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. 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:
a. Endospores- Form inside the vegetative cell.
b. 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 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 includes Bacillus subtilis, Bacillus polymyxa, Bacillus licheniformis, Clostridium, Sporolactobaciltus, Thermoactinomycetes.
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 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 spore. It has inner and outer cortex membrane. It contains 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 high percentage of cysteine, keratin and hydrophobic amino acids. 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 outer loose envelope surrounds endospore known as exosporium. The exosporium is a lipoprotein membrane containing some carbohydrates.
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
- Sporulation process starts with unusual event of formation of axial filament of nuclear material which is usually present in dispersed form. Bacterial chromosome becomes thread like known as axial filament.
- Axial filaments attached to cytoplasmic membrane by mesosome.
- Elongation of cell take places.
- 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 formation of axial filament unequal division of cell takes place due to inward folding of 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 completion of sporulation.
Stage III: Engulfment of forespore
- Mother cell membrane grows around the forespore engulfing it.
- Forespore derives one more membrane called outer core wall.
- Thick layers of peptidoglycan are laid down between the two membrane layers.
Stage IV: Synthesis of exosporium
- Chromosome of mother cell disintegrates.
- Exosporium synthesis occurs.
- Forespore starts forming primodial cortex between two membranes which fills 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 occur forming calcium dipicolonate.
- Further dehydration of cytoplasm. At this stage metabolic activity is very less as compared to vegetative cell and spore starts appearing as refractile body.
- Multi-layered lamellar structure fuses to form 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 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 release of endospore takes place.
- The released highly dehydrated endospore core contains only DNA, small amount of RNA, ribosomes, enzymes and a few important small molecules and contain large amount of organic acid called dipicolinic acid (found in cytoplasm), which is accompanied by large number of calcium ions. These cellular components are essential for resuming metabolism later.
- 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.