Spores and Significance

Spores in the food industry

Members of the gram-positive Bacillus and Clostridium spp.

• Spore forming bacteria and heat- resistant fungi cause problems

for food industry
• respond to slow growth/starvation by initiating sporulation
• Spore formers that produce toxins
– Clos. perfringens, Clos. Botulinum, Bacillus cereus
• The spore formers cause foodborne illness and spoilage:
• of low acid food pH > 4.6 - packaged in cans, bottles, pouches
and canned food (vacuum sealed) – processed by heat
• Some cause spoilage of high acid food pH < 4.6
• Psychrotrophic spore formers cause spoilage of refrigerated
food
• Fungi that produce heat resistant ascospores cause spoilage of
acidic food and beverages
 Spores in the food industry

• Spore-forming bacteria were discovered by Louis Pasteur

– caused food spoilage when he was investigating the butyric
acid fermentation in wine
– He isolated Clos. butyricum
• Robert Koch also discovered spores
• Pasteur and Koch linked spores to food safety
• Appert invented the process appertization – now canning
– placed food in air tight containers and preserved it by
heating
– He thought that removal of air was responsible for stability
in canned food
– Thermal sealing developed to become major industry
– Thermal resistance spores are responsible for food
spoilage in thermally sealed cans C, botulinum type A, B
 Spore forming Bacteria

• The primary sporeformers of significance in foods are:

• Bacillus, Clostridium, Anoxybacillus, Desulfotomaculum,
Geobacillus, Paenibacillus, and Sporolactobacillus.
• Sporeformers are classified in phylum XIII of the Firmicutes
based on sequencing of genes encoding small-subunit rRNA.
• sporeformers are of low DNA mol% G+C
• possess a gram-positive cell wall structure.
• food-related bacteria classified in the Firmicutes include:
Listeria, Staphylococcus, Streptococcus, and Lactobacillus
– it has been hypothesized that these genera have lost the
ability to sporulate during evolution
 Bacteriology of sporeformers of public
health significance
• Three species that cause foodborne illness:
– Clostridium botulinum, C. perfringens B. cereus
– Also Bacillus anthracis – not foodborne but intestinal
anthrax incidents due to ingestion of contaminated raw or
poorly cooked meat
• C. botulinum
– Heterogeneous species that produce distinct toxins A-G
(species specific)
– Divided into 2 groups:
– Type I: mesophiles, produce heat resistant spores, and
proteases that cause spoilage
– Type II: grow at lower temps, non proteolytic, spores are
less heat resistant
 Bacteriology of sporeformers of public
health significance
• C. perfringens
– Wide spread in soil and the GI of certain animals and humans
– Grow rapidly in hi protein food (meats), especially meats that
have been cooked but inadequately cooled
– Generation time of 6-9 minutes in warm foods at 43 -45o C
– Produces an enterotoxin that causes diarrhea
– Produces 2 types of spores:
• Heat resistant with D90o C = 15 -45 minutes
• Heat sensitive spores with D90o C = 3 – 5 minutes
• Both survive during cooking
• Heat shock of 75o to 100o C allows spores to germinate
 Bacteriology of sporeformers of public
health significance
• Genus Bacillus contains
– B. anthracis
– B. cereus
• Produces heat labile toxin that causes diarrhea
• Produces heat stable toxin that causes vomiting
(emetic toxin)
• It must grow to high numbers in food to cause
illness (1x106 cells/g food)
• Spores germinate at the range of 8-30o C
• D95oC = ~24 min
Spore Structure
 Spore Structure

•Exosporium: outermost layer, prominent in spores of B. cereus, B.

anthracis, and some Clos. species,
•may not be present in spores of B. subtilis
•composed of lipid, carbohydrate, protein, glycoprotein
•spore coats: under the exosporium
•composed primarily of protein
•Protect spore PG from attack by lytic enzymes and chemicals. the coats
has no major role in resistance to heat or radiation
•Outer forespore membrane:
•Underlying the coats
•Composed of proteins that is different from that of the inner forespore
membrane.
 Spore Structure

• Cortex Underlying the outer forespore membrane

• PG layer structurally similar to CW PG but with differences, it
contains diaminopimelic acid
• vegetative cell PG contains lysine
• ~65% of the muramic acid in cortical PG lack peptide residues
• responsible for the dehydration of the spore core and for spore
dormancy and resistance
• Germ cell wall and germ CW PG –
– between the cortex and inner forespore membrane
– PG is similar to PG of vegetative cell
• inner forespore membrane - a functional membrane
• extremely strong permeability barrier slowing entry of all molecules
including water into the core
• It has an immobile lipid structure
• phospholipid content is similar to growing cells
 Spore Structure

• Core - the central

• contains DNA, ribosomes, and most enzymes, as well as DPA and
most divalent cations.
•large SASP pool much of which is bound to spore DNA
• the spore core has very low water content
•The core’s low water content plays a major role in spore dormancy
and resistance to various of agents.
•The low water content is the reason for the immobility of ions and
protein in the core
•the water content in other regions of the spore is similar to that
in growing cells.
• Dormant spores germinate if stimulated often by a sugar or
amino acid
• Within minutes of exposure to a germinant, spores lose their
characteristics:
• loss of DPA (excreted)
• loss of the cortex and SASP ( degradation) and loss of spore
resistance
• Completion of germination allows progression into outgrowth,
when metabolism of endogenous and exogenous compounds
begins and macromolecular synthesis is initiated
• Eventually the outgrowing spore is converted back into a growing
cell
 Induction of Sporulation

• In the laboratory
– by nutrient limitation by exhaustion of nutrients during
growth or shifting from a rich to a poor medium.
– Addition of an inhibitor of guanine nucleotide (decoyinine)
• Massive sporulation takes place when cells enter stationary
phase. Stationary-phase events include:
• (i) synthesis and secretion of degradative enzymes such as
amylases and proteases
• (ii) synthesis and secretion of antibiotics such as gramicidin or
bacitracin
• (iii) in some species, synthesis and release of protein toxins
• (iv) development of motility
• (v) killing and cannibalism of sister cells in the population
Bacterial spore structure
• The Spore, Characteristics
– a dehydrated, multilayered structure
– spore is metabolically inert
– remain stable for months to years
• Contains:
– Complete copy of the chromosome
– minimum amount of essential proteins and
ribosomes
– high concentration of calcium bound to
dipicolinic acid
 The Spore

• Dormancy
– Spores have no metabolic activity – due to low water content
– No enzyme action
– Long term survival due to extreme resistance to heat,
radiation, chemicals, desiccation
– Spores resistance is due to
• Low water in the core – dehydrated state
• SASP
• impermeability
 Sporulation

•Sporulation has been most extensively studied in B. subtilis

•The first obvious morphological event in sporulation is an unequal
cell division.
•This creates the smaller prespore or forespore compartment and
the larger mother cell compartment.
•As sporulation proceeds, the mother cell engulfs the forespore,
resulting in a forespore (endospore) within a mother cell
•eventually the mother cell lyses
•Sporulation begins when nutritional conditions become unfavorable
- most significant is depletion of N2 or/and C
 Sporulation

• It occurs at the end of log phase involving specific changes:

– production of new structures, enzymes, metabolites
with the disappearance of vegetative cell components
– Sporulation set of genes become activated while other
genes involved in vegetative cell function are inactivated
– This step is achieved by changing the transcriptional
specifity of RNA polymerase by its association with
promoter-specific protein, called a sigma factor
– Different sigma factors are produced during vegetative
growth and sporulation.
 Sporulation

• Morphological changes
– sporulation begins with the isolation of a terminal nucleus by
the inward growth of the cell membrane
– Enfolding of CM will lead to the formation of a double
membrane structure forming inner membrane and CW facing
outside, which engulf the developing spore
– The remaining layers of the spore will form
• Outer spore membrane and cortex will form between the
facing membranes
• The coat and exosporium will form out side the
membranes
• In the core, all vegetative cell enzymes become degraded
and replaced by spore constituents
 Process of endospore formation

• DNA is replicated
-The spore septum begins to isolate the newly replicated DNA
and a small portion of the cytoplasm
• The plasma membrane starts to surround the replicated DNA
and cytoplasm
• The spore septum surrounds the isolated portion forming a
forespore
• The peptidoglycan layer forms between the 2 membranes
• Thick spore coat forms around the outside membrane, making
endospores resistant to many harsh chemicals
-The new spore loses its water and becomes highly dehydrated
to the point that all that remains in the spore is:
DNA ; small amounts RNA ; ribosomes, enzymes
• Endospore is released from the cell, can remain dormant for
1000s of years
Process of endospore formation
 Spore Resistance

•Resistance to freezing and desiccation

–due to SASP which protect the DNA from damage
– dipicolonic acid protects DNA
•Pressure resistance
–Resist hi pressure >12000 atmospheres (1atm~15 pSi)
–Lower pressure allows spore germination
•gamma-radiation resistance
–Mechanisms are unknown
–Radiation causes damage to the DNA
•Uv radiation
–SASP proteins are responsible for the resistance
•Chemical resistance
–Spore coats provide a barrier
–Impermeability of coats inhibits penetration of chemicals
 Spore Resistance

• Heat resistance
– Implications in industry
– Withstand 100o C for several minutes
– Dt value is used for quantification
• Dt is time in min at temp (t) to kill 90% of population
– The target of heat is not known- DNA is not damaged, no
mutations due to heat, but may be proteins
• C. botulinum spores, the most heat resistant
– Spores must be reduced by 12D
– 12D is the time required in a thermal process for a 12 log
reduction of the spores
– D value: time required for one-log reduction in viability
– one-log = 90% killing of the population
 Germination

• The germination process occurs in three stages:

– Activation
• activation is not triggered by presence of nutrients,
• Triggered by agents that damages the spore coat
• heat, abrasion, acidity, and componds containing free
sulfhydryl groups
– Initiation
– Once activated, a spore will initiate germination if the
environmental conditions are favorable
– receptors bind effector signals of available a rich medium
– Binding of the effector activates an autolysin that degrades
the cortex peptidoglycan
– water is taken up, calcium dipicolinate is released, and spore
constituents are degraded by hydrolytic enzymes
 Germination

– Outgrowth
• new vegetative cell emerges followed by active biosynthesis and
terminates in cell division
• Outgrowh requires a supply of all nutrients essenial for cell
growth.
 Macromolecules

• SASP – small acid-soluble proteins

• play a major role in spore resistance
• Binds to DNA and provide resistance to
chemical/enzymatic cleavage
• Small molecules
• Located in the cortex
• Low amount of spore core water
• Huge amounts of dipicolinic acid
• Divalent cations
• Lower pH
• Little if any hi energy molecules
 Spore stain

• Spores are refractile bodies in unstained cell

suspensions or colorless areas stained cells
• spore wall is impermeable, but dyes (malachite green
or carbolfuchsin) can penetrate by haeting
• Impermeability prevents decolorization
• Counterstain with safranine
 Summary

• Heat resistance is quantified by the D-value

• Commercial sterility is achieved through the application of 12D
• Pharmaceutical preparations must be free of Spore formers
• Sporulation – a process where vegetative cell becomes spore
• Germination/outgrowth allows the spore to resume normal life
• Spore resistance is due to:
– Spore cortex PG
– Dipicolinic acid and SASP
– Low water content in the spore
• during sporulation, gene expression occurs- some are expressed only in
the mother cell or the forespore.
• gene expression is controlled by five new sigma factors for RNA
polymerase and DNA-binding proteins.
• The developing spore becomes encased in 2-3 layers:
• a large layer of peptidoglycan (PG) - the spore cortex, PG structure
differs from that of growing-cell PG,
• a number of spore coat layers,
• in some species, an exosporium.
• Both the coats and exosporium contain proteins unique to spores .
•.
• The spore’s central region or core accumulates
large amounts (10%) of dipicolinic acid and
divalent cations
• the core loses much water
• the developing forespore synthesizes a large
amount of small acid-soluble proteins (SASP),
some of which coat the spore chromosome and
protect the DNA from damage
• the spore becomes metabolically dormant and
resistant to harsh conditions including heat,
radiation, and chemicals