8

 Among major characteristics of bacterial cells are their:

 Size
 Shape
 Structure and
  Arrangement

 These characteristics constitute the Morphology of the cell.

 Depending on the species, individual cells are:

 Spherical
 Rod like, or
 Helical

 Although many variations of these three basic shapes occur.

 In certain species of bacteria the cells are arranged in groups, the most common
of which are:

 Pairs
 Clusters
 Chains
 Trichomes and
 Filaments

 It is important to recognize these patterns of shape and arrangement, since they

are often characteristic of a taxonomic group, e.g. a genus.

 Some bacteria also possess appendages, which can be made visible by:

 Special staining techniques or by

 Electron microscopy.

 All these morphological features are regarded as the gross morphological

characteristics of bacterial cells.

 The bacterial cell possesses a detailed internal structure.

 The discovery of this internal structure was made possible by the development
of electron-microscope techniques and of instruments for slicing a bacterial cell
into extremely thin sections.
 Various structures of a bacterial cell differ from one another not only in their
physical features but also in their chemical characteristics and in their functions.

 The biologists today seek to integrate the structural, chemical, and functional
properties of a bacterial cell.

 This area of research studied by the bacteriologist is referred as biochemical

cytology.

SIZE, SHAPE AND ARRANGEMENT OF BACTERIAL CELLS

Size: Bacteria are very small, most being app. 0.3-1.0 µm in diameter.

 An important consequence of the small size of m/os is that the surface area
/volume ratio of bacteria is exceedingly high as a compared to the same ratio
for larger organisms of similar shape.

 Unusually high rate of growth and metabolism

 No circulatory mechanism needed to distribute the nutrients that
are taken
 So, no cytoplasmic movement within a bacterial cell
  Despite these advantages, a high surface area /volume ratio limits the size of
bacteria to microscopic dimensions.

Biggest Bacterium:

 Epulopiscium fishelsoni, a huge bacterium lives in intestine of brown surgeon fish,

Acanthurus nigrofuscus grows as large as 600x80 μm.

 More recently, even larger bacterium, Thiomargarita namibiensis discovered in ocean

sediment.
Arrangement:
Pasteuria: Pear shaped

Sulpholobus: Lobed spheres

Bacillus anthracis: Rods with square ends.

Caryophanon: Disks arranged like stacks of coins.

Seliberia: Rods with sculptured surface, and

Many others
 Morphology of a Procaryotic Cell.

Structures External To The Cell Wall:

Flagella and motility:
 Flagella (singular, flagellum) are hairlike, helical appendages that protrude through
the cell wall and are responsible for swimming motility.

They are much thinner than the flagella and cilia of eucaryotes, being 0.01-0.02
µm in diameter, and up to 15 or 20 µm long and are also much simpler in structure.

 Their location on cell varies depending on the bacterial species and may be:

 Monotrichous: a single polar flagellum (Fig. A)

 Lophotrichous: a cluster of polar flagella (Fig. B)
 Amphitrichous: flagellas, either single or clusters , at both cell poles (Fig.
C)
 Peritrichous: surrounded by lateral flagella (Fig. D)

 Structure of Flagellum: composed of three parts (Fig. 5-12)

 Basal body :associated with cytoplasmic membrane
 Hook: short and
 Helical filament: usually several times as long as the cell

 Some Gram-negative bacteria have a sheath surrounding the flagellum: this

sheath is in continuation with the outer membrane of the Gram-negative cell
wall.
 The chemical composition of the

 basal body is unknown, but

 the hook and filament are composed of protein subunits (monomers)
arranged in a helical fashion.
 The protein of filament is known as flagellin.

 Unlike hair, a flagellum grows at its tip rather than at the base.

 Flagellin monomers synthesized within the cell are believed to pass along the
hollow centre of the flagellum and are added to the distal end of the filament.

Flagellar Ultrastructure:

 Transmission electron microscope studies have shown that the bacterial

flagellum is composed of three parts.

 The longest and most obvious portion is the flagellar filament, which
extends from the cell surface to the tip.

 A basal body is embedded in the cell; and

 a short, curved segment, the flagellar 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 weight 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,

Bdellovibrio has a membranous structure surrounding the filament.

 Vibrio cholerae has a lipopolysaccharide sheath.

 The hook and basal body are quite different from the filament (figure 3.39).

 Slightly wider than the filament, the hook is made of different protein subunits.

 The basal body is the most complex part of a flagellum.

 In E. coli and most Gram-negative bacteria, the basal body has four rings
connected to a central rod (figure 3.39a,d).

 The outer L and P rings associate with the lipopolysaccharide and peptidoglycan layers,
respectively.

 The inner M ring contacts the plasma membrane.

 Gram-positive bacteria have only two basal body rings—

 an inner ring connected to the plasma membrane and

 an outer one probably attached to the peptidoglycan (figure 3.39b).

Flagellar Synthesis

 The synthesis of bacterial flagella is a complex process involving at least 20 to 30 genes.

 Besides the gene for flagellin, 10 or more genes code for hook and basal body proteins;
other genes are concerned with the control of flagellar construction or function.

 How the cell regulates or determines the exact location of flagella is not known.

 When flagella are removed, the regeneration of the flagellar filament can then be studied.

 Transport of many flagellar components is carried out by an apparatus in the basal body that
is a specialized type III protein secretion system.

 It is thought that flagellin subunits are transported through the filament’s hollow internal
core.

 When they reach the tip, the subunits spontaneously aggregate under the direction of a
special filament cap so that the filament grows at its tip rather than at the base (figure 3.40).

 Filament synthesis is an excellent example of self assembly.

 Many structures form spontaneously through the association of their component parts
without the aid of any special enzymes or other factors. The information required for
filament construction is present in the structure of the flagellin subunit itself.

The Mechanism of Flagellar Movement:

 Procaryotic flagella operate differently from eucaryotic flagella.

 The filament is in the shape of a rigid helix, and the cell moves when this helix rotates.
 Considerable evidence shows that flagella act just like propellers on a boat.

 Bacterial mutants with straight flagella or abnormally long hook regions cannot swim.

 When bacteria are tethered to a glass slide using antibodies to filament or hook proteins, the
cell body rotates rapidly about the stationary flagellum.

 If polystyrene-latex beads are attached to flagella, the beads spin about the flagellar axis due
to flagellar rotation.

 The flagellar motor can rotate very rapidly.

 The E. coli motor rotates 270 revolutions per second;

 Vibrio alginolyticus averages 1,100 rps.

 The direction of flagellar rotation determines the nature of bacterial movement.

 Monotrichous, polar flagella rotate counterclockwise (when viewed from outside the cell)
during normal forward movement, whereas the cell itself rotates slowly clockwise.

 The rotating helical flagellar filament thrusts the cell forward in a run with the flagellum
trailing behind (figure 3.41).

 Monotrichous bacteria stop and tumble randomly by reversing the direction of flagellar
rotation.

 Peritrichously flagellated bacteria operate in a somewhat similar way.

 To move forward, the flagella rotate counterclockwise.

 As they do so, they bend at their hooks to form a rotating bundle that propels the cell
forward.

 Clockwise rotation of the flagella disrupts the bundle and the cell tumbles.

 Because bacteria swim through rotation of their rigid flagella, there must be some sort of
motor at the base.

 A rod extends from the hook and ends in the M ring, which can rotate freely in the plasma
membrane (figure 3.42).

 It is thought that the S ring is attached to the cell wall in Gram-positive cells and does not
rotate.

 The P and L rings of Gram-negative bacteria would act as bearings for the rotating rod.

 There is some evidence that the basal body is a passive structure and rotates within a
membrane-embedded protein complex much like the rotor of an electrical motor turns in the
center of a ring of electromagnets (the stator).

 The exact mechanism that drives basal body rotation is not entirely clear.

 Figure 3.42 provides a more detailed depiction of the basal body in Gram-negative bacteria.

 The rotor portion of the motor seems to be made primarily of a rod, the M ring, and a C ring
joined to it on the cytoplasmic side of the basal body.
 These two rings are made of several proteins; FliG is particularly important in generating
flagellar rotation.

 The two most important proteins in the stator part of the motor are MotA and MotB.

 These form a proton channel through the plasma membrane, and MotB also anchors the Mot
complex to cell wall peptidoglycan.

 There is some evidence that MotA and FliG directly interact during flagellar rotation.

 This rotation is driven by proton or sodium gradients in procaryotes, not directly by ATP as
is the case with eucaryotic flagella.

 The flagellum is a very effective swimming device.

 From the bacterium’s point of view, swimming is quite a task because the surrounding water
seems as thick and viscous as molasses.

 The cell must bore through the water with its corkscrew-shaped flagella, and if flagellar
activity ceases, it stops almost instantly.

 Despite such environmental resistance to movement, bacteria can swim from 20 to almost
90 µm/second.

 This is equivalent to travelling from 2 to over 100 cell lengths per second.

 In contrast, an exceptionally fast 6-ft human might be able to run around 5 body lengths per
second.

 Bacteria can move by mechanisms other than flagellar rotation.

 Spirochetes are helical bacteria that travel through viscous substances such as mucus or mud
by flexing and spinning movements caused by a special axial filament composed of
periplasmic flagella.

 The swimming motility of the helical bacterium Spiroplasma is accomplished by the

formation of kinks in the cell body that travel the length of the bacterium.

 A very different type of motility, gliding motility, is employed by many bacteria:

cyanobacteria, myxobacteria and cytophagas, and some mycoplasmas.

 Although there are no visible external structures associated with gliding motility, it enables
movement along solid surfaces at rates up to 3 µm/second. This kind of movement is
comparatively slow.
Chemotaxis:

 Bacteria do not always move aimlessly but are:

 Attracted by such nutrients as sugars and amino acids, and are

 Repelled by many harmful substances and bacterial waste products.

 Bacteria also can respond to other environmental cues such as temperature (thermotaxis),
light (phototaxis), oxygen (aerotaxis), osmotic pressure (osmotaxis), and gravity.

 Movement toward chemical attractants and away from repellents is known as chemotaxis.

 Such behavior is of obvious advantage to bacteria.

 Chemotaxis may be demonstrated by observing bacteria in the chemical gradient produced

when a thin capillary tube is filled with an attractant and lowered into a bacterial suspension.

 As the attractant diffuses from the end of the capillary, bacteria collect and swim up the
tube. The number of bacteria within the capillary after a short length of time reflects the
strength of attraction and rate of chemotaxis.

 Positive and negative chemotaxis also can be studied with petri dish cultures (figure 3.43).
 If bacteria are placed in the center of a dish of semisolid agar containing an attractant, the
bacteria will exhaust the local supply and then swim outward following the attractant
gradient they have created.

 The result is an expanding ring of bacteria.

 When a disk of repellent is placed in a Petri dish of semisolid agar and bacteria, the bacteria
will swim away from the repellent, creating a clear zone around the disk (figure 3.44).

 Bacteria can respond to very low levels of attractants (about 10-8 µM for some sugars), the
magnitude of their response increasing with attractant concentration.

 Usually they sense repellents only at higher concentrations.

 If an attractant and a repellent are present together, the bacterium will compare both signals
and respond to the chemical with the most effective concentration.

 Attractants and repellents are detected by chemoreceptors, special proteins that bind
chemicals and transmit signals to the other components of the chemosensing system.

 About 20 attractant chemoreceptors and 10 chemoreceptors for repellents have been

discovered thus far.

 These chemoreceptor proteins may be located in the periplasmic space or the plasma
membrane.

 Some receptors participate in the initial stages of sugar transport into the cell.
 The chemotactic behavior of bacteria has been studied using the tracking microscope, a
microscope with a moving stage that automatically keeps an individual bacterium in view.

 In the absence of a chemical gradient, E. coli and other bacteria move randomly.

 For a few seconds, the bacterium will travel in a straight or slightly curved line called a run.

 When a bacterium is running, its flagella are organized into a coordinated, corkscrew-shaped
bundle (figure 3.41c).

 Then the flagella “fly apart” and the bacterium will stop and tumble. The tumble results in
the random reorientation of 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 (figure
3.45a).

 In contrast, when the bacterium is exposed to an attractant, it tumbles less frequently (or has
longer runs) when travelling towards the attractant.

 Although the tumbles can still orient the bacterium away from the attractant, over time, the
bacterium gets closer and closer to the attractant (figure 3.45b).

 The opposite response occurs with a repellent. Tumbling frequency decreases (the run time
lengthens) when the bacterium moves away from the repellent.

 Clearly, the bacterium must have some mechanism for sensing that it is getting closer to the
attractant (or is moving away from the repellent).
 The behaviour of the bacterium is shaped by temporal changes in chemical concentration.

 The bacterium moves toward the attractant because it senses that the concentration of the
attractant is increasing.

 Likewise, it moves away from a repellent because it senses that the concentration of the
repellent is decreasing.

 The bacterium’s chemoreceptors play a critical role in this process.

 The molecular events that enable bacterial cells to sense a chemical gradient and respond
appropriately are presented in chapter 8.

Pili and Fimbriae:

 Many procaryotes have short, fine, hairlike appendages that are thinner than flagella.

 These are usually called fimbriae (s., fimbria).

 Although many people use the terms fimbriae and pili interchangeably, we shall
distinguish between fimbriae and sex pili.

 A cell may be covered with up to 1,000 fimbriae, but they are only visible in an electron
microscope due to their small size (figure 3.37).

 They are slender tubes composed of helically arranged protein subunits and are about 3 to
10 nm in diameter and up to several micrometers long.
 Functions of Fimbriae:

 At least some types of fimbriae attach bacteria to solid surfaces such as rocks in streams
and host tissues.

 Fimbriae are responsible for more than attachment.

 Type IV fimbriae are present at one or both poles of bacterial cells.

 They can aid in attachment to objects, and also

 are required for the twitching motility that occurs in some bacteria such as P.
aeruginosa, Neisseria gonorrhoeae, and some strains of E. coli.

 Movement is by short, intermittent jerky motions of up to several micrometers in

length and normally is seen on very moist surfaces.

 There is evidence that the fimbriae actively retract to move these bacteria.

 Type IV fimbriae are also involved in gliding motility by myxobacteria.

 These bacteria are also of interest because they have complex life cycles that
include the formation of a fruiting body.

 Many bacteria have about 1-10 sex pili (s., pilus) per cell.

 These are hairlike structures that differ from fimbriae in the following ways.

 Pili often are larger than fimbriae (around 9 to 10 nm in diameter).

 They are genetically determined by conjugative plasmids and are required for
conjugation.

 Some bacterial viruses attach specifically to receptors on sex pili at the start of
their reproductive cycle.

 Some pili play a major role in human infection by allowing pathogenic bacteria
to attach to epithelial cells lining the respiratory, intestinal, or genitourinary
tracts.

 This attachment prevents the bacteria from being washed away by the flow of
mucous or body fluids and permits the infection to be established.
  They are thinner, shorter, and more numerous than flagella (Fig. 5-15). They do
not function in motility, since they are found on nonmotile as well as motile
species.

Capsules, Slime Layers, and S-Layers

 Some procaryotes have a layer of material lying outside the cell wall. This layer has
different names depending on its characteristics.

 When the layer is well organized and not easily washed off, it is called a capsule
(figure 3.34a).

 It is called a slime layer when it is a zone of diffuse, unorganized material that is

removed easily.

 When the layer consists of a network of polysaccharides extending from the surface
of the cell, it is referred to as the glycocalyx (figure 3.34b), a term that can encompass
both capsules and slime layers because they usually are composed of polysaccharides.

 Most of bacterial capsules are composed of polysaccharides.

 Capsules composed of a single kind of sugar are termed homopolysaccharides; are

usually synthesized outside the cell from disaccharides by exocellular enzymes.

 The synthesis of glucan (a polymer of glucose) from sucrose by Streptococcus mutans is

an example.

 Other capsules are composed of several kinds of sugars and are termed
heteropolysaccharides; these are usually synthesized from sugar precursors that are
activated (energized) within the cell, attached to a lipid carrier molecule, transported
across the cytoplasmic membrane, and polymerized outside the cell. The capsule of
Klebsiella pneumoniae is an example.

 However, some slime layers and capsules are constructed of other materials.

 For example, Bacillus anthracis has a proteinaceous capsule composed of poly-D-

glutamic acid. This peptide is an unusual one because the glutamic acid is the rare D-
optical isomer rather than the usual L-isomer commonly found in nature.
  Capsules are clearly visible in the light microscope when negative stains or special
capsule stains are employed (figure 3.34a); they also can be studied with the electron
microscope (figure 3.34b).

Figure 3.34 Bacterial Capsules. (a) Klebsiella pneumoniae with its capsule stained for
observation in the light microscope (_1,500). (b) Bacteroides glycocalyx (gly), TEM (_71,250).

Functions of capsule:

 Although capsules are not required for growth and reproduction in laboratory cultures,
they do confer several advantages when procaryotes grow in their normal habitats.

 They help pathogenic bacteria resist phagocytosis by host phagocytes. Streptococcus

pneumoniae provides a dramatic example. When it lacks a capsule, it is destroyed easily
and does not cause disease, whereas the capsulated variant quickly kills mice.

 Capsules contain a great deal of water and can protect against desiccation.

 They exclude viruses and most hydrophobic toxic materials such as detergents.

 They may promote attachment of bacteria to surfaces; for example, Sreptococcus mutans,
a bacterium associated with producing dental caries, firmly adheres to the smooth
surfaces of teeth because of its secretions of a water-insoluble capsular glucan.

 If capsules are composed of compounds having an electrical charge, such as sugar-uronic

acids, they may promote the stability of bacterial suspension by preventing the cells from
aggregating and settling out, because cells bearing similarly charged surfaces tend to
repel one another.

 The glycocalyx also aids in attachment to solid surfaces, including tissue surfaces in plant
and animal hosts (figure 3.35).
  Gliding bacteria often produce slime, which in some cases, has been shown to facilitate
motility.

S-layer:

 Many procaryotes have a regularly structured layer called an S layer on their surface.

 In bacteria, the S-layer is external to the cell wall.

 In archaea, the S-layer may be the only wall structure outside the plasma membrane.

 The S-layer has a pattern something like floor tiles and is composed of protein or
glycoprotein (figure 3.36).
 In Gram-negative bacteria: the S-layer adheres directly to the outer membrane;

 In Gram positive bacteria: is associated with the peptidoglycan surface.

 It may protect the cell against:

 ion and pH fluctuations,

 osmotic stress,
 enzymes, or
 the predacious bacterium Bdellovibrio.

 The S-layer also helps maintain the shape and envelope rigidity of some cells.

 It can promote cell adhesion to surfaces.

 Finally, the S-layer seems to protect some bacterial pathogens against host defenses, thus
contributing to their virulence.

 By light microscopy, capsules appear to be amorphous gelatinous areas surrounding a

cell (Fig. 5-16A): however, special techniques designed to preserve delicate structures for
observation by electron microscopy have revealed that capsules consist of mesh or
network of fine strands (Fig. 5-16B).

 In many instances capsular material is not highly water-soluble and therefore does not
readily diffuse away from the cells that produce it. In other instances
 Sheaths: Some species of bacteria, particularly those from freshwater and marine
environments, form chains or trichomes that are enclosed by a hollow tube called a
sheath.

 This structure is most readily visible when some of the cells have migrated from it. (Fig.
5-17).

 Sheaths may sometimes become impregnated with ferric or manganese hydroxides,

which strengthen them.


 

 Some prosthecate bacteria may form a new bud at the end of a prostheca.

 Others have an adhesive substance at the end of a prostheca that aids in attachment to the
surfaces.

Stalks:

The cell wall

 The cell wall is the layer, usually fairly rigid, that lies just outside the plasma membrane.

 It is one of the most important prokaryotic structures for several reasons:

  it helps determine the shape of the cell
 it helps protect the cell from osmotic lysis;
 it can protect the cell from toxic substances; and
 in pathogens, it can contribute to pathogenicity.

 The importance of the cell wall is reflected in the fact that relatively few procaryotes lack
cell walls.

 Those that do have other features that fulfil cell wall function.

 The prokaryotic cell wall also is the site of action of several antibiotics.

 Therefore, it is important to understand its structure.

 The cell walls of Bacteria and Archaea are distinctive and are another example of the
important features distinguishing these organisms.

Overview of Bacterial Cell Wall Structure

 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-
stain procedure (see table 19.9).

 Gram-positive bacteria stained purple, whereas Gram-negative bacteria were colored pink
or red by the technique.

 The true structural difference between these two groups did not become clear until the
advent of the transmission electron microscope.

 The Gram-positive cell wall consists of a single 20 to 80 nm thick homogeneous layer of

peptidoglycan (murein) lying outside the plasma membrane (figure 3.17).
Figure 3.17 Gram-Positive and Gram-Negative Cell Walls. The Gram-positive envelope is from
Bacillus licheniformis (left), and the Gram-negative micrograph is of Aquaspirillum serpens (right). M;
peptidoglycan or murein layer; OM, outer membrane; PM, plasma membrane; P, periplasmic space;W,
gram-positive peptidoglycan wall.

 In contrast, the Gram-negative cell wall is quite complex. It has:

 a 2 to 7 nm peptidoglycan layer covered by

 a 7 to 8 nm thick outer membrane.

 The walls of Gram-negative archaebacteria are also thinner than those of Gram-positive
archaebacteria.

 Since the chemical composition of the walls of archaebacteria is quite different from that
of eubacteria, wall thickness rather than chemical composition may be the major factor in
the Gram reaction.

 The wall constitutes a significant portion of the dry weight of the cell: depending on the
species and culture conditions.

 It may account for as much as 10-40 %.

 Bacterial cell walls are usually essential for bacterial growth and division.

 Cells whose walls have been completely removed (i.e., protoplasts) are incapable of
normal growth and division.

 Because of the thicker peptidoglycan layer, the walls of Gram-positive cells are more
resistant to osmotic pressure than those of Gram-negative bacteria.

 Microbiologists often call all the structures from the plasma membrane outward the cell
envelope.
  Therefore this includes the plasma membrane, cell wall, and structures like capsules (p.
65) when present.

 One important feature of the cell envelope is a space that is frequently seen between the
plasma membrane and the outer membrane in electron micrographs of Gram-negative
bacteria, and is sometimes observed between the plasma membrane and the wall in
Gram-positive bacteria. This space is called the periplasmic space.

 The substance that occupies the periplasmic space is the periplasm.

 The nature of the periplasmic space and periplasm differs in Gram-positive and Gram-
negative bacteria.

 These differences are pointed out in the more detailed discussions that follow.

 Their walls are usually composed of :

 Proteins,
 Glycoproteins, or
 Polysaccharides.
 A few genera, such as Methanobacterium, have walls composed of pseudomurein, a
polymer whose structure superficially resembles eubacterial peptidoglycan but which
differs markedly in chemical composition

Walls of Gram-Postive Eubacteria:

 Gram-positive bacteria usually have much greater amount of peptidoglycan in their cell
walls than do Gram-negative bacteria; It may account for:

50% or more of the dry weight of the cell wall of some Gram-positive species, but

only 10% of the wall of Gram-negative bacteria.

Structure and Chemical Composition:

 For eubacteria, the shape determining part of the cell wall is largely peptidoglycan
(sometimes called murein), an insoluble, porous, cross-linked polymer of enormous
strength and rigidity.

 Peptidoglycan is found only in prokaryotes; it occurs in the form of a “bag-shaped”

macromolecule surrounding the cytoplasmic membrane.
  Peptidoglycan, or murein, is an enormous meshlike polymer composed of many identical
subunits.

Peptidoglycan Structure:

 Peptidoglycan differs somewhat in composition and structure from one species to

another, but it is basically a polymer which contains:

 two sugar derivatives, N-acetylglucosamine and N-acetylmuramic acid (the

lactyl ether of N-acetylglucosamine),

 and several different amino acids i.e

L-alanine, D-glutamate, D-alanine and

a diamino acid (LL- or meso- diaminopimelic acid, L-lysine, L-

ornithine, or L-diaminobytyric acid).

 Three of these 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.

 The peptidoglycan subunit present in most Gram-negative bacteria and many Gram
positive ones is shown in figure 3.18.
 The backbone of this polymer is composed of alternating N-acetylglucosamine and N-
acetylmuramic acid residues.

 A peptide chain of four alternating D- and L-amino acids is connected to the carboxyl
group of N-acetylmuramic acid.

 Many bacteria replace meso-diaminopimelic acid with another diamino acid, usually L-
lysine (figure 3.19).

 In order to make a strong, meshlike polymer, chains of linked peptidoglycan subunits

must be joined by cross-links between the peptides.

 Often the carboxyl group of the terminal D-alanine is connected directly to the amino
group of diaminopimelic acid, but a peptide interbridge may be used instead (figure
3.20).
 Most Gram-negative cell wall peptidoglycan lacks the peptide interbridge.

 This cross-linking results in an enormous peptidoglycan sac that is actually one dense,
interconnected network (figure 3.21).

 These sacs have been isolated from Gram-positive bacteria and are strong enough to
retain their shape and integrity (figure 3.22), yet they are relatively porous, elastic, and
somewhat stretchable.


 

Gram-Positive Cell Walls

 Gram-positive bacteria normally have cell walls that are thick and composed primarily of
peptidoglycan.

 It may account for 50% or more of the dry weight of the wall of some Gram- positive
species, but only about 10% of the wall of Gram-negative bacteria.

 Peptidoglycan in Gram positive bacteria often contains a peptide interbridge (figure

3.21 and figure 3.23).

 Other substances may occur in addition to peptidoglycan.

 For instance, the walls of Streptococcus pyogenes contain polysaccharides that are
covalently linked to the peptidoglycan which can be extracted with hot dilute HCl.

 In addition, Gram-positive cell walls (e.g. Staphylococcus aureus and Streptococcus

faecalis) usually contain large amounts of teichoic acids, polymers of glycerol or ribitol
joined by phosphate groups (figure 3.23 and figure 3.24).

 Amino acids such as D-alanine or sugars like glucose are attached to:

 Glycerol and ribitol groups.

 The teichoic acids are covalently connected to:

  either the peptidoglycan itself or
 to plasma membrane lipids;
 in the latter case they are called lipoteichoic acids.

 Teichoic acids can be extracted with cold dilute acid.

 Teichoic acids appear to extend to the surface of the peptidoglycan, and, because they
are negatively charged, help give the Gram-positive cell wall its negative charge.

 The functions of these molecules are still unclear, but they may be important in
maintaining the structure of the wall.

 Teichoic acids bind Mg++ ions, and there is some evidence that they help to protect
bacteria from thermal injury by providing an accessible pool of these cations for
stabilization of the cytoplasmic membrane.

 Teichoic acids are not present in Gram-negative bacteria.

 The walls of most Gram positive bacteria contain very little lipid, but those of
Mycobacterium, Corynebacterium, and certain other genera are exceptions, being rich in
lipids called mycolic acids. These compounds have the following general structure:

where R1 and R2 are long hydrocarbon chains.

 The ability of mycobacteria to exhibit acid-fast staining (i.e., when stained, the cells
cannot be decolorized easily despite treatment with dilute acid) is correlated with the
presence of cell wall mycolic acids.

 A mycolic acid derivative called cord factor (trehalose dimycolate) is toxic and play
role in the diseases caused by C. diphtheriae and M. tuberculosis.

 The periplasmic space of Gram-positive bacteria, when observed, lies between the
plasma membrane and the cell wall and is smaller than that of Gram-negative bacteria.

 Even if Gram-positive bacteria lack a discrete, obvious periplasmic space, they may have
periplasm.

 The periplasm has relatively few proteins; this is probably because the peptidoglycan
sac is porous and any proteins secreted by the cell usually pass through it.
 Enzymes secreted by Gram positive bacteria are called exoenzymes. They often serve to
degrade polymeric nutrients that would otherwise be too large for transport across the
plasma membrane.

 Those proteins that remain in the periplasmic space are:

 usually attached to the plasma membrane.

 Staphylococci and most other Gram-positive bacteria have a layer of proteins on the
surface of their cell wall peptidoglycan.

 These proteins are involved in the interactions of the cell with its environment.

 Some are noncovalently attached by binding to the peptidoglycan, teichoic acids, or

other receptors. For example,

 S-layer proteins bind noncovalently to polymers scattered throughout the

wall.

 Enzymes involved in peptidoglycan synthesis and turnover also seem to

interact noncovalently with the cell wall.

 Other surface proteins are covalently attached to the peptidoglycan.

 Many covalently attached proteins, such as the M protein of pathogenic

streptococci, have roles in virulence, such as aiding in adhesion to host
tissues and interfering with host defenses.

 In staphylococci, these surface proteins are covalently joined to the

pentaglycine bridge of the cell wall peptidoglycan.

 An enzyme called sortase catalyzes the attachment of these surface proteins to the Gram-
positive peptidoglycan.

 Sortases are attached to the plasma membrane of the bacterial cell.

Gram-Negative Cell Walls

 Even a brief inspection of figure 3.17 shows that Gram-negative cell walls are much
more complex than Gram-positive walls.

 The thin peptidoglycan layer next to the plasma membrane and bounded on either side by
the periplasmic space may constitute not more than 5 to 10% of the wall weight.

 In E. coli it is about 2 nm thick and contains only one or two sheets of peptidoglycan.

 The periplasmic space of Gram-negative bacteria is also strikingly different than that of
Gram-positive bacteria.

 It ranges in size from 1 nm to as great as 71 nm.

 Some recent studies indicate that it may constitute about 20 to 40% of the total cell
volume, and it is usually 30 to 70 nm wide.

 When cell walls are disrupted carefully or removed without disturbing the underlying
plasma membrane, periplasmic enzymes and other proteins are released and may be
easily studied.

 Some periplasmic proteins participate in nutrient acquisition—for example, hydrolytic

enzymes and transport proteins.

 Some periplasmic proteins are involved in energy conservation. For example,

 the denitrifying bacteria, which convert nitrate to nitrogen gas, and

 bacteria that use inorganic molecules as energy sources (chemolithotrophs)

 have electron transport proteins in their periplasm.

 Other periplasmic proteins are involved in peptidoglycan synthesis and the modification
of toxic compounds that could harm the cell.

 The most interesting difference in the walls of Gram negative bacteria from Gram
positive bacteria is the presence of an outer membrane that surrounds a thin underlying
layer of peptidoglycan.

 The outer membrane lies outside the thin peptidoglycan layer (figures 3.25 and 3.26) and
is linked to the cell in two ways.

 The first is by Braun’s lipoprotein, the most abundant protein in the outer membrane.
 This small lipoprotein is covalently joined to the underlying peptidoglycan, and is
embedded in the outer membrane by its hydrophobic end.

 The outer membrane and peptidoglycan are so firmly linked by this lipoprotein that they
can be isolated as one unit.

 The second linking mechanism involves the many adhesion sites joining the outer
membrane and the plasma membrane.

 The two membranes appear to be in direct contact at these sites. In E. coli, 20 to 100 nm
areas of contact between the two membranes can be seen.

 Adhesion sites may be regions of direct contact or possibly true membrane fusions.

 It has been proposed that substances can move directly into the cell through these
adhesion sites, rather than travelling through the periplasm.

 Possibly the most unusual constituents of the outer membrane are its
lipopolysaccharides (LPSs).

 These large, complex molecules contain both lipid and carbohydrate, and consist of three
parts:
(1) lipid A,
(2) the core polysaccharide, and
(3) the O side chain.

 The LPS from Salmonella has been studied most, and its general structure is described
here (figure 3.27).

 The lipid A region contains two glucosamine sugar derivatives, each with three fatty
acids and phosphate or pyrophosphate attached.

 The fatty acids attach the lipid A to the outer membrane, while the remainder of the LPS
molecule projects from the surface.

 The core polysaccharide is joined to lipid A. In Salmonella it is constructed of 10

sugars, many of them unusual in structure.

 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.

 LPS has many important functions.

 Because the core polysaccharide usually contains charged sugars and
phosphate (figure 3.27), LPS contributes to the negative charge on the
bacterial surface.

 As a major constituent of the exterior leaflet of the outer membrane, lipid A

also helps stabilize outer membrane structure.

 LPS may contribute to bacterial attachment to surfaces and biofilm formation.

 A major function of LPS is that it aids in creating a permeability barrier.

 The geometry of LPS (figure 3.27b) and interactions between neighboring LPS
molecules are thought to restrict the entry of bile salts, antibiotics, and other
toxic substances that might kill or injure the bacterium.

 LPS also plays a role in protecting pathogenic Gram-negative bacteria from

host defenses.

 The O side chain of LPS is also called the O antigen because it elicits an
immune response. This response involves the production of antibodies that
bind the strain-specific form of LPS that elicited the response.

 However, many Gram negative bacteria are able to rapidly change the
antigenic nature of their O side chains, thus thwarting host defenses.
  Importantly, the lipid A portion of LPS often is toxic; as a result, the LPS can
act as an endotoxin and cause some of the symptoms that arise in Gram-
negative bacterial infections.

 If the bacterium enters the bloodstream, LPS endotoxin can cause a form of
septic shock for which there is no direct treatment.

 Despite the role of LPS in creating a permeability barrier, the outer membrane is more
permeable than the plasma membrane and permits the passage of small molecules like
glucose and other monosaccharides. This is due to the presence of porin proteins
(figures 3.25 and 3.26).

 Most porin proteins cluster together to form a trimer in the outer membrane (figure 3.25
and figure 3.28).

 Each porin protein spans the outer membrane and is more or less tube-shaped; its narrow
channel allows passage of molecules smaller than about 600 to 700 daltons.

 However, larger molecules such as vitamin B12 also cross the outer membrane. Such
large molecules do not pass through porins; instead, specific carriers transport them
across the outer membrane.
  Membrane is bilayered st. consisting mainly of :
 Phospholipids
 Proteins
 Lipopolysaccharides (LPS) most unusual constituent of the OM.

Archaeal Cell Walls

 Before they were distinguished as a unique domain of life, the Archaea were
characterized as being either Gram positive or Gram negative.

 However, their staining reaction does not correlate as reliably with a particular cell wall
structure as does the Gram reaction of Bacteria.

 Archaeal wall structure and chemistry differ from those of the Bacteria.

 Archaeal cell walls lack peptidoglycan and also exhibit considerable variety in terms of
their chemical make-up.

 Many archaea have a wall with a single, thick homogeneous layer resembling that in
Gram-positive bacteria (figure 3.30a).
 (a) Methanobacterium formicicum (b) Thermoproteus tenax

CW, cell wall; SL, surface layer; CM, cell membrane or plasma membrane; CPL, cytoplasm

 These archaea often stain Gram positive.

 Their wall chemistry varies from species to species but usually consists of complex
heteropolysaccharides. For example,

 Methanobacterium and some other methane-generating archaea (methanogens) have

walls containing pseudomurein (figure 3.31):

 a peptidoglycan-like polymer that has L-amino acids instead of D-amino

acids in its cross-links,

 N-acetyltalosaminuronic acid instead of N-acetylmuramic acid, and

 β (1→3) glycosidic bonds instead of β (1→4) glycosidic bonds.

  Other archaea, such as Methanosarcina and the salt-loving Halococcus, contain complex
polysaccharides similar to the chondroitin sulfate of animal connective tissue.

 Many archaea that stain Gram negative have a layer of glycoprotein or protein outside
their plasma membrane (figure 3.30b).The layer may be as thick as 20 to 40 nm.

 Sometimes there are two layers—an electron-dense layer and a sheath surrounding it.

 Some

 methanogens (Methanolobus),

 salt-loving archaea (Halobacterium), and

 extreme thermophiles (Sulfolobus, Thermoproteus, and Pyrodictium)

have glycoproteins in their walls.

 In contrast,
 other methanogens (Methanococcus, Methanomicrobium, and Methanogenium)
and
 the extreme thermophile Desulfurococcus

have protein walls.

Macromolecular Surface Arrays:

 Cell walls of some bacteria, both G+ and G-ve, are covered by a mosaic layer of protein
subunits (Fig.).

 Functions of these layers are not well understood, but at least one function is to protect
G-ve bacteria against attack and penetration by other small, predatory bacteria known as
bdellovibrios.
Structures Internal To cell Wall:
 Immediately beneath the cell wall is the cytoplasmic membrane.

 Cytoplasmic membrane: is app. 7.5 nm (0.0075 μm ) thick and is composed primarily of:
 Phospholipids (about 20-30%)
 Proteins (60-70%)

 The phospholipids form a bilayer in which most of the proteins are tenaciously held (integral
proteins) (Fig. 3.5).

 Other proteins are only loosely attached (peripheral proteins).

 The lipid matrix of the membrane has fluidity; allowing the components to move around
laterally.
 This fluidity appears to be essential for various membrane functions and is dependent on factors
such as temperature and on the proportion of unsaturated fatty acids to saturated fatty acids
present in the phospholipids.

Lipids:
 The chemical nature of membrane lipids is critical to their ability to form bilayers.

 Most membrane associated lipids are structurally asymmetric with polar and non polar
ends (Fig. 3.6) and are called amphipathic.

 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 membrane are hydrophilic, whereas hydrophobic ends
are buried in the interior away from the surrounding water (Figure 3.5).

Proteins:
 Two types of membrane proteins have been identified based on their ability to be
separated from the membrane.

 Peripheral proteins are loosely connected to the membrane and can be easily removed
(Figure 3.5) by mild treatments such as osmotic shock.

 They are soluble in aqueous solutions and make up about 20 to 30% of total membrane
protein.
  About 70 to 80% of membrane proteins are integral proteins.

 These are not easily extracted from membranes and are insoluble in aqueous solutions
when freed of lipids.

 These proteins can be removed only by destruction of the membrane, as with treatment
of detergents.

 Integral proteins, like membrane lipids, are amphipathic; their hydrophobic regions are
buried in the lipid while the hydrophilic portions project from the membrane surface
(figure 3.5).

 Integral proteins can diffuse laterally in the membrane to new locations, but do not flip-
flop or rotate through the lipid layer.

 Carbohydrates often are attached to the outer surface of plasma membrane proteins,
where they have important functions.

Difference between Phospholipid of Eubacteria and Archaebacteria:

 In eubacteria the phospholipids are phosphoglycerides in which straight chain fatty
acids are ester–linked to glycerol.

 In archaebacteria, lipids are polyisoprenoid branched-chain lipids, in which long-chain

branched alcohols (phytanols) are ether-linked to glycerol. (Fig. 5.25).
Eubacterial phospholipid, showing two unbranched long-chain fatty acids ester linked to glycerol

Archaebacterial phospholipid, showing two branched phytanol chains that are ether-linked to glycerol

R : any of the several compounds e.g. Ethanolamine, Choline, Serine, Inositol or Glycerol

 The cytoplasmic membrane is a hydrophobic barrier to penetration by most

water-soluble molecules.

 However, specific proteins in the membrane allow, indeed facilitate, the

passage of small molecules (i.e., nutrients and waste products) across the
membrane.

 The cytoplasmic membrane also contains various enzymes involved in

respiratory metabolism and in synthesis of capsular and cell-wall
components;
  moreover, because of its impermeability to protons (hydrogen ions), the
cytoplasmic membrane is the site of generation of the protonmotive force-
the force that drives ATP synthesis in many microorganisms, certain nutrient
transport systems, and flagellar motility.

 Consequently, the cytoplasmic membrane is extremely important functional

structure, and damage to it by physical or chemical agents result in the death
of the cell.

 According to one hypothesis, special membrane proteins might bind the signal peptide at
the inner surface of the cytoplasmic membrane and form a channel by which the protein
can traverse the membrane

Bacterial Membranes
 Bacterial membranes are similar to eucaryotic membranes in that many of their
amphipathic lipids are phospholipids (figure 3.6), but they usually differ from eucaryotic
membranes in lacking sterols (steroid-containing lipids) such as cholesterol (figure
3.7a).

 However, many bacterial membranes contain sterol-like molecules called hopanoids

(figure 3.7b).

 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: it has been estimated that the
total mass of hopanoids in sediments is around 10 11–12 tons—about as much as the total
mass of organic carbon in all living organisms (1012 tons)—and there is evidence that
hopanoids have contributed significantly to the formation of petroleum.

 The emerging picture of bacterial plasma membranes is one of a highly organized and
asymmetric system that also is flexible and dynamic.

 Numerous studies have demonstrated that lipids are not homogeneously distributed in the
plasma membrane.

 Rather, there are domains in which particular lipids are concentrated.

 It has also been demonstrated that the lipid composition of bacterial membranes varies
with environmental temperature in such a way that the membrane remains fluid
during growth.

 For example, bacteria growing at lower temperatures will have fatty acids with lower
melting points in their membrane phospholipids.
  Although procaryotes do not contain complex membranous organelles like mitochondria
or chloroplasts, internal membranous structures can be observed in some bacteria (figure
3.8).

 Plasma membrane infoldings are common in many bacteria and can become extensive
and complex in photosynthetic bacteria such as the cyanobacteria and purple bacteria
or in bacteria with very high respiratory activity, like the nitrifying bacteria.

 These internal membranous structures may be aggregates of spherical vesicles, flattened

vesicles, or tubular membranes.

 Their function may be to provide a larger membrane surface for greater metabolic
activity.

 One membranous structure sometimes reported in bacteria is the mesosome.

 Mesosomes appear to be invaginations of the plasma membrane in the shape of vesicles,

tubules, or lamellae.

 Although a variety of functions have been ascribed to mesosomes, many bacteriologists

believe that they are artifacts generated during the chemical fixation of bacteria for
electron microscopy.

Archaeal Membranes

 One of the most distinctive features of the Archaea is the nature of their membrane
lipids.
 They differ from both Bacteria and Eucarya in having branched chain hydrocarbons
attached to glycerol by ether links rather than fatty acids connected by ester links (figure
3.9).

 Sometimes two glycerol groups are linked to form an extremely long tetraether.

 Usually the diether hydrocarbon chains are 20 carbons in length, and

the tetraether chains are 40 carbons.

 Cells can adjust the overall length of the tetraethers by cyclizing the chains to form
pentacyclic rings (figure 3.9).

 Phosphate-, sulfur- and sugar-containing groups can be attached to the third carbons
of the diethers and tetraethers, making them polar lipids.

 These predominate in the membrane, and 70 to 93% of the membrane lipids are polar.

 The remaining lipids are nonpolar and are usually derivatives of squalene (figure 3.10).
 Despite these significant differences in membrane lipids, the basic design of archaeal
membranes is similar to that of Bacteria and eucaryotes—there are two hydrophilic
surfaces and a hydrophobic core.

 When C20 diethers are used, a regular bilayer membrane is formed (figure 3.11a). When
the membrane is constructed of C40 tetraethers, a monolayer membrane with much more
rigidity is formed (figure 3.11b).

 As might be expected from their need for stability, the membranes of extreme
thermophiles such as Thermoplasma and Sulfolobus, which grow best at temperatures
over 85°C, are almost completely tetraether monolayers.

 Archaea that live in moderately hot environments have a mixed membrane containing
some regions with monolayers and some with bilayers.

 Bacteria normally occur in hypotonic environments (i.e., environments having a lower

osmotic pressure than that within the bacterial cells) and they continually take up water
by osmosis: thus, they tend to expand, pressing the cytoplasmic membrane tightly
against rigid cell wall.
  In the absence of a rigid cell wall, there is nothing to prevent continued expansion and
eventual bursting of a protoplast.

 This bursting can be prevented by preparing protoplasts in an isotonic medium, i.e., in a

medium that has osmotic pressure similar to that of the protoplast.

 Such osmotically protected protoplasts are soft and fragile and are spherical, regardless
of the original shape of the cell.

 Mycoplasmas

Membrane Intrusions and Intracellular Membrane Systems:

 Bacterial cells do not contain membrane-enclosed organelles corresponding to the
mitochondria and chloroplasts of eukaryotic cells. However, bacteria may have
specialized invaginations of the cytoplasmic membrane that increase their surface area
for certain functions.

 Many bacteria, especially Gram-positive bacteria, possess membrane invaginations in

the form of systems of convoluted tubule and vesicles termed as mesosomes.

 Those known as central mesosomes penetrate deeply into the cytoplasm, are located
near the middle of the cell, and seem to be attached to the cell’s nuclear material; they
are thought to be involved in DNA replication and cell division (Fig. 5-26).
  In contrast, peripheral mesosomes show only a shallow penetration into the cytoplasm,
are not restricted to a central location, and are not associated with nuclear material; they
seem to be involved in export of exocellular enzymes such as penicillanase.

 Extensive intracellular membrane systems occur in methane-oxidizing bacteria, in

certain chemoautorophic bacteria (Fig. 5-27), and in nearly all phototrophic bacteria.

 They serve to increase surface area for various metabolic activities.

 For example, in phototrophic bacteria they are the site of the photosynthetic apparatus
of the cell; the infoldings provide a large surface area to accommodate a high content of
light-absorbing pigments.

 In the phototrophs known as cyanobacteria, special intracellular membranes (thylakoids)

occur that seem to be separate from the cytoplasmic membrane.

The Cytoplasm
The cell material bounded by the cytoplasmic membrane may be divided into :
 1) The cytoplasmic area: granular in appearance and rich in the macromolecular RNA-
protein bodies i.e ribosomes, on which proteins are synthesized.

2) The chromatinic area, rich in DNA; and

3) The fluid portion with dissolved substances.

 Unlike animal or plant cells, there is no endoplasmic reticulum to which ribosomes are
bound; some ribosomes are free in the cytoplasm, and others, especially those involved in
the synthesis of proteins to be transported out of the cell, are associated with the inner
surface of the cytoplasmic membrane.

 When the ribosomes of prokaryotes undergo sedimentation in a centrifuge, they have a

sedimentation coefficient of 70 Svedberg units and are composed of two subunits, a 50S
and a 30S subunit.

Cytoplasmic Inclusions and Vacuoles:

 Membrane (2.0-4.0 nm thick) enclosed , which may be

i) single layered e.g. PHB, some glycogen and S granules.

Inclusion body membrane vary in composition. Some are protein and other lipid in
nature.

 Some inclusion bodies are not membrane bound and lie free in cytoplasm e.g.
polyphosphate granules, cyanophycin and some glycogen granules.

1) Volutin Granules: also known as metachromatic granules (as they show the
metachromatic effect i.e they appear red or shades of blue when stained with blue dyes
methylene blue or toludine blue.

 Composed of polyphosphate. Stain intense reddish purple with dil.

methylene blue. Can be observed by light microscopy

 By electron microscopy appear round dark areas.

 Serve as a reserve source of polyphosphate (an important component of cell

constituents such as nucleic acids.

2) PHB (Polyhydroxybutyrate 0.2-0.7 μm diameter): contains ß-hydroxybutyrate molecules

joined by ester bonds between the carboxyl and hydroxyl groups of adjacent molecules.

 Chloroform soluble, lipid like material

  Serve as reserve C and energy source
 PHB granules can be stained with lipid soluble dyes such as Nile Blue.
 By electron microscopy appear as clear round area.

3) Glycogen:
 Polysaccharide granules (20-100 nm diameter) stained brown with I2.

 By electron microscope appear as dark granules

4) Sulfur Elements:

 Intracellular globules of elemental sulfur that may accumulate in certain bacteria

growing in environments rich in H2S. e.g. Purple Phototrophic bacteria.

5) Gas vacuoles: Some bacteria live in aquatic habitats form gas vacuoles that provide
buoyancy.

 By light microscope –are bright, refractile bodies

 By electron microscope- have a regular shape; hollow, rigid cylinders with more or
less conical ends and having a striated protein boundary.

 This boundary is impermeable to H 2O but various dissolved in the culture medium

can penetrate it to fill the cavity.

 The identifying feature of gas vacuole is that they can be made to collapse under
pressure and thereby lose their refractility.

 Gas vacuole walls do not contain lipid but are composed entirely of a single small
protein. These protein subunits assemble to form a rigid enclosed cylinder that is
hollow and impermeable to water but freely permeable to atmospheric gases.

6) Cyanophycin granules:

 Composed of large polypeptides containing app. 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 N2 for bacteria.

7) Carboxysomes:

 are present in many cyanobateria, nitrifying bacteria and thiobacilli.

 They are polyhedral, about 100nm in diameter ,

  and contain the enzyme ribulose-1,5-biphosphate carboxylase in a paracrystalline
arrangement.

 They serve as reserve of this and may be a site of CO2 fixation.

8) Inorganic inclusion bodies can be used for purposes other than storage.

 An excellent example is the magnetosomes which are used by some bacteria to orient
in the earth’s magnetic field. These contain iron in the form of magnetite.

Nuclear material

 In contrast to eukaryotic cells, bacterial cells contain neither a distinct membrane-

enclosed nucleus nor a mitotic apparatus.

 However, they do contain an area near the centre of the cell that is regarded as a nuclear
structure, and the DNA of the cell is confined to this area.

 Because it is not a discrete nucleus, this nebulous structure has been designated by such
terms as the nucleoid, the chromatin body, the nuclear equivalent, and even the
bacterial chromosome, since it consists of a single, cicular DNA molecule in which all
the genes are linked.

 It can be made visible under light microscope by Feulgen staining, which is specific for
DNA.
  By electron microscopy it appears as a light area with delicate fibrillar structure. The
behavior of the nucleoid in growing ,dividing bacteria has been observed by use of phase-
contrast microscopy with a medium having high refractive index.

Membrane Bound Nucleoids in Bacteria

There are a few exceptions:

Membrane bound DNA-containing regions are present in two genera of planctomycetes :

i) Pirellula: has single membrane that surrounds a region, the pirellulosome, which
contains a fibrillar nucleoid and ribosome-like particles.

ii) The nuclear body of Gemmata obscuriglobus is bound by two membranes.

More work is required to know the functions of these membranes and how widespread
this phenomenon is,

Bacterial Endospores
 A number of Gram-positive bacteria can form a special resistant, dormant structure
called an endospore.

 Endospores develop within vegetative bacterial cells of several genera: Bacillus and
Clostridium (rods), Sporosarcina (cocci), and others.

 These structures are extraordinarily resistant to environmental stresses such as heat,

ultraviolet radiation, gamma radiation, chemical disinfectants, and desiccation.

 In fact, some endospores have remained viable for around 100,000 years. Because of
their resistance and the fact that several species of endospore-forming bacteria are
dangerous pathogens, endospores are of great practical importance in food,
industrial, and medical microbiology.

 This is because it is essential to be able to sterilize solutions and solid objects.

 Endospores often survive boiling for an hour or more; therefore autoclaves must be
used to sterilize many materials.

 Endospores are also of considerable theoretical interest. Because bacteria

manufacture these intricate structures in a very organized fashion over a period of a
few hours, spore formation is well suited for research on the construction of complex
biological structures.

 In the environment, endospores aid in survival when moisture or nutrients are scarce.
  Endospores can be examined with both light and electron microscopes.

 Because endospores are impermeable to most stains, they often are seen as colorless
areas in bacteria treated with methylene blue and other simple stains;

 Special endospore stains are used to make them clearly visible.

 Endospore position in the mother cell (sporangium) frequently differs among

species, making it of considerable value in identification.

 Endospores may be centrally located, close to one end (subterminal), or definitely

terminal (figure 3.46).





 Sometimes an endospore is so large that it swells the sporangium.

 Electron micrographs show that endospore structure is complex (figure 3.47).

 The spore often is surrounded by a thin, delicate covering called the exosporium.

 A spore coat:

 lies beneath the exosporium, is composed of several protein layers, and may be
fairly thick.

 It is impermeable to many toxic molecules and is responsible for the spore’s

resistance to chemicals.

 The coat also is thought to contain enzymes that are involved in germination.
 The cortex:

 which may occupy as much as half the spore volume, rests beneath the spore
coat.

 It is made of a peptidoglycan that is less cross-linked than that in vegetative

cells.

 The spore cell wall (or core wall) is inside the cortex and surrounds the protoplast or
spore core.

 The core has normal cell structures such as ribosomes and a nucleoid, but is
metabolically inactive.

 It is still not known precisely why the endospore is so resistant to heat and other lethal
agents.

 As much as 15% of the spore’s dry weight consists of dipicolinic acid complexed with
calcium ions (figure 3.48), which is located in the core.

 It has long been thought that dipicolinic acid was directly involved in heat resistance, but
heat-resistant mutants lacking dipicolinic acid have been isolated.

 Calcium does aid in resistance to wet heat, oxidizing agents, and sometimes dry heat.

 It may be that calcium-dipicolinate stabilizes the spore’s nucleic acids.

 In addition, specialized small, acid-soluble DNA-binding proteins (SASPs), are found
in the endospore.

 They saturate spore DNA and protect it from heat, radiation, dessication, and chemicals.

 Dehydration of the protoplast appears to be very important in heat resistance.

 The cortex may osmotically remove water from the protoplast, thereby protecting it from
both heat and radiation damage.

 The spore coat also seems to protect against enzymes and chemicals such as hydrogen
peroxide.

 Finally, spores contain some DNA repair enzymes. DNA is repaired once the spore
germinates and the cell becomes active again.

 In summary, endospore heat resistance probably is due to several factors:

 calcium-dipicolinate and acid-soluble protein stabilization of DNA, protoplast

dehydration, the spore coat, DNA repair, the greater stability of cell proteins in
bacteria adapted to growth at high temperatures, and others.

 Endospore formation, also called sporogenesis or sporulation, normally commences

when growth ceases due to lack of nutrients.

 It is a complex process and may be divided into seven stages (figure 3.49).
Figure 3.49 Endospore Formation: Life Cycle of Bacillus megaterium. The stages are indicated by Roman
numerals.The circled numbers in the photographs refer to the hours from the end of the logarithmic phase of growth:
0.25 h—a typical vegetative cell; 4 h–stage II cell, septation; 5.5 h–stage III cell, engulfment; 6.5 h–stage IV cell,
cortex formation; 8 h–stage V cell, coat formation; 10.5 h–stage VI cell, mature spore in sporangium.
Abbreviations used: C, cortex; IFM and OFM, inner and outer forespore membranes; M, mesosome; N, nucleoid; S,
septum; SC, spore coats. Bars _ 0.5 _m.

 An axial filament of nuclear material forms (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 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 then are formed around the cortex (stage V),

 and maturation of the endospore occurs (stage VI).

 Finally, lytic enzymes destroy the sporangium releasing the spore (stage VII).

 Sporulation requires about 10 hours in Bacillus megaterium.

 The transformation of dormant spores into active vegetative cells seems almost as
complex a process as sporogenesis.

 It occurs in three stages: (1) activation, (2) germination, and (3) outgrowth (figure
3.50).

 Often a spore will not germinate successfully, even in a nutrient-rich medium, unless it
has been activated.

 Activation is a process that prepares spores for germination and usually results from
treatments like heating.

 It is followed by germination, the breaking of the spore’s dormant state.

 This process is characterized by:

spore swelling,
rupture or absorption of the spore coat,
loss of resistance to heat and other stresses,
 loss of refractility,
release of spore components, and
increase in metabolic activity.

 Many normal metabolites or nutrients (e.g., amino acids and sugars) can trigger
germination after activation.

 Germination is followed by the third stage, outgrowth.

 The spore protoplast makes new components, emerges from the remains of the spore
coat, and develops again into an active bacterium.

Exospores:
 Cells of methane-oxidizing genus Methylosinus form exospores, i.e., spores external to
the vegetative cell, by budding at one end of the cell.

 These are dessication –and heat resistant, but unlike endospores they donot contain DPA.

Conidiospore and Sporangiospores:

 The spores do not have high heat resistance of the endospores, but they can survive long
periods of drying.

Cysts:

 The classic example of a cyst is the structurally complex type produced by the genus
Azotobacter.
 Several other bacteria can differentiate into cystlike forms, but these seem to lack the
degree of structural complexity characteristic of Azotobacter cysts.


 

1. Describe the structure of the bacterial endospore using a labeled diagram.

2. Briefly describe endospore formation and germination.What is the importance
of the endospore? What might account for its heat resistance?
3. How might one go about showing that a bacterium forms true endospores?
4. Why do you think dehydration of the protoplast is an important factor in

1) the ability of endospores to resist environmental stress?