Plasma Membrane

Bacterial plasma membrane is a unit membrane, which contains both protein and lipid. The amount of protein and lipid varies widely and generally bacterial plasma membrane contains more amount of protein as compare to eukaryotic membranes. One major difference between the eukaryotic and prokaryotic plasma membrane is that prokaryotic membranes lack sterols such as cholesterol. On the contrary, bacterial cells contain pentacyclic sterol like molecules known as hopanoids. Hopanoids are synthesized from the same steroids precursor and they play the role similar to that of sterol in eukaryotic cells.

Functions: Plasma membrane acts as a transport system for nutrient uptake, protein secretion and waste excretion. It basically acts as a selectively permeable barrier thereby allowing only particular molecules and ions to pass in and out of the cell and restricting the movement of others. Plasma membrane is also a site for a number of metabolic processes like photosynthesis, respiration, synthesis of lipids etc. It also contains some receptor molecules that help in detecting and responding to the surrounding chemicals. Presence of invagination in plasma membrane helps in chromosome replication or cross wall formation in dividing bacteria. This invagination may be in the shape of tubules, vesicles or lamellae. Lamellae shaped invagination are known as mesosome, which are present in both Gram positive and Gram negative bacteria but are more prominent in Gram positive one. 

Note: Plasma membrane of all the bacteria lacks sterol except Mycoplasma.

Inclusion Bodies

Cytoplasm of the prokaryotic cells contains some organic and inorganic reserve bodies known as inclusion bodies. These inclusion bodies may or may not be surrounded by membranes. Cyanophycin granules, polyphosphate granules and some glycogen granules are a few examples of inclusion bodies which are not surrounded by membrane and lies free in the cytoplasm. Membrane surrounding the inclusion body is usually single layer and is around 2.0 to 4.0 nm thick. These membranes vary in composition; they can be protein in nature whereas other contains lipid. Examples of membrane enclosed inclusion bodies are carboxysomes, gas vacuoles, glycogen, sulfur granules and hydroxybutyrate granule. The main function of inclusion body is to store carbon compounds, inorganic substances and energy. The quantity of the inclusion bodies varies as per the nutritional requirement of the cell. For instance, phosphate granules are depleted in freshwater habitats that are phosphate limited. Inclusion bodies also ties up the molecules in particulate form thereby reducing the osmotic pressure. Some of the inclusion bodies are described below:

Glycogen

It is the polymer of glucose units. In this, glucose monomers are linked linearly by 1−> 4 glycosidic bonds and branching occurs by 1−> 6 glycosidic bonds. They are present in the form of granules in cytosol. When stained with iodine, they appear as reddish brown. They are basically the reservoirs of carbon, supplying materials for energy and biosynthesis. Purple photosynthetic bacteria contains glycogen.

Cyanophycin

Cyanophycin granules are large polypeptides containing equal amount of two amino acids i.e. arginine and aspartic acid. This amino acid polymer is not produced by ribosome and they are commonly found in cyanobacteria and a few heterotrophic bacteria. They can be viewed under light microscope because of their large size. Their main function is to store nitrogen for bacteria.

Carboxysomes

Carboxysomes are polyhedral inclusion bodies and are about 100 nm in diameter. They are mostly found in nitrifying bacteria, cyanobacteria and thiobacilli. They contains CO₂ fixation enzyme ribulose-1,5-bisphosphate carboxylase (RuBisCO) thereby acting as a site of CO₂ fixation.

Poly-hydroxybutyrate (PHB)

PHB are the polymers of the hydroxybutyrate molecules joined by ester bond between carboxyl group of one molecule and hydroxyl group of adjacent molecule. They are the reservoirs of carbon storage thereby supplying materials for energy and biosynthesis. They are commonly found in purple photosynthetic bacteria.

Gas vacuole

They are the aggregates of small, hollow and spindle shaped structure called gas vesicles. Gas vesicles are made up of proteins and they do not contain lipids. These hollow structures are impermeable to water but they are highly permeable to atmospheric gases. Gas vacuole regulates the buoyancy of bacteria and helps them to float at a depth necessary for adequate light intensity, nutrient level and oxygen concentration. They provide buoyancy by decreasing the overall cell density. These organic inclusion bodies are commonly found in purple and green photosynthetic bacteria, many cyanobacteria and in some aquatic forms like Halobacterium and Thiothrix.

Polyphosphate granules

Polyphosphate granules or volutin granules store inorganic phosphate. Orthophosphates are joined by ester bond to form linear polymer i.e. polyphosphate. These granules are basically the energy reservoir and acts as an energy source in many reactions. Volutin granule appears as red or different shades of blue when stained with blue basic dyes like methylene blue or toluidine blue. This is called metachromatic effect and hence granules are sometimes known as metachromatic granules.

Sulfur granules

Another type of inclusion body is sulfur granules. They are used to store sulfur. During photosynthesis in purple photosynthetic bacteria, hydrogen sulfide is used as an electron donor. The resulting sulfur is stored either in periplasmic space or in cytoplasmic globules.

Magnetosome

They are the membrane bound structure having many features similar to eukaryotic organelles. They are not used for storage purpose. But they are used like a compass needle by some bacteria to orient in the earth’s magnetic field. Magnetosome consist of magnetic mineral crystal (i.e. crystals of magnetite or greigite) surrounded by lipid bilayer. They are found in magnetotactic bacteria. These bacteria are motile and mostly aquatic.

Note: Glycogen, cyanophycin, carboxysomes, PHB and gas vacuole are organic inclusion bodies whereas polyphosphate granules, sulfur granules and magnetosome are inorganic inclusion bodies.

Shape & Arrangement of Bacterial Cells

Although bacteria are very small and simple organism, there is huge variation in their shapes.  This variation is due to the differences in genetics and ecology. The basic shapes of bacteria are coccus, bacillus, vibrio and spirilla.

Cocci

Cocci or singular coccus are spherical or ovoid shape cells. When cells divide, they can remain attached and form some arrangements. Diplococci (singular: diplococcus) are one in which cocci remains in pair after dividing as seen in Neisseria. When cocci divides repeatedly and remains attached in a chain like pattern they forms streptococci as seen in genera Enterococcus, Streptococcus etc. Some cocci remains in the group of four i.e. they forms square by dividing in two planes. They are called tetrads as seen in genus Micrococcus. Those cocci that divides in three plane and leads to the formation of cubical packet of eight cells are called sarcinae as observed in genus Sarcina. Staphylococcus divides in multiple planes and forms an irregular grapelike cluster called staphylococci.

Bacilli

Bacilli or singular bacillus are rod shape cells. Bacillus megaterium is a typical rod shape bacteria. The shape of the end of the rod varies from species to species and it can be round, flat, bifurcated or cigar shaped. Diplobacilli are one in which bacilli remains in pair after division. They can also divide repeatedly and forms chain called streptobacilli.

Vibrio

Vibrios are comma shaped cells. They basically look like a curved rod. For example, Vibrio cholerae are comma shaped Gram negative bacteria.

Spirilla

As name implies, they are spiral or helical shaped cells. They basically have rigid bodies as seen in members of genus Spirillum. Another group of spirilla are there which are also helical in shape but they have flexible body; they are known as spirochetes. Example of spirochetes include Leptospira species.

Note: Most of the bacteria maintains single shape i.e. they are monomorphic. However, some bacteria lack the single, characteristics shape and have many shapes. They are known as pleomorphic; for example Corynebacterium.

Size of Bacterial Cells

Bacteria are very small microscopic structures. They vary in size from 0.2-2.0 micrometers (µm) in diameter and 0.5-5.0 µm in length. The smallest known bacteria are the members of the genus Mycoplasma. They are about 0.3 µm in diameter. Recently, nanobacteria or ultramicrobacteria (0.2-0.05 µm in diameter) have been reported.

Escherichia coli, the most commonly studied bacteria is about 1.1-1.5 µm wide and 2.0-6.0 µm long. Some bacteria are very large in size and are visible to unaided eye like Epulopiscium fishelsoni and Thiomargarita namibiensis. E. fishelsoni is rod shaped and about 600 µm in length and 80 µm in diameter. 

As bacteria are very small in size, they have large surface area to volume ratio. Thus all the internal parts of the cell are very close to the surface. This makes nutrients to be easily and quickly available to all the parts of the cell and thereby helping in the rapid uptake and intracellular distribution of nutrients and excretion of wastes.

Endospore

Endospore

Vegetative bacterial cells of several genera like Bacillus and Clostridium forms an exceptionally resistant structure for survival under the harsh or unfavorable environmental conditions. This dormant and non-reproductive structure is known as endospore since it develops within the cell. Endospore formation usually occurs in Gram positive bacteria and they are resistant to environmental stresses like high temperature, gamma radiation, UV radiation, extreme freezing, desiccation and chemical disinfectants.  But when the environmental condition becomes favorable, endospores can revert back to the vegetative state.

The location and morphology of the spore vary with species and are valuable in identification. Endospores vary in shape from spherical to elliptical and they may be smaller or larger in size than that of the parent bacteria. Spore position also differs among the species; it may be terminal or subterminal or centrally located. Terminal endospores are one which is located at the pole of cell; subterminal endospores are located close to one end and central endospores are more and less in the middle. Example of bacteria having centrally located spore is Bacillus cereus and that of bacteria having terminal endospore is Clostridium tetani.

Structure of Endospore:

Endospore structure is very complex and it is composed of following layers:

·      Exosporium

·      Spore coat

·      Spore cortex

·      Core wall

Exosporium is a thin, delicate covering which overlies the spore coat. Spore coat is an impermeable protein layer that is resistance to many chemicals and toxic molecules. Its nature is like sieve thereby excluding large toxic molecules like lysozyme and it also contains the germination enzymes. Beneath the spore coat lies cortex which occupies half of the spore volume and is mainly consist of peptidoglycan. Peptidoglycan in the spore cortex are less cross linked than in the vegetative cells. The core wall is present beneath the cortex and surrounds the core or protoplast. The core contains DNA, normal cell structures like ribosome and other enzymes, but is metabolically quiet.

The endospore contains large amount of dipicolinic acid (pyridine-2,6-dicarboxylic acid or PDC or DPA), upto 15% of endospore’s dry weight. DPA is basically found in complex with calcium ions forming calcium dipicolinate. Earlier it has been thought that DPA is responsible for spores heat resistance property but now DPA lacking mutants have been isolated which are also heat resistance. Presence of calcium helps in providing resistance to oxidizing agents, wet heat and sometimes dry heat also. Endospore also contains some small acid-soluble proteins (SASPs). Recently it has been discovered that this SASPs saturates DNA and are in part responsible for providing resistance from dessication, heat, radiation and DNA-damaging chemicals. Dehydration of the protoplast also aids in the process of heat resistance. By the process of osmosis, cortex removes the water from protoplast, thereby protecting it from damage by heat and radiation. As mentioned above, spore coat also provide protection against enzymes and chemicals like hydrogen peroxide. Also some DNA repair enzymes are present in spore which helps during germination and outgrowth process. In conclusion, the heat resistance property of the endospore is basically due to: presence of calcium-dipicolinate, SASPs stabilization of DNA, dehydration of protoplast, the spore coat, DNA repair, nature of cell proteins to be active even at high temperature and others.

Endospore Staining:

Endospore staining also known as Schaeffer-Fulton staining is a differential staining technique which distinguishes between the vegetative cells and the endospores. In this, malachite green is used as a primary stain and safranin is used as secondary stain. At first, the bacterial cells are heated with malachite green. It is then washed off with water and counterstained with Safranin. This results in green endospores along with pink or red vegetative cells.

In this technique, heat acts as a mordant. So heating the cells with malachite green will help endospores to take up the stain which are otherwise difficult to stain and once they are stained, they resist decolorization. And as the stain binds weakly to the cell wall, water is enough to decolorize the vegetative cells which then takes up the counter stain. Therefore, water here act as a decolorizer.

Endospore Formation:

The process of spore formation is known as sporogenesis or sporulation. Endospore formation is a complex process and usually takes up in seven stages. Unfavorable environmental conditions like lack of nutrients etc. trigger the process of sporulation. In stage I, nuclear material forms an axial filament followed by stage II in which inward folding of the cell membrane occurs which slowly encloses the part of DNA and leads to the formation of forespore septum. In stage III, the growth of membrane continues and it engulfs the immature spore in another membrane. Then follows the accumulation of calcium and DPA and cortex is laid down in the space between the two membranes (stage IV). In stage V, cortex is surrounded by protein coats followed by maturation of spore in stage VI. At last, the release of spore takes place in stage VII by the help of lytic enzymes that destroys the sporangium.

Germination of Endospore:

Endospore germination means the transformation of dormant, resistant spore into metabolically active vegetative cells. This is also a very complex process like that of sporogenesis. It occurs in three stages: activation, germination and outgrowth. Despite the presence of favorable condition, an endospore will not germinate into vegetative cell. Activation like heat treatment is required for germination to take place. The process of activation is reversible and it basically prepares spores for germination. Next, the spore dormancy is broken in process of germination. Germination is triggered by presence of nutrients like amino acids, sugars and other normal metabolites. This process begins with spore swelling followed by rupture or absorption of the spore coat which leads to the release of spore components and loss of refractility and resistance property (i.e. resistance to heat and other stresses). Metabolic activity increases by the process of germination.  Next comes the final stage i.e. outgrowth in which the protoplast makes up the new component. Then protoplast emerges from the spore coat and finally an active bacteria develops.