Bacterial endospore: Structure, Stages of Sporulation and Germination

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  • 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:

                             Bacterial Endospore
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  • 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.
                                                              Stages of Sporulation
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  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