Bacterial Spores (endospore)
Endospores form within the Cell
Endospore is dormant stage of some bacterium that allows it to survive unfavorable conditions that would normally be lethal such as extreme drought or heat
Endospores are resistant against;
Low nutrient conditions
Various chemical disinfectants
The spore is a dehydrated, multishelled structure that protects and allows the bacteria to exist in “suspended animation”
It contains a complete copy of the chromosome, the bare minimum concentrations of essential proteins and ribosomes, and a high concentration of calcium bound to dipicolinic acid.
The Vegetative Cell Gives Rise to One Spore
Endospores are ultimately protection for the bacterial genome
Spores form within the cell and contain a full copy of the bacterial genome
Endospores are not a form of reproduction, because only one new cell germinates from each spore
Spores can be variable in size and location within the cell
Sporulation or Sporogenesis
Process of endospore formation within a vegetative (parent) cell
Germination = return of an endospore to its vegetative state
The sporulation process begins when nutritional conditions become unfavorable, depletion of the nitrogen or carbon source (or both) being the most significant factor.
Sporulation occurs massively in cultures that have terminated exponential growth as a result of such depletion.
Sporulation involves the production of many new structures, enzymes, and metabolites along with the disappearance of many vegetative cell components.
These changes represent a true process of differentiation. A set of genes whose products determine the formation and final composition of the spore are activated, while another subset of genes involved in vegetative cell function are inactivated.
These changes involve alterations in the transcriptional specifity of RNA polymerase, which is determined by the association of the polymerase core protein with one or another promoter-specific protein called a sigma factor. Different sigma factors are produced during vegetative growth and sporulation.
Morphologically, sporulation begins with the isolation of a terminal nucleus by the inward growth of the cell membrane.
The growth process involves an infolding of the membrane so as to produce a double membrane structure whose facing surfaces correspond to the cell wall-synthesizing surface of the cell envelope. The growing points move progressively toward the pole of the cell so as to engulf the developing spore.
The two spore membranes now engage in the activity synthesis of special layer that will form the cell envelope:
the spore wall and cortex, lying between the facing membranes, and the coat and exosporium lying outside the facing membrane.
In the newly isolated cytoplasm, or core, many vegetative cell enzymes are degraded and are replaced by a set of unique spore constituents.
Differences between Endospores and Vegetative Cells
Not all bacterial species can form spores
A few genera of bacteria produce endospore such as Clostridium (gangrene) and Bacillus (anthrax), both of them are gram + rods
Endospore production is associated with Gram Positive bacteria
Since not all bacteria form endospores, we can use this as an identification factor
The shape of the spore is an identifying characteristic
Swelled vs. Not swelled
The location of the spore is also an identifying characteristic
Central, Sub-Terminal, and Terminal spores
Endospores can remain dormant indefinitely but germinate quickly when the appropriate trigger is applied
Endospores differ significantly from the vegetative, or normally functioning, cells
Some spore forming bacteria are capable of causing disease
Clostridium botulinum – botulism
Clostridium perfingens – gas gangrene
Clostridium tetani – tetanus
Bacillus anthracis – Woolsorter’s Disease and wound infections
The Schaeffer-Fulton Stain Procedure is used to differentiate between endospores and vegetative cells
Schaeffer-Fulton Stain Procedure
Make a smear. Air Dry. Heat fix
Flood the smear with Malachite Green stain
Cover the flooded smear with a square of filter paper
Steam slide for 10 minutes (every minute, add a few more drops of Malachite Green stain)
Allow slide to cool (after the 10 min. steam process)
Schaeffer-Fulton Stain Procedure (cont’d)
Drain slide and rinse for 30 seconds with DI water (discard filter paper)
Put slide on steam rack
Flood smear with Safranin (counter stain). This stains the vegetative cell. (Leave for 1 minute)
Drain the slide and rinse with DI water
Use oil immersion objective to view
Endospore Stain Example
Cell: Red or Pink
Activation by heat and nutrients
Ca-dipicolinate and cortex components disappear
Swelling with H2O
Cell begins to divide like normal
Bacillus anthracis (and Clostridium) produces endospores
Easily aerosolized and spread
Relatively easy and inexpensive to prepare in laboratory
Can be easily transported without detection
by John Tyndall (1820-1893)
Boil for 15 min
Keep in warm, humid environment for 1 d
Boil for 15 min
Keep in warm, humid environment for 1 d
Boil for 15 min
Properties of endospores
The core is the spore protoplast.
It contains a complete nucleus (chromosome), all of the components of the proteins-synthetizing apparatus, and an energy-generating system based on glycolysis. Cytochromes are lacking even in aerobic species, the spores of which rely on shorted electron transport pathway involving flavoproteins. A number of vegetative cell enzymes are increased in amount (eg. alanine racemase), and a number of unique enzymes are formed (eg. dipicolinic acid synthetase).
The energy for germination is stored as
3-phosphoglycerate rather than as ATP.
The heat resistance of spores is due in part to their dehydrated state and in part to the presence in the core of large amounts (5 – 15% of the spore dry weight) of calcium dipicolinate, which is formed from an intermediate of the the lysine biosynthetic pathway.
In some way not yet understood, these properties result in the stabilization of the spore enzymes, most of which exhibit normal heat lability when isolated soluble form.
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.
The cortex is the thickest layer of the spore envelope.
It contains an unusual type of peptidoglycan, with many fewer cross-links than are found in cell wall peptidoglycan.
Cortex peptidoglycan is extremly sensitive to lysozyme, and its autolysis plays a key role in spore germination.
The coat is composed of a keratin-like protein containing many intramolecular disulfide bonds.
The impermeability of this layer confers on spores their relative resistance to antibacteral chemical agents.
The exosporium is a lipoprotein membrane containing some carbohydrate.
The germination process occurs in three stages:
Even when placed in an environment that favors germination (eg. nutritionally rich medium) bacterial spores will not germinate unless first activated by one or another agent that damages the spore coat.
Among the agents that can overcome spore dormancy are heat, abrasion, acidity, and componds containing free sulfhydryl groups.
Once activated, a spore will initiate germination if the environmental conditions are favorable.
Different species have evolved receptors recognise different effectors as signaling a rich medium.
Binding of the effector activates an 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.
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.
A period of active biosynthesis follows. This period, which terminates in cell division, is called outgrowth.
Outgrowh requires a supply of all nutrients essenial for cell growth.
The spore stain
Spores are most simply observed as intracellular refractile bodies in unstained cell suspensions or as colorless areas in cell stained by conventional methods.
The spore wall is relatively impermeable, but dyes can be made to penetrate it by haeting the preparation.
The same impermeability then serves to prevent decolorization of the spore by a period of alcohol treatment sufficient to decolorize vegetative cells. The latter can finally be counterstained. Spores are commonly stained with malachite green or carbolfuchsin.