3). Inclusion Bodies:  There are granules of organic or inorganic materials. These bodies are generally for storage( Eg  carbon compound, inorganic substance & energy) & also helps to osmotic pressure. Some of the inclusion bodies are poly-beta- hydroxy butyrate  granules( carbon storage), some glucose(carbon storage), sulphur granules( sulphur storage), gas vacuoles( helps bacteria to regulate buoyancy & float), phosphate granules or volutin granules or meta chromatin granules( storage of phosphates).

Mesosomes:  The cytoplasmic membrane is invaginated  at certain places into the cytoplasm in form of convoluted tubules  & vesicles known as mesosomes. On these surfaces are found enzymes associated to mitochondria of eucaryotic. Most of mesosomes are confined to the periphery showing only a shallow penetration.However, some penetrate deeply into the cytoplasm near to the cell's nuclear material. These  are thought to be involved in DNA replication.

4). Bacterial Endospore:   Endospores or spores are highly resistant dormant structure produced by number of Gram negative bacteria. Eg. Sporasacrium spp , Bacillus spp , Clostridium spp . Bacteria normally grow, matured, & reproduce by somatic cells. When there is nutrient depletion or environmental stress( heat, UV radiation, chemical disinfection desiccation ), spore former bacteria begin spore formation. After return of suitable environmental spores produce vegetative cell. Some endospores remain viable for 100 yrs .Since spores often survive boiling  for an hour or more therefore, autoclave must be used to sterilize any bacteria.

Endospores can be examined with both light and electron
microscopes. Because spores are impermeable to most stains,
they often are seen as colorless areas in bacteria treated with
Methylene blue and other simple stains; special spore stains are
used to make them clearly visible.  Spore position
in the mother cell or sporangium frequently differs among
species, making it of considerable value in identification. Spores may be centrally located, close to one end (subterminal), or definitely
terminal . Sometimes a spore is so large that
it swells the sporangium.

Electron micrographs show that endospore structure is complex
. 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 and responsible for the spore’s resistance
to chemicals. 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 core. The core has the normal cell structures such as
ribosomes and a nucleoid, but is metabolically inactive.

Role Of Calcium Dipicolinate:

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  which is located in the core. It has
long been thought that dipicolinic acid was directly involved in
spore heat resistance, but heat-resistant mutants lacking dipicolinic
acid now have been isolated. Calcium does aid in resistance
to wet heat, oxidizing agents, and sometimes dry heat. It
may be that calcium-dipicolinate often stabilizes spore nucleic
acids. Recently specialized small, acid-soluble DNA-binding
proteins have been discovered 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
during germination and outgrowth after the core has become
active once 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.

Sporulation:  Spore formation, 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
3.43). 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 fore spore septum (stage II). The
membrane continues to grow and engulfs the immature spore in
a second membrane . Next, cortex is laid down in the
space between the two membranes, and both calcium and dipicolinic
acid are accumulated . Protein coats then are
formed around the cortex   and maturation of the spore
occurs . Finally, lytic enzymes destroy the sporangium
releasing the spore . Sporulation requires only 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. Often an endospore will not germinate successfully,
even in a nutrient-rich medium, unless it has been activated.
Activation is a reversible 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.


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