Endospore: Structure, Components and Sporulation process

Structure of Endospore

Spores are unicellular structures. They are highly resistant, dormant metabolically inactive resting structures formed in response to adverse environmental conditions such as lack of nutrients, environmental stress, etc. A spore is a refractile body formed by a vegetative mother cell. They do not have a role in reproduction. Vegetative cells of endospore-forming bacteria begin sporulation when a key nutrient, such as the carbon or nitrogen source, becomes scarce or unavailable. Endospores are highly durable dehydrated cells with thick walls and additional layers. Endospores enable bacteria to lie dormant for extended periods, even centuries. There are many reports of spores remaining viable over 10,000 years.

There are two types of spores:

  1. Endospores- Form inside the vegetative cell.
  2. Exospores – Form in either one of the ends of the vegetative cell.

The diameter of the endospore may be the same as, smaller than, or larger than the diameter of the vegetative cell. Depending on species, the endospore might be located terminally (at one end), sub- terminally (near one end), or centrally inside the vegetative cell. Bacteria show central bulging or non-bulging spores. These are highly resistant to chemical disinfectants, low or high temperature as well as radiations. Sporulating bacteria include Bacillus subtilis, Bacillus polymyxa, Bacillus licheniformis, Clostridium, Sporolactobaciltus, Thermoactinomycetes.

Structure of endospore

Components of Endospores:

1. Core/Protoplast: The core is the spore protoplast. It contains a complete nucleus (chromosome), all the components of the protein-synthesizing apparatus including ribosomes, and an energy-generating system based on glycolysis. Several unique enzymes are formed (e.g., dipicolinic acid synthetase). Spores contain no ATP. The energy for germination is stored as 3-phosphoglycerate rather than as ATP. It takes up a large amount of Ca ions which chelates with dipicolinic acid to form Calcium dipicolinate.

2. Spore germ cell wall: The innermost layer surrounding the inner spore membrane is called the spore wall. It contains normal peptidoglycan and becomes the cell wall of the germinating vegetative cell.

3. Cortex: The cortex is the thickest layer of the spore envelope, accounts for half volume of the spore. It has an inner and outer cortex membrane. It contains a modified form of peptidoglycan. Cortex peptidoglycan makes the spore resistant.

4. Coat: Around the cortex, a layer of spore coat is present which contains proteins with a high percentage of cysteine, keratin, and hydrophobic amino acids. The spore coat contains two layers inner and outer coat. The impermeability of this layer gives spores their relative resistance to antibacterial chemical agents.

5. Exosporium: In some bacteria, the outer loose envelope surrounds the endospore known as exosporium. The exosporium is a lipoprotein membrane containing some carbohydrates.

Sporulation Process:

The process of endospore formation within a vegetative cell takes several hours and is known as sporulation or sporogenesis.

Stage I: Axial filament formation stage

  • The sporulation process starts with an unusual event of formation of the axial filament of nuclear material which is usually present in dispersed form. The bacterial chromosome becomes thread-like known as axial filament.
  • Axial filaments are attached to the cytoplasmic membrane by mesosome.
  • Elongation of the cell takes place.
  • PHBA is the reserved food material in Bacillus spp. is utilized in sporulation.
  • In the first stage of sporulation, a newly replicated bacterial chromosome and a small portion of cytoplasm are isolated by an ingrowth of the plasma membrane called a spore septum.

Stage Il: Forespore formation

  • After the formation of axial filament unequal division of cell takes place due to inward folding of the cytoplasmic membrane at one pole which is known as invagination.
  • The spore septum becomes a double-layered membrane that surrounds the chromosome and cytoplasm. This structure, entirely enclosed within the original cell, is called a forespore.
  • Most of the water present in the forespore cytoplasm is eliminated during the completion of sporulation.

Stage III: Engulfment of the forespore

  • Mother cell membrane grows around the forespore engulfing it.
  • Forespore derives one more membrane called an outer core wall.
  • Thick layers of peptidoglycan are laid down between the two membrane layers.

Stage IV: Synthesis of the exosporium

  • The chromosome of the mother cell disintegrates.
  • Exosporium synthesis occurs.
  • Forespore starts forming primordial cortex between two membranes which fills the gap between two membranes.
  • Dehydration of cell.

Stage V: Synthesis of dipicolonic acid

  • Production of SASPs (small acid-soluble spore proteins) and dipicolinic acid occurs.
  • Incorporation of calcium ions with dipicolonic acid occurs forming calcium dipicolonate.
  • Further dehydration of cytoplasm. At this stage, metabolic activity is very less as compared to vegetative cells and spore starts appearing as the refractile body.
  • Multi-layered lamellar structure fuses to form a thick spore coat.
  • Deposition of spore coat proteins and accumulation of cystine leads to completion of spore coat structure. This coat is responsible for the resistance of endospores to many harsh chemicals.

Stage VI: Maturation

  • At this stage, cortical peptidoglycan synthesis continues forming more homogenous protoplast. Protoplast becomes an electron-dense structure and spore coat synthesis gets completed which makes it heat resistant and refractile.

Stage VII: Release of endospore

  • When the endospore matures, the vegetative cell wall ruptures,  and original cell is degraded, and the release of the endospore takes place.
  • The released highly dehydrated endospore core contains only DNA, a small amount of RNA, ribosomes, enzymes, and a few important small molecules and contains a large amount of organic acid called dipicolinic acid (found in the cytoplasm), which is accompanied by a large number of calcium ions. These cellular components are essential for resuming metabolism later.

Spore Germination:

Bacteria remain dormant by forming spores for years. This dormancy may be retained for years but this condition may be broken up due to changes in the environmental conditions. Under favorable conditions, each endospore germinates to give rise to a vegetative cell. The transformation of dormant spores into vegetative cells is called germination.
The germination process occurs in three stages: activation, initiation, and outgrowth.
  • Activation: Endospores cannot germinate immediately after they have formed, but they can germinate after they have rested for several days. They need certain conditions to be activated.
  • Initiation: Once activated, a spore will initiate germination if the environmental conditions are favorable. Autolysin will be activated, and it will rapidly degrade the cortex peptidoglycan. Water is taken up, calcium dipicolinate is released, and a variety of spore constituents are degraded by hydrolytic enzymes.
  • Outgrowth: Degradation of the cortex and outer layers results in the emergence of a new vegetative cell consisting of the spore protoplast with its surrounding wall. Now, using nutrients around, the cell can multiply again.

Significance of Endospores:

Endospores are important from the clinical viewpoint and in the food industry because they are resistant to processes that normally kill vegetative cells. Such processes include heating, freezing, desiccation, use of chemicals, and radiation. Most vegetative cells are killed by temperatures above 70°C, endospores can survive in boiling water for several hours or more. Endospores of thermophilic bacteria can survive in boiling water for 19 hours.