Bacterial Endospore: Characteristics, Structure and its Formation

What are Endospores?

Bacterial endospores are special tough, dormant and resistant spores produced by some Gram-positive bacteria of Firmicute family during unfavorable environmental conditions. Endospores are developed within the vegetative cells (hence the name, endo = inside). They help the bacteria to endure the unfavorable environmental conditions.

Another importance of endospores is that it can be easily dispersed by wind, water and through the gut of animals. Bacillus and Clostridium are the most studied endospore forming bacterial genera. Bacillus enters into endospore formation cycle when the carbon or nitrogen source is getting limited in the growing medium.

Who discovered endospores

John Tyndal (source cc wikipedia)

Who discovered the endospore?

Endospores were discovered by John Tyndall, a 19th century physicist. He discovered endospores as the heat resistant spores of bacteria which survived even after 100oC. He also discovered a simple cost effective process to kill bacterial endospores called Tyndallization.

What are the characteristics of Endospores?

The endospores are structurally, metabolically and functionally very different from bacterial vegetative cells. The main characteristics of bacterial endospores are giving below:

Learn more: Difference between Endospore and Vegetative Cells

Ø  Endospores are exceptionally resistant to stressful environmental conditions such as heat, ultraviolet radiation, gamma radiation, chemical disinfectants and desiccation.

Ø  Most of the endospores are viable for many years, even for 10, 000 years or more.

Ø  Due to this long viability and their adaptations to stress conditions, most of the endospores producing bacteria are notorious pathogens.

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Ø  Wiping with alcohol or hydrogen peroxide or boiling at 100oC will not kill the bacterial endospores.

Ø  However, endospores can be killed by autoclaving (at 121oC).

Ø  Endospores can be visualized under light and electron microscope.

Ø  Endospores will NOT take the usual bacterial stains such a safranin used in Gram-staining.

Ø  Specific stains and special staining techniques are required to stain the endospores.

Ø  The classically used stain to visualize endospore is Malachite Green and the staining procedure is known as Schaeffer–Fulton Staining.

Ø  The endospore-producing mother cell is called sporangium.

Ø  Sporangium shows distinct differences from other vegetative cells.

Ø  These characteristics are used for the identification purpose in bacterial taxonomy.

Ø  The position of the endospore within the spore mother cell also varies.

Ø  Based on the position of spores, the sporangium/spores may be Central spore, Sub-terminal spore, Terminal spore or Terminal spore with swollen sporangium.

Ø  Both aerobic and anaerobic bacteria (of Gram-positive type) can produce endospores.

Ø  No Archaebacteria are known to produce endospores.

Bacterial Endospore Formation

Bacillus: vegetative cells (pink) and Endospores (green) ( Image Source CC Wikipedia)

Learn more: Difference between Gram Positive and Gram Negative Bacteria

Learn more: Comparison of Archaea, Bacteria and Eukarya

What is the Structure of Endospores?

Ø  The structure of endospore is very complex since they possess multilayered coverings.

Ø  The outermost layer of the spore is called exosporium which is relatively thin and delicate.

Ø  Beneath the exosporium is a Spore Coat composed of several layers of proteins.

Ø  Spore coat is comparatively thick.

Ø  The thickness of the spore coat is one reason for the high resistance of endospores towards heat, radiation and chemicals.

Ø  Inner to the spore coat is the Cortex.

Ø  Cortex is the thicker wall layer in the endospores.

Structure of Endospore

Ø  Cortex is very large and sometimes occupy as much as half of the spore volume.

Ø  The cortex is composed of peptidoglycan

Ø  The peptidoglycan in the cortex is less cross linked than that of vegetative cells.

Ø  The innermost layer of the spore is called the Spore Cell wall or Core Cell Wall.

Ø  Spore cell wall covers the central protoplast or Core of the endospore.

Ø  The endospore core has a normal cell structure as that of a vegetative cell.

Ø  The core contains ribosomes and centrally placed nucleoid (genetic material).

Ø  Unlike the vegetative cells, the core protoplast is metabolically inactive.

Ø  The core only contains about 10 – 25% of water of the normal vegetative cell.

How endospore and formed

Why the Bacterial Endospores are Extremely Resistant to Temperature, Radiations and Chemicals?

The exact reason for the high resistance of endospores towards extreme temperature, radiation and chemicals is still unknown. Several explanations are now prevailing in the scientific community to explain this. Some of the possible explanations are given below:

Ø  Endospores contain high amount of dipicolinic acid in its core (protoplast).

Ø  In some endospores, about 15% of the total dry weight of the spore is contributed dipicolinic acid.

Ø  The dipicolinic acid in bacterial endospore not occurs in free-state rather, it forms a complex with calcium ions (Ca2+).

Ø  For a long time, it was believed that the high concentration of dipicolinic acid is providing the heat resistance to endospores.

Ø  This view is now questioned since mutants lacking dipicolinic acid with heat resistance are isolated and this suggests the involvement of other mechanisms.

Ø  High concentration of calcium ions can impart resistance to wet heat and oxidizing agents.

Ø  The calcium-dipicolinic acid can stabilize the genetic materials of the endospores.

Ø  Large amounts of Small Acid Soluble DNA binding Proteins (SASPs) are reported to occur in the core of endospores.

Ø  These proteins can bind to the DNA of endospores and can prevent the DNA from heat, radiations and chemicals.

Ø  The binding of SASPs to the DNA changes the molecular structure of DNA from its normal B-form to A-form.

Ø  The A-DNA is more compact than B-DNA and thus A-DNA can have higher resistance against pyrimidine dimer formation by UV radiations.

Ø  A-DNA is also comparatively more resistant to the denaturing effects of dry heat.

Learn more: Different forms of DNA (A-DNA, B-DNA and Z-DNA)

Ø  The SASPs can also act as the carbon and nitrogen source of the newly formed vegetative cell during endospore germination.

Ø  The cortex of the endospore can remove water from the core osmotically (cause dehydration).

Ø  Dehydration can provide heat resistance in bacterial cells.

Ø  The thick spore coat can also act as an impermeable barrier against chemical such as hydrogen peroxides.

Ø  Spore coat can also restrict the entry of many hydrolyzing enzymes into the core.

Ø  Bacterial endospores also contain a high amount of DNA repair enzymes.

Ø  These repair enzymes can quickly heal all types of DNA lesions formed in the DNA when the spores are exposed to harsh environmental conditions.

Learn more: DNA Repair Mechanisms with Video Tutorials

Thus, the heat, radiation and chemical resistance of endospores may be contributed by several factors such as the thick spore wall, high concentration of calcium dipicolinic acid, the presence of acid soluble DNA protecting proteins, protoplast dehydration and presence of quick efficient DNA repair mechanisms.

How are Endospores formed in Bacteria?

The process of formation of endospore is called Sporulation or Sporogenesis. Sporulation usually occurs when the bacterial cells face a nutrient deficient condition. The core of the endospore becomes increasingly dehydrated during the sporulation process. The formation of endospore is a complex process and it is completed in seven stages named as State – I (S-I) to Stage – VII (S-VII).

Ø  S-I: Formation of axial filament: The genetic material of the bacterial cell is oriented in the exact center plane of the bacterial cell.

Ø  S-II: Formation of Septa: A plasma membrane invagination grows into the lumen of the cell and forms a septum called forespore septum. The formation of septum results in the separation of a small portion of the DNA from rest of the genetic material.

Ø  S-III: Engulfment of the Forespore: The membrane of the mother cell continues to grow and completely engulf the newly formed immature spore. Thus with the engulfment, the forespore is now covered by two plasma membrane and an inter membrane space.

Ø  S-IV: Formation of Cortex: Cortex formation is started between the inter membrane space of the two membranes. Large amount of calcium and dipicolinic acid is also accumulated in Stage IV.

Ø  S-V: Formation of Protein Coat: Protein coat is laid down over the cortex of the newly formed spore.

Ø  S-VI: Spore Maturation: The core becomes increasingly dehydrated, the cell become metabolically inactive.

Ø  S-VII: Enzymatic destruction of the sporogonium (spore mother cell) and release of the endospores.

Endospore Formation

Formation of Endospores (image source: cc Wikipedia)

Germination of Endospores

The spore germination literally means the transformation of the dormant endospore to a metabolically active vegetative cell. The germination of spores occurs when the environmental conditions are suitable. Similar to the process of sporulation, the endospore germination is also a very complex event.

Ø  The process of germination of endospore is completed in three stages:

(1).   Activation

(2).  Germination

(3).  Outgrowth

(1). Activation

Ø  Activation of the endospore is a pre-request for its germination.

Ø  Spores that are not activated will not germinate even they are placed on the nutrient rich media.

Ø  Activation prepares the endospore for its germination (second step).

Ø  Endospore activation is a reversible process.

Ø  If the environmental conditions are not favorable, the activated spore can go back to its quiescent inactive stage.

Ø  Activation of the endospores can be artificially induced by heat shock.

(2). Germination

Ø  Germination is the breaking of spore’s dormant stage.

Ø  Spore germination is characterized by the following events:

$   Swelling of the spore

$   Rupture of the spore coat

$   Loss of resistance to heat or radiation

$   Loss of refactility

$   The release of spore components

$   The quick increase in the metabolic acidity of spores

Ø  Endospore germination is an irreversible process.

Ø  If the spore sense unfavorable conditions after it germination, it cannot be returned to its quiescent stage, rather it perishes.

Ø  Germination of spores can be triggered by exposing the activated endospores to nutrients such as sugars or amino acids.

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(3). Out-growth

Ø  It is the third stage of endospore germination.

Ø  Spore completely emerges out from the spore coat.

Ø  The protoplast of the spore completely exposed to the outer surroundings.

Ø  They develop into an active vegetative cell.

Examples of bacteria producing endospores:

Aerobic endospore producing bacteria: Bacillus subtilis, B. megaterium, B. anthracis (cause Anthraxes). 

Anaerobic endospore producing bacteria: Clostridium perfringes, C. tetani (cause tetanus) C. botulinum (cause botulism)

Review questions:

(1).  What are endospores?
(2).  Describe the structure of bacterial endospore with a labeled diagram.
(3).  What are the main characteristics of endospores?
(4).  How bacterial endospore is different from vegetative cell?
(5).  Describe the process of endospore formation in bacteria.
(6).  Describe the process of endospore germination in bacteria.
(7).  What are the biological and pathological significance of endospores?
(8).  What are the mechanisms by which the endospores acquire resistance against heat, radiations and chemicals?
(9).  What is meant by Sporulation/Sporogenesis?
(10).  List the layers of endospore wall with its correct sequence.
(11).  Define exosporium
(12).  What is the importance of dipicolinic acid in bacterial endospore?
(13).  Give any two examples of an aerobic endospore forming bacteria
(14).  Give any two examples of anaerobic endospore forming bacteria
(15).  What are SASPs? What is its importance in bacterial endospore?

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