GENERAL MICROBIOLOGY - T.P. Pyrog - 2004

5. STRUCTURE OF THE BACTERIAL CELL

5.4. ENDOSPORES AND OTHER RESTING FORMS IN BACTERIA

5.4.2. Sporulation

Spores are formed inside the bacterial Cell. This process begins with the accumulation of protein material, which increases the refractive index at the site of spore formation. At the same time, reserve substances are consumed (poly-β-hydroxybutyric acid in aerobes and Polysaccharides in anaerobes). During the first 5 hours of sporulation, a significant portion of the mother cell's Proteins is degraded. This process produces dipicolinic acid, a substance specific to spores that is not found in vegetative Cells. During the synthesis of dipicolinic acid, Calcium Ions are taken up; evidently, in mature spores, this acid exists as a calcium chelate and can account for 10-15% of the spore's dry weight. Dipicolinic acid is found exclusively in heat-resistant spores.

Sporulation is one of the most complex processes of bacterial Cell Differentiation. It begins with a distinctive, asymmetrical Cell Division (Fig. 5.12).

Class="center">

Fig. 5.12. Sporulation and Structure of a mature spore:

A, B — process of spore protoplast Separation; C, D, E — Formation of the forespore; F — mature spore; 1 — Cytoplasm; 2 — Plasma Membrane; 3 — germ Cell wall; 4 — spore cortex; 5, 6 — inner and outer spore coats, respectively; 7 — exosporium

As a result of the invagination of the cytoplasmic membrane, a portion of the protoplast separates from the mother cell. This protoplast contains a fraction of the nuclear material—a single genome. A cell wall is not formed between the two protoplasts (unlike ordinary cell division). Instead, the protoplast of the future spore becomes enveloped, or overgrown, by The Plasma Membrane of the mother cell. Consequently, two Plasma Membranes surround the protoplast, and each participates in the Synthesis of the spore wall. The membrane of the spore protoplast synthesizes the germ cell wall outward from itself, while the membrane originating from the mother cell synthesizes the spore cortex inward. The cortex consists of a multilayered peptidoglycan scaffold that differs from the framework of the vegetative cell wall in its degree of cross-linking. The outer spore coat is formed by the mother cell and consists of Polypeptides. The mother cell also produces an additional polypeptide layer—the exosporium—which surrounds the spore like a special sheath. The exosporium is present in only a few Bacteria (e.g., Bacillus cereus).

Induction of sporulation. Spores are not an obligatory stage in the bacterial life cycle. Under favorable conditions, bacteria can multiply by division indefinitely, just like vegetative cells. Spore formation begins only when nutrients become scarce or metabolic products accumulate in excess. In other words, it occurs exclusively when specific environmental triggers are present. Desiccation does not stimulate sporulation. Placing vegetative cells in distilled Water induces 'endotrophic sporulation,' meaning spore formation at the expense of intracellular reserves. In such cases, sporulation is clearly triggered by the lack of an exogenous substrate. The induction of sporulation takes place over several hours. If, for instance, glucose is added to a suspension of Bacillus cereus vegetative cells during the first 5 hours after their transfer to distilled water, spore formation ceases: The addition of a substrate inhibits sporulation. However, if glucose is added later than 6 hours, inhibition no longer occurs. Sporulation induction continues, and within 10–13 hours of immersion in water, about 90% of the cells form spores. Thus, sporulation is regulated by external factors.

In many cases, the proportion of spore-forming cells increases following the addition of manganese salts to the medium.

The ability to form endospores is gradually lost during repeated subculturing of vegetative cells. Because a suspension of spore-forming bacteria contains both spores and vegetative cells, the culture is typically subjected to brief boiling prior to each subculture. This practice helps maintain or enhance the cells' sporulation capacity.

Properties of mature spores. Spores are released upon the autolysis of the mother cells. Mature spores exhibit no metabolic activity whatsoever. They are exceptionally resistant to high temperatures, various types of radiation, and chemical agents. This thermoresistance is attributed to the low water content in spores—15% (comparable to dry casein or wool)—and is roughly proportional to their dipicolinic acid content.

Spore germination. Under appropriate favorable conditions, most spores germinate. Proper pretreatment, specific storage conditions, and heat activation can enhance spore 'viability'—increasing the percentage of germination. For Bacillus subtilis, optimal conditions for stimulating germination are considered to be a seven-day resting period followed by a five-minute heat Treatment in water at 60ºC. Other spores can be activated by brief boiling (10 min, 100ºC). Thermal treatment of spores must be performed immediately before inoculation, as the activation process is reversible.

Spore germination is preceded by Water uptake and Swelling. In some cases, the germination of activated spores requires the presence of glucose, Amino Acids, nucleosides, and other substances. Germination is accompanied by profound physiological changes: Respiration and enzymatic activity increase rapidly, and the release of amino acids, dipicolinic acid, and Peptides begins. Dry weight losses during germination reach 25–30%. As they germinate, spores lose their heat resistance. The germ tube emerging from the spore is surrounded by a very thin, incompletely formed cell wall, allowing even DNA to penetrate the protoplast. The germ tube can form in either a polar or a lateral position (Fig. 5.13). In the former case, the spore coat ruptures; in the latter, the germ tube pierces through it.

Fig. 5.13. Spore germination:

1, 2 — polar; 3 — lateral; a — Bacillus; b — Bacillus subtilis

Lifespan of spores. Bacteria can persist in a state of anabiosis as spores for extended periods. For example, bacilli spores have been detected in soil samples stored for 50–100 years. According to such experiments, up to 90% of spores in dry soil lose their viability over 50 years of storage. Nevertheless, 1 ton of dry soil will still contain viable spores even after 1,000 years.

In the dry state, many bacteria (if not all) retain their viability for many years. For culture collection conservation, vegetative cells are typically subjected to lyophilization (freeze-drying) and stored at room Temperature or low temperatures under a vacuum. Bacteria that cannot withstand lyophilization are preserved for many years as Suspensions at liquid nitrogen temperatures.