Bacterial Endospore

Endospores

Some bacteria produce endospores within their cell by a process called sporulation. Endospore-producing bacteria occur most commonly in the soil and the genera Bacillus and Clostridium are the best studied of endospore-producing bacteria. These spores are extraordinarily resistant to environmental stresses such as heat, ultraviolet radiation, gamma radiation, chemical disinfectants, and desiccation and can remain dormant for extremely long periods of time. Endospores are of great practical significance in food, industrial and medical microbiology due to their resistance and dangerous pathogenic nature of several species of endospore producing bacteria. This is because it is essential to develop adequate methods to sterilize solutions and solid objects.

(i) Structure

The endospore (so named because of its formation within the cell), which is readily seen under the light microscope as strongly refractile bodiesdue to being very impermeable to usual dyes (e.g., methylene blue), is structurally much more complex in that it possesses many layers that are absent in vegetative cells The outermost layer is exosporium, a thin delicate covering made of protein. Beneath the exosporium, there is a thick spore-coat consisting of several protein layers which are spore-specific. The spore-coat is impermeable and responsible for the spores resistance to chemicals.

Bacterial Endospores

A. Intracellular locations of endospores (a = termianl, b = subterminal, c = Central).

B. Diagrammatic Sketch of the sectiones endospore of Bacillus anthracis showing different parts.

1.	Exosporium	5.	Core Wall
2.	Outer Coat Layer	6.	Core Membrane
3.	Inner Coat Layer	7.	Core
4.	Coretx	8.	Spore-Coat

Below the spore-coat is the cortex which may occupy as much as half the spore volume. Cortex consists of loosely cross-linked peptidoglycan. Inside the cortex, there is the core-wall which surrounds the core membrane and the core or spore protoplast. The latter possesses cytoplasm, nucleoid, ribosomes etc. but is metabolically inactive.
The core or spore protoplast of a mature endospore contains abundant dipicolinic acid and calcium ions normally existing in the form of calcium-dipicolinate complex and is in a partially dehydrated state as it contains only 10-30% of the water content of the vegetative cell. Because of it, the consistency of the core cytoplasm is that of a thick gel.

In addition to low water content, the pH of the core cytoplasm is about one unit lower than that of the vegetative cell and contains high levels of core-specific proteins, namely, small acid-soluble spore proteins (SASPs). SASPs are considered to perform at least two important functions: (i) they bind tightly to DNA in the core and protect it from ultraviolet radiation, dessication and dry heat and (ii) they function as a carbon and energy source at the time of endospore-germination to give rise to new vegetative cell

A. Structure of sipicolinic acid (DPA)	B. Cross-linking of Ca++ to DPA form calcium-dipicolinate complex

(ii) Formation of Endospore

Endospore formation (called sporulation or sporogenesis) involves a very complex series of events in cellular differentiation. Endospore formation takes place only in such a vegetative cell which ceases growth due to lack of nutrients. The complex process of sporulation can be divided into seven stages (I to VII).

(iii) Endospore Resistance

Bacterial endospores can retain viability for many years. A few viable endospores of Bacillus subtilis and B. licheniformis were found in the soil attached to plants that had been stored under dry conditions at the Kew Gardens Herbarium for 200-300 years. Endospores can even retain viability for millennia, and viable endospores have been found in geological deposits where they must have been dormant for thousands of years. What factors are responsible for such prolonged viability of endospores? It has long been thought that dipicolinic acid was directly involved in heat-resistance of endospore, but heat-resistant mutants now have been isolated in which dipicolinic acid is absent.

(iv) The Endospore Core and SASPs

Although both contain a copy of the chromosome and other essential cellular components, the core of a mature endospore differs greatly from the vegetative cell from which it was formed. Besides the high levels of calcium dipicolinate, which help reduce the water content of the core, the core becomes greatly dehydrated during the sporulation process. The core of a mature endospore has only 10–25% of the water content of the vegetative cell, and thus the consistency of the core cytoplasm is that of a gel. Dehydration of the core greatly increases the heat resistance of macromolecules within the spore. Some bacterial endospores survive heating to temperatures as high as 1508C, although 1218C, the standard for microbiological sterilization (121°C is autoclave temperature), kills the endospores of most species. Boiling has essentially no effect on endospore viability. Dehydration has also been shown to confer resistance in the endospore to chemicals, such as hydrogen peroxide (H₂O₂), and causes enzymes remaining in the core to become inactive. In addition to the low water content of the endospore, the pH of the core is about one unit lower than that of the vegetative cell cytoplasm. The endospore core contains high levels of small acid-soluble proteins (SASPs). These proteins are made during the sporulation process and have at least two functions. SASPs bind tightly to DNA in the core and protect it from potential damage from ultraviolet radiation, desiccation, and dry heat. Ultraviolet resistance is conferred when SASPs change the molecular structure of DNA from the normal “B” form to the more compact “A” form. A-form DNA better resists pyrimidine dimer formation by UV radiation, a means of mutation, and resists the denaturing effects of dry heat. In addition, SASPs function as a carbon and energy source for the outgrowth of a new vegetative cell from the endospore during germination.

(iv) Germination

The conversion of endospore into active vegetative cell appears a complex process and involves three steps: activation, germination and outgrowth. Activation is the process that prepares endospore for germination. It is most easily accomplished by heating at sublethal but elevated temperature. An activated endospore undergoes germination which is characterized by swelling and rupture or absorption of spore-coat, loss of dipicolinic acid, degradation of small acid-soluble spore proteins (SASPs), loss of resistance to heat and other stresses, loss of refractility, and enhancement in metabolic activity.The final stage is the outgrowth which involves visible swelling due to water uptake and synthesis of new RNA, DNA and proteins. The spore protoplast emerges from the broken spore-coat, develops into an active bacterial cell and begins to divide.

(v) Differences between Endospore and Vegetative Cell

A mature endospore differs greatly from the vegetative cell from which it was formed. These differences are given in.

Differences between Endospore and the Vegetative Cell

Characteristic	Endospore	Vegetative Cell
(i) Structure	Thick spore-cortex, spore-coat, exosporium	Typical gram (+) cell; a few gram (-) cells
(ii) Microscopic appearance	Refractile	Non-refractile
(iii) Dipicolinic acid	Present	Absent
(iv) Calcium content	High	Low
(v) Water content	Low (10-30% in core)	High (80-90%)
(vi) Heat resistance	High	Low
(vii) Radiation resistance	High	Low
(viii) Chemical resistance	High	Low
(ix) Enzymatic activity	Low	High
(x) Synthesis of macromolecules	Absent	Present
(xi) Messenger-RNA	Low or absent	Present
(xii) Oxygen-uptake	Low	High
(xiii) Lysozyme effect	Resistant	Sensitive
(xiv) Small acid-soluble proteins (SASPs)	Present	Absent
(xv) pH of cytoplasm	About 5.5-6.0	About 7

Then, if not DPA, what factors make endospore so resistant to heat and other lethal agents. It is now being believed, however, that there are several factors probably involved in endospore resistance. These are: calcium-dipicolinate and acid-soluble protein stabilization of DNA, protoplast dehydration, the spore-coat, DNA-repair, and the greater stability of cell proteins in bacteria adopted to growth at high temperatures.