Bacterial Endospore

One of the most common  mechanisms for bacteria against environmental stress is forming endospores. Bacterial spores are the most dormant form of bacteria. They exhibit minimum metabolism, respiration and enzyme production. 

Typically, Gram-positive bacteria are best known for producing intracellular spores called endospores. Endospores are highly refractile (scatter light) and thick-walled structures formed inside the bacterial cells. It is most common for Bacillus species as well as Clostridium species to create endospores. 

Bacterial spores help them to overcome adverse environmental conditions, whereas fungal spores are reproductive in nature.

Endospore is named so, since it is seen within vegetative bacterial cells. It is a dormant and highly resistant structure which helps to preserve the cell's genetic material in times of extreme stress.

Spore forming bacteria - Bacillus and Clostridium (rods), Sporosarcina (cocci).

Spores are,

•        resistant (heat, ultraviolet radiation, gamma radiation, chemical disinfectants, and desiccation) & dormant structure

•        Viable for around many thousands of years

•         Several species of endospore-forming bacteria are dangerous pathogens eg., Bacillus anthracis (Anthrax) and Clostridium tetani (Tetanus)

v Practical importance - food, industrial, and medical microbiology (sterilization problems)

•        survive boiling for an hour or more; autoclaves must be used for killing them, if at all

v Theoretical Interest - spore formation -research on the construction of complex biological structures

v In the environment, endospores aid in survival when moisture or nutrients are scarce

Visibility

Examined with light and electron microscopes

v Spores are impermeable to most stains- seen as colorless areas in bacteria treated with simple stains

v special spore stains make them clearly visible- Malachite Green (Primary stain) & Safranin (Counter stain)

v Spore position in the mother cell differs among species; useful in identification

v central, sub terminal – (close to one end ), or terminal

v  some even bulge the sporangium

                                                    

 

Spore - Structure

•        The spore is surrounded by a thin, delicate covering called the exosporium

•        Beneath exosporium, lies the spore coat - fairly thick, made of several protein layers; Impermeable to chemicals - responsible for the spore’s resistance

•        Next is the cortex, which is half the spore volume; made of  peptidoglycan (less cross-linked than in vegetative cells)

•        The spore cell wall (or core wall) inside the cortex, surrounding the protoplast or core

•        The core has the normal cell structures such as ribosomes and a nucleoid, but is metabolically inactive.

Endospore heat resistance contributed by several factors:

Ø  calcium- dipicolinic acid complex

Ø  Stabilization of DNA by acid-soluble proteins

Ø  Dehydrated protoplast

Ø  Spore coat

Ø  DNA repair and others. 

 Ø  Dipicolinic acid complexed with calcium ions - located in the core; contributes to 15% of the spore’s dry weight

•       Dipicolinic acid directly involved in spore heat resistance - ( though, heat-resistant mutants lacking dipicolinic acid now have been isolated)

Ø  Calcium aids in resistance to wet and dry heat, oxidizing agents,

•        Calcium-dipicolinate stabilizes spore nucleic acids

Ø  Specialized small, acid-soluble DNA-binding proteins in the endospore; stabilize spore DNA and protect it from heat, radiation, desiccation, and chemicals.

Ø  Dehydration of the protoplast - important in heat resistance. The cortex osmotically removes water from the protoplast-protection from both heat and radiation damage

Ø  Spore coat - protect against enzymes and chemicals such as hydrogen peroxide

Ø  DNA repair enzymes – present in spores - which perform repair during germination

Sporogenesis

Ø  Spore formation, sporogenesis or sporulation; when growth ceases due to lack of nutrients.

Ø  Requires only about 10 hours in Bacillus megaterium.

Ø  It is a complex process and may be divided into seven stages

•        DNA replicates and aligns along the axis of the cell - axial filament of nuclear material forms (stage I)

•        Inward folding of the cell membrane to enclose part of the DNA to produce the forespore septum (stage II)

•       The membrane continues to grow and engulfs the immature spore in a second membrane (stage III)

•        Cortex laid down in the space between the two membranes, and both calcium and dipicolinic acid are accumulated (stage IV)

•        Protein coats then are formed around the cortex (stage V)

•        Maturation of the spore occurs (stage VI).

•        Lytic enzymes destroy the sporangium releasing the spore (stage VII).

 

Germination of Spore

Transformation of dormant spores into active vegetative cells - as complex as sporogenesis.  Reactivation of the endospore occurs when conditions are more favourable and involves activation, germination, and outgrowth.

Even if an endospore is located in plentiful nutrients, it may fail to germinate unless activation has taken place. This may be triggered by heating the endospore.

•        Three stages: (1) activation, (2) germination, and (3) outgrowth.

•        Often an endospore will not germinate successfully, even in a nutrient-rich medium- it has to be activated

•        Activation is a reversible process that prepares spores for germination - usually results from treatments like heating

•        Germination - the breaking of the spore’s dormant state-      characterized by spore swelling, rupture or absorption of the spore coat, loss of resistance to heat and other stresses, loss of refractility, release of spore components, and increase in metabolic activity;  triggered by many normal metabolites or nutrients (e.g., amino acids and sugars)

•        Germination is followed by the third stage, outgrowth-  The spore protoplast makes new components-  emerges from the remains of the spore coat

 

Thus, germination involves the dormant endospore starting metabolic activity and thus breaking hibernation. It is commonly characterized by rupture or absorption of the spore coat, swelling of the endospore, an increase in metabolic activity, and loss of resistance to environmental stress.

Outgrowth follows germination and involves the core of the endospore manufacturing new chemical components and exiting the old spore coat to develop into a fully functional vegetative bacterial cell, which can divide to produce more cells.

Endospores can stay dormant for a very long time. For instance, endospores were found in the tombs of the Egyptian pharaohs. When placed in appropriate medium, under appropriate conditions, they were able to be reactivated.

 In 1995, Raul Cano of California Polytechnic State University found bacterial spores in the gut of a fossilized bee trapped in amber from a tree in the Dominican Republic. The bee fossilized in amber was dated to being about 25 million years old. The spores germinated when the amber was cracked open and the material from the gut of the bee was extracted and placed in nutrient medium.