Food Microbiology: Spores

• Food microbiology focuses on spores and their significance.

Learning Outcomes

• Identify how sporulation is induced
• Describe the structure of the spore and macro & micro components
• Describe the morphological, biochemical, and physiological changes during sporulation
• Differentiate between dormant and vegetative cells
• Recognise the importance of spores to the food industry

Contents

• Fundamental bases of sporulation
• Spore structure and composition
• Spore dormancy and resistance
• Spore activation and germination

Spore-Forming Bacteria

• They present a major threat in heat-treated food plants.
• They can exist in both spore and vegetative forms.
• Clostridium botulinum causes botulism, which leads to paralysis and death.
• Bacillus cereus causes diarrhoea.
• Spores are resistant to chemicals like disinfectants, and physical treatments, both thermal and non-thermal, in the food processing industry.
• Cooking heat activates the germination of spores, turning them into vegetative cells.

Spores

• Endospores are produced inside the bacterial cell.
• Examples of endospore-producing genera are Bacillus and Clostridium.
• Spore development has been extensively studied.
• Bacillus subtilis was the first spore-forming bacteria to have its genome sequence reported.
• Endospore-forming bacteria can thrive in different parts of food processing plants.
• Exospores are produced outside the cell and are less established in food, with Methylosinus being an example.

Sporulation

• Sporulation is the survival strategy under harsh environmental conditions.
• Bacillus spp. spores are 10 to 50 times more resistant to UV radiation than growing cells.
• This allows them to adapt to changes and survive in time and/or space.
• Bacteria can form endospores in about 6-8 hours after exposure to adverse environments.
• Foods left at moderate temperatures for extended times after cooking can allow for the growth of large amounts of vegetative cells within 90 minutes.

Induction of Sporulation in the Laboratory

• Sporulation can be induced by limiting one or more nutrients, such as through the exhaustion of nutrients during growth.
• This can occur through shifting cells from a rich to a poor medium.
• Adding decoyinine, an inhibitor of GMP synthetase, can induce sporulation by affecting guanine nucleotide biosynthesis.
• Xanthosine monophosphate (XMP) is converted to guanosine monophosphate (GMP), a precursor for DNA and RNA synthesis and other cellular processes in bacteria.
• Laboratory methods can cause most cells in a culture to sporulate.
• These methods may not reflect how sporulation is induced in nature.

Induction of Sporulation

• Massive sporulation occurs only when cells enter the stationary phase.
• Sporulation is not an obligatory outcome of entering the stationary phase.
• Early events in the stationary phase involve attempts to access new nutrient sources, which, when unsuccesful, leads to sporulation.

Events in the Stationary Phase

• Events include the synthesis and secretion of degradative enzymes, such as amylases and proteases.
• Antibiotics like gramicidin or bacitracin are synthesized and secreted.
• Protein toxins active against insects or animals are synthesized and released in some species.
• The development of motility, killing and cannibalism of sister cells, and genetic competence occur.
• These phenomena are not always necessary for sporulation but are often regulated by mechanisms that modulate gene expression during sporulation.

Induction of Sporulation - Metabolism Changes

• Changes in the metabolism of the stationary-phase cell extend into sporulation, including:
  • Catabolism of polymers like poly-β-hydroxybutyrate (PHB), formed in vegetative growth and stored in bacterial cells as granules, can make biodegradable plastic.
  • Aerobic bacteria oxidize glucose to carbon dioxide and reduce oxygen to water, producing 38 ATP through oxidative metabolism in the TCA cycle. -Anaerobic bacteria use fermentation to generate 1-2 mol of ATP per mol of hexose catabolized.

Induction of Sporulation - Dipicolinic Acid

• Heat-resistant endospores contain dipicolinic acid (DPA), appearing as white, highly refractive bodies under phase-contrast microscopy.

Morphological, Biochemical, and Physiological Changes during Sporulation

• Stages are based on morphological characteristics of cells throughout development, taking as little as 8 hours.
  • Stage I: Axial filamentation
  • Stage II: Asymmetric cell division
  • Stage III: Forespore engulfment
  • Stage IV: Cortex formation
  • Stage V: Coat formation
  • Stage VI: Spore core maturation
  • Stage VII: Release of fully mature spore

Stage 0: Growing Cells

• Spore development begins with growing cells

Stage I: Axial Filamentation

• It is a discrete stage in sporulation.
• It involves the presence of two nucleoids in an axial filament, observable in electron micrographs.
• This stage includes the formation of an elongated chromosome structure.

Stage II: Asymmetric Cell Division

• The cell membrane forms an asymmetric septum, dividing the sporulating cell into the mother cell (MC) and forespore (FS) compartments.
• Both compartments contain complete and identical single chromosomes.

Transition from Stage II to III

• This involves the subdivision of Stage II into three substages.

Stage II to III: Engulfment of Forespore

• The MC membrane grows and engulfs the FS during the transition from Stage II to III.
• The FS has two membrane layers, inner and outer FS membranes with opposite polarities.
• In late Stage III, the forespore pH falls by 1-1.5, and FS dehydration begins.

Stage III to IV: Formation of Cortex

• A thick cortex forms with peptidoglycan (PG) between the inner and outer FS membranes.
• The cortex PG structure is similar to that of the cell wall PG.
• The FS synthesizes glucose dehydrogenase and small acid-soluble proteins (SASP).

Stage IV to V: Formation of Coat

• The proteinaceous coat forms outside the outer FS membrane.
• There are over 30 proteins, almost all of which are unique to spores.
• FS acquires gamma-radiation resistance, and furhter chemical resistance.
• FS dehydration continues.

Stage V to VI: Maturation of Spore Coat

• The core's depot of DPA accumulates after DPA synthesis in the MC.
• There is DPA uptake with M2+ in a 1:1 complex with DPA in the core.
• M2+ is predominantly Ca2+ with Mg2+ and Mn2+ (CaDPA).
• This involves the final dehydration process in the core.
• The spore becomes metabolically dormant, and further gamma-radiation and chemical resistance acquired.

Stage VI to VII: Release of Fully Mature Spore

• Autolysins are produced in the mother cell, leading to lysis and release of the spore.

B. subtilis Sporulation

• This process details the different stages of the production of spores

Spore Structure

• The structure of a dormant spore differs from a growing cell
• Many spore structures have no counterparts in growing cells

Structures and Compositions of Endospores

• Lipid, carbohydrates and proteins make up the exosporium
• There are 30 unique proteins form the coats
• The cortex is composed of Petidoglycan (PG)
• There is also a germ cell wall and core
• SASPs, proteins, DNA, RNA, DPA and Calcium make up the core

Exosporium

• Exosporium size varies significantly between species
• It is very large in spores os:
• Bacillus cereus
• B. anthracis
• B. thuringiensis
• Some Clostridium spp
• It may not be present in spores of species such as B. subtilis
• It is an additional layer encased in the spore in some pathogenic organisms
  • Protection of the spore and in its environmental interactions
• Its function is currently unclear

Spore Coat

• The coat is resistant to environmental conditions
• It protects spore PG from attack by lytic enzymes as well digesting the spores from predatory protozoa
• B. Subtillis underlies the exosporium

Outer Forespore Membrane

• This membrane underlies the spore coats
• It is a functional membrane in a developing FS
• Composition protein is distinct

Cortex

• Underlying the outermost forespore membrane, the PG layer is structurally similar to all cell walls
• It is responsible for spore dormancy

Germ Cell Wall

• This portion is between the coretex and the inner
• The PG layer is indentical to PG's in vegetative cells

Inner Forespore Membrane

• This layer is functional membrane
• In the spore core, it has extremely strong permeability
• It has a relatively freozen stricture

Core

• The core is 4g of dehydrated water
• It is high in SASP, around 10% of spore protein

Spore Marcomolecules

• SASPs are major parts of pores resitence and is synthesized in Stage III

Three Kinds of SASP

• Alpha/Beta: similar functions and confer resistance to UV radiation

Spore Small Molecules

• Spores differ from growing cells their molecules:
  • In the spore, the ph is low
  • The molecules are "high-energy"

Spore Germancy

• The metabolism with no detection
  • It is related to the lower water
• The core contains stable enzyme substrate pairs
  • The pairs interact in the first 30 minutes

Spore Resistance

• There is an ability to survive long periods of time
  • It is extremely resistant to lethal treatments

Resistance Types

Freezing and desiccation: - Some growing bacteria have resistance Radiation - SPORES CAN HAVE IT!

y-Radiation Effects

UV Radiation Effects

Spores are 7-50 times resistant to UV

Spore Germs

• The activate enzymes are regulated at the pH levels

Spore Germination

Germination is triggered by a variety of compounds amino acids

• Metabolism is not a requirement
• In the first few minutes RNA synthesis in the first few minutes

Summary

• Under stressful environmental conditions, such as exposure to toxic chemicals and biocidal agents, pressure and temperature extremes, and UV or ionizing radiation, bacteria produce spores that can stay dormant for extended periods
• Spores possess thick layers of highly cross-linked coat proteins, a modified peptidoglycan spore cortex, a low core water content, and abundant intracellular constituents including the calcium dipicolinate and acid-soluble spore proteins
• The processes of spore gemination into vegetative cells are very fast, creating a high food safety problem