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.