MICROBIOLOGY - M.H. Serhiichuk - 2008

Chapter 2. MORPHOLOGY AND STRUCTURE OF THE BACTERIAL CELL

ULTRASTRUCTURE OF THE PROKARYOTIC CELL

Despite its seemingly simple outward appearance, a bacterial Cell is a complex living entity (Fig. 2.17) whose Structure in many respects resembles that of plant or animal Cells.

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Fig. 2.17. Schematic combined diagram of a Introduction/4.html">Prokaryotic Cell

1 - nucleoid; 2 - Ribosomes; 3 - Cytoplasm; 4 - cytoplasmic membrane; 5 - Cell wall; 6 - capsule; 7 - flagella;

8 - mesosomes; 9 - aerosomes (gas vacuoles); 10 - lamellar structures; 11 - tubular thylakoids; 12 - plate-like thylakoids;

13 - chlorosomes; 14 - chromatophores; 15 - sulfur inclusions; 16 - carboxysomes; 17 - poly-β-hydroxybutyric acid granules;

18 - polyphosphate granules; 19 - polysaccharide granules

The bacterial cell is enclosed by a rigid envelope known as The Cell wall. In many Bacteria, the cell wall is surrounded externally by a capsule or slime layers, pili (fimbriae), and flagella. Internal to the cell wall lies the cytoplasmic membrane (an essential structural component of all bacterial cells), which surrounds the cytoplasm. The cytoplasm contains the bacterial chromosome (nucleoid), ribosomes, reserve nutrients, and other bacterial structures. Structures located outside the cytoplasmic membrane are referred to as surface structures.

The cell wall is an obligatory STRUCTURE OF THE bacterial cell. The exceptions are Mycoplasmas and L-form bacteria, which lack this structure.

The cell wall is rigid yet somewhat elastic. It determines the shape of the bacterial cell, which can be easily demonstrated experimentally: disrupting the integrity of the cell wall leads to The formation of morphologically altered spheroplasts or protoplasts.

The cell wall protects the internal Contents of the cell from mechanical and osmotic influences of the external environment and plays a vital role in regulating Cell Growth and Division. The chemical Composition and Structure of the cell wall vary among different eubacteria, which accounts for their differential Gram-staining behavior (Table 2.2).

Table 2.2. Chemical composition of cell walls in Gram-positive and Gram-negative prokaryotes

An essential structural component of the cell wall in both Gram-positive and Gram-negative bacteria is the biopolymer mucopeptide (peptidoglycan, glucosaminopeptide, glycopeptide, or murein; Lat. murus - wall) (Fig. 2.18). It is this biopolymer that imparts rigidity to the Bacterial cell wall. Its content in the cell walls of Gram-positive prokaryotes reaches 50-90% by dry weight. In most species of Gram-negative bacteria, the peptidoglycan content ranges from 1 to 10%. In the cell walls of cyanobacteria, which exhibit a Gram-negative type of cell wall Organization, murein accounts for 22-52%.

Fig. 2.18. Model of cell wall organization in Gram-negative (a) and Gram-positive (b) bacteria:

1 - peptidoglycan; 2 - periplasmic space; 3 - cytoplasmic membrane; 4 - outer membrane; 5 - lipoteichoic acid; 6 - teichoic acid; 7 - lipoproteins; 8 - porin proteins; 9 - lipopolysaccharides

Electron Microscopy has demonstrated that the cell wall of Gram-positive prokaryotes appears as a homogeneous, electron-dense layer composed of peptidoglycan interspersed with teichoic acids, polysaccharides, etc. The thickness of this layer varies among different bacterial species from 20 to 50 nm.

The cell wall of Gracilicutes (Gram-negative bacteria) is a multilayered (heterogeneous) structure containing:

- an inner electron-dense layer 2-3 nm thick, composed of peptidoglycan;

- an outer layer, 8-10 nm thick, adjacent to it, which consists of two electron-dense bands separated by an electron-lucent space. This three-component cell wall structure of Gram-negative bacteria, resembling a unit membrane, is called the outer membrane, and the space between the cytoplasmic membrane and the outer membrane is termed the periplasmic space.

Thus, the cell walls of Gram-positive and Gram-negative prokaryotes differ significantly in their ULTRASTRUCTURE AND CHEMICAL composition.

The method of bacterial Cell Differentiation proposed in 1884 by the Danish scientist H. Gram (1853–1938) entered microbiological practice as the Gram stain. Its essence lies in the fact that in certain species of bacteria, specific cell wall components form a stable complex when interacting with gentian violet (or crystal violet) in the presence of iodine, which is not washed out by alcohol or acetone. Such bacteria are called Gram-positive (or firmicutes). Bacteria that do not retain the Gram stain are called Gram-negative (or gracilicutes). Gram staining is one of the essential Methods for bacterial differentiation. Theoretically, all bacteria can be divided into two groups: Gram-positive and Gram-negative. In reality, instances occur where the same microorganism is characterized as Gram-variable, meaning that depending on the age of the culture, it stains either positively or negatively.

Thus, the cell walls of Gram-positive and Gram-negative bacteria differ in ultrastructure and chemical composition, yet peptidoglycan remains a mandatory and fundamental component for both. This biopolymer is not found in the cells of other organisms, making it a unique prokaryotic structure.

The peptidoglycan molecule features a backbone formed by residues of N-acetylglucosamine (N-Ac.Gl.) and N-acetylmuramic acid (N-Ac.M.).

N-Ac.Gl. is a glucose derivative in which the hydroxyl group at the second carbon atom is replaced by an amino group attached to an acetyl residue.

The N-Ac.M. molecule is an ether of N-Ac.Gl. and lactic acid. This compound is exclusive to Prokaryotic Cells, rendering it unique.

Both molecules are interconnected by β-1,4-glucosidic bonds, forming a linear structure known as glycan (Fig. 2.19).

Fig. 2.19. Structure of the glycan molecule

The elements forming the glycan are similar to Disaccharides such as cellobiose or Chitin. However, unlike cellobiose and chitin, the glycan structure contains carboxyl groups that determine its acidic properties.

A peptide is attached to the carboxyl group of lactic acid in the N-Ac.M. molecule, frequently taking the form of a tetrapeptide. The peptide residue contains alternating L- and D-Amino Acids (an unnatural configuration, given that biological activity is generally exhibited solely by L-forms).

The tetrapeptide composition often includes an Unusual amino acid as well—meso-diaminopimelic acid (DAP), which can occur in the meso- or L-form. This amino acid is found exclusively in prokaryotes and solely within their cell wall structure.

The amino group of The amino acid at the first position of the peptide binds to the carboxyl group of N-Ac.M. in the glycan molecule (Fig. 2.20). Most commonly, this amino acid is L-Alanine, and occasionally the L-form of Serine or Glycine.

Fig. 2.20. Structure of the murein molecule

The second position contains D-glutamic acid. It subsequently links to the diamino acid occupying the third position in the peptidoglycan peptide residue. This diamino acid (third position) is most frequently m-diaminopimelic acid (m-DAP). Alternatively, the third position may contain LL-DAP, D-Lysine, L- or D-Ornithine, m-2,6-diamino-3-hydroxy-β-pimelic acid, 2,4-diaminobutyric acid, or homoserine.

The fourth amino acid in many peptidoglycans is D-alanine.

The peptide residue of peptidoglycan does not necessarily consist of exactly four amino acid residues. Sometimes a peptide may feature two terminal D-Ala residues, while in other cases it contains only three amino acid residues.

D-glutamic acid possesses two carboxyl groups. One of them remains free or serves as the attachment site for the amino acid residue of a second peptide, thereby acting as the cross-linking site between two Peptides.

Studies of peptidoglycans from various bacteria have demonstrated that they all share a uniform organization comprising a glycan chain, peptide chains, and cross-linking bridges connecting the peptide residues.

All of this creates a net-like structure for this biopolymer, the spatial organization scheme of which is shown in Fig. 2.21.

Fig. 2.21. Spatial Organization of the peptidoglycan molecule

When studying enzymatic hydrolyzates of peptidoglycans, several minor components were isolated:

- a derivative of muramic acid in which the hydroxyl group at C₆ is replaced by an O-acetyl or phosphate group. The latter links peptidoglycans to other cell wall polymers, such as teichoic acids;

- in some muramic acid residues, N-acetylmannosamine is found instead of the N-Ac.Gl. residue;

- sometimes muramic acid is detected with a free, i.e., non-acylated, amino group;

- a small amount of muramic acid is identified that contains N-glycolyl groups instead of N-acetyl groups.

The thickness of a single peptidoglycan layer ranges from 1.5 to 2.0 nm, meaning that the peptidoglycan of Gram-negative bacteria can be considered single- or double-layered, whereas in Gram-positive bacteria it has 20–40 layers, depending on the cell wall thickness.

The Main Functions of peptidoglycan are considered to be:

- providing rigidity (mechanical strength) and thus determining the shape of the bacterial cell;

- counteracting the turgor pressure of the cell contents, thereby preventing osmotic lysis;

- largely determining the antigenic Specificity of the bacterial cell;

- certain elements of the cell wall exhibit a nonspecific adjuvant effect. Adjuvants are substances that enhance the Immune Response to an antigen. In the case of peptidoglycan, these are typically tripeptides (L-Ala - D-Glu - m-DAP) linked to N-AcM;

- the presence of free carboxyl (СОО^-) or amino groups (NH⁺) determines the surface charge of the cell.

Thus, peptidoglycan is one of the essential Components of the bacterial cell wall, although its structure as a biopolymer may vary.

Since the peptidoglycan of certain bacteria possesses antigenic properties, The ability to inhibit its synthesis is of great importance for medicine. The murein layer of Gram-positive and Gram-negative bacteria can be destroyed by lytic Enzymes or its synthesis can be inhibited by penicillin Antibiotics, such as benzylpenicillin, 2-hydroxybenzylpenicillin, n-heptylpenicillin, n-amylpenicillin, etc. The inhibition of cell wall synthesis is also exhibited by bacitracin, vancomycin, ristomycin, and cycloserine.

Enzymes that degrade the peptidoglycan structure were first described by A. Fleming in 1922 and are known as lysozymes.

Lysozyme (acetylmuramidase) is a bactericidal enzyme found in lacrimal fluid, nasal mucus, egg albumen, and milk. This enzyme has also been isolated from the cells of certain bacteria (e.g., E. coli, some Representatives of the genus Streptomyces) and Bacteriophages. It cleaves the glucosidic bonds in the murein molecule between the first carbon atom of N-Ac.M. and the fourth carbon atom in the N-Ac.Gl. molecule. As a result of this action, polysaccharide chains are cleaved into disaccharide fragments.

The Cleavage site in the peptidoglycan molecule depends on the enzyme used for this purpose (Fig. 2.22):

- endo-N-acetylglucosaminyl-N-acetylmuramidase (glucosidase) - cleaves the bonds following N-acetylglucosamine;

- endo-N-acetylmuramyl-N-acetylglucosylamidase (glucosidase) - acts on the bonds following N-muramic acid;

- N-acetylmuramyl-L-alanine amidase - cleaves the tetrapeptide from the glycan molecule.

Fig. 2.22. Scheme of the action of lytic enzymes on the peptidoglycan molecule

The integrity of the peptidoglycan structure can also be disrupted by the action of Endopeptidases—enzymes that cleave bonds within the tetrapeptide molecule or between two peptides. This action results in the formation of spheroplasts, which are structures partially deprived of a cell wall, or protoplasts, which are structures completely devoid of a cell wall.

The function of peptidoglycan was first demonstrated experimentally by W. Weidel in 1953. To this end, he placed B. megaterium cells in an isotonic sucrose solution and then added lysozyme. The rod-shaped cells transformed into spherical cells—protoplasts—which retained respiratory activity and synthesized proteins and NUCLEIC ACIDS.

Because protoplasts lack a cell wall, they acquire A wide variety of shapes: elongated, swollen, pear-shaped, spiral, etc.

Thus, it was proven that lysis of the cell wall does not lead to the disruption of metabolic functions: protoplasts respire similarly to intact cells, and if sporulation had been previously initiated, it culminates in the formation of a spore.

In hypotonic solutions, protoplasts undergo lysis. Because protoplasts lack a cell wall, they are exceptionally sensitive to chemical agents, making them a convenient model for physiological research.

It is believed that hypertonic solutions act as a stabilizing factor for cell wall-deficient cells, causing them to assume a spherical shape. Furthermore, it has been established that the specific substance used to create the hypertonic medium plays a crucial role in spheroplast formation. When spheroplasts are derived from E. coli, a sucrose-based hypertonic solution effectively preserves the protoplasts, whereas a cellobiose-based hypertonic solution causes them to rupture.

Once protoplasts are washed free of lysozyme and placed in an optimal medium, they increase in size and exhibit a limited capacity for reproduction. However, they do not regain the ability to synthesize cell wall components. Cell wall regeneration occurs only under specific specialized conditions, allowing the cell to recover its original shape. Protoplasts remain relatively stable in hypertonic or isotonic solutions, whereas hypotonic conditions induce osmotic lysis.

The Hydrolysis of murein in Gram-negative bacteria by lytic enzymes does not remove the outer membrane. Instead, structures sensitive to changes in osmotic pressure, known as spheroplasts, are formed. Spheroplasts, much like protoplasts, are 3 to 10 times larger than the parental cells from which they originate. However, unlike protoplasts, they retain remnants of muramic and diaminopimelic acids and revert more readily to their original forms.

L-forms are bacteria that lack a cell wall yet retain the capacity for GROWTH AND DEVELOPMENT. They were first observed by scientists at the Lister Institute (UK) in 1935, which is THE ORIGIN OF their name. It was noted that cultures of Streptobacillus moniliformis could form atypical colonies containing abnormal cells. When transferred to a serum-supplemented medium, these cells are capable of indefinite proliferation. Initially, they were thought to be contaminants, symbionts, or parasites of Streptobacillus moniliformis, but it was later demonstrated that some of these cells can revert to their original rod-shaped Morphology.

On Agar media, L-forms produce colonies that grow deep into the nutrient agar, bearing a resemblance to a fried egg. The growth of such colonies is characteristically slow. Microscopic examination of the colonies reveals structures that vary considerably in both morphology and size: elementary bodies (0.2–1.0 µm) with minimal reproductive capacity; spherical or irregularly shaped bodies ranging from 1.0 to 5.0 µm; filamentous structures of various dimensions; large bodies (5.0–10.0 µm); and amorphous masses.

In Gram-positive bacteria, peptidoglycan forms a multilayered structure. The murein matrix is covalently linked to other macromolecules, among which are teichoic acids (from the Greek teixos meaning wall).

Teichoic acids were discovered by Baddiley in 1958 within the cell walls of Bacillus subtilis, Staphylococcus aureus, and Lactobacillus arabinosus. It was subsequently shown that teichoic acids are present in the cell walls of the majority of Gram-positive bacteria. Their content can reach up to 50% in eubacteria and 20–30% in actinomycetes.

The backbone of a teichoic acid molecule consists of a polymer chain comprising alternating ribitol phosphate or glycerol phosphate residues joined by phosphodiester bonds. A single teichoic acid molecule typically contains 7 to 15, and occasionally up to 50, alcohol residues.

Certain hydroxyl groups of these polyalcohols are substituted with D-alanine, glucose, N-acetylglucosamine, or N-acetylgalactosamine (R).

Depending on The Nature of the polyhydric alcohol (ribitol or glycerol), teichoic acids are divided into two main groups: ribitol teichoic acids and glycerol teichoic acids (Fig. 2.23), and a single cell wall may contain both types.

The free hydroxyl groups of the phosphoric acid impart polyanionic properties to teichoic acids. In glycerol teichoic acids, the phosphate residues are located in close proximity to one another, enabling them to bind divalent cations efficiently. The phosphate groups of ribitol teichoic acids are spaced further apart than those of glycerol teichoic acids, resulting in a lower affinity for divalent cations. The free amino groups of alanine confer amphoteric properties upon teichoic acids and can neutralize their internal charge.

Fig. 2.23. Cytology/cytology/92.html">SCHEMATIC STRUCTURE OF ribitol teichoic acid (a) and glycerol teichoic acid (b)

The bacterial cell wall may contain either glycerol teichoic acids, ribitol teichoic acids, or both. All membrane-associated teichoic acids are exclusively glycerol phosphate polymers.

Teichoic acids are considered the primary Antigens of the bacterial cell. Their antigenicity is enhanced upon partial hydrolysis of the murein.

The glycerol teichoic acids of the bacterial membrane are covalently linked to Glycolipids. Such complexes are referred to as lipoteichoic acids.

Cell wall teichoic acids perform a variety of crucial functions. They act as supplementary elements in the spatial ORGANIZATION OF THE cell wall structure; teichoic and lipoteichoic acids exhibit antigenic properties and largely determine the antigenic specificity of the cell surface; the carbohydrate constituents of teichoic acids serve as receptors for bacteriophages; as polyanions, teichoic acids dictate the surface charge of the cell and influence its cation exchange; in certain bacteria, teichoic acids participate in regulating The activity of autolytic enzymes (Hydrolases) capable of lysing the Organism's own peptidoglycan. Frequently, teichoic acids bind their own lytic enzymes, and any disruption of these bonds can lead to cell lysis.

Thus, In addition to peptidoglycan, the cell walls of Gram-positive bacteria contain teichoic acids, which serve as structural elements for spatial organization and perform a range of other essential biological functions.

A specific component of the cell walls of Gram-negative bacteria is lipopolysaccharide (LPS). It is localized in the outer leaflet of the outer membrane and comprises three distinct regions—lipid A, the core oligosaccharide, and the O-specific chain—which exhibit diverse biological properties.

The principal functions of LPS are as follows:

- stabilization of the outer membrane (structural role);

- Prevention of the penetration of detergents, antibiotics, and other toxic substances into the cell (barrier function);

- receptor function (LPS acts as a receptor for bacteriocins and phages);

- Determination of the serological specificity of the microbial cell (O-antigen);

- exhibits toxic properties – an endotoxin.

The Biochemical Composition of LPS varies significantly among different bacterial species, yet its structural backbone remains similar across all Gram-negative prokaryotes.

Lipid A consists of both hydrophilic and hydrophobic domains. The hydrophilic domain is represented by a disaccharide composed of D-glucosamine residues linked by a β-1,6-glycosidic bond (in *Salmonella*, *Pseudomonas*, *Serratia*, etc.) or a β-1,4-glycosidic bond (in *Shigella*, *Escherichia*, etc.). The disaccharide unit, in turn, is attached to fatty acid residues that form the hydrophobic region of the lipid A molecule. These Fatty acids are interconnected via ester bonds and are primarily represented by hydroxy acids (such as hydroxycaproic, hydroxymyristic, and lauric acids). LPS molecules featuring an atypical (altered) structure of lipid A are non-toxic.

The core (core oligosaccharide) is an oligomer comprising 3 to 11 monosaccharide residues. It is conventionally divided into two structural regions: the inner core, or KDO-heptose region (2-keto-3-deoxyoctulosonic acid), which is directly linked to lipid A; and the outer core, or hexose region, which connects to the O-antigen chain.

The O-specific polysaccharide chain is a polymer built from repeating oligosaccharide units. These units contain monosaccharide residues that vary in composition and structure across different species and even strains of microorganisms. The structural CHARACTERISTICS OF THE O-chain determine the serotype of a given strain.

Depending on the completeness of its structure, several LPS phenotypes are distinguished:

- S-phenotype (from smooth) – bacteria that synthesize the complete (fully developed) LPS structure. On agar media, bacteria of this type form colonies with a smooth, glossy surface;

- R-phenotype (rough) – the LPS core is complete or partially defective, and the O-specific chain is entirely absent. Bacteria with this LPS phenotype form rough, wrinkled colonies. Pathogenic bacteria exhibit reduced toxicity upon forming the R-phenotype;

- RS-phenotype – the O-chain is represented by a single repeating unit.

In terms of chemical composition, the cell envelopes of archaebacteria differ from the cell walls of Gram-positive and Gram-negative eubacteria, yet they also exhibit differential staining with the Gram stain. The fundamental structural difference between the cell walls of eubacteria and archaebacteria lies primarily in the absence of true peptidoglycan in the cell envelopes of archaebacteria. Only in the cell walls of certain archaebacteria (e.g., representatives of the genus *Methanobacterium*) is a murein-like substance detected, which is referred to as pseudomurein (Fig. 2.24).

Fig. 2.24. Schematic structure of the pseudomurein molecule

The pseudomurein molecule features a backbone composed of N-acetyl-D-glucosamine and N-acetyl-L-talsaminuronic acid residues linked together by β-1,3-glycosidic bonds. This structure resembles a glycan. A peptide (a penta- or tetrapeptide) is attached to the N-acetyl-L-talsaminuronic acid. The peptide fragments include exclusively L-amino acids: glutamic acid, alanine, lysine, etc.

Muramic acid is not detected in The structure of pseudomurein, nor are D-amino acids present. Electron microscopy demonstrates that the cell wall of such bacteria appears as a homogeneous layer 15–40 nm thick (similar to Gram-positive eubacteria). Cases have also been described (e.g., in certain methanogenic bacteria) where the cell wall thickness reaches 500 nm. Archaebacteria containing pseudomurein in their cell walls stain as Gram-positive during differential Gram staining.

In the cell walls of other archaebacteria (such as extreme halophiles), murein is absent. More frequently, the cell wall consists of a heteropolysaccharide (in representatives of *Halococcus*) or a glycoprotein (*Halobacterium*). The cell walls of *Methanosarcina barkeri* also possess a heteropolysaccharide nature. In such cases, the cell wall substance contains sugars, aminosugars, and uronic acids. The cell wall of certain halobacteria and methanobacteria is formed by protein, and sometimes these proteins are linked to aminosugars. *Methanospirillum hungatii* possesses a remarkably unusual Cell Structure (Fig. 2.25). It is encased in a cylindrical protein sheath that covers the entire spiral multicellular filament. Spacers are located between individual cells, where filament breakage or fragmentation can occur. Archaea lacking pseudomurein stain as Gram-negative.

Fig. 2.25. Structure of the archaebacterium Methanospirillum hungatii

ч – protein sheath; во – inner envelope; к – terminal spacer; с – intercellular spacer; н – nucleoid; в – inclusion; мв – membrane inclusion; цпм – cytoplasmic membrane

Thus, the cell walls of different bacterial groups differ from one another in both Chemical Composition and structural organization. They incorporate substances belonging to various classes of chemical compounds, which ensures the multifunctional Properties of the cell wall.

Cells of many bacteria are capable of synthesizing organic substances that are deposited on the outer side of the cell wall as slime layers or capsules (Fig. 2.26).

Fig. 2.26. Schematic representation of the arrangement of microbial cell surface layers:

1 – cytoplasmic membrane; 2 – cell wall; 3 – microcapsule; 4 – capsule; 5 – slime layer

Occasionally, a cell possesses both a capsule and amorphous slime of identical chemical composition. For instance, cells of *Leuconostoc mesenteroides* feature a capsule

of dextran nature alongside slime of the exact same composition, which dissolves into the surrounding medium.

A capsule is a layer that serves as the outer covering of the cell and remains connected to the cell wall. Depending on its thickness and consistency, capsules are classified into macrocapsules, microcapsules, slime layers, and soluble slime.

Macrocapsules are structures that are clearly visible under a Light Microscope after staining the preparation with India ink or nigrosin. Under such Treatment, the capsules appear as light structures against a dark Background (Fig. 2.27). Thinner capsules are identified by treatment with homologous sera. The resulting picture resembles Swelling.

Fig. 2.27. Capsules of Bacillus megaterium

Water accounts for 95–98% of the capsule substance, while other components make up 2–5%. The chemical composition of the dry matter is quite diverse, but all dry residue components are hydrophilic, negatively charged, and have a high molecular weight (~1,000,000).

The most common are polysaccharide capsules (Streptococcus, Aerobacter, and some members of the genus Bacillus).

For example, the capsular polysaccharide of B. polymyxa has mannose, glucose, galactose, and glucuronic acid residues in its structure in equal ratios of 1 : 1 : 1 : 1. The capsule material can be represented by both homo- and Heteropolysaccharides. The polysaccharide of S. pneumoniae consists of alternating glucose and glucuronic acid residues. Other polysaccharide capsules contain galactose, rhamnose, 2-keto-3-deoxygalactonate, mannose, and other sugars.

In many bacilli (B. anthracis, B. subtilis, etc.), the capsule consists of Polypeptides built from D- and L-glutamic acid residues. In some representatives of the genus Bacillus, the capsule substance is formed solely by poly-D-glutamine. The capsule substance of B. megaterium has a more complex structure, comprising both polypeptide and polysaccharide components.

The polysaccharide envelope of representatives of the genus Aerobacter or Klebsiella also qualifies as macrocapsules in terms of size. It contains a complex heteropolysaccharide consisting of repeating oligosaccharide units. The oligosaccharide of Aerobacter aerogenes has the following composition:

In some microorganisms, notably Mycobacterium tuberculosis, the capsule material consists of heteropolysaccharides, lipids, and other high-molecular-weight compounds. Such a COMPOSITION OF THE capsule helps these bacterial cells survive in adverse environmental conditions.

Under bright-field microscopy, the capsule appears as a homogeneous structure. Electron micrographs reveal fibrils arranged perpendicularly or parallel to the cell wall. Sometimes these fibrils form a mesh-like structure.

On solid nutrient media, encapsulated cells form shiny S-type colonies. Upon mutation, encapsulated forms transform into non-encapsulated ones, and the colonies acquire an R-type.

The synthesis of capsule substance in various groups of microorganisms is quite diverse. In most cases, capsular polysaccharides are synthesized from sugar nucleotide precursors. Glycosyl residues are then sequentially transferred to the polysaccharide chains via a lipid carrier of the cytoplasmic membrane. It is believed that the sugars forming the capsule substance are synthesized by the cell itself, and the composition of the medium has little effect on their Biosynthesis.

If the capsule substance is dextran (polyglucose) or levan (polyfructose), its synthesis proceeds at the expense of an exogenous substrate, the disaccharide sucrose ($\alpha$-glucosyl-$\beta$-fructose).

During levan biosynthesis, sequential attachment of fructosyl units to the acceptor sucrose molecule takes place:

The synthesis of levans is catalyzed by extracellular levansucrase. The levan-type capsule is characteristic of many microorganisms (Bacillus subtilis, B. cereus, Streptococcus salivarius, S. mutans, Azotobacter chroococcum, etc.).

During dextran synthesis, successive addition of glucosyl units to the sucrose molecule residue takes place.

Thus, dextran is a heteropolysaccharide consisting of α-D-glucose residues linked at the 1,6 position; in other words, dextran is an α-1,6-glucan whose parallel chains form a network.

It has been observed that many microorganisms produce slime particularly intensely in media containing sucrose. One such microorganism is Leuconostoc mesenteroides, a representative of Heterofermentative lactic acid bacteria. In a short period of time, this microorganism transforms a sugar solution into a dextran gel, which has earned it the name "frog spawn bacterium" in the sugar industry.

Microcapsules are sometimes considered part of the cell wall. They are less than 0.2 μm thick. Microcapsules have been best studied in representatives of the family Enterobacteriaceae; they are heteropolysaccharide in nature.

Capsules can be detached from the microbial cell surface by shaking or homogenizing bacterial Suspensions, or acapsular mutants can be obtained. These Procedures do not lead to cell death, meaning the capsule is not a vital structure. Based on this, one might think the capsule is an inert cell structure, but this is not the case. The capsule performs A number of important functions:

- it protects (shields) the cell from the action of toxic substances;

- since the capsular substance carries a negative charge, cations concentrate on the cell surface, creating a cationic pool that can be utilized for the cell's needs;

- being a strongly hydrophilic substance, it promotes water absorption by the cell, which is crucial under moisture-deficient conditions;

- enhances the adhesive properties of the cell;

- the capsular substance exhibits antigenic specificity, which is used for serotyping many groups of bacteria (K-antigen);

- in some bacteria, the capsular substance serves for immunological mimicry—the microorganism produces an antigen similar to the antigens of the macroorganism, i.e., the host organism. In such cases, the host's immune system is unable to produce Antibodies against this microorganism because it fails to recognize them. For instance, the capsular substance of Yersinia pestis possesses an antigen related to the anti-

gen of Blood group O erythrocytes, leaving a person defenseless upon infection.

The attachment of the capsule to the cell wall can vary. Some bacteria synthesize mucous substances that easily detach from the cells, especially when cultivated in a liquid nutrient medium. In other cases, the bond between the capsule and the cell wall is so strong that it is sometimes considered part of the cell wall. The presence of a capsule depends on the microbial strain and its cultivation conditions.

The substance surrounding the cell may have an amorphous, structureless appearance and easily detach from the cell surface. In such cases, it is referred to as a slime layer.

Sometimes slime binds individual cells together, forming specific groupings. For example, Acetobacter xylinum secretes Cellulose, which binds individual cells into a dense pellicle. In Sarcina ventriculi, cells are connected by cellulose into regularly shaped "aggregates." Cellulose acts as a binding agent in these cases. In its Structure and function, it differs from the capsular substance. The loss of cellulose-synthesizing ability in mutants does not disrupt Microbial growth, but the pattern of cell groupings changes.

Sheaths, unlike capsules, generally have a fine structure. Often, several distinct structural layers can be identified within them. Sometimes a sheath may enclose multiple cells. This is particularly characteristic of filamentous bacteria, notably the iron-oxidizing bacterium Sphaerotilus natans. In water, this microorganism forms filaments that create clumps capable of clogging pipes, settling basins, drainage pits, and the like. Such a filament consists of several cells enclosed in a common sheath. In Sphaerotilus natans, this structure contains (in %): CARBOHYDRATES (36); hexosamines (~11); proteins (~27); lipids (~5); phosphorus compounds (~0.5). The sheaths of S. natans are often encrusted with iron oxides.

Spikes can also be found On the surface of certain bacteria. Spikes are cylindrical protein structures anchored to The surface of the outer membrane. Their length ranges from 1-3 mkm, and their thickness is ~65 nm.

The spike protein, spinin (from Lat. spina - thorn), has a Molecular Weight of 19,000.

Spikes are found on the cell surface of certain pseudomonad-like planktonic marine bacteria. They can be easily sheared (disrupted) by mechanical factors and organic Solvents. Up to 10 spikes can be found on the surface of a single cell. Cells bearing spikes are generally non-motile. They have a higher density and settle more easily. The functions of spikes are currently under investigation.

In some bacteria, directed secretion of slime occurs, resulting in the formation of stalks (Fig. 2.28) that can provide bacteria with limited mobility (e.g., Nevskia, Gallionella).

Fig. 2.28. Bacteria with mucous stalks:

a - Nevskia; b - Gallionella

Many transitional forms have been identified among these supra-Membrane structures in prokaryotes, which is why it is sometimes impossible to clearly demarcate a capsule from the cell's slime secretions or a capsule from a sheath.

Pili (fimbriae) are straight, cylindrical protein structures that protrude from the cell surface and are anchored in the cell wall (Fig. 2.29). The pilus tubule passes through the murein layer and the outer membrane of the cell to its outer side.

Fig. 2.29. E. coli cells:

F - flagella; P - pili

The pilus filament consists of the protein pilin with a molecular weight of 14,500–26,200. Protein monomers are assembled into helical chains around a hollow core.

A distinction is made between common pili and sex pili (F-pili).

Pili have been studied in the greatest detail in E. coli cells. These bacteria possess both common pili and sex pili.

Common pili are divided into two groups. Type 1 pili are rather firmly bound to the cell and are resistant to chemical Reagents. They can be destroyed by boiling in low-pH solutions, which is accompanied by Protein Denaturation. The protein of this pilus type has a molecular weight of 17,000. From 50 to 400 type 1 pili can be found on the surface of a bacterial cell. The length of a pilus reaches 1.5 mkm, the outer diameter is ~7 nm, and the diameter of the inner channel is 2.0-2.5 nm.

Type 1 pili are peritrichously arranged and perform specific functions, namely:

- they induce erythrocyte agglutination, which is driven by the ability of pili to bind to red Blood Cells;

- they determine the adhesive capacity of the cell;

- they confer cell Hydrophobicity, causing such bacteria to form a surface pellicle when grown in a liquid nutrient medium;

- they reduce the electrophoretic mobility of cells.

Type 2 pili are antigenically and structurally similar to type 1 pili, but they do not promote pellicle formation during Bacterial growth in liquid media and do not cause erythrocyte agglutination. They may represent a mutant form of type 1 pili.

In enteropathogenic E. coli strains, pili act as a virulence factor, ensuring the ability of cells to attach to the intestinal epithelium. This leads to epithelial colonization by bacteria, thereby amplifying The Effect of bacterial enterotoxin on intestinal epithelial cells and disrupting tissue Water METABOLISM. Consequently, bacteria proliferate intensively in the Small Intestine and are subsequently shed in large numbers into the environment via feces.

Sex pili are proteinaceous cylinders oriented perpendicularly to the cell surface. The molecular weight of the protein identified in this type of pili is 11,800. The diameter of a sex pilus filament ranges from 8.5 to 9.5 nm, and its length can reach 1.1 µm. They are easily sheared upon agitation of the bacterial suspension.

Sex pili are formed on cells of donor strains, a characteristic feature of which is the presence of a specific genetic determinant known as the sex factor or transmissible factor. The latter can exist as an autonomous replicon (the F-factor), be part of an autonomous replicon, or integrate into the bacterial chromosome.

F-pilin synthesis occurs on ribosomes bound to the cytoplasmic membrane (CM). It is not detected in the cytoplasm. A pool of pilin accumulates in the CM. Each molecule contains a signal peptide that is cleaved off during pilin Transport Across the CM. The formation of sex pili is inhibited by the presence of cyanide, sodium azide, or dinitrophenol in the medium. F-pilin is a hydrophobic structure that readily dissociates in sodium dodecyl sulfate solutions and is disrupted by organic solvents. Cells bearing F-pili have reduced motility and tend to autoagglutinate, for example, upon a decrease in environmental pH.

The F-factor harbors genes responsible for conjugation as well as genes determining F-pili. F-pilus synthesis is controlled by a Gene complex (~13 genes). Under F-factor depression, 1-2 sex pili are formed. Under anaerobic conditions or in an optimal nutrient medium, 4-5 pili of this type are produced.

F-pili feature a central channel (pore) through which genetic material is transferred from one cell to another during conjugation. Furthermore, the free

The end of the F-pilus attaches to the recipient cell at the receptor site. An outer membrane protein of the recipient cell serves as this receptor. Initially, the contact is fragile and can be disrupted by hydrodynamic forces. Over time (within a few minutes), the contact stabilizes, and a cytoplasmic bridge forms between the two cells.

Sex pili are most abundantly produced by cells in the active growth phase. Cells in the stationary phase typically do not form pili and serve as poor Donors of genetic material.

In addition to E. coli, F pili are produced by other members of the Enterobacteriaceae family, including Vibrio, Pasteurella, Aeromonas, and Pseudomonas.

Some bacteria produce retractable pili, such as Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Streptococcus sanguis, and certain species of the genera Moraxella and Pasteurella. Cells equipped with this type of pili are capable of twitching motility, which ultimately results in the translocation of individual cells (and occasionally entire colonies) across a solid substrate.

Thus, the Surface structures of a bacterial cell include the cell wall, capsule, sheaths, stalks, prosthecae, pili, spines, and flagella.

The cytoplasm is the cell content enclosed by the cytoplasmic membrane (CM). It acts as a medium that integrates all intracellular structures into a unified system. The internal cavity of the cell is filled with the Cytosol—a semi-liquid colloidal mass consisting of 70–85% water, RNA, proteins (including enzymes), and metabolic products and substrates. The cytoplasm contains cellular structures (ribosomes, nucleoid, etc.), cytoplasmic membrane formations (gas vacuoles, mesosomes, chromatophores, thylakoids), inclusions enclosed by a protein shell (chlorosomes, phycobilisomes, aerosomes, magnetosomes, carboxysomes), as well as membrane-free structures regarded as reserve Materials.

The cytoplasmic membrane is an essential and vital structural element of every cell. Any disruption of the CM integrity leads to a loss of cell viability. The CM performs several critical functions:

- it acts as a selective barrier between the cell's internal contents and the external environment;

- in many groups of prokaryotes, the CM plays a major role in cellular Energy Metabolism, which is not the case in Eukaryotic cells. For example, in aerobic bacteria, the Respiratory Electron Transport system is embedded within the CM (in eukaryotes, this system is localized in Mitochondria). Consequently, bacteria with an intensive respiratory metabolism feature a highly complex CM structure characterized by numerous invaginations penetrating into the cytoplasm;

- it is believed that specific sites on the inner surface of the CM serve as attachment points for DNA. This is further supported by the fact that membrane growth (elongation) is accompanied by genome partitioning;

- the CM exhibits a certain degree of elasticity while remaining stretchable. For instance, when Acholeplasma laidlawii cells are placed in a hypotonic NaCl solution, their surface area increases by approximately 50% without compromising membrane integrity.

The CM accounts for 8–15% of the cell's dry weight. Transmission ELECTRON MICROGRAPHS OF ultrathin sections reveal a trilaminar structure of the CM (Fig. 2.30): two electron-dense layers flanking an electron-lucent space. Each osmiophilic electron-dense layer is 2–3 nm thick, while the electron-lucent interspace measures 4–5 nm.

Fig. 2.30. The "fluid-mosaic" membrane model

In terms of its chemical composition, the cytoplasmic membrane (CPM) is a protein-lipid complex containing 50–75% proteins, 15–45% lipids, and a small amount of carbohydrates. The Membrane Lipids of many prokaryotes contain several specific fatty acids that are entirely absent from the membranes of eukaryotic cells.

Microbial membrane lipids are the most thoroughly studied component. Membrane lipids consist of phospho- and neutral lipids. Phospholipids are the principal lipids of the bacterial CPM, accounting for 70–90% of its dry mass.

The molecular Skeleton of membrane phospholipids is formed by phosphatidic acid (PA), which consists of glycerol and fatty acid residues (R) (Fig. 2.31). Alcohol residues, amino acids, and Other Compounds (X) are attached to the PA.

Fig. 2.31. Structure of the phosphatidic acid molecule

The most common membrane phospholipids include: phosphatidylethanolamine (PE): PA-CH₂-CH₂NH₂; phosphatidylglycerol (PG): PA-CH₂-CHOH-CH₂OH; and diphosphatidylglycerol (cardiolipin - CL): PA-CH₂-CHOH-CH₂-PA.

Although the composition of phospholipids can vary significantly without generally disrupting Membrane Functions, a reduction in phosphatidylethanolamine content by more than 50% halts cell growth. Interestingly, this effect is not observed even in the complete absence of cardiolipin.

Proteins occupy roughly 50% of the membrane surface. Some of these are loosely bound to the CPM and can be released by altering the Ionic strength of the solution; these are termed peripheral proteins. Other proteins form stable bonds with CPM lipids to create protein-lipid complexes, making up about 10% of the membrane mass. Such proteins are referred to as integral proteins.

The protein composition of the CPM is quite diverse. For instance, about 120 different proteins have been identified in the membrane of E. coli. Among them, a special role is played by transport proteins (such as permeases) and Active Transport systems, as well as biosynthetic enzymes that catalyze the final stages of synthesis for membrane lipids and cell wall macromolecules (murein, teichoic acids, and lipopolysaccharides).

The prokaryotic membrane contains components of the ATP regeneration apparatus, while in purple bacteria (Rhodospirillum), the cell's photosynthetic apparatus is also localized within the CPM.

Most microbial membranes contain 2–5% carbohydrates. In some cases, these may simply be remnants of the cell wall adhered to the membrane. Evidence suggests that the membranes of Gram-positive bacteria contain glycolipids; for example, the membranes of Micrococcus lysodeikticus contain mannosyldiglyceride, whereas Streptococcus faecalis contains monoglucosyldiglyceride and galactosylglucosyldiglyceride.

The surface area of the CPM can change through invaginations, which typically form at the center of the Cell Division site. It is believed that these invaginated structures participate in the Formation of the cross-wall (septum) during cell division.

Invaginations are also required to house centers of respiratory and photosynthetic activity. This is supported by the fact that such invaginations are particularly prominent in the membranes of bacteria with high respiratory activity, such as Azotobacter and nitrifying bacteria.

Thus, while the CPM of a prokaryotic cell is generally similar to that of a eukaryote, it is notably richer in proteins.

The differences between prokaryotic and eukaryotic cells regarding their membrane systems were outlined earlier. The absence of typical Organelles in prokaryotes—meaning structures completely segregated from the cytoplasm by unit membranes—is a fundamental characteristic of their Cellular Organization.

Intracytoplasmic membranes can be classified into several types (Table 2.3). A well-developed system of intracytoplasmic membranes is characteristic of most photosynthetic prokaryotes. Because the photosynthetic apparatus of the cell is localized within these membranes, they are collectively referred to as photosynthetic membranes. All photosynthetic membranes, like all intracellular membranes, derive from the cytoplasmic membrane through proliferation and deep invagination into the cytoplasm. In some bacteria, such as purple bacteria, photosynthetic membranes maintain a close connection with the CPM and are readily visible in ultrathin cell sections via electron microscopy. In cyanobacteria, this connection is less pronounced.

Table 2.3. Prokaryotic membranes

* not found in green bacteria or the cyanobacterium Gloeobacter violaceus;

** highly developed in nitrifying, nitrogen-fixing, and methane-oxidizing bacteria.

The intracytoplasmic membranes of photosynthetic bacteria can appear as tubules, vesicles (chromatophores), or flattened closed disks (thylakoids) formed by two closely appressed membrane plates (lamellae). The photosynthetic membrane system is highly dynamic. Its morphology and degree of development within the cell are influenced by numerous environmental factors (light intensity, oxygen concentration, nutrient availability) as well as the age of the culture. However, in green bacteria and the cyanobacterium Gloeobacter violaceus, intracellular photosynthetic membranes are absent. The Main Components of their photosynthetic apparatus are localized in the CPM, and only light-harvesting pigments (chlorosomes in green bacteria and phycobilisomes in cyanobacteria) reside in specialized structures adjacent to the CPM that lack a unit membrane.

Prokaryotic organisms also feature local invaginations of the CPM known as mesosomes, which are well-developed and complexly organized in Gram-positive bacteria. In Gram-negative prokaryotes, these structures are rarer and simpler in organization.

Mesosomes are relatively simple, and occasionally quite complex, invaginations of the CPM. They vary in size, shape, and cellular localization. Three MAIN TYPES OF mesosomes are distinguished: lamellar (plate-like), vesicular (vesicle-like), and tubular (Fig. 2.32).

Fig. 2.32. Types of structure of true mesosomes:

a - lamellar; b, c, d - tubular

Mesosomes of a mixed type are frequently observed; they can consist of lamellae, tubules, and vesicles. Based on their cellular localization, the following types are distinguished: mesosomes formed in the zone of cell division and transverse septum formation; mesosomes to which the nucleoid is attached; and mesosomes formed as a result of the invagination of peripheral Regions of the cytoplasmic membrane (CPM).

The term "nucleidosomes" refers to chromosome-associated mesosome-like structures. The attachment of DNA to a specific membrane region is essential for genome function; however, much like true mesosomes, nucleidosomes are not found in all bacteria nor at all times, meaning their presence in the cell is not obligatory.

There are various Perspectives regarding The Role of mesosomes in the prokaryotic cell.

Some authors believe that they are not essential structures of prokaryotes, but rather serve to enhance certain cellular functions by increasing the overall "working" surface area of the membrane. There is also evidence linking mesosomes to an enhancement of cellular energy metabolism. It is believed that these structures play a certain role in Chromosome Replication and its segregation between daughter cells. It has been established that mesosomes of certain Gram-positive bacteria are involved in secretory processes. Another view suggests that mesosomes do not actively participate in Cellular metabolic processes, but instead perform a structural function by ensuring the compartmentalization of the prokaryotic cell—that is, the spatial Separation of intracellular contents into relatively discrete regions, which creates more favorable conditions for specific links of enzymatic reactions.

Thus, the coexistence of different hypotheses concerning the role of mesosomes in the prokaryotic cell indicates that their functions remain unresolved.

A well-developed system of intracytoplasmic membranes, morphologically distinct from mesosomal ones, has been described in representatives of three groups of Gram-negative chemotrophic bacteria (nitrogen-fixing, nitrifying, and methane-oxidizing), which exhibit high respiratory activity as well as the ability to metabolize gaseous compounds dissolved in liquid media.

Chromatophores. Some groups of microorganisms are characterized by the presence of membrane systems that are derivatives of the CPM and spatially associated with it, although such a connection is not always detectable.

In photosynthetic purple bacteria, Photosynthesis processes are localized on intracellular membrane systems defined as chromatophores. In Rhodospirillum, Thiocystis, Chromatium, and many other purple bacteria, the membrane structures of the chromatophore appear as tubules and vesicles with a diameter of 20–100 nm. The tubules and vesicles form a complex membrane network within the cell and retain connections with the CPM in numerous sites. Replicas of freeze-fractured such membranes reveal particles 7 and 10 nm in diameter on the inner surface.

In some purple bacteria, notably Thiocapsa pfennigii, the chromatophore is formed by a system of tubules arranged in parallel or branched rows. The chromatophores of Rhodopseudomonas gelatinosa and R. tenue represent doubled membrane lamellae that are invaginations of the CPM. Such doubled membranes are called thylakoids. In the cells of certain purple bacteria, thylakoids are assembled into stacks. Stacks of disc-shaped thylakoids are found in cells of some Rhodopseudomonas and Ectothiorhodospira species. Budding purple bacteria (e.g., Rhodomicrobium, Rhodopseudomonas viridis) have chromatophores in the form of lamellae arranged parallel to the cell wall. Such a chromatophore sometimes consists of several thylakoids and features an opening at the cell pole through which DNA can pass into the bud after nucleoid division. During budding, thylakoids remain in the mother cell, whereas in buds they are formed de novo.

Unlike plant Chloroplasts, bacterial chromatophores can disappear and form anew. This phenomenon is observed in bacteria capable of heterotrophic growth in the dark, during which chromatophores disappear in the dark and reappear when the bacteria are cultivated in the light.

The Development of an intracellular membrane system is characteristic of methane-oxidizing bacteria. Two Types of such membranes are found in them. Type I membranes consist of stacks of densely packed vesicular discs distributed throughout the cytoplasm. This type of structure is found in Methylomonas, Methylobacter, and Methylococcus. These structures are formed through the invagination of the CPM at one of the poles, although the contact point between the intracellular structures and the cytoplasmic membrane may be slightly shifted from the cell pole. Type II membranes are characteristic of Methylosinus, Methylocystis, and Methylobacterium; these are paired membrane layers located along the periphery of the cytoplasm.

The functional role of the intracellular membrane systems in methylotrophs is not yet fully understood, but it is believed that their presence increases the local concentration of substrates (methane and oxygen) for methane monooxygenase and provides spatial approximation of all components involved in methane oxidation.

Bacterial ribosomes serve as the site of Protein Synthesis. These are rounded ribonucleoprotein particles 16–18 nm in size, located in the cytoplasm. Ribosomes contain 80–85% of all bacterial RNA.

The ribosome consists of two subunits: in bacteria, these are the 30S and 50S subunits, which form a 70S-type ribosome—meaning it sediments during ultracentrifugation at a rate of about 70 Svedberg units (S), hence they are called 70S ribosomes. A bacterial cell contains approximately 5,000 to 90,000 ribosomes, depending on the Rate of protein synthesis. Eukaryotes possess 80S-type ribosomes.

The 30S subunit contains one 16S RNA molecule and, in most cases, one molecule of each of 21 protein species. Protein molecules are designated S1–S21 according to their position in Polyacrylamide gel Electrophoresis during Chromatography. The 50S subunit consists of two RNA molecules (23S and 5S) and one copy of each of 34 protein species—L1–L34. Most ribosomal proteins are present in a single copy and perform a structural function.

The structure of eubacterial, archaebacterial, and eukaryotic ribosomes is generally similar (Fig. 2.33). At the same time, certain differences exist: archaeal and eukaryotic ribosomes feature a beak (Nose), which is absent in eubacterial ribosomes; the eukaryotic ribosome differs from the bacterial one by the presence of lobes.

Fig. 2.33. Ribosome structure:

a-c - small subunits of eubacteria (a), archaebacteria (b), eukaryotic cytoplasmic ribosome (c); d - eubacterial ribosome associated with the CPM; I - translating region; II - protein exit region; III - bacterial CPM; IV - synthesized protein molecule; EF-G - elongation factor; 1 - beak (nose); 2 - HEAD; 3 - platform; 4 - body; 5 — lobes

Thus, the bacterial cell contains all the necessary structures in its cytoplasm to ensure its vital activity.

Reserve substances exist in an osmomotically inert form, but when conditions change and become favorable for growth, they enter metabolic processes (Table 2.4). Based on their consistency, these substances can be divided into liquid (e.g., poly-β-hydroxybutyric acid - PHBA), semi-liquid (sulfur), and solid (Glycogen).

For microorganisms, as for animals, carbohydrates are not the primary building material. This is characteristic only of plants, which serve as a source of carbohydrates for all other living beings.

Microorganisms are characterized by the accumulation of certain carbohydrates as reserve nutrients. These substances include glycogen, which is referred to as animal starch because it is synthesized in the bodies of humans and animals and used as a reserve energy material.

Bacterial Reserve Polysaccharides (glycogen) are glucans which, unlike cell wall polysaccharides, are formed from α-D-glucose. The molecular backbone features α(1-4)-type glucosidic bonds and α-(1-6)-type branching bonds. Branches emerge on average every 12 glucose residues in the main chain.

When reacting with Lugol's solution, polysaccharides stain red-violet (or brown). The molecular weight of glycogen can reach 1,000,000. Glycogen, or animal starch, shares similarities with amylopectin, but its chains are more heavily branched. It is found in Yeast cells, certain bacilli (B. polymyxa), many members of the Enterobacteriaceae family (Escherichia, Salmonella), as well as in Sarcina and Arthrobacter.

Under microscopy, glycogen appears as round, electron-dense granules up to 200 nm in diameter. The granules can be dissolved by treatment with hot KOH.

Due to the α-glucosidic bonds, polyglucose chains are not stretched out linearly, but rather coiled into a helix.

The accumulation of polysaccharide granules is stimulated by nitrogen deficiency in the presence of an excess of carbon and Energy Sources in the medium. When bacteria are cultivated under optimal conditions, glycogen accumulates during the transition of the culture into the stationary phase of growth.

In the cells of certain bacteria, the polysaccharide content can reach up to 25% of their dry mass.

Fat-like substances. Fat droplet granules are very frequently found in microorganism cells in the form of poly-β-hydroxybutyric acid (PHBA), a polyester of β-hydroxybutyric acid where individual monomers are linked by an ester bond formed between the carboxyl and hydroxyl groups of neighboring β-hydroxybutyric acid molecules.

PHBA as a reserve substance is found in the cells of Azotobacter, Rhizobium, Bacillus, and phototrophic bacteria, and is practically absent in obligate anaerobic chemotrophs. In some hydrocarbon-oxidizing bacteria, poly-β-hydroxybutyric acid accounts for up to 70% of the dry cell mass. Lipid accumulation in the cell occurs under conditions where the medium is rich in a carbon source and poor in nitrogen.

PHBA in cells appears as round, sometimes elongated granules measuring 200-800 nm. Their content within cells can reach up to 80% of the dry mass. Lipophilic Dyes are used for their detection: Sudan III (granules stain red) and Sudan Black (PHBA granules appear black). Such sudanophilic granules are soluble in chloroform, alcohols, pyridine, dioxane, toluene, 1 M NaOH, and camphor, and are practically insoluble in water, ether, methyl and ethyl alcohols, and acetone.

PHBA granules are surrounded by a protein membrane ranging from 2.2 to 8.0 nm in thickness. Enzymes responsible for the Synthesis and Breakdown of this reserve substance are bound to the membrane.

In the cells of certain bacteria, such as B. cereus and members of the genus Azotobacter, enhanced PHBA synthesis is observed upon The addition of glucose to the medium, while in Micrococcus halodenitrificans it occurs in the presence of glycerol, Pyruvate, or acetate.

An important factor stimulating PHBA synthesis is the limitation of the exogenous nitrogen source.

If cells containing PHBA granules are placed in a medium lacking a carbon and energy source, they begin to vigorously utilize PHBA.

Eukaryotic organisms lack the enzymes for PHBA synthesis or degradation; therefore, bacteria that accumulate this substance in their cells are undesirable for use in The production of food or feed additives.

In the cells of mycobacteria, nocardiae, and actinomycetes, other fat-like substances can accumulate in vacuoles and may even be secreted into the medium. For instance, Waxes (esters of Higher Fatty Acids and alcohols) can make up to 40% of the dry mass in mycobacterial cells.

Polyphosphates. Many bacteria and green Algae exhibit the ability to store phosphoric acid in the form of polyphosphate granules. They were first discovered in the cells of Spirillum volutans, and are therefore referred to as volutin granules. Another name—metachromatic granules—was given because polyphosphates cause certain dyes (methylene blue, toluidine blue) to undergo characteristic color changes, known as metachromasia. The detection of volutin is best performed at low pH values, as they retain their stain in an acidic environment, which is a key indicator that the inclusions belong to volutin. The basis of metachromatic staining is the interaction of polycations (basic dyes) with polyanions (polyphosphates), which causes the dyes to absorb

longer wavelengths of light. The Essence of this reaction lies in the polymerization of the dye on the polyanion macromolecules.

Polyphosphate grains dissolve in hot water and weak alkalis. They are insoluble in alcohols, ether, and chloroform.

Volutin accumulates in significant quantities in the cells of certain coryneform bacteria. In the cells of Corynebacterium diphtheriae, these inclusions are called Babes-Ernst bodies or granules (named after the scientists who first drew attention to them).

Volutin forms granules up to 1 µm in diameter, which consist primarily of polyphosphates, though RNA, DNA, and protein are also detected in granule preparations.

It is believed that volutin granules function as a phosphate depot, enabling the cell to divide even under conditions of phosphorus deficiency in the medium. In some cases, the accumulation and breakdown of volutin depend on the Stages of the Cell Cycle of the bacterial population. For example, studies of synchronous cultures of Corynebacterium diphtheriae have established that volutin accumulates in cells prior to division and is consumed during the process. Volutin is synthesized particularly vigorously when bacteria are transferred from a phosphorus-limited medium to one rich in phosphates. Polyphosphates are utilized by cells as a source of phosphorus. The question of whether they can serve as an energy source in prokaryotes remains a matter of debate.

A specific reserve substance of cyanobacteria is cyanophycin granules. Chemical analysis has shown that they consist of a polypeptide containing equimolar amounts of arginine and aspartic acid. The molecular backbone is built of aspartic acid residues linked by peptide bonds, with arginine residues attached to its β-carboxyl group. The initiation of cyanophycin synthesis requires ATP molecules, as well as K⁺ and Mg²⁺ ions. The appearance of cyanophycin granules when cyanobacteria are cultivated in a nitrogen-rich medium and their disappearance upon its depletion indicates that they serve as a nitrogen reserve, which is mobilized in the event of environmental scarcity.

Bacteria whose metabolism is linked to sulfur compounds are capable of depositing sulfur molecules within their cells. Sulfur accumulates when hydrogen sulfide is present in the environment and is oxidized to sulfate once all the hydrogen sulfide is depleted. For aerobic thionic bacteria that oxidize H₂S, sulfur serves as an energy source, whereas for anaerobic photosynthetic bacteria, it acts as an electron donor.

Gas vacuoles are membrane-bound structures formed by an accumulation of gas vesicles. They are found exclusively in prokaryotic cells. A gas vesicle is cylinder-shaped, 200–1000 nm in length and about 75 nm in diameter. The protein shell surrounding the vacuole is up to 2 nm thick and is built from protein subunits with a molecular weight of 14,000. The hydrophobic amino acids of the shell face the interior of the vacuole, while the hydrophilic ones face the outside. This specific arrangement of shell amino acids prevents water from penetrating into the gas vacuole. The composition of the air found within the vacuoles matches that of the surrounding environment.

Under bright-field microscopy, gas vacuoles appear as strongly light-refracting, optically empty spaces. Under electron microscopy, they look like hollow cylinders with conical ends arranged in parallel rows.

The formation of gas vacuoles (aerosomes) is characteristic of many aquatic bacteria, especially phototrophic ones, although a number of colorless bacteria (Pelonema, Peloploca), halobacteria (Halobacterium halobium), and certain members of the genus Clostridium also contain gas vacuoles. They provide the cell with the ability to alter its average density and remain in suspension, allowing some bacteria in stratified lakes to maintain their position within the water layer that offers optimal conditions for their growth. Anoxygenic phototrophic bacteria, notably purple bacteria (Lamprocystis, Amoebobacter, Thiodictyon) and green bacteria (Pelodictyon), grow in the anaerobic zone of water bodies (the hypolimnion). It is believed that the buoyancy of these organisms is sufficient for them to remain suspended in the cold (heavier) water layer of the hypolimnion, but does not provide enough lifting force to keep them in the warm (lighter) water layer. Oxygenic cyanobacteria (Oscillatoria agardhii, Aphanizomenon flosaquae, Microcystis aeruginosa) reside in warmer water layers. It is thought that the buoyancy of these bacteria is regulated via photosynthesis, cell turgor, and Changes in the number and size of gas vesicles.

Parasporal bodies. In the cells of Bacillus thuringiensis and related species (B. laterosporus, B. medusa), special crystal-like inclusions known as parasporal bodies are found. Under bright-field microscopy using aniline black, they appear as black crystals located near unstained spores (Fig. 2.34). Chemically, they consist of a protein that is highly toxic to blood-sucking mosquitoes. Such bacteria have found Practical Application as biological control agents not only against blood-sucking mosquitoes but also against many agricultural insect pests.

Fig. 2.34. Parasporal bodies in Bacillus thuringiensis

Magnetosomes are found in the cells of bacteria capable of magnetotaxis, meaning they can move along the magnetic field lines. They consist of membrane-enclosed particles of _Fe3O4. For instance, in the cells of Aquaspirillum magnetotacticum, magnetosomes are cubic in shape with sides measuring 40–50 nm and are arranged in a row along the cell. The shape, number, and arrangement pattern of these structures vary among different groups of microorganisms.

Locomotory Organs of bacteria. The motility of a bacterial cell can be achieved through various mechanisms, with swimming and gliding types of movement being the most characteristic.

The locomotory organ of bacteria with a swimming type of movement is the flagellum. Flagellated forms are found among all known groups of bacteria, indicating that flagella are very ancient structures of the bacterial cell.

Several types of flagellar arrangement on the surface of the microbial cell are distinguished (Fig. 2.35):

- monotrichous flagellation, in which a single flagellum is located at one of the cell poles (Vibrio comma, Pseudomonas aeruginosa, Neurospora europea, Thiobacillus ferrooxidans), or a single flagellum is localized subterminally (Rhizobium lupini);

- lophotrichous, where a tuft of flagella is located at one of the cell poles. This type of flagellation is characteristic of Pseudomonas fluorescens, P. putida, Chromatium okenii, and members of the genus Sphaerotillus (subpolar arrangement of the flagellar tuft);

- amphitrichous, where tufts of flagella are found at both poles of the microbial cell (bipolar polytrichy) – Spirillum;

- peritrichous flagellation, where flagella are localized across the entire surface of the cell. This is typical, for example, of representatives of the genera Proteus, Escherichia, and Salmonella.

Fig. 2.35. Main types of flagellar arrangement in bacteria:

a – monotrichous; b – lophotrichous; c – amphitrichous; d – peritrichous

The number of flagella and their arrangement pattern can serve as a differential taxonomic feature. However, it should be kept in mind that this trait is influenced by cultivation conditions and that flagella may break off during specimen preparation. Special techniques, such as phase-contrast or dark-field microscopy, are used to visualize bacterial flagella (Bdellovibrio, Chromatium okenii, Pseudomonas, Spirillum). Electron microscopy yields excellent results. In bright-field microscopy, special staining methods that thicken the flagellar filament are employed.

In prokaryotes, two types of flagella are distinguished: simple and complex. The thickness of a simple flagellum ranges from 12 to 18 nm, and its length is 3–15 µm. Complex flagella are covered by an additional protein sheath. The sheath protein, with a molecular weight of 55,000, differs both from the cell envelope proteins and from the flagellar proteins themselves. The thickness of a complex flagellum is approximately ~18 nm. This type of flagellum is found in certain representatives of the genera Pseudomonas (P. rhodos) and Rhizobium (R. lupini).

The rotational speed of a flagellum is relatively high. For example, the flagella of spirilla rotate at a speed of 40–60 rev/s (~3,000 rev/min).

The swimming speed of a flagellated cell varies among different groups of microorganisms and does not depend on the number of flagella. For instance, cells of B. megaterium, which have a peritrichous flagellation type, move at a speed of ~27 µm/s, whereas Vibrio comma (monotrichous) move at ~200 µm/s. The movement speed of a flagellated cell ranges from 300 to 3,000 body lengths per minute (900–9,000 µm/min) given a cell body length of ~3 µm. To put this in perspective, if a human 1.5 m tall were to move at a proportional speed, it would translate to 27–270 km/h.

The flagellar apparatus consists of a filament, a hook, and a basal structure (Fig. 2.36).

Fig. 2.36. Diagram of the flagellar apparatus structure in Gram-negative bacteria

Chemically, the flagellar filament is composed of the protein flagellin with a molecular weight of 25,000–60,000. Protein subunits (monomers) of the flagellum are assembled into helical chains wound around a hollow core. A distinctive structural feature of flagellin is the complete absence of Histidine, Proline, Tyrosine, Tryptophan, and Cysteine in its Amino Acid Composition.

The flagellar protein exhibits antigenic specificity. It is referred to as the H-antigen and is widely used in bacterial identification.

The hook protein differs somewhat from the flagellar filament protein in both molecular weight and antigenic specificity. It is more resistant to low pH values, heat, and urea treatment.

The basal body of the flagellum consists of a central rod (axis) embedded in a system of rings. In Gram-negative bacteria, this structure features two pairs of rings: the inner (M and S) and the outer (P and L); in Gram-positive bacteria, there is only one pair of rings (M and S).

The inner pair of rings is located at the level of the cytoplasmic membrane (CPM). Specifically, the M-ring is embedded within the cytoplasmic membrane, while the S-ring is positioned slightly higher, nearly abutting the inner surface of the peptidoglycan layer. The L-ring is situated at the level of the lipopolysaccharide (LPS) layer of the outer membrane in the cell wall of Gram-negative bacteria, whereas the P-ring lies at the level of the murein layer.

The length of the basal structure ranges between 25–30 nm, depending on the thickness of the cell wall. The connection between the rings and the rod-axis is relatively weak, as evidenced by electron micrographs showing that some rings may be absent.

The primary function of the basal body is the fixation and anchoring of the flagellum. Given The complexity of this structure, each of its elements serves a specific purpose. Since L and P rings are found exclusively in the flagellar structure of Gram-negative bacteria, it is believed that they provide additional anchorage for the flagellum, whereas the inner pair alone (the S and M rings) is sufficient for the basic operation of the flagellar apparatus.

Flagellar motility has been studied in the greatest detail in bacteria with lophotrichous and amphitrichous flagellation. In most lophotrichous bacteria, the flagella (acting like a ship's propeller) push the cell through the medium.

In bacteria with peritrichous flagellation, individual laterally positioned flagella are thought to function as a single integrated unit (Fig. 2.37). They lie closely appressed along the cell body, extending beyond its posterior end, and rotate to propel the cell through the medium. Peritrichous bacteria swim less efficiently than mono- or lophotrichous ones and frequently tumble; however, some species move very effectively across moist solid surfaces, sometimes crawling better than they swim.

Fig. 2.37. Movement of a bacterium with peritrichous flagellation

Flagella can change their direction of rotation spontaneously or in response to external stimuli. In some polar-flagellated bacteria, this results in the cell reversing its direction of movement. For example, in Chromatium okenii, in response to a light flash, the Rotation of the flagella reverses—transforming the flagellar bundle into a pulling apparatus. In Thiospirillum jenense, a giant phototrophic spirillum, the polar flagellar bundle during reverse motion beats ahead of the cell. The sweeping space of the flagella encompasses the sides of the cell, effectively turning it inside out (much like an umbrella inverted by a wind gust). In amphitrichous spirilla, depending on the circumstances, either one or the other of the polar flagellar bundles assumes this position.

The flagellum forms a helical structure, typically left-handed (counterclockwise coiling). The flagellum itself also rotates counterclockwise (when viewed from behind a swimming cell), which is accompanied by the rotation of the cell body in the opposite direction

of motion. The direction of flagellar rotation can reverse. For instance, if a left-handed helix begins to rotate clockwise, the cell stops or tumbles. When the direction of flagellar rotation changes in E. coli, its conformation and helical pitch are altered. The flagellar filament transforms into a right-handed structure with a decreased pitch, forming "curly" flagella. Such flagella are incapable of bundling together, causing the cells to tumble or quiver without making net forward progress.

Certain bacteria (representatives of the genus Bdellovibrio) possess an unusually thick, polarly localized flagellum (~50 µm) that allows this bacterium to move significantly faster than other bacteria. Upon encountering a host bacterium, the parasite attaches to its surface via the end opposite to the flagellum. Occasionally, Bdellovibrio rotates around its longitudinal axis. Subsequently, the prey cell rounds up and resembles a spheroplast. After penetrating the host cell, the Bdellovibrio rapidly increases in volume and multiplies. The progeny cells of Bdellovibrio remain within the spheroplast until its contents are completely digested. Following the lysis of the infected bacterium's cell wall, the bdellovibrios emerge to infect new cells (Fig. 2.38). Bdellovibrio species predominantly lyse Gram-negative bacteria, primarily pseudomonads and enterobacteria.

Fig. 2.38. Bdellovibrio bacteriovorus, a bacterium that parasitizes other bacteria:

a — Life Cycle of B. bacteriovorus; b, c — penetration of a B. bacteriovorus cell into the host cell

The Mechanism of the flagellar apparatus remained a mystery for a long time. It was eventually discovered that the flagellar "motor" is located in the inner pair of rings of the basal body, which rotate and transmit their motion to the filament.

A hypothetical model of the flagellar motor mechanism was proposed by O. Gogolev and V. Skulachev in 1978. One of a series of amino groups opens into the upper proton-conducting pathway (channel) (Fig. 2.39). An anionic group (COO^-) is located on the cytoplasmic membrane near the lower proton-efflux pathway. Upon protonation, the NH₂ group acquires a positive charge (-NH₃⁺), which leads to electrostatic attraction between the anionic and cationic groups. This forces the M-ring to move—causing a rotation of the ring, which in turn shifts the next amino group toward the upper proton pathway. The expulsion of H⁺ cations occurs through the lower proton-efflux channel into the cytoplasm.

Theoretical calculations indicate that a single rotation of such a motor consumes approximately 10³ H⁺ protons.

Fig. 2.39. Model of the proton motor in the basal body of the bacterial flagellar apparatus: p — rod-axis; m — M-ring

Spirochetes possess a uniquely structured motility apparatus (Fig. 2.40). A spirochete cell consists of a protoplasmic cylinder bounded by a cytoplasmic membrane and enclosed in an outer sheath formed by a murein layer and an outer membrane. In other words, the envelope of spirochetes shares the same structural organization as the envelopes of other Gram-negative bacteria.

Fig. 2.40. Schematic structure of a spirochete cell

In the space between the peptidoglycan layer and the outer membrane of the envelope lie axial fibrils (axial filaments). Depending on the species, the number of fibrils can range from 2 to 100. A bundle of such filamentous structures wraps around the protoplasmic cylinder. Thus, the motility organelle of spirochetes is located within a space enclosed by cellular structures, meaning it is an intracellular structure.

The axial fibril is somewhat smaller (shorter) than the cell body, spanning approximately 2/3 of its length. One end is anchored at the pole of the protoplasmic cylinder, while the other remains free. The free ends of opposing fibrils overlap.

The attachment of the axial fibril to the protoplasmic cylinder involves a hook and a basal structure. The latter is a rod-like axis bearing 1–2 rings.

Spirochetes can perform various types of movement: the cell body can bend, forming a secondary wave (spiral); rotate in place; or exhibit translational motion.

Two types of cell movement are possible in leptospires. In the first case, it is assumed that the outer sheath and the protoplasmic cylinder are fixed relative to each other. Here, the rotation of the filaments is accompanied by an oscillation of the cell ends. The outer sheath and protoplasmic cylinder bend but do not rotate.

The second type of movement is associated with the rotation and bending of both the protoplasmic cylinder and the outer sheath, driven by the rotation of the axial filaments. Leptospira cells (Leptospira interrogans) execute translational motion, effectively corkscrewing their way through a viscous medium.

Any movement has its biological purpose. Motile bacteria move under The Influence of certain external forces or stimuli. Such movement is called taxis (from Greek *taxis* – arrangement). In other words, taxis is movement oriented relative to the direction of a stimulus, i.e., directed movement.

Depending on the external factors driving the movement, a distinction is made between chemotaxis, phototaxis, aerotaxis, magnetotaxis, and viscositaxis.

Chemotaxis is a prime example of behavioral responses for which not only the phenomenology of the phenomenon, but also the molecular processes ensuring its realization, have been relatively well studied. In a suspension of flagellated bacteria, cells are in a state of continuous yet random motion. When a concentration gradient of certain chemical substances is established within a bacterial population, the cells migrate and accumulate in the region where the concentration of the substance is optimal for them. Certain substances (mainly nutritional substrates) act as attractants: cells accumulate in the zone of highest concentration of this substance. Other substances, most of which are toxic, act as repellents, meaning cells avoid zones with high concentrations of them and gather in the gradient region where the concentration is lowest. Aerotaxis, caused by the influence of oxygen, and osmotaxis, caused by the action of salts, can also be attributed to chemotaxis.

Changes in effector concentrations can be directed via several mechanisms. It has been established that more than 20 chemoreceptors are localized in the cytoplasmic membrane of *E. coli* and *S. typhimurium*, each binding a specific set of effector molecules and/or periplasmic proteins that serve as primary acceptors of these effectors.

The ability to exhibit taxis confers certain advantages upon the cell, primarily enabling it to actively search for an optimal living environment. As was established in the case of *E. coli*, such a search has a logical Conclusion: upon reaching the source of a glucose gradient, flagellin synthesis is repressed via catabolite repression. Beyond searching for universal substrates such as glucose and amino acids, taxis helps bacteria locate their specific ecological niches.

Thermotaxis is the movement of bacteria toward areas with elevated temperatures. It was described as early as 1920; however, quantitative studies of this phenomenon were undertaken only recently. The authors used a simple approach: a linear Temperature gradient was established in a Glass tube containing an *E. coli* suspension. Within just 1 hour, the cells gathered in a narrow band in the region where the temperature was 34⁰C (which is optimal for the growth of these bacteria). A sharp drop in temperature on the microscope slide from 34 to 20⁰C caused the cells to enter a trembling state, followed by subsequent adaptation. When the temperature was raised to the optimal value, the speed of cell movement in the medium increased.

Aerotaxis is the movement of bacteria driven by oxygen concentration gradients. This phenomenon is easily observed in "hanging drop" or squashed preparations, where an oxygen gradient is established by diffusion directed from the edges of the coverslip toward the center of the preparation. In motile bacteria, the type of metabolism (aerobic or anaerobic) can be determined by their aerotactic movement and the accumulation of cells at specific distances from the edge of the coverslip or near air bubbles.

Phototaxis is the movement of bacteria toward or away from light. Motile phototrophic bacteria, such as purple bacteria, can respond to light intensity gradients—a phenomenon known as phototaxis. This behavior is easily demonstrated by projecting a narrow beam of bright light onto a poorly illuminated suspension of motile phototrophic bacteria. Within 10–30 minutes, a significant portion of the population gathers in the best-illuminated area, which acts as a "light trap." A swimming cell enters the light spot due to random motion, but once there, it is unable to leave it. As soon as the cell enters the dark zone, the direction of flagellar rotation instantly reverses, and the cell returns to the illuminated area. The change in flagellar apparatus operation occurs so rapidly that this response has been termed the "scuttling reaction" (phototaxis). Even a minor difference in illumination between two areas is sufficient to trigger this response. Cells of bacteria of the genus *Chromatium* accumulate in a specific spot if its illumination is merely 0.7% higher than that of the surrounding areas.

If a wet mount of motile purple bacteria is illuminated not with white light but with a spectrally dispersed spectrum, and the rays are focused on the preparation, the cells rapidly gather in areas corresponding to the main absorption maxima of these bacteria's pigment systems. Precise Quantitative determination of the relative effectiveness of different wavelengths in eliciting phototaxis has shown that its action spectrum in purple bacteria closely matches the photosynthetic action spectrum.

Magnetotaxis is the ability of bacteria to move along the magnetic field lines. Bacteria capable of orienting themselves in a magnetic field and moving along its lines have been isolated from surface mud layers. They contain a high amount of iron (0.4% of dry weight) in the form of ferromagnetic iron oxide (magnetite)—specifically 10–20 microcrystals measuring 40–90 nm—contained in granules (magnetosomes) located near the flagellar attachment sites. Bacteria isolated in the Northern Hemisphere "seek" the south; here, the magnetic field lines run downward at an angle of 70⁰ to the horizon, deep into the body of water. Magnetotactic behavior directs bacteria deep into the mud, where oxygen is scarce or absent. Since magnetotactic bacteria are anaerobes or microaerophiles, their response to the magnetic field is ecologically adaptive. Such cells, if transported to the Southern Hemisphere, usually perish; only a few "erroneously" polarized cells survive and can subsequently reproduce. Polarity is presumably not genetically fixed.

Viscositaxis is the movement of bacteria in the direction of increasing or decreasing solution density. This feature has adaptive significance, particularly for spirochetes that move along the surface of mucous membranes. The mechanism of this process remains unclear.

Morphological differentiation in prokaryotes. According to F. Jacob and J. Monod, "one cell can be considered differentiated relative to another if, given identical genomes, the set of proteins synthesized by these cells differs."

A characteristic feature of certain prokaryotes is the ability to form structures that differ significantly from the original forms, as well as from similar structures in other biological objects; microorganisms exhibit morphological differentiation accompanied by substantial biochemical and physiological changes. Morphological differentiation is underpinned by specific biochemical processes controlled by the cell's genetic apparatus. Morphologically differentiated structures in prokaryotes include exospores, endospores, cysts, heterocysts, and akinetes (Table 2.5).

An endospore is a dormant stage of a bacterial cell (Fig. 2.41) that facilitates the survival (preservation) of bacteria under adverse environmental conditions; that is, an endospore does not serve a reproductive function.

Fig. 2.41. Endospore of a prokaryotic cell

Table 2.5. Morphologically differentiated structures of prokaryotes

Endospore formation is characteristic exclusively of prokaryotes. Bacterial endospores are a special type of dormant cell forms in bacteria that are formed endogenously, i.e., within the cytoplasm of the "mother" cell (sporangium). Endospores exhibit a unique physical state of the protoplast.

It is generally accepted that endospore formation represents one of the developmental phases of a microbial population. However, this stage is not mandatory for The life cycle of spore-forming bacteria. Under favorable nutritional conditions, cells can multiply indefinitely without entering the dormant stage.

The sporulation process can also be inhibited, for instance, by adding glucose to the medium or through frequent subculturing. The role of the inhibiting factor depends on the timing of its introduction. Thus, if glucose is added to a suspension of vegetative cells of B. mycoides within the first 5 hours after placing them in water, spore formation is repressed. Glucose added later than 6 hours has no effect—spore formation is no longer inhibited. At the same time, induction (derepression) of sporulation is maintained between 10 and 13 hours after placing the bacilli in water, with approximately 90% of the cells forming spores. Thus, the sporulation process is regulated to a certain extent by environmental factors.

The sporulation process can also be induced. Factors that induce endospore formation include nutrient limitation, accumulation of Metabolic waste products, the presence of Mn²⁺ and Ca²⁺ ions in the medium, changes in cultivation temperature, pH levels, O₂ concentration, etc.

Consequently, sporulation occurs when general environmental conditions permit it. If vegetative cells at a certain stage of development are placed in distilled water, so-called endotrophic sporulation can be observed, i.e., spore formation at the

expense of the cell's reserve substances. In such cases, spore formation is indeed triggered by an exogenous substrate deficit.

The duration of the sporulation process varies among different endospore-forming bacteria. In aerobic mesophilic bacilli, in particular, sporulation takes about 8 hours.

The cellular capacity for spore formation is determined by a complex of genes known as the sporulon, although it is not a structurally integrated entity. Nevertheless, the existence of several groups—operons located in different regions of the chromosome—has been established. It has been shown that B. subtilis possesses 42 sporulation operons, each containing ~3 genes. Overall, B. subtilis has 150–200 genes controlling the sporulation process. The events occurring at each stage of sporulation may depend on the activity of Genes from different operons.

Sporulation has been most thoroughly studied in representatives of the genera Bacillus and Clostridium, although recent data indicate that this process is fundamentally uniform across all species of endospore-forming bacteria.

Sporulation proceeds in several phases (stages). The transition to each successive stage is driven by the activation of corresponding genes. In this process, one of the products of the currently "functioning" gene induces the activity of the next gene.

The initiation of sporulation requires the completion of bacterial chromosome replication. The signal for sporulation must be received during the replication period itself. If chromosome replication finishes in a nutrient-rich medium, this signal is not sent, and the population continues vegetative division. During spore formation, the chromosome is not replicated; instead, one or more previously fully replicated Chromosomes pass into the spore.

A cell preparing for sporulation contains at least two nucleoids that form a strand extending almost the entire length of the cell and incorporating all of its DNA (Fig. 2.42).

Fig. 2.42. Schematic representation of endospore formation in spore-forming bacteria:

a — vegetative cell; b — invagination of the cytoplasmic membrane; c — formation of the spore septum; d — formation of the forespore double-membrane system; e — mature forespore; f — cortex formation; g — spore coat formation; h — lysis of the mother cell; i — free mature spore; j — spore germination; 1 — nucleoid; 2 — cytoplasm; 3 — cytoplasmic membrane; 4 — cell wall; 5 — spore septum; 6 — outer spore membrane; 7 — inner spore membrane; 8 — cortex; 9 — spore coats

In some species of the genus Clostridium, it is formed by only a fraction of the cellular DNA and localizes at one of the cell poles, occupying a minor portion of its length. Subsequently, the region corresponding to the chromosome pinches off from the axial strand and migrates to one of the cell poles.

Next, the cytoplasmic membrane begins to invaginate, forming two differently sized structures separated by a membrane. At this stage, the sporulation process is still reversible. If the bacterial cells are transferred to favorable growth conditions, cell division will resume, yielding two vegetative cells of unequal length. This process has been found to be controlled by genes located in eight operons across various regions of the chromosome.

During the Initial Stages of sporulation, reserve substances and a portion of the mother cell's proteins are utilized. In most species of the genus Bacillus, specific proteases are synthesized, which are attributed various functions, including participation in protein turnover, modification of DNA-dependent RNA polymerases, and precursor proteins for the spore coats.

Subsequently, the cytoplasmic membrane of the larger cell portion begins to engulf the smaller structure, resulting in a structure bounded by two Cytoplasmic membranes—the forespore. The two membranes surrounding the forespore are oriented in a mirror-like fashion relative to each other: the outer layer of the inner membrane (previously facing the environment) now faces outward, while the outer membrane faces inward. At the next stage, the forespore detaches from the mother cell's cytoplasmic membrane and floats freely within it, as it were. At this point, sporulation becomes irreversible, accompanied by altered membrane properties, particularly its transport functions. The process becomes irreversible not only for the forespore but also for the mother cell, which can no longer return to vegetative growth. If such a system encounters unfavorable conditions, it will either complete spore formation or perish.

In the forespore, the uptake of Ca²⁺ ions intensifies, and the synthesis of Certain amino acids and dipicolinic acid (pyridine-2,6-dicarboxylic acid) begins. Vegetative cells are incapable of synthesizing the latter.

The resulting forespore migrates to the center of the cell or remains near one of its poles, depending on the species-specific spore Location pattern.

An additional shell—the cortex—is formed between the two cytoplasmic membranes in most representatives of the genus Bacillus. This process occurs in two stages. First, the primordial layer is synthesized, which gives rise to the germ cell wall during spore germination. Over the primordial layer, a modified murein layer is synthesized, forming the cortex. Cortex substance synthesis continues until spore maturation. This biopolymer is more acidic than its counterpart in the cell wall, which is explained by the fact that many muramic acid residues lack peptides and therefore possess more free carboxyl groups.

In B. cereus, the exosporium forms simultaneously with the cortex synthesis. Initially, it is synthesized at one of the poles of the forespore and subsequently envelops it entirely. In most species of the genus Clostridium, coat deposition begins prior to cortex formation.

In cross-sections of the spore, the cortex appears as a massive electron-lucent layer. In a dormant spore, it exhibits a homogeneous structure, whereas in germinating spores, it appears fibrillar.

The exosporium resembles a sac enclosing the spore. Its structure varies among different species, with that of B. cereus being the most thoroughly studied. The exosporium is composed of proteins, lipids, and carbohydrates, and it is believed to protect the spore from adverse environmental factors.

The exosporium of C. botulinum consists of 15 parallel layers with a total thickness exceeding 100 nm. In C. pasteurianum, it forms a delicate structure featuring a large pore at one of its poles. In some cases, the outer exosporium is covered with appendages or surface projections of various shapes.

Toward the end of exosporium formation, the young spore remains osmotically unstable and non-viable, yet Certain physical properties already begin to change. Notably, light refraction increases, indicating the synthesis of protein compounds, since light refraction does not rise in the presence of Protein Synthesis Inhibitors.

The formation of the spore coat proceeds subsequently. The spore coat develops on the exterior side of the outer membrane, while the outer membrane itself is barely discernible in cross-sections. Calcium dipicolinate continues to accumulate within the spore, enhancing its resistance to organic solvents such as chloroform. Although the spore attains osmotic stability, it is not yet thermoresistant.

Spore development is governed by the activity of two genomes: its own and that of the mother cell. The maternal portion of the cell—the sporangium—maintains metabolic activity until the final stages. The Genome of the mother cell dictates the synthesis of dipicolinic acid, proteins, spore coat materials, the exosporium, and spore appendages, whereas the spore's own genome regulates dehydration and the formation of the calcium dipicolinate complex.

The spore cytoplasm is enclosed by the cytoplasmic membrane (CM), which transitions into the primordial layer and subsequently into the cortex. The cortical substance accounts for 20–60% of the spore volume, yet does not exceed 15% of its mass.

During maturation, the spore undergoes dehydration. Water content in the mature spore drops to 10–15%, leading to Enzyme inactivation and, consequently, the suppression of metabolic processes. The spore thus acquires thermoresistance.

The structure of the mature spore is largely uniform across various endospore-forming bacteria. The cytoplasm contains nuclear material, and proteins and nucleic acids comprise 50–60% of the spore's dry mass. Dipicolinic acid constitutes a significant portion of the spore; in mature spores, it is present as calcium dipicolinate, which can account for up to 10–15% of the dry weight.

The cortex is bounded by the outer spore membrane, which borders the outer coat. The substance of the outer coat provides the spore with resistance to lysozyme.

Sporulation is accompanied by the active synthesis of proteins which, unlike vegetative cell proteins, are rich in cysteine and hydrophobic amino acids—a feature also linked to the spore's resilience against unfavorable environmental factors.

The spore coats ensure resistance against enzymatic degradation, permeability to organic solvents, Surfactants, antibiotics, and dyes, among other agents. Once the spore is fully formed, the cell wall undergoes lysis, releasing the spore.

A schematic diagram of a mature spore structure is shown in Fig. 2.43.

Fig. 2.43. Schematic structure of a mature spore:

1 - exosporium; 2, 3 - outer and inner spore coats, respectively; 4 - cortex; 5 - germ cell wall; 6 - cytoplasmic membrane (CM); 7 - cytoplasm with nuclear material

Spores possess distinct characteristics: first, they are metabolically inactive; second, they exhibit high resistance to elevated temperatures, chemical agents, and ionizing radiation.

The exact nature and mechanism of spore thermoresistance remain fully unresolved. According to H. Schlegel, this phenomenon can be attributed to the low water content. Another widely held view is that thermoresistance is roughly proportional to the concentration of dipicolinic acid. Resistance to chemical agents is further reinforced by the impermeability of the outer layers, while spore coat proteins protect the spore from lytic enzymes.

Bacterial spores vary in shape, size, and intracellular location within the mother cell (Fig. 2.43). In many species, the spore is situated in the center of the cell with a diameter not exceeding the cell's width—known as the central (bacillary) localization type (B. subtilis, B. anthracis, B. megaterium). Many bacterial spores are oval-shaped yet have a diameter greater than the cell width, representing the clostridial localization type, which gives the cell a spindle-like appearance (C. pasteurianum). Alternatively, the spore may locate at one of the poles of the microbial cell, swelling the sporangium and causing the cell to resemble a drumstick—the plectridial arrangement type (C. tetani). The spore may also occupy the central region of the sporangium while distorting one of its sides, known as the lateral position (B. laterosporus).

A characteristic feature of sporulation in anaerobic bacteria is the formation of specialized surface appendages of diverse structures on the spores (Fig. 2.44). This trait is species-specific, genetically fixed, and highly stable. Even in defective spores that have lost the ability to form a coat, these appendages are retained without structural alteration.

Fig. 2.44. Types of spore localization within the cell:

a - central (bacillary); b - subterminal (bacillary); c, d - plectridial; d - clostridial

These appendages may take the form of fine filaments or flagella, tubes of varying thickness distributed across the entire spore surface, or bottle-brush-like rod clusters, long broad ribbons, spines, or structures resembling deer antlers.

The development of appendages begins during the Early stages of sporulation—prior to cortex formation—after which they grow and elongate, and the cytoplasm of the mother cell surrounding them undergoes lysis. Once mature spores are released from the sporangium, their appendages unfold.

Fig. 2.45. Structure of surface spore structures in certain anaerobic bacteria:

a - spore without appendages and exosporium; b - spore without appendages, but with an exosporium; c-h - spores with various types of appendages and an exosporium

The appendages are composed primarily of proteins, along with minor amounts of polysaccharides and lipids.

The functions of these appendages are currently under investigation. Some researchers believe they act as specifically sensitive (chemosensory) organelles that trigger spore germination under favorable conditions. Others suggest that the appendages play a crucial role in spore maturation, participating in the formation of the spore coats and cortex. There is also a hypothesis that spore appendages are merely the consequence of certain Metabolic Disorders.

The spore germination process can be divided into three stages: initiation, activation, and outgrowth.

An endospore may fail to germinate even under favorable conditions; this process must be initiated. Activation denotes the readiness of the spore for germination while preserving its heat resistance, light refraction, and other characteristics. One of the initiating factors is heat treatment. It has been found that the higher the temperature, the faster the activation process occurs. For instance, at 65 ⁰C activation takes about 45 min, whereas at a drop to 34 ⁰C it takes approximately 48 hours.

Activation is an irreversible process that lasts several minutes and proceeds in a specific sequence: first, thermoresistance decreases and calcium dipicolinate is released into the medium, after which the spore becomes stainable; next, the spore loses diaminopimelic acid and glucosamine, followed by a decrease in light scattering.

The initiation process depends on temperature, environmental humidity, pH, and other factors.

Prior to the onset of germination, the spore intensively absorbs water and swells. Within the first minutes of germination, it loses nearly 25% of its proteins—specifically those synthesized during sporulation. Protein Hydrolysis is carried out by a specific spore protease that acts exclusively on these proteins.

During spore germination, its coats undergo lysis or rupture, allowing the germling to emerge from the envelopes.

Outgrowth is an active growth phase characterized by intensive synthesis of RNA and proteins, followed by the initiation of DNA replication within 1–2 hours.

Actinomycete spores are formed predominantly on the aerial mycelium (sporophores), and in some cases on the substrate mycelium. The arrangement of sporogenic branches in certain actinomycetes is a rather regular and constant feature. In terms of shape, spiral sporophores are most common (featuring multiple coils, primitive spirals, stretched coils, or tightly wound clusters). Based on their arrangement along the sporogenic branches, they are classified into verticillate, opposite, and sequential types.

Spore formation in actinomycetes can occur through various mechanisms. The most common and well-studied method is spore formation via fragmentation or segmentation of the sporogenic branches.

The fragmentation process is characterized by the concentration of cytoplasm around nuclear elements uniformly distributed along the hypha, which then acquires its own envelope and transforms into a mature spore. The envelope of the sporogenic filaments persists for some time but eventually breaks down, releasing the spores that can then germinate under appropriate conditions to form hyphae of new actinomycete colonies.

Segmentation involves a simple division of the sporogenic branches. Transverse septa form along the entire length of the hypha, dividing the sporophore into a series of uniform spore-cells. Prior to segmentation, the nuclear material within the sporogenic branch divides to form as many Chromatin granules as there are future spores. Cytoplasm also concentrates around each chromatin grain. Once the spore matures, the sporophore breaks apart into individual spores.

In certain actinomycete genera (Actinoplanes, Ampullariella, Streptosporangium, etc.), sac-like sporangia containing spores are formed (Fig. 2.46).

Some actinomycetes form unusual structures. For instance, representatives of the genera Dermatophilus, Frankia, and Geodermatophilus produce numerous spores through the division of hyphae in multiple planes rather than perpendicularly to the axis. Such structures are referred to as multilocular sporangia.

Fig. 2.46. Sporangia of Actinoplanes (a), Ampullariella (b), and Streptosporangium (c) containing spores

Certain actinomycetes are capable of forming sclerotia—spherical structures that contain lipid-filled cells rather than spores. A sclerotium germinates as a single unit, producing a single germ tube.

Representatives of the genera Actinobifida, Microbispora, Micromonospora, and Micropolyspora lack sporangia. Single spores, pairs, or chains (of up to 20 spores) serving as resting and reproductive forms are located directly on the mycelial hyphae or on very short sporogenic branches (sporophores).

Actinomycete spores vary in shape, appearing spherical, cylindrical, rod-like, or pyriform. The surface morphology of spores also differs among actinomycete species. Spore surfaces may feature spine-like, tuberculate, or Hair-like structures, while in some cases the envelope surface remains smooth. This characteristic is utilized for taxonomic identification of actinomycete groups.

Actinomycete spores are more resistant to adverse environmental conditions than vegetative cells. For instance, air-dried cultures of actinomycetes can remain viable for several years. Spores are also resistant to various mechanical stresses: when cultures are ground in a mixture with sand particles, the spores retain their viability, whereas similarly sized fragments of the vegetative mycelium are destroyed.

The germination pattern of actinomycete spores is uniform. Upon contact with a fresh nutrient medium, they first swell slightly, after which small bumps or buds appear on their surface; these elongate and grow into long filaments. The filaments branch, eventually forming a mycelium—the characteristic actinomycete colony.

Cysts function as resting forms in various bacterial groups, including Azotobacter, spirochetes, myxobacteria, and rickettsiae. In most myxobacteria, cyst (myxospore) formation represents a stage of the life cycle. Following the active growth phase, myxobacterial cells aggregate to form fruiting bodies, which consist of a mass of mucus in which the cells are embedded. Within these fruiting bodies, the cells enter a resting state (Fig. 2.47).

Fig. 2.47. Morphological differentiation in myxobacteria:

a - diagram of the Myxococcus developmental cycle: 1 - actively dividing vegetative cells; 2 - aggregation of fruiting body precursor cells; 3 - fruiting body; 4 - myxospores; b - fruiting bodies: 1 - Myxococcus; 2 — Chondromyces

The formation of myxospores is accompanied by the synthesis of a specific protein. DNA is not synthesized de novo, but rather transferred from the vegetative cell. A myxospore may contain three to four copies of the chromosome. Myxospore cysts are significantly more resistant to heat, desiccation, and various physical factors than vegetative cells; therefore, this structure can be regarded, to a certain extent, as a resting form.

In some bacterial species, cysts are morphologically indistinguishable from vegetative cells, but in most cases, their formation is accompanied by marked morphological and structural changes: the vegetative cell wall thickens, resulting in the formation of optically dense, highly light-refractive structures (Fig. 2.48).

In representatives of the genus Azotobacter, cyst formation is accompanied by changes in cell morphology, loss of flagella, accumulation of PHB granules in the cytoplasm, and the synthesis of additional outer (exine) and inner (intine) layers. These protective layers differ from each other both structurally and in chemical composition.

Fig. 2.48. Schematic diagram of the Structure of Prokaryotic resting forms:

a - myxospores of myxobacteria; b - Azotobacter cysts; c - cyanobacterial akinetes; d - endospores: 1 - nucleoid; 2 - cytoplasm; 3 - cytoplasmic membrane; 4 - cell wall; 5 - capsule; 6 - reserve material granules; 7 - inner layers (intine); 8 - outer layers (exine); 9 - thylakoids; 10 - sheath; 11, 12 - inner and outer spore membranes, respectively; 13 - cortex; 14 - multi-layered spore coats; 15 — exosporium

The resting forms of certain cyanobacteria are called akinetes. They are larger than vegetative cells and possess an elongated or spherical shape and a thick cell wall. Akinetes are formed during the culture growth lag phase. During this process, the cell enlarges, reserve material granules (glycogen, polyphosphates, cyanophycin granules) accumulate in the cytoplasm, and carboxysomes are formed. Concurrently, the peptidoglycan layer thickens, and the mucilaginous sheath becomes denser. Overall, the akinete walls contain more lipids and polysaccharides, whereas the cytoplasm contains less water compared to a vegetative cell. During akinete formation, DNA content and the number of ribosomes increase, while the amounts of chlorophyll and phycobilin pigments decrease.

Once formed, these resting forms can retain their viability for varying periods (Table 2.6) and germinate under favorable conditions to give rise to metabolically active cells.

Table 2.6. Resistance of prokaryotic resting forms to extreme conditions