Bacterial spores

Bacterial spores

Dormant cellular forms of Gram-positive bacteria

• High potential of stability

• Endure extreme conditions

• Can survive for years

Sporulation life cycle

1. Vegetative cell Growth occurs via standard binary fission

2. When the bacteria face starvation, they enter the sporulation cycle

3. Formation of a mother cell and a forespore

4. Once conditions improve, the spore undergoes germination to return to a vegetative state

Spore structure

• Size: 0.8-1.2 um

• Spherical or ellipsoidal

• Different positions depending on the species (central, terminal, subterminal)

• Several layers: core, internal membrane, primordial wall, cortex, external membrane, coat, exosporium

Endospores

Spores formed inside the mother cell

Spore core

Contains DNA, RNA, enzymes, ATP, NADH, DPA and SASP

Dipicolinic acid (DPA)

Decreases water content in the spores, not don’t in the vegetative cells

Small acide-soluble proteins (SASP)

Saturate spore DNA → protection against damaging agents (UV light)

Internal membrane (IM)

• Composed of phospholipids and fatty acids

• Low permeability

• Linked to membrane-like structures → allow IM surface to increase during germination

Primordial wall

Or germ cell wall

• Thin

• Retained during spore germination and outgrowth

Cortex

• Thick peptidoglycan layer

• 50% of the NAM residues in the cortex lose their peptide side chains and cyclize to form muramic-δ-lactam

• Fully degraded during spore germination

Muramic-δ-lactam formation

Requires the enzymes CwlD (for peptide cleavage) and PdaA (for deacetylation and cyclization)

Coat

• Found in all species

• Made by several protein layers: basal layer, inner, and outer coat

• Composed of proteins unique to spores

• The number of layers depends on the species

• Higher electron density

Exosporium

Layer away from the spore outer coat

• Some species

• Glycoproteins with polysaccharide

• Resist chemical and enzymatic attack

• Provide surface for adhesion

• Harbour enzymes that modulate spore germination

• Protection from macrophage-mediated killing

Spore formation

Triggered by prolonged nutrient deprivation

• Development of an asymmetric division of the sporulation cell → generation of forespore and larger mother cell

Stage I and II of spore formation

• Duplication of the chromosome

• Asymmetric division with a septum → mother cell and forespore

• Chromosome translocated to the forespore by SpoIIIE

• σF and σE activation

Stage III of spore formation

• Engulfment

• Mother cell completely engulfs the forespore in a phagocytic-like process mediated by a peptidoglycan degradation complex

• σG activation

Stage IV of spore formation

• Cortex synthesis

• σK activation → regulation of coat formation

Stage V and VI of spore formation

Coat synthesis

Stage VII of spore formation

Programmed autolysis releasing the mature spore to the environment

Spatio-temporal sporulation regulation

• Activation of transcriptional factors: Spo0A and alternative sigma factors cascade

• 4 sigma factors are post-translationally activated in the spore-forming cell

Alternative sigma factors

• Other sigma factors than the sigma factor used for the transcription of constitutive genes (sigma70 or A)

• Replace to form ARN polymerase

• Permit the recognition of promoters

Sporulation initiation

• Activation of Spo04 starts sporulation

• Spo04 activated via five sensor kinases (Kin A to E)

• SpoOf → SpoB → Spo0A - gene transcription → σ alternative factors cascade → physical sporulation changes

Spore germination

Spore → vegetative cells

• Response to an inducer: chemical (germinant) or physical (pressure)

Physical germination changes

Release of H+, Na+, and K+ ions and Ca-DPA → activation of cortex-lytic enzymes (CLEs) → hydrolyze the cortex → continuous water uptake and full core rehydration

Core rehydratation

Nucleic material is visible

Spore germination examples

• B. subtilus induced by L-alanine

• Clostridioides difficile induced by taurocholate( bile salt) or glycin

Bacillus cereus

• Toxic infections → diarrheal and emetic symptoms

• Vegetative cells and spore ingestion → reach intestine → spores’ survival higher → toxins production

Bacillus cereus

• Botulism → flaccid paralysis

• Spores contaminate food → survive pasteurization → germinate and outgrow into neurotoxic cultures → germinate in the gur

Clostridium perfringens

• Gas gangrenous

• Spores in environment → contaminate food or wound → germinate in the digestive tract or wound → produce toxins

Clostridium tetani

• Tetanus → generalized muscle spams (lockjaw)

• Spores in feces of animals → contaminate wound → germinate → toxins production

Clostridioides difficile

• Toxic infections → pseudomembranous colitis

• Spore contamination in aerosol or food → take advantage of gut dysbiosis to germinate → toxin production

Factors involved in spore resistance

• Low water content → protection against heat damage

• SASP

• Many DNA repair proteins

• Detoxifying enzymes (superoxide dimutase and catalase)

• Inner membrane → protection against chemical products

• Coat and exosporium → against chemical products and hydrolytic enzymes

Sterilization in a medical context

• Heat: steam heated (110º, 20 min) and dry-air sterilizer (120º, 45 min)

• Chemical: glutararaldehyde with phenol, hydrogen peroxide with peracetic acid

Spore inactivation in the food industry

• Thermal processing → reduce nutrient content, modify fresh like qualities

• Omic heating: current passed through the food

• Radio frequency heating: electromagnetic wave

• Microwave non-ionizing electromagnetic waves

• Infrared heating or radiation

• Superheated or dry steam

• Cold plasma: plasma generated from ionize gases

Non thermal processing technique

• High pressure or ultra-high pressure processing (100-1000 MPa)

• Ultrasonic technology (20-100 kHz) → induce cavitation

• UV with pulse light

• Ionizing radiation: gamma, X-ray, electron-beam

Chemical control methods

• Neutral electrolyzed water

• Electro-activated solution

• Gaseous ozone

• Organic acid salts

• Gaseous acetic acid