Cytoplasmic Inclusion # 2.

Polyphosphates (Volutin Granules or Metachromatin Granules):

Many bacteria and microalgae accumulate inorganic phosphates in the form of granules of
polyphosphates. Polyphosphate is a liner polymer of orthrophosphates joined by ester bonds (Fig. 5.23).

Because they were first described in Spirillum volutans and because they bring a about metachromatic
effect (i.e., appear red or a different shade of blue when stained with methylene blue or toluidine blue
dyes), they have also been given the name ‘volutin granules’ and ‘metachromatin granules’, respectively.

Dark-field microscopic view of polyphosphate granules present in the cells of spirillum volutans

These granules are composed of polymetaphosphate and are common in diphtheria bacillus and in
certain lactic acid bacteria. The chemical structure of polyphosphate is shown in fig. 5.24.

These granules refractive and hence arc easily observable under light microscope. The polyphosphates
represent intracellular phosphate reserve when nucleic acid synthesis does not occur, and when the
letter starts, the polyphosphate granules are degraded and used as sources of phosphate for nucleic
acids.

Polyphosphates are also used as source of phosphate for phospholipids. In some cells the
polyphosphates act as an energy reserve and can serve as energy source in reactions.

Polyphosphate structure

Cytoplasmic Inclusion # 3. Poly-β-hydroxybutyrate (PHB):

Poly- β -hydroxybutyrate (PHB), one of the most common inclusion bodies in bacteria, is a lipid formed
from β -hydroxybutyrate monomers (units) joined by easter-linkages between the carboxyl and hydroxyl
groups of adjacent molecules resulting in long PHB polymer (Fig. 5.25) which aggregate into granules of
around 0.2-0.7 µm in diameter.
The length of the monomer in the polymer can vary considerably, from a short as C4 to as long as C18 in
certain bacteria.

Poly-β hydroxybutyrate granules are readily stained with Sudan black for light microscopy and are
clearly visible in the electron microscope (Fig. 5.26).

PHB is accumulated by aerobic and facultative bacteria when the cells are deprived of oxygen and must
carry out fermentative metabolism. On return of aerobic conditions, PHB, which is a long-term energy
storage, is used as an energy and carbon source and incorporated into the oxidative metabolism.

Structure of poly-β-hydroxy butyrate

Some bacteria produce co-polymers of PHB often referred to as poly-β-hydroxy-alkanoate (PHA). The
latter can be thermo-plastically moulded and used as new plastics that shows advantage over
conventional plastics (polypropylene or polyethylene) of being biodegradable.

However, a copolymer containing approximately equal amounts of poly-β-hydroxybutyrate (PHB) and

poly-β- hydroxyvalerate (PHV) has had the greatest market success thus far. But, since they are more
cost-effective, the conventional petroleum-based plastics still make up virtually the entire plastics
market today.

Electron microscopic view of cells of a phototrophic bacterium showing poly-β-hydroxybutyrate granules

Cytoplasmic Inclusion # 4. Glycogen:

Glycogen (Fig. 5.27) like PHB, is another storage product formed by prokaryotes. 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 a(1 → 6) glycosidic bonds.
Glycogen is dispersed more evenly throughout the cytoplasmic matrix as small (about 20 – 100 nm in
diameter) and is a storage reservoir tor carbon and energy. Glycogen is also known as ‘animal starch’
and, besides prokaryotes, is found in fungi.

Glycogen structure

Cytoplasmic Inclusion # 5. Gas Vacuoles:

Gas vacuoles, the most remarkable organic inclusion bodies, are formed as a result of the aggregation of
enormous number of small, hollow, cylindrical structures called gas vesicles. These structures confer
buoyancy on cells by decreasing their density and live a floating existence within the water column of
lakes and the oceans.

Each gas vacuole appears about 75 nm in diameter with conical ends and about 200-1,000 nm in length.
The most dramatic instances of floatation due to gas vacuoles are seen in cyanobacteria that form
massive accumulations (blooms) in lakes.

Gas vacuoles also characteristically occur in many aquatic bacteria such as purple and green
photosynthetic ones, and a few non-photosynthetic aquatic bacteria such as Halobacterium and
Thiothrix.

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.

Each gas vesicle is a spindle-shaped, single membrane-bound gas-filled structure made of protein; the
protein subunits assemble to form the wall of the gas vesicle which encloses the hollow cylinder and is
impermeable to water but freely permeable to atmospheric gases.

Two different proteins, GvpA and GvpC (Fig. 5.28), 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.

Isolated gas vesicles and details of a portion of gas vesicle showing the arrangement of proteins GvpA
and GvpC

Cytoplasmic Inclusion # 6. Magnetosomes:

Magnetosomes are the inorganic inclusion bodies of iron usually in the form of intracellular chains of
magnetite (Fe3O4). Some species from sulfidic habitats possess magnetosomes containing greigite
(Fe3S4) and pyrite (FeS2). Magnetosome (Fig. 5.29) containing bacteria are called magnetotactic
bacteria (e.g., Aquaspirillum magnetotacticum).

Magnetosomes vary in shape from square to rectangular to spike-shaped as their morphology is species-
specific. They are around 40 to 100 nm in diameter and bounded by a monolayer membrane made up of
phospholipids, proteins, and glycoproteins.

Magnetosome membrane is a non-unit membrane similar to that surrounding granules of poly- β-

hydroxybutyrate (PHB) and its proteins probably play a role in precipitating F3+ as Fe3O4 in the
developing magnetosome.

Magnetosome

Most of the magnetotactic aquatic bacteria grow best at very low O2 concentrations the main function
of magnetosomes is probably to guide such bacteria toward the sediment where O2 concentration is
lower. Magnetotacic bacteria exhibit magnetotaxis, the process of orienting and migrating along earth’s
magnetic field lines, and hence are referred to as the living magnets.
For convenience, magnetotactic bacteria in the Southern hemisphere use their magnetosome chain to
determine southward and downward directions and swim down to nutrient-rich sediments or locate the
optimum depth in fresh water and marine habitats. Magnetotactic bacteria in Northern hemisphere
orient northward and downward for the same purpose.

Despite magnetotactic bacteria, magnetosomes also occur in the heads of birds, dolphins, tuna, green
turbles, and other animals, presumably to aid navigation. Magnetotactic bacteria and animals therefore
share more in common behaviourally than previously thought.

Cytoplasmic Inclusion # 7. Sulphur Globules:

Sulphur globules (Fig. 5.30) are present in the bacterial cells growing In H2S rich environment such as
photosynthetic purple sulfur bacteria and filamentous non-photosynthetic bacteria (Beggiatoa and
Thiothrix). These bacteria oxidize H2S into elemental sulfur (H2S → S°) which accumulates inside the cell
in visible sulfur globules.

These sulfur globules of elemental sulfur remain until the H2S source is reduced. In the latter condition
the stored sulfur in these granules is oxidized to sulfate (S° → SO42-) and the globules slowly disappear.

It is reported that the sulfur globules occur in the periplasm rather than the cytoplasm of the bacterial
cell. The periplasm expands outwards to accommodate the globules and contracts when the sulfur of
the globules is oxidized.

Bright-field photomicrograph showing sulfur globules present inside the cells of a people sulfur
bacterium

Cytoplasmic Inclusion # 8. Carboxysomes:

Carboxysomes are polyhedrical bodies surrounded by thin, non-unit membrane and range about 100 nm
in diameter. They contain, apart from a little DNA, the enzyme ribulose-1, 5- bisphosphate carboxylase
(RUBISCO) in a paracrystalline arrangement.
It is thought that carboxysomes are a mechanism to increase the amount of RUBISCO in the bacterial
cell to allow for more rapid CO2 fixation without causing any effect on the osmolarity of the cytoplasm;
the osmotic pressure of the cytoplasm is not affected as the carboxysome is insoluble.

Photoautotrophic (cyanobacteria) and chemolithoautotrophic (sulfur bacteria, nitrifying bacteria) that

use Calvin cycle for CO2 fixation produce carboxysomes. The latter do not occur in facultative
autotrophic bacteria (photoorganoheterotrophic), which grow either as autotrophs or as heterotrophs.
Thus, the carboxysomes appear to be an evolutionary adaptation to bacteria under strict autotrophic
environment.