Sporulation endospore formation in Grampositive bacteria

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Sporulation (endospore formation in Gram-positive
bacteria

• Typical feature of some Gramve bacteria such as
  Bacillus, Clostridium and Sporosarcina, including
  the pathogenic bacteria Bacillus anthracis (cause
  of anthrax), Clostridium botulinum (botulism),
  Clostridium tetani (tetanus), Clostridium
  perfringerns (gas gangrene and food poisoning),
  and Clostridium difficile (antibiotic-associated
  and pseudomembranous colitis)
• Not found in Gram-ve bacteria

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Endospore
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Sporulation A survival strategy

• Sporulation usually occurs as a response to
  adverse environmental conditions. In the
  laboratory, sporulation can be induced by
  starvation for a carbon, nitrogen, and/or
  phosphorus source. In these conditions,
  sporulation begins immediately following
  exponential growth
• Sporulation is not a reproductive process, that
  is 1 vegetative cell 1 spore. When
  environmental conditions return to to normal, one
  spore will transform into one vegetative cell
  which then undergoes cell division

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A survival strategy

• Spores are metabolically dormant and highly
  resistant to a variety of environmental insults,
  including UV and gamma radiation, free radicals,
  high and low temperature, acid and alkali
  conditions, hydrolytic enzymes, and organic
  solvents. For example, spores can survive at
  120oC,which kills all bacterial cells
• Spores can remain dormant for long periods of
  time (perhaps indefinitely) but will germinate
  when they encounter a nutrient-rich environment

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Growth phase of bacteria
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Features of Spores

• Spores can be seen with a light microscope due to
  the high protein levels causing a high
  refractivity
• Spore represents almost the entire dry mass of
  the cell although it occupies only 10 of the
  volume of the vegetative cell

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Spores under light microscope
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Spore structure
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Spore core
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Spore Structure

• Spore coat
• The outmost structure common to spores of all
  species
• Consists of a series of one or more
  morphologically distinct layers that protect
  spore from a variety of toxic molecules and from
  mechanical damage
• Is largely protein, e.g., 20 proteins in B.
  subtilis

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Spore structure

• Spore cortex
• Cortex surrounds the spore core and is composed
  predominantly of peptidoglycan (PG) with a
  different structure from that of vegetative cell
  wall
• 50 of N-acetyl muramic acid (NAM) residues in
  the cortex are present as muramic acid ?-lactam
  (MAL)
• 25 of NAM residues carry only a single
  L-alanine, therefore there are only 25 of many
  DAP residues available to participate cross-link
  formation in spore contex
• Total cross-linking in cortex PG is 3, 10- to
  12- fold below that is vegetative cell wall PG
  (35)
• Cortex is important for maintenance of spore core
  dehydration, which increases spore wet-heat
  resistance

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Core wall

• Immediately underlying the cortex is a second
  layer of PG with a different structure from that
  of cortex. This second PG layer makes up only a
  small amount of total spore PG is the spores
  nascent, or germ cell wall (core wall).
• Core wall PG is similar to that of vegetative
  cell wall
• Cortex is targeted for degradation early in spore
  germination, the germ cell wall is not degraded
  and provides the template for new cell wall
  synthesis during spore outgrowth
• Unlike the walls of growing cells, in which
  teichoic acids are a major component, teichoic
  acids are not present in spores

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Spore core

• Contains cytoplasmic membrane, cytoplasm and
  nucleoid
• The core cytoplasm
• Is highly dehydrated- the water content is 1/3
  of growing cells
• Contains high concentration of dipicolinic acid
  (DPA) and calcium, which forms calcium
  dipicolinic acid. The presence of DPC is
  associated with low water contents and heat
  resistance
• pH is 1 unit lower (6.3-6.5) than that in
  growing cells (7.5-8.2)
• Spore nucleoid is highly condensed and bound by
  SASPs (small acid-soluble spore proteins), which
  protect DNA from potential damage from UV
  radiation, desiccation and dry heat

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Differences between endospores and vegetative
cells
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Spore formation

• One of the most complex differentiation processes
  in bacteria
• gt200 genes are involved
• Spore formation is a highly controlled process,
  many checkpoint mechanisms exist to ensure that
  sporulation initiates only under conditions in
  which cells are capable of faithfully replicating
  and segregating their chromosomes

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Initiation of sporulation

• Initiation of spore formation requires multiple
  conditions or signals, including nutrient
  deprivation, high cell density, the absence of
  DNA damage
• Multiple environmental and physiological signals
  control the activation of Spo0A, which is
  required for the initiation of sporulation

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Spo0AP

• Activates genes required for later events in
  sporulation
• Represses genes involved in stationary-phase
  growth
• A phosphorelay pathway produces Spo0AP, which
  initiates sporulation

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