Bacterial endospore: Structure, Stages of Sporulation and Germination

• Spore is a resting or dormant cell which is metabolically inactive and is produced during unfavorable conditions like nutrition deficiency, extreme temperature and pH, presence of antimicrobials etc.
• The return of favorable nutritional conditions and activation lead to the germination of spore producing a single vegetative cell.
• The process of formation of spores is called sporulation, and it occurs during the late log phase or early stationary phase.
• The average size of spore is about 0.2 µm in diameter.
• Basically there are two types of bacterial spores:
  • Endospore:
    • It is produced within the bacterial cell.
    • Bacteria like Bacillus, Clostridium, Sporosarcina, Thermoactinomyces produce endospores.
  • Exospore:
    • It is produced outside the cell.
    • Very few bacteria produce exospores. E.g. Methylosinus

Structure and properties of Endospore:

• An endospore consists of the following 5 layers:
  1. Core:
     • Core is the innermost part of the endospore and is also known as spore protoplast.
     • It consists of chromosome (nucleoid), all the components of the protein synthesizing apparatus (ribosomes and other cellular materials) in the cytoplasm surrounded by a cytoplasmic membrane.
     • It is a gel-like dehydrated state (10-25% water) and provides heat resistance to the endospore.
     • Calcium dipicolinate present in high amount (10-15% of the spore dry weight) also enhances the heat resistance of endospores.
     • Core also contains high percentage of small acid soluble proteins (SASP) which are synthesized during sporulation.
     • SASPs prevent the potential damage of core DNA from UV radiation, desiccation and drying.
     • In addition, SASPs also provide nutrition and energy for spore germination.
  2. Spore wall:
     • It is the innermost layer surrounding the core and lies outside the core cytoplasmic membrane.
     • It contains normal peptidoglycan and becomes the cell wall of the germinating vegetative cell.
  3. Cortex:
     • Cortex lies outside the spore wall.
     • It is the thickest layer of the spore envelope.
     • Cortex contains an unusual layer of peptidoglycan; with many fewer cross links than are found in cell wall peptidoglycan.
     • Cortex peptidoglycan is comprised of alalnine (55%), tetra-peptide (15%) and muramic lactam (30%).
     • It is extremely sensitive to lysozyme, and its autolysis plays a role in spore germination.
  4. Spore coat:
     • Spore coat is a thick double layered membrane that encloses the cortex.
     • It consists of spore specific keratin like proteins containing many intra-molecular disulfide bonds.
     • The proteins are rich in cysteine and hydrophobic amino acids.
     • Due to the presence of these amino acids, spores are resistant to adverse environmental conditions including antibacterial chemical agents.
  5. Exosporium:
     • It is the outermost layer covering the spore coat.
     • It is made up of lipoprotein and some carbohydrate.

Different stages of sporulation:

• The sporulation process begins when environmental conditions become unfavorable.
• Deficient carbon and nitrogen source, extreme pH and temperature, antimicrobials trigger and support this process.
• There are several stages in the formation of an endospore. In Bacillus subtilis, the entire process of sporulation completes in 8 stages: stage 0 to stage VII and takes about 7-8 hours to complete.

1. Stage 0: Normal vegetative cell
   • This is a normal condition of a vegetative cell prior to sporulation.
2. Stage I: Axial filament formation
   • The bacterial chromosome becomes long thread-like called axial filament.
   • Mesosomes attach these axial filaments to the cytoplasmic membrane.
   • The bacterial cell elongates.
   • Bacillus subtilis has a reserved food material, para-hydroxy benzoic acid (PHBA), which is utilized for sporulation.
3. Stage II: Asymmetric septation and formation of forespore
   • The cell divides asymmetrically as the cell membrane undergoes in-folding near one end to produce a small double membrane structure called forespore.
   • The forespore encloses a small portion of DNA.
4. Stage III: Engulfment of forespore
   • The mother cell membrane grows continuously towards and around the forespore and engulfs it.
   • The result is a double-membrane bound forespore in the mother cell cytosol.
5. Stage IV: Cortex synthesis
   • Forespore starts forming primordial cortex between the two membranes.
   • Chromosome of the mother cell disintegrates and the cell dehydrates.
   • Specialized peptidoglycan is synthesized that resides between the two membrane layers surrounding the forespore; outer cortex and inner germ cell wall.
   • The synthesis of exosporium occurs.
6. Stage V: Coat deposition
   • Dipicolinic acid and SASPs are synthesized in the spore core, which later on incorporate together to form calcium dipicolinate.
   • The cytoplasm undergoes further dehydration.
   • An outer shell called coat is deposited outside the cortex forming a coat layer.
7. Stage VI: Maturation
   • In the newly isolated cytoplasm, or core, many vegetative cell enzymes are degraded and are replaced by a set of a set of unique spore constituents.
   • The endospore matures.
8. Stage VII: Release of endospore
   • The mother cell undergoes lysis and releases a matured endospore into the environment.

Germination of endospore:

• Bacterial endospores germinate to vegetative cells when placed in favorable condition, but most endospores cannot germinate after they have formed.
• They either need to remain dormant for some time or get activated before germination.
• The processes involved in germination of endospores are:
  1. Activation:
     • The endospore is activated in a nutritionally rich medium.
     • The spore coat must be damaged for which agents like heat, abrasion, acid and compounds containing free sulf-hydryl groups are used.
  2. Initiation:
     • Once activated, a spore will initiate germination if the environmental conditions are favorable.
     • Initiation is triggered by binding of endospore receptors with the effector molecules present in the medium.
     • Binding of effector activates autolysin that rapidly degrades the cortex peptidoglycan.
     • Water is taken up, calcium dipicolinate is released, and a variety of spore constituents are degraded by hydrolytic enzymes.
  3. Outgrowth:
     • Uptake of water makes the endospore swell.
     • A period of active biosynthesis follows, where DNA, RNA and proteins are synthesized.
     • A new germ cell emerges out breaking the spore coat and becomes a vegetative cell.
     • The vegetative cell grows and finally terminates in cell division with continuous supply of essential nutrients.

Bacterial endospore: Structure, Stages of Sporulation and Germination

References:

1. https://www.brainkart.com/article/Sporulation—Structure-and-Functions-of-Bacterial-Cell-Envelope_17830/
2. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC107777/
3. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4078662/