Microbial growth part 2 - spores

Which genera of bacteria produce spores?

How long can bacterial spores survive?

Spores can persist for hundreds to millions of years.

How many bacterial genera can form spores?

Only about 35 out of approximately 40,000 known genera.

Why do only a few bacteria form spores?

Sporulation is a survival strategy but energetically costly — it requires over 200 genes and a long commitment (10 hours to 5 days).

Bacillus vs Clostridia spores

Bacillus: Facultative anaerobes — can grow with or without oxygen.

Clostridium: Obligate anaerobes — cannot survive in the presence of oxygen.

white part is the spore

What is sporulation?

The transition from vegetative growth to forming a highly resistant spore in response to environmental stress.

• More than 10 hours — it is a very slow process. bc It involves complex gene regulation, structural reorganization, and synthesis of protective spore layers — requiring over 200 genes to complete.

What triggers sporulation in bacteria?

Exhaustion of nutrients and accumulation of metabolic byproducts (e.g., acid buildup, pH drop).

Do all bacterial cells undergo sporulation?

No, not all cells sporulate — only a portion of the population does, even under stress.

• Autoinducer molecules produced by cells can inhibit sporulation, signaling cells to delay it.

What are the 7 stages of bacterial sporulation and what happens in each?

1. Triggering: Sporulation begins when nutrients are exhausted or stress conditions occur.

2. Stage 1 – Axial Filament Formation: DNA twists and aligns along the cell’s axis, usually toward one end rather than the center.

3. Stage 2 – Asymmetric Cell Division: The cell divides unevenly, creating a pre-spore and a mother cell. The mother cell acts as a power source, feeding and supporting the developing spore.

4. Stage 3 – Forespore Engulfment: The mother cell engulfs the pre-spore, forming a cell within a cell—each with its own membrane.

5. Stage 4 – Cortex Formation: A thick peptidoglycan cortex with uronic acid forms between the inner and outer membranes. This layer begins dehydrating the spore core.

6. Stage 5 – Spore Coat Synthesis: A keratin-like coat forms around the cortex, creating an impermeable barrier. Dipicolinic acid (DPA) and calcium enter the core, replacing water and stabilizing DNA in a glass-like state—this gives spores their “phase brightness.”

7. Stages 6–7 – Spore Maturation & Release: The spore matures, the mother cell lyses, and the free endospore is released. Some species also form an exosporium (outermost layer).

Extra Note: In Clostridium perfringens, spore release can be accompanied by toxin production, which helps the spore survive and spread.

Spore structure

Term: Cortex
Definition: The thick peptidoglycan layer that dehydrates and protects the spore core.

Term: Spore coat
Definition: The impermeable outer protein layer that shields the spore from chemicals and enzymes.

What is found in the spore core?

The spore core contains the DNA, stabilized by small acid-soluble proteins (SASP) that twist and coat it for protection.

SASP protect DNA from damage by binding and twisting it, allowing it to form a spore photoproduct that can be repaired after germination.

What does DPA (dipicolinic acid) do?

DPA binds calcium and dehydrates the spore core, helping stabilize it and maintain dormancy.

What is the function of the inner spore membrane?

It becomes the new cell membrane during germination.

What is the role of germination receptors?

They detect germinant molecules, triggering DPA release and degradation of the cortex to start germination.

What happens if germination receptors are inactivated?

The spore cannot respond to signals and remains dormant.

Why are spores more resistant than vegetative cells?

Spores have protective layers, low water content, and DNA-binding proteins (SASP) that make them resistant to heat, UV, and chemicals.

Psychrotropic spores

Spores that grow at cold temperatures (around 4°C); more heat-sensitive than thermophilic spores.

Thermophilic spores

Spores that thrive around 38°C and are highly heat-resistant.

Endospore

A dormant, highly resistant structure formed by certain bacteria to protect genetic material until favorable growth conditions return.

What is the main purpose of an endospore?

To protect the bacterial DNA and ensure survival during harsh conditions.

Where is the DNA of a spore located?

It is contained within the spore core.

What is the main component of the spore core?

The core contains a dipicolinic acid and calcium complex (DPA:Ca).

What is the function of the DPA:Ca complex?

It dehydrates the spore, setting the DNA into a solid, glass-like matrix for stability and protection.

What happens during the first 10 minutes of germination?

The cell is autonomous and uses its own stored resources while transitioning out of dormancy.

What is the purpose of the spore coat?

It forms an impermeable barrier that protects the spore from sanitizers and environmental damage

What does the spore core contain besides DNA?

Enzymes, RNA, NADH, and 3-phosphoglyceric acid—molecules essential for early germination

Is there metabolic activity during dormancy?

No, the spore is metabolically inactive and completely inert until germination begins.

Spore cortex

What is the spore coat made of?

It is composed of protective proteins that form an impermeable outer layer.

What is the main function of the spore coat and outer forespore membrane?

To prevent antimicrobial and sanitizer molecules (like iodine) from diffusing into the spore’s inner structures.

What is spore germination?

The process where a dormant spore is activated and begins returning to a vegetative (growing) state.

What triggers spore germination?

Activation by mild heat (around 70°C for 10 minutes), high pressure (e.g., 400 MPa), or oxidizing/reducing agents like chlorine.

What effect does activation have on spores?

It increases the permeability of the spore coat, allowing germination agents to enter.

What happens if no germination agents are present after activation?

The spore reverts back to its dormant state.

What is the most common germinating agent?

L-alanine, the simplest and most abundant amino acid, which effectively triggers spore germination

What inhibits the action of L-alanine during germination?

D-alanine, a competitive inhibitor that blocks L-alanine’s germination signal.

What are other germinating agents besides L-alanine?

Inosine, asparagine, glucose, fructose, and potassium chloride (collectively known as IAGFR).

What special germinating agent triggers Clostridium difficile spores?

Bile acids.

super-dormant spores

Spores that germinate very slowly or show delayed response to germination signals.

Steps of Spore Germination (Sequence of Events)

1. Activation – Triggered by heat, pressure, or chemicals.

2. DPA:Ca Release – Dipicolinic acid and calcium exit the core.

3. Rehydration Begins – Core absorbs water, spore becomes phase dark.

4. Cortex Degradation – Enzymes break down the cortex peptidoglycan layer.

5. SASP Breakdown – Small acid-soluble proteins are degraded to release energy and form NADH.

6. 3-Phosphoglyceric Acid Metabolism – Provides initial internal energy for 10 minutes.

7. External Nutrient Uptake – After 10 minutes, the spore begins using external metabolites.

8. Loss of Resistance – Heat, UV, and sanitizer resistance are lost.

9. DNA Expression and Repair – DNA replication and protein synthesis begin.

10. Outgrowth – The cell elongates and divides; the germ cell wall becomes the new outer membrane.

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How long does germination take compared to sporulation?

Germination is fast—about 45 minutes—while sporulation takes 10 hours to several days.

What indicates the completion of germination?

The spore fully rehydrates, resumes metabolism, and divides into new vegetative cells.

Does germination require external energy sources?

No, the process uses stored internal energy and does not require exogenous energy sources.

Is DNA expression needed in the early stages of germination?

No, DNA remains inactive during the initial stages of germination.

What are the main targets of spore inactivation methods?

Most focus on damaging spore DNA or inactivating germination systems to prevent revival.

Why are spores significant in microbiology and food safety?

They are the most resistant biological structures and serve as indicators to verify sterilization methods like heat or irradiation

• Because of their extreme resistance to heat, radiation, and disinfectants, proving effective sterilization requires killing them.

What types of spores are particularly important in food safety?

Clostridium botulinum (botulism), Clostridium perfringens (common food poisoning), Bacillus cereus (found in rice), and Bacillus anthracis (anthrax)