MICROBIOLOGY Study Guide - 2012

CHAPTER 3. BACTERIAL MORPHOLOGY

3.4. DORMANT FORMS OF PROKARYOTES

Many Bacteria are capable of forming structures that help them survive adverse conditions for extended periods and return to vegetative forms once they encounter environments suitable for reproduction. Such structures include endospores, cysts, and akinetes.

Endospores are formed by bacteria of the genera Bacillus, Clostridium, Desulfotomaculum, Sporosarcina, Sporolactobacillus, Sulfobacillus, and several others. Endospore formation in bacteria is not associated with reproduction, since a single vegetative Cell produces only one spore. Bacterial endospores are resting Cells of Gram-positive bacteria formed within the Cytoplasm of the mother cell, and they exhibit high resistance to elevated temperatures and radiation doses.

Bacterial sporulation should be viewed as a resting stage in the Cell life cycle, typically triggered by adverse environmental factors. Under favorable conditions, the vegetative cells of these microorganisms can multiply indefinitely by division.

The factors inducing sporulation vary: nutrient depletion in the medium, changes in pH or Temperature, accumulation of Metabolic waste products, and others. The sporulation process consists of several stages (Fig. 9).

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Fig. 9. Diagram of bacterial endospore formation and The Structure of a mature spore (Schlegel, 1987): a, b — Separation of the spore protoplast; c, d, e — Formation of the forespore; f — mature spore: 1 — cytoplasm with nucleoid; 2 — Plasma Membrane; 3 — germ Cell wall; 4 — spore cortex; 5 — inner membrane; 6 — outer membrane; 7 — exosporium

Stage 1: Preparatory stage. In vegetative bacteria transitioning to sporulation, growth processes cease, nucleoid Morphology changes, and METABOLISM is restructured. Enhanced accumulation of protein material begins, causing the refractive index to increase at the site of spore formation. This region of The Cell, featuring condensed cytoplasm and genetic material represented by a replicated chromosome, is called the sporogenic zone. During this same period, a new compound specific solely to spores appears — dipicolinic acid (Fig. 9). It is synthesized from meso-diaminopimelic acid.

Dipicolinic acid forms a chelate with calcium, known as calcium dipicolinate, the presence of which ensures the high thermostability of the mature spore.

Stage 2: Forespore formation. The cytoplasmic membrane invaginates, resulting in a portion of the protoplast becoming separated from the mother cell. The protoplast of the future spore becomes surrounded by The Plasma Membrane of the prospective cell, forming a forespore — a structure located within the cell and completely isolated from it by two membranes: the outer (exine) and inner (entine).

Stage 3: Formation of spore coats. The inner membrane of the forespore protoplast synthesizes the germ cell wall on its outer side. A multi-layered spore coat is formed over the outer membrane of the forespore. The spore cortex is synthesized between the inner and outer membranes; this cortex is a peptidoglycan framework that differs from murein in its degree of cross-linking. In some bacteria, an additional structure called the exosporium forms over the spore coat. The exosporium is often multi-layered and surrounds the spore like a loose sheath (e.g., in B. cereus).

Stage 4: Spore maturation. The spore acquires a shape characteristic of the given species and occupies a specific position within the cell. The Location may be central, eccentric (slightly offset from the center), or terminal (at the cell pole). Spore-forming aerobic bacteria in which the spore diameter does not exceed the cell diameter are called bacilli, whereas spore-forming anaerobic bacteria with a spore diameter greater than the cell diameter are called clostridia (from Greek closter, meaning spindle). Clostridia with terminally positioned spores are termed plectridia (Fig. 10).

Fig. 10. Arrangement of spores in spore-forming bacteria:

1, 2 — bacillary; 3, 4 — clostridial; 5 — plectridial; 6 — lateral

As the spore matures, the mother cell lyses, releasing the spore. The STRUCTURE OF THE mature spore is roughly similar across different bacterial species. The central part of the spore, containing the nucleoid, is enclosed by the cytoplasmic membrane; this is adjacent to the murein layer of the germ cell wall, followed by a thick cortex layer. The outer membrane lies On the surface of the cortex. Externally, the spore is covered by a multi-layered coat. The spore coat consists primarily of Proteins with small amounts of Lipids and glycopeptides.

In chemical composition, spores do not differ significantly from vegetative cells. The sole exception is dipicolinic acid, the content of which can reach up to 15% of the spore's dry weight. Spores differ from vegetative cells by having a higher lipid content, with an especially high concentration in their coats.

Spore germination. Upon encountering favorable conditions, spores germinate and give rise to a vegetative cell. The germination process lasts 4–5 hours and can be divided into several stages:

1) activation. Spore germination is induced by various nutrients (Alanine, glucose), certain Metal Ions, an acidic environment, and brief heating ("heat Shock");

2) initiation. During this period, spores lose up to 1/3 of their initial mass, which is associated with the release of dipicolinic acid. The spore rapidly absorbs Water, swells, and loses its refractility. Profound physiological changes occur within the spore; Respiration and enzymatic activity increase rapidly;

3) outgrowth. Lytic Enzymes degrade the multi-layered spore coats, and a germ tube surrounded by a very thin cell wall emerges at the rupture site. It increases in size until it adopts the shape of a vegetative cell. This completes the spore germination process.

Spore resistance. Mature spores exhibit no metabolic activity whatsoever. They are exceptionally resistant to adverse factors. Spores withstand high and low temperatures, prolonged desiccation, radiation, high osmotic pressure, and toxic substances. Spores can survive in soil for decades and centuries, withstand boiling for 1 hour and short-term sterilization at 125 °C, as well as exposure to high concentrations of disinfectants. Viable spores have been isolated from the remains of mammoths and Egyptian mummies thousands of years old.

The thermoresistance of spores is attributed to their very low water content. Water in spores exists in a bound state and cannot participate in metabolic processes. Resistance to high temperatures is also conferred upon the spores by the presence of calcium dipicolinate.

Radioresistance of spores is also higher than that of vegetative cells. The spore coat proteins are similar in composition to keratin and are rich in Cysteine, which provides the spore with enhanced mechanical strength and radiation resistance. The chemical resistance of spores is due to the impermeability of their coats to many substances.

The high heat resistance of bacterial spores poses major challenges for the food industry. Spores survive pasteurization temperatures and, remaining in pasteurized products, cause spoilage under favorable conditions. The problem of food sterilization is primarily the problem of destroying Spore Forms. Sterilization regimens depend on The chemical composition of the product, its pH value, package size, the species COMPOSITION OF THE initial microflora, etc.

Cysts are dormant survival forms found in various groups of eubacteria (spirochetes, rickettsiae, mycobacteria, azotobacter) designed to withstand adverse environmental conditions. Cysts feature additional cell layers, their cytoplasm contains little free water, and their metabolic and energy-producing processes are sharply reduced.

Akinetes are resting cells of certain cyanobacteria characterized by increased resistance to desiccation and low temperatures. The walls of akinetes contain more lipids and Polysaccharides, and their cytoplasm contains less free water than those of vegetative cells.