, although the sulfur
globules appear to be in the cytoplasm they
actually reside in the periplasm. The
periplasm expands outward to
accommodate the globules as H2S is
oxidized to S0 and then contracts inward as
S0 is oxidized to SO42-.
Cyanophycin granules:- Many cyanobacteria have two
distinctive organic inclusion bodies. Cyanophycin granules
(given as ‘pb’ in the figure) are composed of large
polypeptides containing approximately
equal amounts of the amino acids arginine and aspartic
acid.

The granules often are large enough to be visible in the

light microscope and store extra nitrogen for the bacteria.
Microcompartments
:
Some bacterial inclusions are unique and serve functions other
than simply storing substances for later use by the cell. These
inclusions are called microcompartments. Microcompartments
share several characteristics. They are relatively large
polyhedrons formed by one or more different proteins.
Enclosed within the protein shell are one or more enzymes.

Microcompartments include:
1. Ethanolamine utilization (Eut) microcompartment
2. Propandiol utilization (Pdu) microcompartment and
3. Carboxysomes
Carboxysomes:
Carboxysomes are present in many cyanobacteria and other C02-
fixing bacteria. Their polyhedral coat is composed of about six
different proteins and is about 100 nm in diameter. Enclosed by
the shell is the enzyme carbonic anhydrase, which converts
carbonic acid into C02.
Biological membranes allow the free diffusion of C02;
however, the carboxysome shell prevents C02 from
escaping so it can accumulate within. Also enclosed
within the polyhedron is the enzyme ribulose-1, 5-
bisphosphate carboxylase/oxygenase
(RubisCO). RubisCO is the critical enzyme for C02
fixation, the process of converting C02 into sugar.
Thus the carboxysome serves as a site for C02
fixation.
Magnetosomes:

Some bacteria can orient themselves specifically within a magnetic field

because they contain magnetosomes. These structures are
intracellular particles of the iron mineral magnetite-Fe3O4 or
greigite-Fe3S4 or pyrite-FeS2. These are about 35-125 nm in diameter
and are enclosed within invaginations of plasma membrane.
Magnetosomes impart a magnetic dipole on a cell, allowing it to
respond to a magnetic field. Each iron particle acts as a tiny magnet.
Bacteria that produce magnetosomes exhibit magnetotaxis, the process
of orienting and migrating along Earth‟s magnetic field. For the cell to
move properly within a magnetic field, magnetosomes must be arranged in
a chain. A cytoskeletal protein called MamK is currently thought to be
responsible for establishing a framework upon which the chain can form.
 Magnetosomes:
The alignment of magnetosomes in the cell
simply imparts a magnetic moment that
orients the cell in a particular direction in its
environment. Magnetosomes are surrounded
by a thin membrane containing
phospholipids, proteins, and glycoproteins.
This membrane is not a true unit (bilayer)
membrane and the proteins present play a role in
precipitating Fe3+. The major function of
magnetosomes is unknown. one function of
magnetosomes may be to guide these primarily
aquatic cells downward (the direction of Earth‟s
magnetic field) toward the sediments where
O2 level is low.
Gas vacuoles/vesicles:

Some prokaryotes are planktonic,

meaning that they live a floating existence
within the water column of lakes and the
oceans. These organisms can float because
they contain gas vesicles.
The most dramatic examples of gas-
vesiculate bacteria are cyanobacteria
that form massive accumulations called
blooms in lakes or other bodies of
water. Many primarily aquatic bacteria
have gas vesicles and the property is
found in both Bacteria and Archaea.
Gas
vacuoles/vesicles:

Gas vesicles are spindle-shaped structures made of protein; they

are hollow yet rigid and of variable length and diameter. Gas
vesicles in different organisms vary in length from about 300 to
more than 1000 nm and in width from 45 to 120 nm. Gas vesicles
may number from a few to hundreds per cell and are impermeable
to water and solutes but permeable to gases. Clusters of
vesicles, called gas vacuoles, appear as irregular bright
inclusions in TEM.
These structures confer buoyancy on cells, allowing them to
position themselves in a water column in response to
environmental cues.
Molecular Structure of Gas vacuoles/vesicles:
Bacteria possessing gas vacuoles can regulate their buoyancy to float at the
depth necessary for proper light intensity, oxygen concentration, and nutrient
levels. They descend by simply collapsing gas vesicles and further float upward
when new gas vesicles are formed and join them.

Two different proteins, GvpA and GvpC,

compose the gas vesicle wall. GvpA
composes 97% of total gas vesicle protein
and is the major gas vesicle protein. It is a
small highly hydrophobic and very rigid
protein. The rigidity of the gas vesicle wall
is essential for the structure to resist the
pressures exerted on it from outside.
GvpC, the protein in minor amount of
3%, functions to strengthen the wall of the
gas vesicle.
Ribosomes are the site of protein synthesis, and large
numbers of them are found in nearly all cells. The cytoplasm of
bacterial cells is often packed with ribosomes, and other
ribosomes may be loosely attached to the plasma membrane.
The cytoplasmic ribosomes synthesize proteins destined to
remain within the cell, whereas plasma membrane-associated
ribosomes make proteins that will reside in the cell envelope or
are transported to the outside. Translation, the process of
protein synthesis, is amazingly complex and is evidenced in part
by the structure of ribosomes, which are made of numerous
proteins and several ribonucleic acid (RNA) molecules.
Bacterial ribosomes are called 70S ribosomes and are
constructed of a 50S and a 30S subunit. The S in 70S and similar
values stands for Svedberg unit. This is the unit of the
sedimentation coefficient, a measure of sedimentation
velocity in a centrifuge.

Bacterial ribosomes are composed primarily of ribosomal RNA

(rRNA) molecules.
The small subunit contains 16S rRNA;
23S and 5S rRNA molecules are in the large subunit.
Approximately 55 proteins make up the rest of the mass of the
ribosome: 21 in the small subunit, and 34 in the large subunit.
The nucleoid is an irregularly shaped region that contains the cell's chromosome
and numerous proteins. The chromosomes of most bacteria are a single circle
of doublestranded deoxyribonucleic acid (DNA), but some bacteria
have a linear chromosome. E. coli's circular chromosome measures
approximately 1,400 μm, or about 230-700 times longer than the cell. Thus the
chromosome must be organized and packaged in a manner that decreases its
overall size. Supercoiling is thought to be important. It produces a dense,
central core of DNA with loops of DNA extending out from the core.
Several nucleoid-associated proteins (NAPs) cause the chromosome to
bend and fold, thereby also helping to pack the DNA into a smaller space. One
NAP found in many bacteria is the protein HU. During cell division, bacterial
chromosomes are further compacted by proteins called condensins. This extra
level of packing is important for proper segregation of daughter chromosomes
during cell division. For most bacteria, the nucleoid is simply a region in the
cytoplasm; it is not separated from other components of the cytoplasm by a
membrane. However, there are a few exceptions.
Many bacteria contain additional DNA molecules other than
nucleoid which are called plasmids. Plasmids are small,
circular, double-stranded DNA molecules that can exist
independently of the chromosome and provides
extrachromosomal inheritance property to the bacteria.
Both circular and linear plasmids have been documented, but most
known plasmids are circular. Plasmids have relatively few genes.
Their genetic information is not essential to the bacterium,
and cells that lack them usually function normally. However, many
plasmids carry genes that confer a selective advantage to the
bacterium in certain environments.
Plasmids use the cell's DNA-synthesizing machinery to replicate,
but their replication is independent of chromosomal DNA
replication.
Episome: Some plasmids are able to integrate into the
chromosome. Such plasmids are called episomes and when
integrated are replicated as part of the chromosome.
Plasmid curing: Plasmids are inherited stably during cell
division, but they are not always equally distributed into daughter
cells and sometimes are lost.The loss of a plasmid from the cell is
called curing. It can occur spontaneously or be induced by
treatments. Some commonly used curing treatments are
acridine mutagens, ultraviolet and ionizing radiation,
thymine starvation, antibiotics, and growth above
optimal temperatures.
Certain species of Bacteria produce special, resistant,
dormant structures called endospores during process
called sporulation. Endospores (the prefix endo
means “within”) are highly differentiated cells that are
extremely resistant to heat, harsh chemicals, and
radiation. Endospores function as survival structures
and enable the organism to withstand unfavorable growth
conditions, including extremes of temperature, drying, or
nutrient depletion and many more. Endospores can thus
be thought of as the dormant stage of a bacterial life
cycle.
Endospore forming bacteria:
Bacillus subtilis, Bacillus anthracis
Clostridium tetani, Clostridium botulinum
Sporosarcina
• Endospore is structurally quite complex and doesn‟t resemble
with the structure of vegetative cell. Endospore structure can be
studied with the help of electron microscope.

• Endospores can also be seen by light microscopy by using

special dyes as the spores are impermeable to most dyes.

• Most commonly malachite green is used for staining spore

and the dye is infused into the spore by steam. Safranine is
used as a counter stain. Spores take green colour (malachite
green) and the vegetative cells remain red (safranine).
As revealed in electron microscopy an endospore contains the following
parts:
 Exosporium: The outermost layer of an endospore. It is a thin
delicate covering.
 Spore coat: The layer next to exosporium. It is composed of 50
different types of spore specific proteins which are highly cross-linked
to each other.
 Cortex: Below the spore coat is the cortex. Cortex may occupy half
of the spore volume. It contains less cross-linked peptidoglycan. The
cortex is surrounded by a phospholipid bilayer called the outer
membrane.
 Core: Inside the cortex there is the core. Core is surrounded by an
inner membrane which in turn is covered by a core wall. Core contains
cytoplasm, nucleoid, ribosomes, and other cellular essentials. The core
has very low water content.
The ability of the spore to survive heat, radiation, and damaging chemicals
requires that its enzymes and DNA be protected. The various layers of the
spore contribute to this resistance in several ways. The spore coat protects
the spore from chemicals and various lytic enzymes such as
lysozyme. The inner membrane is extremely impermeable to various
chemicals, including those that cause DNA damage. The core has very low
water content, high amounts of dipicolinic acid (DPA) complexed with
calcium ions (Ca2+-DPA), and a slightly lower pH, all of which contribute
to the spore‟s resistance to harsh conditions. The water content of the core
is low enough and the consistency of the core is like a gel. Dehydration
greatly increases the heat resistance of the macromolecules. The spore's DNA
is protected by two main mechanisms: Ca2+-DPA complexes bind to free
water molecules and thus helping in dehydration, these complexes
intercalate between the nitrogenous bases of DNA, which helps to stabilize
it. The DNA is further stabilized by small, acid-soluble DNA-binding
proteins (SASPs), which saturate spore DNA.
SASPs:
The endospore core contains high levels of small acid-soluble
proteins (SASPs). These proteins are made during the
sporulation process and have at least two functions. SASPs
bind tightly to DNA in the core and protect it from potential
damage from ultraviolet radiation, desiccation, and dry heat.
Ultraviolet resistance is conferred when SASPs change the
molecular structure of DNA from the normal “B” form to the
more compact “A” form. A-form DNA better resists
pyrimidine dimer formation by UV radiation and resists the
denaturing effects of 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.
The mature endospore is located in a characteristic location in the mother cell
(referred to as the sporangium), depending on the species of bacteria.
Endospores may be centrally located, close to one end (subterminal), or
terminal. Sometimes an endospore is so large that it swells the sporangium.
Sporulation may be divided into seven stages.

The cell's DNA is replicated (stage I), followed by an inward

folding of the cell membrane to enclose part of the DNA and
produce the forespore septum (stage II). The mother cell
membrane continues to grow and engulfs the immature
endospore in a second membrane (stage III). Next, cortex is
laid down in the space between the two membranes, and both
calcium and dipicolinic acid are accumulated (stage IV).
Protein coats are formed around the cortex (stage V), and
maturation of the endospore occurs (stage VI). Finally,
lytic enzymes destroy the sporangium, releasing the spore
(stageVII).
The transformation of dormant spores into active vegetative cells is
almost as complex as sporulation. It occurs in three stages: (1)
activation, (2) germination, and (3) outgrowth. Activation is a
process that prepares spores for germination and can result from
treatments such as heating. This is followed by germination, the breaking
of the spore„s dormant state. It begins when proteins called germinant
receptors, located in the inner membrane, detect small molecules such as
sugars and amino acids. Upon detection of these molecules, a series of
events occur, including release of the Ca-DPA complexes, breakdown of
the peptidoglycan in the cortex, and water uptake. Eventually water levels
inside the germinating spore reach those characteristic of vegetative cells
and enzymes in the core become active. This allows the spore to begin
synthesizing the various molecules it needs to initiate spore outgrowth
and return to a vegetative state.